METHODS AND COMPOSITIONS FOR THE TREATMENT OF GASTROINTESTINAL DiSORDERS

TECHNICAL FIELD

This disclosure relates to methods and compositions for treating gastrointestinal disorders, obesity, congestive heart failure, benign prostatic hyperplasia (BPFI) and other disorders.

BACKGROUND

irritable bowel syndrome (LBS) is a common chronic disorder of the intestine that affects 20 to 60 million individuals in the US alone (Lehman Brothers, Global 11 ealth care- Irritable Bowel Syndrome industry Update, September 1999). !BS is the most common disorder diagnosed by gasiroenterologists (28% of patients examined) and accounts for 12% of visits to primary care physicians (€armileri 20(51 Gastroenterology 120:652-668). In the US, the economic impact of IBS is estimated at $25 billion annually, through direct costs of health care use and indirect costs of absenteeism from wost (Talley 1995 Gastroenterology 109: 1736- 1741 ). Patients with IBS have three times more absenteeism from work and report a reduced quality of life. Sufferers may be unable or unwilling to attend social events, maintain employment or travel even short distances (Drossman 1993 Dig Dis Sci 38: 1569- 1580). There is a tremendous unmet medical need in this population sinee few prescription options exist to treat IBS.

Patients with IBS suffer from abdominal pain and a disturbed bowel pattern. ^' Three subgroups of IBS patients have been defined based on the predominant bowel habit: eonstipaiion-predorninant Cc-IBS), diarrhea -predominant (d-IBS) or alternating between the two (a-! BS). Estimates of individuals who suffer from C-(BS range lfom 20-50% of the IBS patients with 30% frequently cited, in contrast, io the other two

subgroups that have a similar gender ratio, c-iBS is more common in women (ratio of 3: 1) fTalley et aJ. 1995 Am , J Epidemiol 142:76-83).

The deiiπilion and diagnostic criteria for IBS have been formalized in the ^{^} ' Rome Criteria" (Orossrnan et al. 1999, Gut 45 :Supp! II; 1 -S 1 ), which are well accepted m clinical practice. Briefly, the criteria specify that for at least 12 weeks (consecutive or non-consecutive in the preceding 12 months of abdominal discomfort or pain at least iwo of the following three features must occur: ( ! ) relieved with defecation, (2) onset associated with a change in frequency of stool, and {3} onset associated with a change in form (appearance) of stool. The Rome ^' !! criteria also state that the symptoms thai cumulatively support the diagnosis of irritable bowel syndrome include: abnormal stool frequency ("abnormal" may be defined as greater than 3 bowel movements per day and less than 3 bowel movements per week), abnormal stool form (lumpy/hard or loose/watery stool), abnormal stool passage (straining, urgency, or feeling of incomplete evacuation), passage of mucus, and bloating or feeling of abdominal distension. However, the complexity of symptoms has not been explained by anatomical abnormalities or metabolic changes. This lias led to the classification of IBS as a functional (JI disorder, which is diagnosed on the basis of the Rome criteria and limited evaluation to exclude organic disease (Ringel et aϊ. 2001, Annu Rev Med 52: 31 y-338}. !BS is considered to be a ^{^} biopsychosociaf disorder resulting from a combination of three interacting mechanisms: altered bowel motility, an increased sensitivity of the intestine or colon to pain stimuli (viscera! sensitivity) and psychosocial factors (CaniiHeri 200! . Gastroenterology 120:652-668), Recently, there has been increasing evidence for a role of inflammation in etiology of IBS. Reports indicate that subsets of (IJS patients have small but significant increases in colonic inflammatory and mast ceils, increased inducible nitric oxide (NO} and synthase (iNOS) and altered expression of inflammatory cytokines (reviewed by Tailey 2000, Mεdscape Coverage of DDW week).

The present disclosure features peptides that activate and/or bind the guanylaie cyelase-€ (GC-C) receptor (reviewed by Lucas et al. 2000 Pharmacol. Rev 52:375-

414 and Vaamirager el al. 2002 Molecular and Cellular Biochemistry 230:73-83} and any of its variarscs, including but not limited to insertion, deletion, mutation, and splice variants. GC-C is a key regulator in mammals of intestinal function (although low levels of GC-C have been detected in other tissues). GC-C responds to the endogenous hormones, guaπyim and uroguanylin, and to enteric bacteria] peptides from the heal stable enterotoxin family (ST peptides). When agonists bind to GC-C. there is an elevation of the second messenger, cyclic GMP, and an increase in chloride and bicarbonate secretion, resulting in an increase in intestinal fluid secretion. The Genbank. protein CJI accession number for guanyly) cyclase C hαrnologs from multiple organisms are;

Particularly useful peptides bind to and/or activate the human GC-C receptor in th« assay described belovv using the T84 human colon carcinoma cell line.

SUMMARY

The present disclosure features compositions and related methods for treating IBS and S oϊher gastrointestinal disorders and conditions (e.g., gastrointestinal motility disorders, inflammatory bowel disease (IBD). chronic intestinal pseudo-obstruction, colonic pscudo-ohsiαietion, Crohn's disease, duodenogastrie reflux, dyspepsia, functional dyspepsia, nonulcer dyspepsia, a functional gastrointestinal disorder, functional heartburn., gastroesophageal reflux disease (GERD), gastroparesis. irritable 0 bowel syndrome, post-operative ileus, ulcerative colitis, chronic constipation, and disorders and conditions associated with constipation (e.g. constipation associated with use of opiate pain killers, post-surgical constipation, and constipation, associated with neuropathic disorders as well as other conditions and disorders are described herein,

5 The present disclosure also features compositions and related methods for treating obesity, congestive heart failure (including congestive heart failure at any of stages I- IV according to New York Heart Association (NYHA) Functional Classification} and benign prostatic hyperplasia (BPlI).

Without being bound by any particular fceory, in the case of IBS and other 0 gastrointestinal disorders the peptides are useful because they can increase gastroimes-inal .motility.

Without being bound by any particular theory, in the case of IBS and other gastrointestinal disorders the peptides are useful, in part, because they can decrease inflammation.

Without being bound by any particular theory, in the case of IBS and other gastrointestinal disorders the peptides are also useful because they may decrease

gastrointestinal pain, visceral pain, chronic visceral hypersensitivity, or hypersensitivity to colorectal distension.

Without being bound by any particular theory, in the case of salt retention., fluid. retention disorders and combinations thereof the polypeptides are also useful because they may elicit one or more of diuresis, naturesis and/or kalmresis. Thus the peptides described herein may be diuretics.

The disclosure features pharmaceutical compositions comprising certain peptides that are capable of activating the guanylate-cydase C (GC-C) receptor. Also within the disclosure are pharmaceutical compositions comprising a peptide or GC-C ^' agonist of die disclosure as well as combination compositions comprising a peptide of the disclosure and one or more additional therapeutic agents including, without limitation, the agents described herein. The other agents can be administered with the peptides of the disclosure (simultaneously or sequentially). They can also be linked to a peptide of the disclosure to create therapeutic conjugates.

Described herein is a polypeptide comprises the amino acid sequence:

A'-B'-C wherein:

A' is an amino acid sequence comprises a pre sequence depicted in PIG. 4 or is missing; B" is an amino acid sequence comprises a pro sequence depicted m FIG. 4 or is missing;

Cr is an amino acid sequence comprises a GC-C receptor agonist polypeptide amino acid sequence, wherein one or more Asn having the structure:

- *! -

is optional ly repl aced by a group having a structure selected from (a), (b) (c):

provided thai an Asa at the carboxy terminus is not replaced by structure (a) or structure (c).

In some embodiments: C comprises die amino acid sequence: Xaaj Xaa> Xaaj C ^' ysα Xaa~ Xaa _ft Xaa- X&& _% Xaay Xaaso Xaaπ CyS=; Xaa _s j X.aa _;; < Xaa. j Xa& _{! 6} (SIiQ ID NO: ! } wherein:

Xaa ₅ Is Ser. Asn, Tyr. Ala, GIn, Pro, Ly s. Giy. or Thr, or is missing-. Xa« ₂ is Mis, Asp, GIu, Aia, Scr, Asn, GIy, or is missing: Xaa;, is Thr, Asp. Ser. GIu, Fro, VaI or Leu: Xa& ₅ is Asp, Ue or GIu;

Xaa _& is lie, Irp or Leu; Xaa j is Cys, Ser, or Tyr; Xa% is Ak, VaK Thx, Ue, Me? or is missing:

Xaa-j is a _? any amino acid, b) Phe, Tyr, Asn, Trp, e) an amino acid other than Phe, Trp, or Tyr. d) non-aromatic amino acid or e) is missing; Xaajo is Ala, VaL Met, ^' fhr or He; Xaai t h Ala or VaI;

XiXiπ la Aia ur Thr;

Xaaj. _! is GIy, Ala or Ser; Xaas5 is Cys, Tyr or is missing; and

Xaajή is: a) Tφ, Tyr or Phe; b) Lys or Arg; c) is missing or d) His or Leu or Ser. In other embodiments: the polypeptide is selected from:

5 (a) a polypeptide comprises A', B' and C Vherein one or more Asn is optionally replaced by a group havmg a structure selected from (a), (h) and (c):

(h) a polypeptide comprises B' and C, wherein one or more Asn is optionally replaced by a group having a structure selected from (a), (b) and (e);

(cϊ a polypeptide comprises A" and C wherein one or more Asn is -■Q optionally replaced by a group having a structure selected from (a), (b) and (c); and

(d) a polypeptide comprises C wherein one or more Asn is optionally replaced by a group having a structure selected from (a), (b) and fc),

Io other embodiments: the polypeptide is selected from: ^■ :5 (a) a polypeptide consists essentially of A ^" , B' and C wherein, one or more Asn ∑s optionally replaced by a group having a structure selected from (a), (b) and (c);

(b) a polypeptide consists essentially of B' and C ⁵ , wherein one or more

Asn is optionally replaced by a group having a structure selected from (a), (b) and (e); 20 (C) a polypeptide consists essentially of A' and C wherein one or more

Asn is optionally replaced by a group having a structure selected from (a), (b) and (c); and

(d) a polypeptide consists essentially of C wherein one or more Asn is optionally replaced by a group having a structure selected from (a} _> (b) and (e). 25 in other embooirnenls: the polypeptide is selected from:

(a) a polypeptide consists of A', B' and C 'wherein one or more Asn is optionally replaced by a group having a structure selected from (a), (b) and (c);

(b) a polypeptide consists of B' and C\ wherein one or more Asn is ^■ JO optionally replaced by a group having a structure selected from (a), (b) and (c);

(c,s s polypeptide consists of A ¹ and C wherein one or more Asn is optionally replaced by a group having a structure selected from (ah (b) and (cϊ; and (d) a p ^o lypeptide consists oi ^' C wherein one or more Asn is optionally replaced by a group having a structure selected from (a), (b) and (c).

hi other embodiments; the polypeptide is selected from:

(a} a polypeptide comprises A ', S' and C'wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c);

(b) a polypeptide comprises B ^* and C, wherein one or more Asn is 10 replaced by a group having a structure selected from (a), Sb) and (c);

(c) a polypeptide comprises A' and C wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); and

(d) a polypeptide comprises C wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (e),

15 in other embodiments: the polypeptide is selected from:

(a) a polypeptide consists essentially of A', B' and C wherein one or ^■ more Asn is replaced by a group having a structure selected from (a), (b) and Ce);

(b) a polypeptide consists essential!)' of B' and C, wherein one or more :?0 Asn is replaced by a group having a structure selected from (a), (b) and (c); fc) a polypeptide consists essentially of A' and C wherein one or more Asn is replaced by a group having a structure selected from (a), (h) and (ej; and (d) a polypeptide consists essentially of C wherein one or more Asn is replaced by a group having a structure selected from (a _. ), (b) and (c).

2b

Io other embodiments: the polypeptide is selected from:

(a) a polypeptide consists of A\ B" and C", wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (e); ih} a polypeptide consists of B' and C, wherein one or more Asn is .30 replaced by a group having a structure selected from (a), (b) and (c);

• s -

(c) a polypeptide consists of A' and C\ wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (e): and Cd) a. polypeptide consists of C" wherein one or more Asn is replaced by a group having a structure selected from fa), Cb) and Cc),

In various embodiments: C comprises an amino acid sequence depicted in Figure 1 , wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); C consists essentially of an amino acid sequence depicted in Figure I ₅ wherein one or more ASK is .replaced by a group having a structure selected from (a), (b) and (c); C consists of an amino acid sequence depicted in Figure ! , wherein one or more Asn is replaced by a group having a structure selected from Ca), (b> and ϊc); C comprises an amino acid sequence depicted in Figure 2, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and Cc); C consists essentially of an amino acid sequence depicted in Figure 2, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); C consists of an amino acid sequence depicted in Figure 2, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and {c}; C comprises an amino acid sequence depicted in Figure 3, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); C" consists essentially of an amino acid sequence depicted in Figure 3, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); C consists of an amino acid sequence depicted in Figure 3, wherein one or more Asn is replaced by a group having a structure selected from {a), Cb) and (c); the polypeptide comprises an amino acid sequence depicted in FIG. 4, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); the polypeptide consists essentially of an amino acid sequence depicted in FlO. 4, wherein one or more Asn is replaced by a group having a structure selected from (a), (b) and (c); the polypeptide consists of an amino acid sequence depicted in FIG. 4, wherein one or more Asn is replaced by a group having a structure selected from (a), Cb) and (c); the polypeptide comprises an amino acid sequence depicted in FiG. 4, wherein one or more Asn is replaced by a

- 1> .

group having a structure selected from (a), (b) and (c); the polypeptide consists essentially of an amino acid sequence depicted in FIG. 4.

Ia various embodiments: one or more Asn is replaced by a group having a structure selected from (a), (fa) and fc); the polypeptide consists of ail amino acid sequence depicted in FIG. 4, wherein one or more Asn is replaced by a group having a structure selected frnni (a), (b) and (c); one or more Asn is replaced by a group having a structure selected from (a) and (c); one or more Asn is replaced by a group having structure (a); one or more Asn is replaced by a group having structure (c); wherein one or more Asn is replaced by a group having structure (b); an Asn at the amino terminus of the polypeptide is replaced by a structure selected from (a), (b) and (c); an Asn at the carboxy terminus of the polypeptide is replaced by a structure Cb); an Asn that is neither at the carboxy terminus of the polypeptide nor at. the amino terminus of the polypeptide is replaced by a structure selected from (a), (b) and (e); all Asn are replaced by a structure selected from (a), (b) and Ce) at least two Asn are replaced by a structure selected from (a), (b) and (e); at least three Asn are replaced by a structure selected from {a), (b) and (c); wherein at least four Asn arc replaced by a structure selected from (a), (b) and (e); at least five Asn are replaced by a structure selected from (a), (b> and (c); at least six Asn are replaced by a structure selected from Ca), (b) and (c); all Asn replaced by a structure selected from (a), (h) and (c) are replaced by structure (a); all Asn replaced by a structure selected from (a), Cb) and (c) are replaced by structure (b); all Asn replaced by a structure selected from (a), (b) and (c) are replaced by structure {c}; at least one Asn within A', when A' is present, is replaced by a structure selected from (a), (b) and (c); at least one Asn within B\ when B' is present, is replaced by a structure selected from (a), (b) and (c); at least one Asn within C is replaced by a structure selected from (a), (b) and (c); all Asn within C are replaced by a structure selected from fa), Ib) and (c); at least one Asn within A\ when A" is present, Ls replaced by structure (a); at least one Asn within B\ when B' is present, is replaced by structure (a); at least one Asn within C. when C is present, is replaced by structure (a); at least one Asn within A ', when A' is present, is replaced by structure (b); at least one Asn within B', when B' is present, is replaced by

- 50 -

structure (b): at least one Asn within C\ when C is present is replaced by structure (b); at least one Asn within A\ when A' is present, is replaced by structure (c); and at least one Asn within B', when B' is present is replaced by structure (c); at least one Asn within C\ when C is present is replaced by structure (c).

In some cases the polypeptide is selected from the polypeptides in Table A:

TABLE A

FGTCEiCASAACTGC (SEQ ID NO; } PGTCHICATAACTGC CSEQ ID NO: )

PGTCEICANAACTGC (SEQ !D NO: }

I ⁵ GTCEICAQAACTGC (SEO ID NO: }

PGTCEiCARAACTGC (SEQ IE) NO: )

PGTCE!CABAACTGC (SEQ ID NO: ) PGTCEiCADAACTGC (Sl-Q IU NO: }

PGTCEiCAGAACTGC (SEQ SD NO: )

TH 'Gl ^' CEiCAAAACTGC (SEQ ID NO; )

XX GTCEICAMAACTGC (SEQ ϊD NO: )

PGTCEICAiAACTGC (SEQ ID NO: ) PGTCEiCALAACTGC (SEQ [D NO: }

PGTCEICAVAACTGC (SEQ 03 NO: )

PCiTCEiCAHAACTGC (SEQ ID NO: )

PGTCEGϊCAYA ACTGC (SEQ ID NO; )

PGTO-IGCAYAACTGC (SEQ ID NO; ) PGTCEϊCGAYAACTGC (SEQ ID NO: )

PGTC E 1C: AG YAACTGC (SEQ ID NO: )

FGTCEiCAYGAACTGC (SEQ ϊD NO; )

PGTCBICAYAGACTGC (S EQ ID NO; }

PGTOπCA YAAGCTGC (SEQ ID NO: } PGTCεϊCA YAACGTGC (SEQ ID NO: } i'GTCE ICA YAACTGGC (SEQ ID NO: }

FGTCAEICAYAACTGC (SEQ ID NO: )

PGTCEAICAYAACTGC (SEQ ID NO: )

PGTCEϊACλY AACTGC (SEQ ID NC): )

PGTCEiC AλYAλ€TGC (SEQ ID NO: ) PGTCEICAYAAACTGC CSEQ ID NO: )

PGTCEICAY AACATGC (SEQ ID NO; )

PCJTCEICAY AACTAGC (SEQ FD NO: )

PGTCEiCAYAACTGAC (SEQ ID NO: )

PGTCAEICAAYAACTGC (SEQ ID NO: ) PGTC EAlC A AY AACTGC (SEQ ID NO: )

PGTCEIACAAYAACTGC (SEQ ID NO: )

KDDCELCVNVACTGCL

KDECHLCVNVACTGCL

KDDCI;LC~ ^" VNVACTGC KDECELCVNVACTGC

ECELCINVACTGC

ECELCVNYACTGCL

ECELCVN VACTGC

EKTLRl IA N DDCB LC VN VACTGC FKTLRTϊANDDCELCVNVACTGCL

FKTLRTIANDECELCVNVACTGCL

FKTLRl !ANDECELC VN VACTGC

NDDCELCVNVACTGC

NDDCELCVNVACTGCL NDECELCVNIACTGC

NDECELCVNVACϊGCL

NDECELCVNVACTGC

PNTCEiCANAACTGC

FNTCEiCAYAACTGC TDECE LCIN VACTGC

TIANUDCbLCVNVACTGCL

- ϊ 2 -

TϊANDDCELCVNV ACTGC

TiANDECBLCVNVACTGCL

IIANDECELCVN VACTGC

TfAlOECELCIN VACTGC TfATDECELCfNVACTGC;

MNAWLLS VLCLLGALAVLVEGVTVODGDLSFFLESVKQLKi ϊLREVQEPϊ LM

SHKKFALRLPICPVAPELCSQSAFPEALRPLCEfCPNAEE)LORLEAIAQDFNTCE!

CAYAACTGC;

EDPGTCEICA YAA(TGC; PSTCEICAYAACAC]C;

PNTCGtCAYAACTGC:

NDDCEEXVNBACTGCL;

FKTLRTIANDI)CEtCVNVACTGCL;

FKTiRlTANDDCLCVNVACTGCL; LO/ALRTMDNDECELCVNiACTGC;

FKTLRTiANDUCELCVNVACTCCL

N DDCH LC V N V ACTGC L

MDDCELCVNVACTACL

NDDCELCVNYAC ¹ AGCL NDDCELCVNAACTGCL

NDDCELCVAVACTGCL

NDDCELCANVACTGCL

NDDCE ACVN V ACTGCL

NDDCALCVNVACTGCL NDACELCVNVACTGCL

NADCELCVN VACTGCL.

λDDCELCVN ^' VACTGCL

NDDCELCAYAACTGCL

N DDC E LCV NPACTGC L NiDDCELCVNVACTGCLKiC

NDDCELCVNVACTACLKK.

N DDC ELCVN VACTGCl

NDBCE XVNV ACTCK:: L NDECE ..CVNVλCTACL

NDECE ,CVNVACAGC E NDECE LCVNAACTGCL NDECELCVAVACTCCL

NDECELCANVACTGCL N DEC EACVN VACTGCL NDECALCVNVACTGCL N D A C IiLC VN VACTGC L NADCELCVNVACTGCL ADECBLCVNVACTGCL

NDECE XAYAACTGCL

NDECE XVNPACTGCL NDECE XVNVACTGCLKK

NDECELCVNVACTACLK-K NDECELCVNVACTGCI

NDDCELCVNVACTGC N DDC ELCVN VACTAC

NDDCELCVNVACAGC

NDDCELCVNAACTGC

NDDCELCVAVACTGC

NDDCELCANVACTGC

NDDCEACVNVACTGC

NDDCALCVNVACTGC

NDACELCVNVλCTGC

NADCELCVNVACTGC

ADDCELCVNVACTGC

NDDCELCAVAACTGC

NDDCΈLCVNPACTGC

NDECELCVNVACTGC

- 1-1

Nf)HCELCVNVACTAC

NDBCELCVNVACAGC

NDECELCVNAACTGC

NDECRLCVλVACTGC NOEC BLCAN VACTGC

NDECEACVNVACTGC

NDECALCVNVACTGC

NDACELC VN VACTGC

NADCELCVNVACTGC ADECELCVNVACTGC

NDECELCAYAACTGC

NDECELCVNPACTGC

N DDC E LCV N V ACTGC A

NDECELCVNVACTGCA PGTCEICAYAACTAC

PGTCElC AYAACTGC L FGTCE iCAYAACTGCLKK PGTCEICAYAACTGCl wherein one or more Assi Ls optional Iy replaced by a group having a structure seiecied from (a), (b) and (c).

in some embodiments: C comprises an amino acid sequence selected from: PGTCEiCAYAACTGC (SEQ ID NO: ); and NDDCELCVNVAtTGCL (SEQ ID NO; ), wherein cmc or more Asn is replaced by a group having a structure selected from (a), fb) and (c).

Also disclosed is a polypeptide produced by the hydrolysis of structure Cb) within a polypeptide; a polypeptide produced by the hydrolysis of structure (a) within a polypeptide; a polypeptide produced by the hydrolysis of structure (c) within a

polypeptide; a polypeptide wherein none of the Asa are replaced by a structure selected from (a), (b) and (c); and a polypeptide that is purified.

Also describee! is a pharmaceutical composition comprises an aforementioned polypeptide.

A method of treating a gastrointestinal disorder comprising administering a pharmaceutical composition comprising any oi ^' the forgoing polypeptides is described.

In various embodiments of the method: the gastrointestinal disorder is selected, from: a gastrointestinal motility disorder, chronic intestinal pseudo-obstruction, colonic pseudoobstruction, Crohn's disease, duodenogastrie reflux, dyspepsia, functional dyspepsia, normlcer dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GERD), g&stroparesis, irritable bowel syndrome, post-operative ileus, inflammatory bowel disorder, ulcerative colitis, constipation, chronic constipation, chronic idiopathic constipation.

A method of treating heart failure comprising administering a pharmaceutical composition comprising any of the forgoing polypeptides is described.

A method of treating obesity comprising administering a pharmaceutical composition comprising any of the forgoing polypeptides is described. In various embodiments: me disorder is constipation; the disorder is chronic idiopathic constipation; the disorder Ls irritable bowel syndrome (e.g., diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable, bowel syndrome; alternating-irritable bowel syndrome); the disorder is inflammatory bowel disorder; the disorder is Crohn's disease; the disorder is ulcerative colitis.

A method of treating heart failure disorder comprising administering a pharmaceutical composition comprising any of the forgoing polypeptides is described.

- l ή -

λ method of treating benign prostatic hyperplasia comprising administering a pharmaceutical composition comprising any of the forgoing polypeptides is described.

A method of decreasing gastrointestinal pain or visceral pain comprising administering a pharmaceutical composition comprising any of the forgoing polypeptides is described.

Also described is a method of preventing or treating a side-effect associated with opioid administration, the method comprises administering to a patient that is being treated with an opioid a forgoing polypeptide, in various embodiments; none of the Asp are replaced by a structure selected from (a), (b) and (c); the patient is being treated with an opioid selected from the group consists of alfemanil, buprenorphine, butorphaπoi, codeine, dexocine, dihydrocodeine, fentam-1, hydrocodone, hydroiπorphone, levorphanol, meperidine (pethidine), methadone, morphine. nalbuphine, oxycodone, oxymorphone, pentazocine, propiraro, propoxyphene, sufentanil and tramadol; the patient is being treated with, an opioid selected from the group consists of: morphine, codeine, oxycodone, hydrocodone. dihydrαcodeine, propoxyphene, fentanyi and tramadol; the side effect is selected from the group consists of constipation, nausea and vomiting; the side effect is constipation; the side effect is nausea; the side effect is vomiting; the method further comprises administering an opioid antagonist (e.g., naloxone or naltrexone); the polypeptide is one in Table A.

Also described is a method of treating pain or preventing pain comprises administering an opioid and a CJCC receptor agonist, hi various embodiments: none of the Asp arc replaced by a structure selected from (a), (b) and Cc); the patient is being treated with an opioid selected from the group consists of aifentanil, buprenorphirse, buiorphanol codeine, dezoeine, dihydrocodeine, fentanyl, bydrocodone, hydromorphone, levorphanol, meperidine (pethidine), methadone, morphine, nalbuphine, oxycodone, oxymorphone, pentazocine, propiratπ, propoxyphene, sufentanil and tramadol; the patient is being treated with arc opioid

selected iron; the group consists of: morphine, codeine, oxycodone, hydrocodone, dihydroeαdeiπe, propoxyphene, fentaayl and tramadol; the side effect is selected from the group consists of constipation, nausea and vomiting; the side effect is constipation; the side effect is nausea; the side effect is vomiting; the method further comprises administering an opioid antagonist (e.g., naloxone or naltrexone); the polypeptide is o.πe in Table A,

Also described is a method of treating or preventing pain comprises administering a pharmaceutical composition comprises an opioid and a GCC receptor agonist. In various embodiments: none of the Asp are replaced by a structure selected from (a), (b) and (c); the patient is being treated with an opioid selected from the group consists of aifentanii, buprenorphine, butorphanol, codeine, dezoeme, dihydroeodeine, fentanyl, hvdrocodone, hydromorphonc, levorphaπoi, meperidine (pethidine); methadone, morphine, nalbuphine, oxycodone, oxymorphone, pentazocine, propirarn, propoxyphene, sufentanil and tramadol; the patient is being treated with an opioid selected from the group consists of: morphine, codeine, oxycodone, hydroeαdone, dihydroeodeine, propoxyphene, fentanyl and tramadol; the aide effect is selected from the group consists of constipation, nausea and vomiting; the side effect is constipation; the side effect is nausea; the side effect is vomiting; the method further comprises administering an opioid antagonist (e.g., naloxone or naltrexone); the polypeptide is one in Table A,

in various embodiments of the forgoing methods; the pain is viscera! pain; the pain is gastrointestinal pain; the pain is gastrointestinal pain; the pain is acute pain; the pain is inflammatory pain; the pain is neuropathic pain; the pain is post surgical pain; the pain is bone pain; and the pain is chronic pain;

Also described is a pharmaceutical composition comprises an opioid and a GCC receptor agonist. In various embodiments: the GCC receptor agonist is & forgoing polypeptide; none of the Asp in the polypeptide are replaced by a structure selected from (a), (b) and (c); the opioid is selected from the group consists ofaifentaniL

huprenorphine, buiorphanol, codeine, dezoeine, dihydrocodeine, fentanyl, bydrocodoπe, bydrorøorphone, levorphanol, meperidine (pethidine), methadone, morphine, nalbuphine, oxycodone, oxymαrphone, pentazocine, propiram, propoxyphene, sufentanil and tramadol; the opioid is selected from the group consists of: morphine, codeine, oxycodone, hydrocodone, dihydroeodeine. propoxyphene, feniαπy! and tramadol; and the polypeptide is one m ^' Table A.

Described herein is a pharmaceutical kit comprising: (a) a first container containing pharmaceutical dosage units comprises an effective amount of an opioid; and Cb) a second container containing pharmaceutical dosage units comprises an effective amount of a GCC! receptor agonist. In various embodiments: the GCC receptor agonist, .is a forgoing polypeptide; none of the Asp in the polypeptide are replaced fay a structure selected from (a), (h) and (c); the opioid is selected from the group consists of aifenianii, huprenαrphine, butorphanol, codeine, dezoeme, dihydrocodeine, fentanyl, hydrocodone, hydromorplione. levorphanol, meperidine (pethidine), methadone, morphine, nalbuphine, oxycodone, oxymorphoπe. pentazocine, propiram, propoxyphene, sufentanil and tramadol; the opioid is selected from she group consists of: morphine, codeine, oxycodone, hydrocodone, dihvdrocodeme, propoxyphene, fentanyl and tramadol; and the polypeptide is one in Table A,

Also described is a polypeptide comprises the amino acid sequence:

A'-B'-σ wherein:

A ⁵ is an amino acid sequence comprises a pre sequence depicted in FIG. 4 or is missing; B" is aB amino acid sequence comprises a pro sequen.ee depicted m FiG. 4 or is missing;

C is an amino acid sequence comprises a GC-C receptor agonist polypeptide amino acid sequence.

In various embodiments: C comprises an amino acid sequence selected from;

PGTCBiCASAACTGC (SEQ ID NO: ) PGϊCEICATAACTG€ (SEQ ID NO; } PGTCEICA NA ACTGC (SEQ ID NO: } FGTCBICAQAACTGC (SEQ ID NO: ) FGTCB!CARAλCTGC (SEQ ID NO: } PGTCElC AEA ACTGC (SEQ ID NO; } PGTCEICADAAC'KIC (SEQ ID NO: ) PGTCEICAGAACTGC (SEQ ID NO: ) PGTCEfCAAAACTGC (SEQ ID NO: ) PGTCEICAMAACTGC (SEQ ID NO; } PGTCElCAiAACTGC (SEQ ID NO; ) PGTCEiCALAACTGC (SEQ ID NO: ) PGTCEiCAVAACTGC (SEQ ID NO: } PGTCEICAHAACTGC (SEQ ID NO: ) PGTCEGlCAYAACTGC (SEQ iD NO: } PGTCElGCA YA ACTGC (SEQ IO NO; ) PGTCElCGA YAACTGC (SEQ JD NO: ) PGTCEICAGY AACITJC (SEQ ID NO: ) PGTCOCA YGAACTGC (SEQ ID NO: ) PGTCBICAYAGACTGC (SEQ ID NO: } PGTCElCAY AAGCTGC (SEQ ID NO; } PGTCmCλYλACGTGC (SEQ ID NO: } PCiTC I* iCA Y AACTGGC (SEQ ID NO: } PGTCAEiCAYAACTGC (SEQ ID NO; ) PGTCEAICAYAACTGC (SEQ ID NO: } PGl'CEIACAYAACTGC (SEQ ID NO: } PGTCEICAAYAACTGC (SEQ ID NO: } PGTCEICAYAAACTGC (SEO ID NO: ) PGTCEICAYAACATCK; (SEQ ID NO: } PGTCEiCAYAACTAGC (SEQ iD NO: ) PGTCEiCAYAACTGAC (SEQ ID NO; )

PGTCABICAAYAACTGC (SEQ JD NO: ) PGTCEAlC AA YAACTGC (SEQ ID NO: ) and

FGTCEiACAAYAACTGC (SEQ ID NO: ); C composes an amino add sequence selected from the processed active sequences shown hi FlG. 4; A' is missing and B' is an amino acid sequence comprises a pro sequence depicted in FKl, 4; A ^s is an amino acid sequence comprises a pre sequence depicted in FfG. 4 and B' is an amino acid sequence comprises a pro sequence depicted in FiG. 4; and the polypeptide is purified.

The disclosure includes methods for treating various gastrointestinal disorders by administering a peptide that acts as a partial or complete agonist of the GC-C" receptor. Unless otherwise specified, the term "agonist" used herein refers to an agonist of die GC-C receptor, The peptide contains up to four cysteines that form one or two disulfide bonds, ϊn certain embodiments the disulfide bonds are replaced by other covaleπt cross-links and m some cases the cysteines are substituted by other residues Io provide for alternative eovaJeπt cross-links. The peptides may also include at least one trypsin or ehymotrypsin cleavage site aπ.d/or a earhoxy-ierrnina] analgesic peptide or small molecule, e.g., AspPhe or some other analgesic peptide. When present within the peptide, the analgesic peptide or small molecule may be preceded by a diymotrypsm or trypsin cleavage site that allows release of the analgesic peptide or small molecule. The peptides and methods of the disclosure are also useful for treating pain and inflammation associated with various disorders, including gastrointestinal disorders. Certain peptides mciude a functional ehymo trypsin or trypsin cleavage site located so as to allow inactivation of the peptide upon cleavage. Certain peptides having a functional cleavage site undergo cleavage and gradual inactivation in the digestive tract, and this is desirable in some circumstances, in certain peptides, a functional chymotrypsin site is altered, increasing the stability of the peptide in vivo (e.g.. guanyiirt).

Toe disclosure includes: a method for increasing intestinal motility comprising administering a GC-C receptor agonist, eg,, a peptide described herein, to a patient HI need thereof.

5 The disclosure includes a method for treating a disorder associated with reduced gastrointestinal transit rates or reduced gastrointestinal motility comprising administering a GC-C receptor agonist, e.g., a peptide described herein, to a patient in need thereof

^• ;o The disclosure also includes a method for treating a gastrointestinal hypomotility disorder comprising administering a GC-C receptor agonist, e.g., a peptide described herein, to a patient in need thereof.

The disorders which can be treated by administering a GC-C ^' receptor agonist, e.g., a 16 peptide described herein, include constipation, constipation dominant irritable bowel syndrome and pelvic door dyssyπergia.

The disclosure features a method treating a noπ-infiarrsrnatory gastrointestinal disorder comprising administering a CJOC receptor agonist e.g., a peptide described 20 herein, to a patient in need thereof.

^' The disclosure includes a method treating a gastrointestinal disorder other than Crohn's disease and ulcerative colitis comprising administering a GC-C receptor agonist, e.g., a peptide described herein, to a patient in nccό thereof.

25

The disclosure includes methods for treating other disorders such as congestive heart failure and benign prostatic hyperplasia by administering a peptide or small molecule (parenteral!)-- or orally) that acts as an agonist of the GC-C receptor. Such, agents can be used in combination with natriuretic peptides (e.g., atrial natriuretic peptide, brain

30 natriuretic peptide or C- type natriuretic peptide), a diuretic, or an inhibitor of angiotensin converting euxyrae.

. ?2 -

The disclosure features methods and compositions for increasing intestinal motility. Intestinal motility involves spontaneous coordinated distentions and contractions of the stomach, intestines, colon and rectum to move food through the gastrointestinal tract during the digestive process.

In certain embodiments the patient has been diagnosed as suffering from IBS according to the Rome criteria. In certain embodiments the patient is female.

The peptide can contain additional carboxy terminal or amino terminal ammo acids or both. For example, the peptide can include an amino terminal sequence thai facilitates recombinant production of the peptide and is cleaved prior to administration of the peptide to a patient. The peptide can also include other amino terminal or carboxy terminal anii.no acids. In some eases the additional amino acids protect the peptide, stabilize the peptide or alter the activity of the peptide. In some cases sums or all of these additional amino acids are removed prior to administration of the peptide to a patient. The peptide can include I ₅ 2, 3, 4, 5, H) ₅ 15, 20. 2.5, 30, 4O ₅ 50. 60, 70 80. 90, 100 or more amino acids at its amino terminus or carboxy terminus or both. The number of flanking amino acids need not be the same. For example, there can be 10 additional amino acids at the amino terminus of the peptide and none at the carboxy terminus.

in certain embodiments the peptides include either one or two or more contiguous negatively charged amino acids (e.g.. Asp or GIu) or one or two or more contiguous positively charged residues {e.g., Lys or Arg) or one or two or more contiguous positively or negatively charged amino acids at the carboxy terminus, in these embodiments all of the flanking amino acids at the carboxy terminus are either positively or .negatively charged, hi other embodiments the carboxy terminal charged amino acids are preceded by a Leu, For example, the following amino acid sequences can be added to the earboxy terminus of the peptide: Asp; Asp Lys; Lys Lys Lys Lys Lys Lys; Asp Lys Lys Lys Lys Lys Lys; Leu Lys Lys; and Leu Asp, Il is also possible to simply add Leu at the carboxy terminus,

In a first aspect, the disclosure features a polypeptide comprising, consisting of. or consisting essentially of the amino acid sequence: Xaa-. Xaa^ Xaa;s Cys* Xaa* Xaa _h Xaa? Xa&x Xaa ^, > Xaaκ _> Xaan Cysj; Xaa ^ Xaaμ Xaa^ Xaaj<- _> (SEQ ID NQ: 1} wherein:

XΆΆ _\ is Scr, Asn, Tyr, Ala, Gin. Pro, Lys, GIy, or Tbr, or is nnssiojr, 5 Xaa? is His, Asp, GIu, Ala, Scr, Asn, GIy, or is missing:

XatM is Thr, Asp, Ser. GIu, Pro, VaI or Leu;

Xaas is Asp, Oe or GIu;

X&ά' _d is lie, Trp or Leu;

Xaa> is €ys, Scr, or TyT: i D Xaax is Ala, VaL Thr, He, Met or is missing;

Xaa. ₉ is a) any ammo acid, b) l ^J hc, IVr, Asn, Trp, c) an amino acid other than P he, Tip, or ^' Tyr, d) non-aromatic amino acid or e) is missing;

Xaaio is Ala, VaI, Men, Thr or lie;

Xasa is Ala or YaI; 15 Xa.as _. -i is Aia or Thr;

Xaa ^ is GIy, Aia or Sen is €ys, Tyr or is missing; and

Xauiή is: a) Trp, Tyr or Phe to create a chymoirypsin cleavage sue; b) Lys or Arg to create a trypsin cleavage site; e) is missing or d) FHs or Leu or Ser.

20 !rs some embodiments, Xaaj is preceded by Lys or Tyr.

In certain embodiments, a Cys is replaced by any amino acid other than Cys. Certain such polypeptides will have fewer disulfide bonds.

in a related aspect the disclosure features a composition comprising a polypeptide composing, consisting of, or consisting essentially of the amino acid sequence: Xaa.) 25 Xaa> Xaa^ Cys ₄ Xaa _? Xaa ₆ Xaa ₇ Xaa ₈ Xaa^ Xa^ ₀ Xaa^ Cys^ Xaa^ Xaa _S4 Xaa _i5

Xaa _H . (SiϊQ ID NO:! ) wherein; Xaa] is Ser, Asn, Tyr, Ala, GIn ₅ Pro, Lys, GIy; or Tin; or is missing; Xaa ₃ is His, Asp, GIu, Aia, Ser, Am, GIy, Pro or is missing; Xa ^; .u is Thr, Asp, Ser, GIu ₅ Pro, Va! or Leu; Xaa _s is Asp, He or GIu; Xaa^is He, Trp or Leu; Xaa? is

Cys, Ser, or ϊyπ Xaa$ is Ala, VaI, Thr, Oe. Met or is missing; Xaa^ is Plκ _\ Tyr, Asn. IVp, an amino acid other than Phe, Tip, or Tyr, is a uou-aromafic amino acid or is missing; Xaa«; is Ala. VaL Met, Th? or He; Xaaj ; is Ala or VaI; Xaa, _:; is Ala or Thr; Xua:.f is GIy, Ala or Ser; Xaais is Cys, Tyr or is missing; and Xaa^is; a) Tip, Tyr or Phe to create a ehyrnotrvpsin cleavage site; b) Lys or Aig to create a trypsin cleavage site; e) ss missing or d) His or Leu or Ser and a pharmaceutically acceptable earner. \n related aspects, the disclosure features a pharmaceutically acceptable tablet, pill, capsule comprising the peptide,

In a related aspect, the disclosure- features a polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence: Xaaj Xaa ₂ X^ Qy-S ₄ Xatu Xaa _(i Xaih Xaas Xaa< _; . Xaa _Kf Xaa _n CySj ₂ Xaa _{f .} ; Xaa^ Xaa _{f 5} Xaa^, (SEQ ID NO: i) wherein:

Xaaj is λsn, any amino acid or is missing;

Xaa _? is Asp, C)Iu, any amino acid or is missing; Xaa _. ; is Asp or GIu;

Xaa _s is any amino acid or GIu;

Xaa _f , is as\y amino acid or Leu;

Xaa?is Cys;

Xaasis any amino acid or VaI;

Xaairt is is any amino acid or VaI;

Xaan is any amino acid or Ala;

Xaa. ₃ i? is any amino acid or Thr;

Xaa;4 is is any amino acid or GIy; Xaa^ is Cys;

Xaa;,' ₍ is any amino acid. Leu or missing

hi a related aspect, the disclosure features a polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence: Asti; Xatb Xaa^ Xatu G!u ₅ Leu^ Xaa? Vais Aso^ Xaa _{j 0} Xaan Xaa _{i 2} Thfss Xaaμ Xaa _S5 Lcu^ (SBQ ID NO; } Xaa? is Asp or Glυ;

Xaa? is Asp or GIu;

Xaa.} is Cys or MpC (mercaptoproline) or Pea (penicillamine) or Dpi" (dianiinopropionic acid) or Asp or GIu;

Xaa? is Cys or M ^' pt (mercaptoproline} or Pen (penicillamine) or Dpr (dirøiinopropiome acid) or Asp or GIu;

Xaauj is Va! or Fro;

Xaai i is Ala or Aib (aipha-aminoisobutyric acid); Xaa i j is Cys or Mpt (mercaptoprolnie) or Pen (penicillamine) or Dpr (diaiinnopropionic acid) or Asp or GIu; Xaa ₅ .< is GIy or Ala;

Xauj-s is Cys or Mpt (mereapioproline) or Pen { ^' penicillamine) or Dpr (diaminopropiαπic acid) or Asp or GIu; and

In certain embodiments, where Xaa ^ is other than Cys or is missing, Xaa ₇ is Ser or an amino acid other than Cys.

In certain embodiments 1 , 2, 3, 4, 5, 6, 7. 8. 9, 10, I i or 12 of Xaa _f , Xaa, Xaa ₃ , Xaa^ Xiuv,, Xaa _? , Xaas, Xaao, Xaa so, Xaau, Xaas?, Xaa^, and Xa a ^ are any amino acid other than C'ys.

In certain embodiments, Xaa<> is any amino acid other than GIn. In other embodiments where Xaa ₂ and Xaa^ MC GIU, Xaa<;. is any amino acid other than Gin,

hi certain embodiments Xaa; and Xaa _: > are missing; Xaa^ is Thr; Xaa-? is GIu; Xaa ₍ , is Ue or Leis; Xaas is AIa, Va!, or lie; Xaa<.> is Phe or Tyr; Xaa _i(} is Ala or VaI; Xaa ₅ ! .is ASa; Xaa.) ₃ is Ala or Thr; Xaa _u ss GIy; and Xaa _κ> is Trp, Tyr, Phe, Lys, Arg or is missing.

(π certain, embodiments the polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence: Xaa _s Xaa? Xaa.? Cysa Xaiu Xtuv> Xaa? Xaag X&'άa Xaaio Xaau Cysu X&&u Xaa ^ Xaa _f 5 Xaa _t 6 (SEQ ID NCH ) is not cleaved alter Xaa- _. , by chymoirvpsin. In these embodiments wherein:

Xaa-. is Ser, Asn, Tyr, Ala, GIn, Pro, Lys, GIy, or Tin; or is missing;

Xaa? is His, Asp, Gk, Ala, Ser, Asn, or GIy, or is missing;

Xaa-ξ is Thr, Asp, Ser, GIu ₅ Pro, VaI or Leu or is missing;

Xaa? is Asp, Ik- or GIu;

Xaa? is Cya, Ser, or Tyr;

Xaa _s is Ala, Va!, Thr, JIe, Met or is missing;

Xa&> is eiiher; a) any amino acid other than Ph e and T>τ, b) any arni.no acid other than Phe. Tyr, and Trp, c) any amino acid other than Phe, Tyr, Trp. He. Leu and Vai; d) any amino acid other than Phe, Tyr, Trp. lie, Leu. VaI, and His; d) any πon- arømatte amino acid or e) .is missing:

Xaa _U i is Ala, VaK Met, Thr or lie;

Xaa-:; is Ala or Va!;

Xaasj is Ala or Thr;

Xaa _{S 5} is Cys, Tyr or i$ missing; and

Xaa K. is; a) Trp, Tyr or Phc to create a ehymotrypsin cleavage site; b) Lys or λrg to create a trypsin cleavage site; c) is missing or d) His or Leu or Ser.

!π additioji, the disclosure features variants of Xaa; Xaa^ Xaa? Cys4 Xa;u Kaa _< ; Xaai Xaa^ Xi ^' uk. Xaajo Xaaπ Cyss _^ Xaa^ Xaau Xaa^ Xaa^ (SEQ ID NO:] } that is not cleaved alter Xaa^ by chyiπotrypaiu due to the addition of an amino terminal lysine. An example of such a molecule is a human guanyiin variant having an amino terminal lysine: KPGTCEICAYAACTGC (SEQ ϊD NO: ).

In certain embodiments of the peptide comprising, consisting of, or consisting essentially of the amino acid sequence: Xaa§ Xaα _. > Xaa _? Cys^ Xaas Xaa,, Xaa? Xaas Xaao Xaaso Xaa^ Cysj? Xaa^ X&&a Xaa^ Xaa,i ₆ (SEQ ID NO:1 ) thai is not cleaved after Xaa^ by chyiπotrypsin, Xaa- _? and Xaaij are both Cys.

77 „

Also within the disclosure are variants of PGTC EICAYA ACTGC (human gυanylin) (SEQ ϊD NO; ) wherein Y is substituted by any amino acid other than, a) Phe; b) any amino acid other than Phe and Trp; c) any amino acid other than Phe, Trp, He, Lets and VaI; d) any amino acid other than Phc\ Trp, Ik, Leu, VaI and His; e) any rκ>.o-- aromatic amines acid or t) is missing.

In certain embodiments the polypeptide comprising, consisting of, or consisting essentially of toe ammo acid sequence: Xaa _f Xaa^ Xaa-j CyS ₄ Xaa ₅ X a a* Xas-..- Xaa _s Xaa,) Xaa ₁₀ Xaa _H Cys _s > Xaa _π Kna^ Xaa _{i S} Xaa^ (SEQ ID NO: i) is not cieaved after Xaa^ by either ehymotrypsin or trypsin, hi these embodiments wherein:

Xaai is Sex, As.n. Tyr, Ala, GSn. Pro, Lys, GIy, or Thr, or .is missiTig;

Xau; is His, Asp, GIu, Ala, Sen Asii, or GIy, or is missing:

Xaai is Thr, Asp, Ser, GIu ₅ Pro, VaI or Leu or is missing;

Xaa$ is Asp, He or GIu; Xd' & _t , is He, Trp or Leu;

Xaa; is (\'s, Ser, or Tyr;

Xasx ϊS AIa, VaI, Thr, lie, Met or is missing;

Xaa< _; . is either: a) any amino acid other than Lys, Arg, Phe and Tyr, b) any amino acid other than Lys, Arg, Phe, Tyr, and Trp, c) any amino acid other than Lys, Arg, Phe, Tyr, Trp, He, Leu and Vai; d) any amino acid other than Lys, Arg, Phe, Tyr, Trp, fie, Leu, VaI, and His; or e) is missing;

Xaat _t ; is Ala, VaI, Met, Thr or He;

Xiisj ; is AIa or VaI;

Xaa.} _. * is Ala or Thr; Xaaj.s is G!y, Ala or Ser;

XiUiss Ls C YS, Tyr or is missing; and

Xaasc is: a) Trp, Tyr or Phe to create a chymotrypsin cleavage site; b) Lys or Arg to create a trypsin cleavage site; e) is missing or d) His or Leu or Ser.

Ln certain embodiments of the peptide comprising, consisting of, or consisting essentially oi ^' lhe amines acid sequence; Xaa>, Xm ₂ Xaa ₅ Cys^ Xaa- _; Xaot _f . Xa.a ₇ Xaag

Xaiiq Xaa _it! Xaa _n Cys;.? Xaa _i3 Xaa ^ Xaa^ Xaa _if> (SEQ (D NO: J ) that is not cleaved alter Xaa<; by ehymotrypsin or trypsin, Xaa _? and Xaa _{! ?} are both Cys.

Useful variants of PGTCEICAYA ACTGC {human, guanyiin) (SEQ ID NO: } that should Dot be cleaved by ehymotrypsin include: PGTCH1CASAλCTGC (SEQ H ⁾ NO; }

PGTCEiCAmACTGC (SEQ ID NO: )

PGTCεICANAACTGC (SEQ ID NO: )

PGTCOCAQAACTGC (SEQ ID NO: )

PGTCBICARAACTGC (SEQ ID NO: ) PGTCEiCAEAACTGC (SEQ ID NO; )

PGTCEI€ADAACTGC (SEQ ID NO: }

PGTCElCAG AACTGC (SEQ ϊD NO: )

PGTCEICAAAACTGC (SEQ ID NO; ?

PCiTCEICAMAACTGC (SEQ ID NO: ).

Additional variants which are not likely to be cleaved by ehymotrypsin under certain conditions include:

PGTCIϊICAiAACTGC (SEQ ID NO: ) PGTCEiCALAACTGC (SEQ ID NO; ) PGTCEICAVAACTGC (SEQ iD NO: ) PGTCEiCAHAACTGC (SEQ ID NO: )

The disclosure also features deletion variants of any of the peptides describee! herein Sn which one, two, three or four amino acids, other than a Cys, arc deleted. Where two (or inert*} amino acids are deleted and the peptide comprises the sequence: Cys _a Xaa Xaa Cysh Xaa Xaa Xaa Xaa Cys _e Xaa Xaa Cysa, in some embodiments two or snore deletions ears be located between Cys _s and Cys _b or between C.y% and CyS ₄ - or between Cys _< ; and Cys _d . Thus, there can be two or more deletions between two Cys. However, in other embodiments there Is at most one deletion between each Cys, i.e., there is no .more than one deletion between each of Cys _a ami Cys _t , Cys _b and Cys _v -, and

Cys _t and Cys _< j. Thus, the disclosure includes any of the peptides described herein comprising the sequence Cys _a Xaa Xaa Oysb Xaa Xaa ^' Xaa Xaa Cys,.. Xaa Xaa Cys wherein: a) one amino acid between Cys _a and Cys^ is deleted; b) one amino acid between Cys _b aiici Cys _c is deleted: c) one amino acid between Cys _c and Cys,-- is deleted; ά) one amino acid between Cys _a and Cysj, is deleted and one ammo acid between Cy.% and Cys^ is deleted; e) one amino acid between Cys _cS and €y$ _b is deleted and one amino acid between Cys- and Cys,j is deleted; f) one amino acid between Cys _b and Cys _i: is deleted and one amino acid between Cys _e and CyS ₄ J is deleted; or g) one amino acid between CyS ₁₁ and Cys^is deleted, one amino acid between €y% and Cys _e is deleted, and one amino acid between Cys^ and Cys _< j is deleted. In addition, one or more amino acids preceding Cys _a and/or one or more amino acids following €ySii can be deleted, in certain embodiments, the deletion variants are peptides that bind to and/or activate the GC-C receptor. Sn certain embodiments, the deletion variants increase cGMP levels.

The disclosure also features deletion variants of any of the peptides described herein in which one, two, three or four amino acids (or non-natural amino acids or natural or non-natural amino acid analogs), other than a Cys (or an amino acid substituted tor Cvs. e.g., an amino acid capable of forming a covalent bond to another amino acid) is deleted. Thus, additional variants include those in which a Cys is substituted by an amino acid capable of fomsing a covaSent linkage with another amino acid (e.g., a Cys or a substitute there fore). Such ammo acids include: Mpt (mercaptoprolme) or Pen (penicillamine) or Dpr (dianunopropionic acid).

no, I includes deletion variants of human guanylin in which one, two, three or four amino acids are deleted. The deleted amino acids are between Cys _a and Cys.j as well as amino terminal to Cys ₃ .

The disclosure also features insertion variants of any of the peptides described herein in which one, two, three or four amino acids are inserted.

Where two (or more) amino acids are inserted and the peptide comprises the sequence: Cys _a Xaa Xaa Cys _h Xaa Xaa Xaa Xaa Cys _c Xaa Xaa Cy%, in some embodiments two or more insertions can be located between Cys _; , and Cy%, or between Cys^ and Cys _c or between Cys _c and Cysj. However, in other embodiments there is at most one insertion between each of Cys ₃ and Cys*, or between Cys _b and Cys _c or between Cys _c and Cys^. Thus, the disclosure includes any of the peptides described herein comprising the sequence Cys _ft Xaa Xaa Cysj _> Xaa Xaa Xaa Xaa €ys _c Xaa Xaa Cys^ wherein; a) one amino acid is inserted between Cys _a and Cy%; b) one amino acid is inserted between Cyst, and Cys _c ; e) one amino acid is inserted between Cys _c and Cys,,; d) one amino acid is inserted between Cys,, and CyS _b and one amino acid is inserted between Cysj, and Cys _ς: ; e) one amino acid is inserted between Oys _a arsd Cys&anci one amiao acid is inserted between Cys _c and Cysa; 0 one- amino acid is inserted between Cysb and Cys _c and one amino acid is inserted between Cys _>v and Cys _t j or g) one amino acid is inserted between Cys _a and Cys> _> , one amino acid is inserted between CySb and Cys _e , and one amino acid is inserted between Cys _c and CyS _d • In addition, one or more amino acids can be inserted preceding Cys _a and/or one or more amino acids cars be inserted following Cys _c j. The insertions can be any natural or non- natural occurring amino acid (e.g., GIy or Ala) or amino acid analog and where there are more than one insertions present, they can be the same or different in certain embodiments, the insertion variants are peptides that bind to and/or activate the GC-C receptor. In certain embodiments, tbe insertion variants are peptides thai increase cGM P levels,

For example, the disclosure includes the following variants of FCiTCCIEICAYAACTGC (human guanylin) (SEQ ID NO: }

PGTCEGICAYAACTGC (SEQ ID NO: )

PC]TCB KiCAYA ACTGC (SEQ ID NO: ) PGTCEICGAYAACTGC (SEQ ID NO: )

PGTCEiCAGYAACTGC (SEQ ID NO: )

PGTCεiCAYGAACTGC (SEQ JD NO: }

PGTCEICAYAGACfGC (SHQ ID NO: )

PGTCEiCAYAAGCTGC (SEQ ID NO: }

PGTCEiCAYAACGTGC (SEQ ID NO: ) PGTCEiCAYAACTGGC (SEQ ID NO: )

PGTCAHiCAYAACTGC (SEQ ID NO: )

PGTCEAϊC AYAACTGC (SEQ ID NO: )

PGTCEtAC AYAACTGC (SEQ ID NO: )

PGTCEICAAYAACTGC (SEQ ID NO: } PGTCEICAYAAACTGC (SEQ iD NO; )

PGTC Jϊ ECAYAACATGC (SEQ ID NO: )

PGTCEiCAYAACTAGC (SEQ ID NO: )

FGTCEICAYλACTGAC (SEQ ID NO: }

FGTCAE1CAAYλACTGC (SEQ ID KO; ) PGTCEA1CAAYAACTGC (SEQ !D NO: )

PGTCEIACAAYAACTGC (SEQ ID NO: )

Other insertion variants of human guaπylin can have up to four amino acids (i.e., O, 1, 2, 3 or 4 natural or non-natural amino acids) inserted after each of the 15 amino acids m human gmmymi. Thus, the disclosure includes peptides having the sequence: Pro Xa%_.|;Gly Xaa(o.4 _? Thr Xaa^ ₎ Cys Xa%- _4J Ala Xaa ₍ o-s) Tvr Xa^o-n Ala Xaa^ Ala Xa%u; Cys Xaa^-;} Thr XaS ₁ ^ ₄ ) GIy Xa% _M ) Cys Xaa _(< ',4) (SEQ ID NO: ). Tbe inserted amino acids can be any amino acid and can be the same or different. In certain embodiments the inserted amino ackis arc all GIy or all Ala or a combination of G)y and Ala,

FIG. 2 depicts insertion variants of human guanylin in which one, two, three or four amino acids are inserted. The inserted ammo acids are between Cys _a and Cys^as well as amino terminal Io Cys _a and earboxy terminal to Cys _d .

The disclosure also features variants of peptides having the sequence Xaaj Xaa;> Xa& ₃ C vs.* Xatu Xaa,, Xaa ₇ Xαa* Xaa? Xaaio Xaaπ Cysu Xaa Xaa>a Xaa^ Xaa _5fj (SEO ID NO: i h e.g., variants of PGTCEiCAYAACTGC human guanyϋn (SEQ IO NO: } in which iφ U) four amino acids art; deleted and/or up to four amino acids are inserted, 5 The insertions and deletions can be between Cys4 and Cysϊ2 m SEQ ID NO: i or they can be amino terminal to €ys4 and/or carboxy terminal to Cysi2 m SEQ ID NO: 1.

When XaiHt, is Trp, Tyr or Phe, the peptide has a diymotrypsin cleavage site that is located at. a position where cleavage will liberate the portion of the peptide carboxy- teπiuna! to Xaa _it( . When Xaa^ is Lys or Arg, the peptide has a tπpsin cleavage site

1C that is located at a position where cleavage will liberate portion of the peptide earboxy-imninai to Xaau,. Thus, if the peptide includes an analgesic peptide carboxy-ierniinal to Xaa _!t j. the peptide will be liberated in the digestive tract upon exposure to the appropriate protease. Among the analgesic peptides which can he included Ln the peptide are: λspPhe, endomorphin- i , endomorphin-2, .πoeistatin, fβ dabrgm, lupron, and ziconotide, substance P and other analgesic peptides described herein.

When Xaaj or the ammo-terminal amino acid of the peptide of the disclosure (e.g., Xa& ₂ or Xaaj) is Trp, lyi or Phe, the peptide has a chymotrypsio. cleavage site that is located at a position where cleavage will liberate the portion of the peptide amino- 0 terminal to Xaa; (or Xaa ₂ or Xaa,0 along with Xaa ₃ , Xaa _? . or Xaa- _S . When Xaai or die amino-tcrminal amino acid of the peptide of the disclosure (e.g., Xaa? or Xaa$) is Lys or Arg, the peptide has a trypsin cleavage site that is located at a position where cleavage will liberate portion of the peptide ammo-terminal to Xaa.j along with Xaa ₅ , Xaa _. i or Xaa _. y, Thus, fur example, if the peptide includes an analgesic peptide amino- 5 terminal io Xaa _} , the peptide -will be liberated in the digestive tract upon exposure to the appropriate protease. Among the analgesic peptides which can be included in the peptide are: AspPhe, endomorphin- 1 , endomorphin-2, noeistatin, dalargin, lupron, /ieonotide, arid substance p and other analgesic peptides described herein.

^' The peptides cars be coadministered with or linked, e.g., covaleπtly linked to any of a variety of other peptides or compounds including analgesic peptides or analgesic compounds including, without limitation, the agents described herein

5 Amino acid, non-ammo acid, peptide and non-peptide spacers can be interposed between a peptide that is a GC-C receptor agonist and a peptide that has some other biυlαgieai function, e.g., m analgesic peptide or a peptide used to treat obesity. The linker can be one that is cleaved from the flanking peptides in vivo or one thai remains linked to ύia flanking peptides in vivo. For example, glycine, bela-aiaoirse,

10 glyeyl-gtychic. giyey.-beta-aiarmie, garama-aπiinobutyric acid, o-aniinocaprok acid, L -phenylalanine, L -tryptophan and glycil-L-valil-L-phenyklanine can be used as spacers (Chaltin et ai. 2003 Helvetica Chimica Acta 86:533-547; Caϋceti et al. 1993 FAIlMCXi 48:919-32) as can polyethylene glycols (BuUerworth et al. 1987 J. Med. Cheni 30: 1295-302) and makimide derivatives (Kiag et at 2002 Tetrahedron Lett

15 43:1987-1990). Various other linkers are described in the literature (Nestler 1996 Molecular Diversity 2:35-42; Finn et al. 1984 Biochemistry 23:2554-8; Cook et al. 1994 Tetrahedron Lett. 35:6777-80; Brokx et al. 2002 Journal of Controlled Release 78; H 5-123; Griffin et al. 2003 .!. Am. Chero. Soc. 125:6517-6531 ; Robinson et al. 1998 Proc. Natl Acad. Sci. USA 95:5929-5934), Linkers are also described in

20 US20U50171014, for example, amino acid linkers such as FALA, VLALA, ALAL, λLALA, 2-oyclohexyi-L-alanine-LALA, 2-cyclohexyl-L-aiamBe~2~cycIohex>i~f.,- αlaπine-LAL _f 1 -naphtyϊ-aianinc-ChaLAL and i-αaphtyi-alanine-LALA, Peptides and agonists of The disclosure can also be conjugated to: an affinity tag (such as (histidiue 6) H6), a HiV tai peptide residues 49-57, H IV tat peptide residues 49-56, the tat

25 sequence YGRKKRRQR RR, a polyarginine peptide having from 6 to 20 residues (such as Rfi J and the following peptide sequences: YARKARRQARR, YARAAARQARA, YARAARRAARR _? YARAARRAARA, ARRRiIRRRRE, and YAAAR RIlRRRR, which are disclosed in WO 99/29721 and in US patent No. 6,221 ,355 (SEQ. ID. NOs. 3-8).

,JU

The peptides of the disclosure can. be attached to one, two or more different moieties each providing the same or different functions. For example, the peptide can be linked to a molecule that is an analgesic and to a peptide that is used to treat obesity. The peptide and various nioieti.es can be ordered in various ways. For example, a peptide of the disclosure can have an analgesic peptide linked to its amino terminus and an anti-obesity peptide linked to its earboxy terminus. The additional moieties can be directly covalently bonded to the peptide or can be bonded via linkers.

The peptides of the disclosure can be a cyclic peptide or a linear peptide, In addition, multiple copies of the same peptide can be incorporated into a single cyclic or linear peptide.

The peptides can include the amino acid sequence of a peptide that occurs naturally in a vertebrate (e.g., mammalian) species or in a bacterial species, in addition, the peptides can be partially or completely non-naturally occurring peptides. Also within the disclosure are peptidormraetics corresponding to the peptides of the disclosure.

When fully folded, disulfide bonds are present between the first and third cysteines and between the second and fourth cysteines, e.g.. there is a disulfide bond between Cy§4 and OySs; and a disulfide bond between Xaa-.? and Xaais (when Xaa ₇ is a Cys and Xaa _!5 is a Cys). ϊn some embodiments, the peptide has only one disulfide bond, e.g., between the first arid third cysteines {i.e., CyS ₄ and Cys^; corresponds to the first and second cysteines when Xaa? is other than Cys). In certain embodiments one or more Cys can. be replaced by Mpt (mereaptoproline) or Pen (penicillamine) or Dpr (diarninopropiome acid) or some other amino acid that can eovalently link to another amines acid (e.g., Cys, Mpt, Pen or Dpr}, In otlit-r embodiments; the peptide is a reduced peptide having no disulfide bonds.

In some embodiments, one or both members of a pair of Cys residues which normally form a disulfide bond can be replaced by homocysteine, penicillamine, 3- rnereaptoproline (Kokxføiej et al. 1.996 iniJPept Protein Res 48:274); β, jj-

duneShylcysieine (Hunt et al. i ^c >93 Im J Pe.pt Protein Res 42:249} or diamiπopropionic acid (Smith et al, 1978 J MeJ Chem 21 :1 17) to ibrπi alternative internal cross-Links at the positions of the normal disulfide bonds.

in addition, one or more- disulfide bonds can be replaced by alternative covaieni cross- links, e.g., an amide linkage (-CH ₂ CH(O)NHCHr or -CI-S ₂ NHCH(O)CH ₂ -), aw ester linkage, a thin ester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage, a thiourea linkage, a phosphoπaie ester linkage, an alky! linkage (-Q bCThCf IbCI-I?-- _. ), an aikeny! Hnkagei.-CH;CH<:HCH _r ), an ether linkage (-CH ₂ CH ₂ OCHr- or - OH-Oα-hCHr), a thioether linkage (-CH ² CH ₂ SQl ₂ - Or -CHjSCH ₂ CH ₂ -), an amine linkage (-CH ₂ CK ₂ NKCH ₂ - or -CB ₂ NJ-ICH ₂ CS-J ₂ -) or a thioamide linkage C-

CH ₂ CH(S)HNHCHr or -CFhNHCH(S)CHr)- For example, Ledu et al. (Proe Nai'I λead. Sci. 100: 11263-78, 2003) describe methods for preparing lactam and amide eross-imks. Scha&neistcr et al (J. Am. Chem. See. 122:589! , 2000) describe stable, hydrocarbon cross-links. Hydrocarbon cross links can be produced via metathesis (or methaihesis fol lowed by hydrpgenation in the case of saturated liydrocai'boαs crosslinks) using one of another of the Grubbs catalysts (available from Materia, Inc. and Sigma-AkJrich and described, for example, in U.S. Patent No. 5,831 ,108 and 6 J 1 IJ 21 }. In some cases, the generation of such alternative cross-iinks requires replacing the Cys residues with other residues such as Lys or GIu or non -natural Iy occurring amino acids. In addition the lactam, amide imά hydrocarbon cross-links can be used to stabilize the peptide even if they link amino acids at positions other than those occupied by Cys, Such cross-links can occur between two ammo acids that are separated by two amino acids or between two amino acids that are separated by six amino aeids (see. e.g., Sehafrneister et al. (J. Am. Chem. Soc. 122:5891 , 2000.)}.

lα certain embodiments, one or more amino acids can be replaced by a norvnaturalϊy recurring amino acid or a naturally or non-natural iy occurring amino acid analog. There are many amino acids beyond the standard 20 (Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, Mis, Ue, Leu, Lys, Met, Flic, Pro, Set, Thr, Trp, Tyr, and VaI). Some are natural fy-oeeurring others are not (see. for example. Hunt, The Non-Protein Amino

Acids: in Chemistry and Biochemistry of the Amino Acids, Barrett. Chapman and Ball, 1985). For example, an aromatic amino acid can be replaced by 3,4-dihydroxy- L-phenyiaiamne. 3-iodo-L-tyrosiπe, triiodothyronine, [.-thyroxine, phenylglyeine S Phg) or nor -tyrosine (norTyr). Phg and nor fyr and other amino acids including Phc ixnά Tyr can be substituted by, e.g., a halogen, -CH3, -OH, -CH^NFh, -C(O)H ₅ - CH?CIL, -CN, -SI-I, or another group. Any anii.no acid can be substituted by the D-fonn of the amino acid.

With regard to ϊion-naturaliy occurring amino acids or a naturally and non-πaiuraiiy occurring amino acid analogs, a number of substitutions in the peptide and agonists of the disclosure are possible alone or in combination,

For example, glutaπύne residues can he substituted with gamma-Hydroxy-Glu or garama-Csrboxy-Glu. Tyrosine residues can be substituted with an alpha substituted amino acid such as L-alpha-inethylphenyhilanine or by analogues such as: 3-Amino- !yr; 'Fyr{CH3); 7yr(POi{CH ₃ ) ₂ ); Tyτ{SO _.; B); beta-Cyciohexyl-AIa; beta-(l - Cyclopentenyl)-Ala; heia-Cyclopentyi-Ala; beta-Cyclopropyl~Aia; bota-Quinolyi-Ala; betα-(2- ^" rhiazoJyl)-Ala: bcta-(lriazoSe-I-yl)-Aia; beta-(2-Fyπdyl)--Ala; beta-(3~ Pyridy!)-λla; Amino-Phe; Fluorø-Phe; Cydohexyl-GIy; tBu-Giy; beta-(3~ beπzothieny!)-A3a; beta-(2~thienyl)-Aia; 5-Methyl-Tφ; aπd 4-Methyl-I ^' rp. Proline residues can be substituted with homopro (L-pipecolic acid); hydroxy-Pro; 3,4- Dehydro-Pro; 4-flαoro-Pro; or alpha-methyi-Pro or an N(alpha)-C(aipha) cyclized amino acid analogues with thø structure:

π * 0, 1 , 2, 3 Alanine residues can be substituted with alpha-substitυed or N -methylated amino acid such as aipha-amino isobυtyric acid (aib), L/D-alpha-ethylalanine (U ^' D-isovaUne), L/D-meihylvaline, or L/D-aipha-inethylJeueine or a noπ-natui'al aiTiino acid such as beta- tluoro- Ala. Alanine can also substituted with:

π - 0, 1 , 2, 3

Glycine residues can be substituted with alpha-amino isobirtyric acid (aib) or UD- alpha-ethylalanine f L/D-isovaline).

Further examples of unnatural amino acids include; an unnatural analogue of tyrosine; an unnatural analogue of giυtaniinc; an unnatural analogue of phenylalanine; an unnatural analogue of serine; an unnatural analogue of threonine; an aikyi, aryl, aevL azido, eyano, halo, hydrazine, hydraxide, hydroxy!, alkeπyl, alkynl, ether, thiol, sulfønyl xeleno, ester, thioacid, borate, boronate, phospho. phosphono, phosphine, heterocyclic, enone, irnrne. aldehyde, hydroxy} amine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross- linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel Rmctional group; an amino acid that covaientiy or noncovaleπtiy interacts with another molecule; a metal binding amino acid; an amino acid that is ami dated at a site that is not naturally amklated, a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid, a biotin or biotio-anaiogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyctheπ a heavy atom substituted amino acid (e.g., an amino acid containing deuterium, trϊtmm, ⁰ C, ^{f >} N, or ^{i R} O); a chemically clcavable or photocleavable amiπu acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox -active amino acid; an «.-hydroxy containing acid; an amino thio acid containing amino acid; an «, α disubstituted amino acid; a β- ammo acid; a cyclic amino acid other than proline; an O-methyl-L-tyrosine; an 1,-3- (2-naplithyl}akιniflc; a 3-methyl-phenyklanme, a^-acetyl-L-phenyklanme; an 0-4- aliyl-L-tyrosine; a 4-propyl-L-tyrosine; a tri-O-aeetyl~GlcN ^? λcβ-serinc; an L-Dopa; a

- 33 -

lluorinaied phenylalanine; an isopropyl-L-phenylaiamne; a p-azido-L-phenylaianine; a p-acyl-L-phenylalanine; a p-benzoyl-L-phenyialaπiπe; an L-phosphoserine; a phosphonoserine; a phosphonotyrosine; a p-iodo-phersylahmine; a 4~ lluoiOphcnylglyciiie; a p-bromophenyialamne; a p-amino-L-pherrykikrώie: an isupropyl-L-phenylalanine; L-3-(2-naρlithyI)aIanme; an amino-, isopropyl-, or O aliyi-eontahinig phenylalanine analogue; a dopa, O-methyl-L-tyrosine; a glycosylated amino acid; a p-(propargyioxy)phenylalanine; dimethyl-Lysme; hydroxy-proline; nicrL'apttjprυpit>nic acid; metbyi-lysine; 3 -nitro- tyrosine; norleiicine; pyro-giutaraie Licid; Z fCarbobenzoxy!); £~Aeety!-Lysine; β-aianine; aminobenxoy! derivative; aminobut>τic acid (λbu); citruHine; aminohexanoic acid; aminoisobutyrsc acid; cyclohexyϊalaniπe; d-cyclobexyiaianinc; hydroxy-proline; nitro-aj-ginine; πifro- pbenyialariinc; nitro4yτosine; norvaline; octahydroimiole carboxylate; ornithine; peniciϋamine; telrahydroisoquinolme; acetamidoractiiy! protected amino acids and pegylatetl amino acids. Further examples of unnatural amino acids and amino acid analogs can be found in U.S. 200301.08885, U.S. 20030082575, US2006001934? (paragraphs 410-418} and the- references cited therein. The polypeptides of the disclosure can include further modifications including those described in US20060019347, paragraph 589,

hi some embodiments, an amino acid can be replaced by a naturally-occurring, nonessential amino acid, e.g., taurine.

Methods to manufacture peptides containing unnatural amino acids can be found in, for example, U.S. 2003010S885, U.S. 20030082575, US20060019347, Dciters ct al, J Ant Chem Soc. (2003) 125 ; 1 ! 782-3 , Chin et aL Science (2003 } 301 :964-7, and the references cited therein.

Peptides that include non-natural amino acids can also be prepared using the methods described in WO02086075.

- IQ -

I ^' he peptides of the disclosure can have one or more conventional peptide bonds replaced by an alternative bo&d. Such replacements cars increase the stability of the pepude. For example, replacement of the peptide bond between a residue amino terminal to an aromatic residue (e.g. Tyr, Phe, Tip) with an alternative bond can reduce cleavage by carboxy peptidases and may increase half-life in the digestive tract. Bonds that can replace peptide bonds include: a retro-in verso bond (C(O)-NH instead of NH-C(O); a reduced amide bond (NH-OH); a thiomcthylene bond (S-CH;. or CHrS); an oxomethylene bond (0-CH ₂ or CH _. r0}; an ethylene bond (CIi ₂ -CI-I-); a traoarπide boud (C(S)-NH); a trans-olefinc bond (CB-CH); a iϊunro substituted trans- πbtine bond (CF-CH); a ketomethylene bond (C(O)-CI-IR or CHR-C(O) wherein R is 1 1 or CH3; and a bond (C(O)-CFR or CFR-C ¹ CO) wherein R is ϊ f or F or CH _? .

The peptides of the disclosure? can be modified usmg standard modifications. Modifications may occur at the amino (N-). carboxy (C-) terminus, internally or a combination of any of the proceeding. In one aspect, of the disclosure, there may be more than one type of modification on the peptide. Modifications include but. are not limited to; aeetylation. amidation, biotinylation, einnamoylaikm. farnes ylation, lormylaiioo, myristoylation, palmitoyiation, phosphorj'lation (Scr. Tyr or Thr), stearoylation, succinylation, suifurylation and e>clisation (via di≤υliϊde bridges or amide eyelisation), and modificaiϊoπ by Cy3 or CyS. The peptides oft.be disclosure may also he modified by 2 ₅ 4-dinitroplieiiyi (DNP), DNP-!ysin, modification by 7- λmino-4-πiethyl-comτιaτiϊi (AMC), flourescdn, NBD { ^' 7-Nitvobeπκ-2-Oxa-!,?- Diavcoie), p-nitro-anihde, rhodamine B, EDANS (5-((2- i.iπuπoetJiγf)aniiπo)πaρhthaleric~S - sulfonic acid), dabcvL dabsyL toxas red, i ^' MOC, and lamra (Tetrarnctbyϊrhodamine). The peptides of the disclosure may also be conjugated to. for example, polyethylene glycol (PRO)-, aikyl groups (e.g.. C1-C20 straight or branehetl alkyl groups); tatty acid radicals; combinations of PFXI alky! groups and tatty acid radicals (see U.S. Patent 6,309,633: Soltero et aL, 20Oi Innovations hi Pharmaceutical Technology 106-110); BSA and KLIl (Keyhole Limpet

- -JO -

JHcmoeyanirs), The addition of PEG and other polymers which can be used to modify polypeptides of the disclosurejis described in US2006019347 section FX,

The peptides of the diselosurejbear some sequence similarity to uroguarsylin, 5 guanvlm, lymphoguanylin arκ| renoguanylin peptides. However, they may include amino acid changes and/or additions that in some instances, improve functionality. These changes can, for example, increase or decrease activity (e.»., increase or decrease the ability of the peptide to stimulate intestinal motility), alter the ability of the peptide to fold correctly, alter the stability of the peptide, alter the ability of the 10 peptide to bind the GC-C receptor and/or decrease toxicity, in some cases the peptides may function more desirably than wild-type uroguanylin, guanylin, iyπφhoguanyiin and renoguarjyUn peptides. For example, they may limit undesirable side effects such as diarrhea ajid dehydration.

i b The peptides and agonists of the disclosure can be chemically modified to increase therapeutic activity by synthetically adding sugar moieties (WO 88/02756; WO 89/09786; DE 3910667 Al , φ 0 374 089 A2; and U.S. 4.861 ,75S), adding eationic anchors (EP03635S9), lipid lϊjσieties (WO91/09S37; U.S. 4,837,303} or the substiiuents described as compounds L IK and ill in US555252O.

20 I

The disclosure also features ajpurified polypeptide comprising, consisting of or consisting essentially of the ajnmo acid sequence: Xaaj Xaib X aa.^ Cys4 Xaa^ Xaa-; Xaa _N Xaao Xaa _H} Xsa^ φys _n Xaan Xaa _!4 Xaa _{i S} Xaa _ltJ (SEQ iD N ^Q : 1 ) wherein:

Xaai js any amino aciφ or is missing; 25 Xaa^ is any an\ino acitjt or is missing;

Xaa^ is any amino acib or is missing;

Xaa _? is GIu; \

Xaa«« is l.\τ, TVp, Phe όy Leu;

Xaa _? is Cys; ! ao Xaas is any of the 20 Naturally-occurring amino acids other ιhaτi C ^' ya or .is missing; I

XiSSy is any oi ^' thc 20 naturally-occurring amino acids;

Xaaji is any of the 20 fsatutaily-occurring amino acids; Xiuii - is Thr, VaI or GIy;

Xafiis is Cys; and Xaat _t ; is any of the 20 naturally-occurring amino adds or is missing,

in various ymbodimerits: Xaa>s is Asm Xaaj j is Ala or Thr; Xaag is missing; and Xaa^ is Tyr.

In other embodiments Xaa* is immediately preceded by an amino acid sequence seieted from: Ser His Thr; Pro Scr Thr; Thr; Pro Asp Pro; lie Ala Giu Asp Ser I Iis Thr; ilo /via Gin Asp Pro Ser Thr; Ala Asm Thr; Asn Thr; Asp Fro Asn Thr; tys Asn Thr; Pro Asn Thr; Hc Ala GIn Asp Pro Asn Thr; Lys Pro Asn Thr; Asp Pro GIy Thr; GIu Asp Pro Giy Thr; Pm GIy Thr: Pro Ala Thr; VaI Ala Ala Arg Ala Asp Leu; GIy Asp Asp; Asn Asp GIu; GIn GIu Asp; Asn Asp Asp; Arg Thr He AIa Asn Asp Asp; Thr He Ala Asn Asp Asp; Asp Asp; Arg Thr Met Asp Asa Asp GIu; Arg Thr Ik- Ala GIy Asp Asp; Arg Thr He Ala Asn Asp; Asp; GIu Asp; Arg Ser lie Ser Gin GIu Asp; Thr Asp GIu; Arg Thr Ue Ala Thr Asp GIu; GIu; Uc He Thr Pro Pro Asp Pro; GIn GIu Leu; Lys λsp Asp; GIn GIu GIu; Arg Tyr He Asn GIn GIu GIu; Ala Ser Scr Tyr λla Scr; and Thr Scr Scr Tyr Ala Ser,

The disclosure further features a purified polypeptide- comprising, consisting of or consisting essentially the amino acid sequence: Xaaj Xaa-> Xaah CyS ₄ Xa;.κ Xaa _ή Xaa? Xaaji Xa&; Xaa _{! β} Xaa _H CySi ₂ Xaa _r j Xa&u Xaa _i5 Xaa _ih (SEQ ϊD NO:i ) wherein ^;

Xaa; is: a) Ser, Asn. Tyr, Ala, GIn. Pro. Lys, GSy, or Thr, or is missing; b) preceded by Lys or Tyr; c) arxy amino acid; ϋ) missing; e) any amino acid other than Cys; or t) Lys or Arg;

Xa;i> Ls: a) His. Asp, GIu, Ala, Ser. Asn, GIy, or is missing; b) His, Asp, GIu, ASa, Scr, Asn, GIy, Pro or is missing; c) Asp, GIu, any amino acid or is missing; d)

Asp or Giu; c) any amino acid: other than Cys; e) GIu; 1) missing; g) IVp, Tyr or Phe; or h) Lys or Arg; ^'

Xaa ₃ .is: a) Thr, Asp, Sα\ GJu, Pro, VaI or Leu; Asp or GIu; b) any amino acid other thars Cys; c) GIu; d) Thrj e) Thr, Asp _s Ser, Gki, Pro. VaI or Leu or is missing; f) Trp, Tyr or Phe; or g) Lya or Arg;

Xiut-j is: a) Cys, Mpt (raercaptoproline). Pen (penicillamine), Opr (dsarøinopropionic acid), Asp _5! or GIu;

Xaa _? is: a) any amino acid; b) GIu, Asp. Gm, GIy or Pro; c) Giu; d) GIu or Asp; e) Asp, lie or Giu; f) any. amino acid; or g) any amino add other ih&n Cys; Xaa ₆ is: a) Leu, Oe ₅ VaL Ala, Lys, Arg, Trp, Tyr or Phe; b) Leu, lk\ VaK Lys,

Arg, Trp, Tyr or Phe; Leυ, ϊh\ Lys. Arg, IVp, Tyr or Phe; c) Leu, He, VaL Tφ, Tyr oτ Phe; d) Tφ, Tyr, Phe or Leu; e) Leu, Ik or VaI; t) He, Trp or Leu; g) Tφ, Tyr or Phe; h} Uc or Leu; i) Tyr; j) any aniino acid; k) tirrv amino acid except ϊ,εu: i) any natural or json-nahsral aromatic amino acid; or rn) any amino acid other than Cys; Xaa ₇ is: a) Cys, Ser. or- Tyr; Cys; b) Cys, Mpt (mereaptoproϋne), Pen yxmicillamine), Dpr (dianiinoipropionic acid). Asp or Giu; c) Ser; or d) an amino acid other than Cys; ;

Xaaa b: a) Ala, VaL oir De; b) Ala, VaL Tbr, He, Mel or is missing; c) any aniino acid; d) VaI; e) any amino acid other than Cys; or f) missing; Xaa _v is: a) any amino acid; b) any amino acid other than Phe and Tyr; c) any amino acid other than Phe, Tyr, and Tip; d) airy amino acid other than Phe, Tyr, Trp,. lie, Leu and YaI; e) any amrøφ acid other than Phe, ϊyr, Trp, lie, Leu, VaI, and His; f) any ammo acid other than Ohi; g) any amino acid other than Lys, Arg, Phe, Tyr, and Trp; h) any amino acid othw tlian Lys, Arg, Phe, Tyr, Trp, lie, Leu and Va!; i) any amino acid other tliars Lys. Aijg, Phe, Tyr, Trp, He, Leu, VaI, and His; j} any non- aromatic amino acid; k) missing; i) Phe, Tyr, Asn, or Tφ; m) Asn, Tyr, Asp or λia; n) Asn, G hi, or Tyr; o) Phe or Tir; p) Asn; or q) any amino acid other than Cys;

Xά- ά' u) is; a} Ala, Pro or GIy; b) Pro or GIy; c) Pro; d) Ala, VaL Met, Thr or lie; c) any amino acid; I) VaI; g) ^" Va! or Fro; h) Ala or VaI; i) any amino acid other than CysJ _. ) Pro; or k) GIy: ^

Xasj j is; a) any amino iacid; b) Ala, Leu. Ser, GIy, VaK Gks, GIn, lie, Leu, Lys, Arg, or Asp; c) Ak or GIy; d)jAla; a) Ala or VaI; i) arty amino acid; g) AIa or Aib iarpha-arninϋisobuiyric acid) ¹ . Ih) any amino acid other than Cys; i) A!a or Thr; or j) Thr. i

Xaa,i is: a} Cy tj, Mpϊ (jmercaptoproUne}, Fen (penicillamine), Dpr (dianiinopfopionic acid), Aspj or GIu; or b) any amino acid oilier than Cys;

Xaa^ is; a) Thr, Ala, λsn, Lys, Arg, or Trp; b) Thr, A! a, Lys, Arg, or Trp; c) any amino acid; d) arsy πori-aφrnatie amino acid; e) Thr, Ala, or Trp; f) Trp, Tyr or Phi'; g) Thr or Ala; h) any amino acid; i) Thr; j) any amino acid other than Cys; k) Thr. VaI, or GIy; 1} Thr or VaIL m) Thr or GIy ₅ n) VaI or Thr; o) VaI; p) Thr; or q) GIy;

Xaau is; a) GIy, Pro φ Ala; b) GIy; c) any amines acid; d) GSy, Ala or Ser; e) GIy or Ala; r) any amino acid bther than Cys; or g) Ala;

Xaaj _? ; is; a) Cys, Tyr or is missing; b) Cys; c) Cys, Mpt (mercaptoprolinc). Pen (penicillamine), Dpr {diaminφropionic acid). Asp, GIu; or d) any amino acid other than Cys or is missing; and

Xaa _ff , is-, a) Trp, Tyr, ^he. Asrs, He, VaI, His or Leu; b) IYp, Tyr, Fhe, Asn or [,cu; c) Trp, Tyr, Phe or Leu; d) Trp, Tyr, or Phe; e) Leu, lie or VaI; t) His, Leu or Set; g) Tyr or Leu; Lys or Arg; h) His; i) any amino acid, j) Leu, or missing; k) Trp, lyτ _f Phe, Lys. Arg or is missing; I) missing; m) any amino acid other than Cys; or n) i yr.

Also featured is purified pohφc jptide comprising, consiting of or consisting essentially of the amino acid sequence; X aai Xaa _?. Xaaj Xaa* Xaa ₅ Xaa^ Xaa- _/ Xaa« Xaa<; Xaaπj Xaaπ Xaa _: : Xaa^ Xaa;4 Xaa;i Is Xaai« (SEQ ID NO: I ) wherein: Xaa: is any amino acid or is missing; Xa22 is any ammo acid or is missing; Xa a ₃ is any amino acid or is missing; Xaa,; is Cys, Mpt (met aptoproime). Pen (penicillamine), Dpr

(diamirsopropϊonic acid), Asp jor GIu; Xaajis GIu; Xaa _f! is Tyr, Tip, Phe oi" Leu;

Xaa- is? t ^' vs, Mpt (mcreaptopiolme). Pen (penicillamine). Dpi sdsammt'pufmnπe aesd). Asp or GIu;

>«m _s is απ> ammo acid other than C) s or is missing;

XtKi , κs <m\ ammo acid, f* Va ,) :s Pu* Oϊ Gl) ,

Xui ^ any ammo acid:

Xεa _d > :s ( \ \ Mpt (mereaptoprohnc). Pen (penicillamine). Dpr <dianimopr^fu>nic acul). Asp or GIu;

\aa _s ! ^s thr, \ al oi GIy:

PeB (pcniciliamme). Dpr f cliarrjuopmphM-v acidi Asp Of GIu, and

Mtj.ii, iϊ> tun αrrano acid or is missing.

"5 1 ho discϊrsυie aKo features peptides whicb ma> mclmk one or -m»rc oi lie peptide modiikaisons, OJIO or r.iort? noti-naUiπil amino acid or amino acid analogs, one or πϊOIC O^ ihe αisullkle bond, altoiiatives or one more of the alternath c peptide bonds

Ji'Secbci: hcseiπ

?Q 5.f{ ^' -( ^' aguiα.-tλ oϊ the dise'usute L.an also comprise, consist essunUiily υf, or consist oi peptides dem ed Irons the C ^' -temiinal domain of an> of tlie peptides deserted hei ctn Uujs, ώes ^an contam, f<>r example, anywhere from 13-75 ammo acids including 13. 14, L\ U\ r. l 5>, i^ 20, 21, 22, 25. 24, 25, 26, 27, 28. 2^, 30, 31, ?2, H. 34, 35, 36, 37, 18, 39, 40. 41 . 42, 43, 44, 45, 46. 4 ^~ , 48, 49, 50, ;> i, 52, 53, 54, 55. 56. 5 ^" , 58, 59, is *0, 01 . o2. o3, 64, 65, 66, 67, oK, 69, U\ 71 , "2. 73. 74, and or "5 amino acids of the O -tømπuil di _' TJi ₁ J ui of ans of ihe peptides described herein.

The ^aπou^ peptides can be present vuth a counter Son Useful eoimteπnns inelυdc salts >« ⁽ i; .uctarc, bonxcncsuifonate. beivoatc, calcium cdetaie. casn>> We carbonate. otO Lilt ate, edftatv' ( HfTFA), edssyjatc, emboudte, esyidte, lumarau', gluceptαte, gluαnjate, giutaniiite, μl>coll}lαrsaiiπatc, hcxvlresorαaate, iodide, bromide, irbloπde,

5^

hydroxynaphthoate, isethionate, lactate, lactobionate, estokfe, maleate, maiate, mandehu ^' e, mesylate, mucate, napsyiate, nitrate, pantothenate, phosphate, salicylate, sieaπite, succinate, sulfate, tartaπite, tbeoclate. aeetarmdobenzoate, adipate, alginate, aminosalicylate _; anhydrømethyiene-citratc, ascorbate, aspartate, caraphoratc, oapτate, eaprαaie, capryiate, emn&mate, cyclamate, dichloroacetate, formate, gerttisate, gbacuvonate, glycerophosphate, »]ycolate, hippurate, fluoride, raalonate, napadisyiate, nicotinate, oleate, orotate, oxalate, oxogluiarate, paimiiate, pectmate, pectinate polymer, phenyl ethyibαrbif urate, pi crate, propionate, pidoSate, sebacate, rhoάaoide, tosylate, and tavinate.

In a second aspect, the disclosure also features a therapeutic or prophylactic method comprising administering a composition comprising a purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ IO NO: I or another peptide or agonist of the disclosure. For the treatment of gastrointesimai disorders, ihe peptide cars be administered orally, by rectal suppository or parersleraiiv.

In various embodiments, the patient is suffering from a gastrointestinal disorder; the patient is sυftering from a disorder selected from the group consisting of; gastrointestinal motility disorders, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, functional dyspepsia, nonuiccr dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GHRD), gastroparesis, irritable bowel syndrome, post-operative ileus, ulcerative colitis, chronic constipation, and disorders and Condi lions associated with constipation (e.g. constipation associated with use. of opiate pain killers, post-surgical constipation, and constipation associated with neuropathic disorders as well as other conditions and disorders are described herein); the patient is suffering from a gastrointestinal motility disorder, chronic intestinal pseudo-obstruction, coionϊe pseudo-obstruction, Crohn's disease, duodenogastrie reflux, dyspepsia, functional dyspepsia, nonuicer dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GERD), gastroparesis, inflammatory bowel disease, irritable bowel syndrome, post-

operative ileus, ulcerative colitis, chronic constipation, and disorders _* and conditions associated with constipation (eg. constipation associated with use of opiate pain killers, post-surgical constipation, and constipation associated with neuropathic disorders ϊIS well as other conditions and disorders art- described herein); the composition is administered orally; the peptide comprises 30 or fewer amino acids, the peptide composes 20 or fewer amino acids, and the peptide comprises no more than 5 amino acids prior to Xaii _f ,; the peptide comprises I SO, 140. 130, 120, 1 10, 100, * ^• >{), 80. 70, 60. 50, 40, or 30 or fewer amino acids. In other embodiments, the peptide comprises; 20 or fewer amino acids, ϊn other embodiments the peptide comprises no muα* than 20, 15, 10, or 5 peptides subsequent to Xaa^. in certain embodiments XisSj „ is a chyme trypsin or trypsin cleavage site and art analgesic peptide is present immediately following Xaa$s _> .

Among She useful peptides are those comprising, consisting of or consisting essentially of any of the following amino acid sequences;

SHTCEϊCλFAACAGC (opossum guaαylin) (SEQ ϊD NO: };

PGTCεϊC AYAACTGC (human guanylin) (SEQ (D NO; );

PSTCHICAYAACAGC (pig guanylin) (SEQ iD NO: );

PNTClϊϊCλYAACTGC (rat guanylin} (SEQ ID NO; );

PDPCKICAN ^' λACTGCL (European eel guanylin, inferred) (SEQ ID NO: );

NDDCULCVNVACTGCL (human uroguanylin) (SEQ ID NO: };

QEECELCiNMACTGY (opossum lymphoguanylin) (SEQ ID NO: };

GDDCEI-CVNVACTGCS (pig uroguanylin} (SEQ ID NO: );

NDECELCVNIACTGC {guinea pig urøguanylifi) (SEQ ID NO: >;

TDECBLCiNVACTGC (rat uraguanylin) (SEQ ID NO: );

QEOCO CIKVAClGC (opossum uroguanylin) (SEQ ID NO: K

MPSTQyiRRrλSSYASCIWC ^' rTλC ASCHORfTKPSLAT ( EAST S ) (SEQ ID NO: K

MPSTQMRRPASSYASCIWCATACASCHGRTTKPSLAT (SEQ ID NO: };

MFSIX)YLRRPTSSYASCIWCATACASCi-IGRITKl ⁵ SLAT (SEQ ID NO; };

MPSTQYIRRPTSSYASCIWCArfυCASCliGRTTK.PSLAr (SEQ ID NO: };

MPSTQYfRRPASSYASClWυATACASCηGRTTiϊPSlAT (SEQ (D NO: };

QEECELSi(NMACTGY (opossum ϊyraphoguanyliπ analog) (SEQ ϊD NO: ):

YPBCOCMPAACTGC (Japanese eel guanyiin) (SEQ ID NO: };

VCKICλFAACTGC (Zebrafish guanyiin, inferred) (SEQ ID NO: );

λDI.CE1CλF AACTGCL {Japencsc eel renoguanylin. inierred) (SEQ ID NO: );

PGTCEICAYA ACTGCL (SEQ ID INO: K

PGTCEICA VAACTCJCLKK (SEQ ID NO; );

PNTCε1CλY AACTGC K.KKKKK (SEQ iD NO: K

PNTCEICAYAACTGCD (SEQ ID NO; );

PNTCEif'λ YAACTGCDK (SEQ ⁽ D NO: );

YPNTCEICAYAACTGC (SEQ ID NO: );

KNITεICAYAACTGC (SEQ ID NO: );

- 4S ^■■

KPNT€.εICAYAACTGC (SEQ ID NO: };

tDPGTCϊϊlCAY AACTGC (SEQ ID NO: );

VTYODG NFSFSLfSVR KLKi)IQBPQE PRVGKLR NFA PIPGEPVVP! [.CSNPNFPLE LKPLCKEPNA QEIIQRLEEIAEDPGTCEICAYAACTGC (SEO ID NO: };

DPGTCLtOVYA ACTGC (SbQ ID NO: };

MNAFLLSALC LLGAWAALAG GVTVQDGNFS FSLBSVKkLK DLQEPQEPRV CJKLRNFAPIP GEPWPiLCS NPN FPEHLKP LCKEPNAQE! LQRLEEIAED PGTCEICAYAACTGC (SEQ ID NO: };

MNAFLLhA LC LLGA WAALAG GVTVQfXINFS ESLEPRVGKL RNF APlPGEP VVPILCSNPN FPEELKPLCK EPNAQEILQR LEEϊAEDPGTCEϊC AYAACTGC

(SEQ ID NO: };

TGSMNAKLLF ALCLLGAWAA LAGGVTVQDG NFSFSLEPRV GKLRNEAPIP GEPVVPILCS NPNFPEELKP LCKEPNλQEI LQRLEEJ AEDPGTCHiCAY AACTGCLEG (SEQ ID NO: };

MDECELCVNVACTGCL (SEQ ϊD NO: );

HCELCVNVACTGCL (SEQ ID NO: };

EDCELC1NVACTGC (SEQ ID NO; );

NDDCELCVλCTGCL (SEQ ϊD NO: );

FKTLRTIANDDCEL-CVNVACTGCL (S BQ ϊD NO: K

FKTLRl IA NDDCLCV N VACTGCL (SEQ ID NO: ):

DDCELCVNV AClGCL (SEQ ID NO: );

DCELCVN VACTGCL (SEQ ID NO: );

CELCVNVACTGCL (SEQ SD NO: );

K-DDCELCVNVACTGCL (SEQ ID NO: };

FNTCEICANPACTGC (SEQ ID NO. ):

NDDCELCVN'VACTGCS (cow uroguanyiin) (SEQ ID NO:....);

PDVCDVCAFAACSGC (Xenopus guanylin) (SEQ ID NO....);

LDLCElCAi-AACTGC <Fugu guany!in) (SEQ ID NO....};

VDVCEICAFAACTGC (Zebrafish guanylin) (SEQ ID NO...};

LPICBICAFAACTGC (PuSerfish guanylin) (SEQ !D NO... );

ADLCi;' iCANAACSC^CF (chicken uroguanyiin) (SEQ IO NO...};

LDPCEICANPSCFGCLN (fugu uroguanyiin) (SEQ ID NO...);

!DPC OCA NVACTGC (øel uroguanyiin) (SEQ ID NO..);

SDPCEICANPSCFGCLD (killifisϊi uroguaπylin) (SEQ ID NO..);

PGTCEICAYAACTAC (SEQ ID NO. );

PGTCEICAYAACAGC (SHQ ϊD SO. }:

PGTCεTCAλA ACTGC (SEQ ID NO. );

PGTCEACAYAACTGC (SEQ ID NO. );

PσrCAiC AYA ACTGC (SEQ ID NO. )

PGACH ICAYA ACTGC (SEQ ID NO. );

FλTCE (CAVA ACTGC (SEQ FD NO. );

AGTC ¹ EICAYAACTGC (SEQ ID NO. };

PTCEtCAYAACTGC (SEQ ID NO. };

PGTCEiCVNVACTGC (SEQ ID NO, ):

PCrrCEICA NPACTGC (SEQ ID NO. };

PGTCBiCAVAACTCC (SEQ ID NO. );

FϋTCεICAYλACTDC (SEQ ID NO. );

PGTC! : !CAYAACTHC (SBQ ID NO. );

PGϊCEICAYAACTFC (SEQ [D NO. ):

PGTCBC AYAACTHC (SEQ ID NO, );

PGTCEICAYAACTIC (SEQ ID NO, );

PGTCEFCAYAACTKC (SEQ ID NO. );

PGTCEiCAYAACTLC (SEQ ID NO. );

PGTCHICAYAACTMC (SEQ TD NO. };

PGTCIiIC ¹ AYAACTMC (SEQ ID NO. };

PGTCEICAYAACTPC (SEQ ID NO. );

PGTCEICAYAACTQC (SEQ ID NO. );

PGϊCEICAYAACTRC (SEQ ID NO, );

PGTCRICAYAACTSC (SEQ ID NO. };

I ⁵ C]TCKICAYAACTTC (SEQ JD NO, );

δ PGTCEICAYAACI'VC (SEQ ID NO, );

PGTCmCλYAλCTWC CSEQ ID NO. };

PGTCE !CAYAACTYC (SEQ ID NO. );

NDDCELCVNVACTGCA (SEQ ID NO.,}:

MDDCEECVNVACTACL (SEQ SD NO..);

10 NDDCELCV NVACAGCL (SEQ ID NO..):

NDDCELCV NAACTGCE (SEQ I D NO..};

NDDCKLCVAVACTGCE (SEQ ID NO.,};

NDOCELCANVACTGCE (SEQ ID NO.,);

NDDCEACVNVACTGCL (SEQ ID NO..);

?5 NPDCλLCVNVACTGCL(SEQ ID NO..);

N Dλθπ..C VN VACTGCL (SEQ ID NO..);

NADCELCVNVACTGCL (SEQ ID NO,.};

ADDCELCVN VACTGCL (SEQ ID NO..);

NDDCELCAYAACTGCL (SEO SO MO..):

NDDC£LCVNPACTGCL (SEQ ID NO..);

! .RTIATDECe.CINVACTGC (SEQ ID NO. ).

Additional guunvlin/uroguanylin-like sequences include;

TiATDECELCINVACTCC;

M ^' NAWLlSVLCLlGAbAVLVEGVTVQDGDLSFPLESYKQLOILkE VQEFTLM Sϊ-iKKPALRLPK.PVAPiϊLCSQSAPPEALRPLCEK.PNAEEILQRLϋAiAQDPNT Cε.

CAYAACTGC;

ϊϊDPGTCEiCAY AACTGC; PSTC'EICAYAACAGC; FNTCB IC AYA ACTGC;

NDDCELCYNBACTGCL;

FKTLRTlA NDDCELCVNVACTGCL;

F KTLR ^' πAN DDCLCVNVACTGCL; LQALRTMDNDECELCVNIACTGC; and FKTLRTIANDDCELCVNVACTGCL

Further useful guanyliu-'uroguanylm-like sequences which may either exhibit slower or quicker iritroconversion between the A and B iso forms, described in greater detail below, when compared to wild-type sequences include:

NDDCELCVNVACFGCL

NDDCELCVNVACTACL

NDDCHLCVNVACAGCL NDDCELCVNAACTGCL

N DDCHLC VA VACTGCL NDDCIiLCANVACTGCL NDDCEACVNYACTGCL NDDCALCVNVACTGCL NDACLLCVNVACTGCL

NADCELCYNVACTGCL.

ADDCΈLC V NVACTGCL

NDDCELCAYAACTGCL

NDDCELCVNPACTGCL NDDCRLCVNVACTGCLKK

NDDCELCVN VACTACLKK

NDDCELCVNVACTGCI

NDHCHLCVNVACTGCL

NDHCELCVNVACTACL NDECεLCVN VACAGCL

NDECELCVNλACTGCL

NDECB LCY AVACTGCL

NDLCBLCANVACTGCi.. NDECEACVNVACTGCL NDECALCVNVACTGCL

NDACB LCVN Y ACTGC L

NADCELCVNVACTGCL

ADLCBLCVNVACTGCL

NDECELCAYAACTGCL NDHCELCVNPACTGCL

NDECELCVNVACTCiCLKK

N DlX £LC VN VACTACLKK

N DBC ELC VN VACTGCl

NDDCELCVNVACTGC NDDCELCYNVACTAC

N DDCELCYNVACACTC

NDDCε LCVNAACTGC NDDCELCVAVACTGC NDϊXεLCλNVACTGC NDDCEACVNVACTGC NDDCALCVNVACTGC NDACELCVNVACTGC NADCELCVNVACTGC ADDCELCVNVACTGC

^DDCHLCAYAACTGC 0 NDDCHLCVNPACTGC NDHCELCVNVACTGC NDECELCVNVACTAC NDECELCVNVλCAGC NDHCFLCVNAACTGC 5 NDHCELCVAVACTGC NDECELCANVACTGC .NDECEACVNVACTGC NDECλLCVNVACTGC NDACELCVNVACTGC α NADCELCVNVACTGC ADHCELCVNVACTGC N DECELCAY AACTGC NDHCELCVNPACTGC NDDCELCVNVACTGCA 5 NDECELCVN VACTGCA I ⁵ GTCBICAYAACTAC PGTCEfCAYAACTGCL PGTCHiC AY AACTGCLKK and 0 PGTCeCAYAACTGCI

si -

The peptides can include the amino acid sequence of a peptide that occurs naturally in a vertebrate (e.g., mammalian) species or in a bacteria! species. In addition, the peptides ears be partially or completely ϊion-naluralϊy occurring peptides.

in a third aspect the disclosure features a method for treating a patient suffering from constipation, the method comprising administering a composition comprising a peptide comprising, consisting essentially of or consisting of the amino acid sequence of SIiQ IB NO: 1 or another peptide or agonist of the disclosure. Clinically accepted criteria ^[ hat define constipation range {torn the .frequency of bowel movements, the consistency of feces and the ease of bowel movement. One common definition of constipation is less than three bowel movements per week. Other definitions include abnormally hard stools or defecation that requires excessive straining (Schiller 200I Ailment Pharmacol Thcr 15:749-763). Constipation may be idiopathic (functional constipation or slow transit constipation) or secondary to other causes including neurologic, metabolic or endocrine disorders. These disorders include diabetes rπdlitus, hypothyroidism, hyperthyroidism, hypocaicaenua, Multiple sclerosis,

Parkinson's disease, spina! cord lesions. Neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung disease and cystic fibrosis. Constipation may also be the result of surgery or due to the use of drugs such as analgesics (like opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics,

hi various embodiments, the constipation is associated with use of a therapeutic agent; the constipation is associated with a neuropathic disorder; the constipation is postsurgical constipation; the constipation is associated with a gastrointestinal disorder; the constipation is idiopathic (functional constipation or slow transit constipation); the constipation, is associated with neuropathic, metabolic or endocrine disorder (eg,, diabetes rnellitus. hypothyroidism, hyperthyroidism, hypocalcaemia. Multiple Sclerosis, Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung disease or cystic fibrosis). Constipation rnay also be the result of surgery or due to the use of drugs such as analgesics (e.g.,

opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics.

in s fourth aspect, the disclosure features a method for treating & patient suffering from a gastrointestinal disorder, the method comprising administering to the patient a composition comprising a purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: I or another peptide or agonist of the disclosure.

in various embodiments, the patient is suffering from a gastrointestinal disorder; the patient is suffering from a disorder selected from the group consisting of gastrointestinal motility disorders, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, functional dyspepsia, nonulcer dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (CJEIID) ₅ gastroparesis, irritable bowel syndrome, post-operative ileus, ulcerative colitis, chronic constipation, and disorders and conditions associated with constipation (e.g. constipation associated with use of opiate pain killers, post-surgical constipation, and constipation associated with neuropathic disorders as well as other conditions and disorders are described herein), obesity, congestive heart failure, or benign prostatic hyperplasia

Ia a lϊtth aspect, the disclosure features a method for increasing gastrointestinal motility in a patient, the method comprising administering to the patient a composition comprising a purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SHQ JD NO: \ or another peptide or agonist of the disclosure.

hi a sixth aspect, ihe disclosure features a method for decreasing gastrointestinal pain or visceral pain in a patient, the method comprising administering Io the patient a composition comprising a purified peptide comprising, consisting essentially of or

•- 5'? -

consisting of ihe amino acid sequence of S EQ ID NO; 1 or another peptide or agonist υf the disclosure.

in a seventh aspect, the disclosure features a method for increasing the activity of an intestinal guanylate cyclase (GC-C?) receptor in a pa.ieni, the method comprising administering to the patient a composition comprising a purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SBQ ID NQ: 1 or another peptide or agonist of the disclosure,

In an eighth aspect, the disclosure features an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: I or another rxtntide or a "Cgo ¹ nist of the disclosure.

in a ninth aspect, the disclosure features a composition comprising a punfied polypeptide comprising, consisting essentially of or consisting of the amnio acid sequence of SEQ ID INO: ! or another peptide or agonist of the disclosure, In an embodiment, the composition is a pharmaceutical composition.

in a tenth aspect, the disclosure features a method for treating obesity, the method composing administering a composition comprising a purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: I or another peptide or agonist of the disclosure. The peptide can be administered in combination with one or more agents for treatment of obesity, including, without limitation, the anti-obesiiy agents described herein. A peptide use&S for treating obesiiy can be administered as a co-therapy with a peptide of the disclosure either as a distinct molecule or as part of a fusion protein with a peptide of the disclosure. Thus, for example, FYY-J .^ can be fused to the carboxy or amino terminus of a peptide of the disclosure. Such a fusion protein can include a chymostrypsin or trypsin cleavage site (hat can permit cleavage to separate the two peptides.

hi an eleventh aspect, the disclosure features a method for treating congestive heart failure, ihc method comprising: administering to the patient a composition comprising <j purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: I or another peptide or agonist of the disclosure. The peptide can be administered in combination with one or more agents for treatment of congestive heart failure, for example, a natriuretic peptide such as atrial natriuretic peptide, brain natriuretic peptide or C-type natriuretic peptide), a diuretic, or an inhibitor υf angiotensin converting enzyme.

in a twelfth aspect, the disclosure features a method for treating benign prostatic hyperplasia, the method comprising: administering to the patient a composition comprising a purified peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 1 or another peptide or agonist of the disclosure. The peptide can be administered in combination with one or more agents for treatment of BPH, for example, a 5-alpha reductase inhibitor (e.g., finasteride) or an alpha adrenergic inhibitor (e.g., doxazosine).

In a thirteenth aspect, the disclosure features a method for treating a patient suffering from a gastrointestinal disorder, the method comprising administering to the patient a composition comprising a complete or partial agonist of the CJC-C receptor, including but not limited ϊ.o the peptides and agonists described herein, in various embodiments, the disorder is a gastrointestinal motility disorder, chronic intestinal pseudoobstruction, colonic pseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, functional dyspepsia, rrønuleer dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GERD), gasirβparesis, irritable bowel syndrome, post-operative ileus, ulcerative colitis, chronic constipation, and disorders and conditions associated with constipation _* (e.g. constipation associated with use of opiate pain killers, post-surgical constipation, and constipation associated with neuropathic disorders as well as other conditions and disorders are described herein), obesity, congestive heart failure, or benign prostatic hyperplasia. In various embodiments the composition comprising an agonist of the

- 5 ⁽ ϊ -

intestinal guanylate cyclase (GC-C) receptor is administered orally, by rectal suppository, cr parenieraily, In various embodiments: the agonist is a peptide, the peptide includes two Cys that form one disulfide bond, the peptide includes two Cys, the peptide includes four Cys that form two disulfide bonds, the peptide includes four Cys, two of which form a disulfide bond.

hi a fourteenth aspect, the disclosure features a method for treating a patient suffering from constipation, the method comprising administering a composition comprising a complete or partial agonist of the GC-C receptor, in various embodiments; the agonist is a peptide, the peptide includes two Cys that form one disulfide bond, the peptide includes two Cys, the peptide includes four Cys that fbrra two disulfide bonds, Hu: peptide includes four Cys, two of which form a disulfide bond. In various embodiments, the constipation is associated with the use of a therapeutic agent (e.g. anti hypertensi ves, anticonvulsants, antispasmodics, analgesics, anticholinergics, antidepressants, antipsychotics, cation-containing agents, anticonvulsants, ganglion blockers, vinca alkaloids); associated with a muscular, neuropathic, metabolic or endocrine disorder (including but not limited to myotonic dystrophy, derrnanrryoskis, sysiernie sclerosis, sclerodoma, amyloidosis (neurologic or muscular), ischemia, tumor of the central .nervous system, autonomic neuropathy, Chagus disease, cystic πbrosis, diabetes meϋitus, Hirschsprung disease, hyperthyroidism, hypouakaeraia, hypcuhyroidisrn, Multiple Sclerosis, neurofibromatosis, Parkinson's disease, and spinal cord lesions {for. example, related to sacra! nerve damage related to trauma or a tumor or the enteric nervous system)); post-surgical constipation (postoperative ileus); associated with a structural colon alteration (for example that associated wilh Neoplasm, stricture, volvulus, anorectal, inflammation, prolapse, rectoeele, or fissure); associated with the a gastrointestinal disorder; associated with a systemic illness or disorder (for example, electrolyte abnormalities, thyroid disease, diabetes roeliitus, panhypopituitarism, Addison's disease, pheochromocytoma, uremia, porphyria); chronic constipation; associated with the use of analgesic drugs (e.g. opioid induced constipation); associated with megacolon; idiopathic constipation; functional constipation; functional constipation associated with norma! transit, slow

transit (e.g. ana or fewer bowel movements per week) or pelvic Door dyssynergia; associated with bloating and abdominal pain,

in a fifteenth aspect, the disclosure features a method for increasing gastrointestinal motility in a patient, the method comprising administering to the patient, a 5 composition comprising a complete or partial agonist of the GC-C receptor, including bui not limited to the peptides and agonists described herein.

!n a sixteenth aspect, the disclosure features a method for decreasing gastrointestinal pain or visceral pain in a patient, the method comprising administering to the patient a composition comprising a complete or partial agonist of the GC-C" receptor, including iθ but not limited to the peptides and agonists described herein.

!rs a seventeenth aspect, the disclosure features a method for treating congestive heart failure, the method comprising administering a complete or partial agonist of the GC- C receptor, including but not limited to the peptides and agonists described herein. GC-C agonists can set in the kidney and adrenal gland to control natri uresis,

"6 kaliuresis, and diuresis thereby reducing the build-up of fluid associated with congestive hear* failure (LoretK et al. J CUn Invest 112; i 138, 2003: Caπithers et ai. Kidney Int 65.40, 2004). The agonist can be administered in combination with one or more agents for treatment of congestive heart failure, including but not limited to the agents useful for combi therapy described herein. For example, the agonist can be 0 administered in combination with a natriuretic peptide such as atrial natriuretic peptide, brain natriuretic peptide or C-fype natriuretic peptide), a diuretic, or ars inhibitor of angiotensin converting enzyme. In various embodiments the congestive heart failure is categorized as Class II congestive heart, failure: the congestive heart failure is categorized as (."lass III congestive heart failure; and the congestive heart 5 failure is categorized as Class IV congestive heart failure. The New York Heart

Association (NYiIA) functional classification system relates congestive heart failure symptoms to everyday activities and the patient's quality of fife. The NYFiA defines the classes of patient symptoms relating to congestive heart failure ΆS: Class II-slight

limitation of physical activity, comfortable at rest, but ordinary physical activity results in fatigue, palpitation, or dyspnea; Class 111- marked limitation of physical activity, comfortable at rest, but less than ordinary activity causes fatigue, palpitation, or dyspnea and Class IV- unable to carry out any physical activity without discomfort, symptoms of cardiac insufficiency at rest, it ^* any physical activity is undertaken, discomfort is increased. Heart failure treatment using the polypeptides am! methods described herein can also be classified according to the ACCVAHA guidelines (Stage A ^; At risk for developing heart failure without evidence of cardiac dysfunction; Stage B: Bvidenoe of cardiac dysfunction without symptoms; Stage C: Evidence of cardiac dys-unetion vds.h symptoms; and Stage D; Symptoms of heart failure despite maximal therapy).

in an eighteenth aspect, the disclosure features a method for treating BPH, the method comprising administering a complete or partial agonist of the GC-C receptor, including but not limited to the peptides described herein. GC-C agonists acting in the prostate can reduce cellular hypertrophy and complications associated with cellular hypertrophy. The agonist can be administered in combination with one or more agents for treatment of BPH, for example, a 5-alρha reductase inhibitor {e.g., finasteride) or an alpha adrenergic inhibitor (e.g., doxazosine).

In a nineteenth aspect, the disclosure features a method for treating obesity, the method comprising administering a complete or partial agonist of the GC-C .receptor, including but not. limited Lo the peptides and agonists described herein. The agonist. easϊ be administered alone or in combination with one or more agents for treatment of obesity, including but not limited to the ami -obesity agents described herein. Thus, for example, can be fused to the earboxy or amino terminus of a peptide of the disclosure. Such a fusion protein can include a ehymostrypstn or trypsin cleavage site that can permit cleavage to separate the two peptides.

In various embodiments: the ago.ni.st is a peptide, the peptide includes two €ys thai form one disulfide bond, the peptide includes two Cys, the peptide includes four €ys

that form two disulfide bonds, the peptide includes four Cys, two of which form a disulfide bond.

The peptides and agonists of the GC-C receptor, including but not limited to the peptides and agonists described herein can be used to treat, for example, constipation, decreased intestinal motility, slow digestion, slow stomach emptying. The peptides can be used to relieve one or more symptoms of IBS (bloating, pain, constipation), GHRD (acid reflux into the esophagus), duodeno gastric reflux, functional dyspepsia, or g-isiroparesis (nausea, vomiting, bloating, delayed gastric emptying} and other disorders described herein.

Clinically accepted criteria that define constipation range from the frequency of bowel .movements, the consistency of feces and the case of bowel movement. One common definition of constipation is less than three bowel movements per week. Other definitions include abnormally hard stools or defecation that requires excessive straining (Schiller 20Oi, Aliment Pharmacol Ther 15:749-763). Constipation may be idiopathic (furicύonal constipation or slow transit constipation) or secondary to other causes including neurologic, metabolic or endocrine disorders. ^' These disorders include diabetes raellitua, hypothyroidism, hyperthyroidism, hypocalcaemia. Multiple Sderosb, Parkinson's disease, spinal cord lesions, Neurofibromatosis, autonomic neuropathy, Chagas disease. Hirschsprung's disease and cystic fibrosis Constipation may also be the result of surgery or due to the use of drugs such as analgesics (like opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics.

fo a iwemieth aspect, the disclosure features isolated nucleic acid molecules comprising or consisting of a sequence encoding a peptide of the disclosure. The disclosure also features vectors, e.g., expression vectors that include such, nucleic acid molecules and can be used to express a peptide of the disclosure in a cultured ceil (e.g., a eukarvoiic cell or a prokaryoiic cell). The vector can further include one or more regulatory elements, e.g., a heterologous promoter or elements required for

translation operably linked to the sequence encoding the peptide, in. some cases the nucleic acid molecule will encode an amino acid sequence that includes die amino acid sequence of a peptide of the disclosure. For example, the nucleic acid molecule can encode a prcproteiα or a preproprotein that can be processed to produce a polypeptide described herein. In cases where unnatural amino acids are present in the polypeptides described herein, selector codons can be utilized in the synthesis of such polypeptides similar to that described in US20060019347 (for example. paragraphs 398-408, 457-499, and 576-588} herein incorporated by reference.

λ vector that includes a nucleotide sequence encoding a peptide of the disclosure or a peptide or polypeptide comprising a peptide of the disclosure may be either RNA oτ DNAj single- or double-stranded, prokaryotie, eukaryotic, or viral. Vectors can include iraπsposons, viral vectors, episomes, (e.g., plasmids), chromosomes inserts, and artificial chromosomes, {e.g. BACs or YACsK Suitable bacterial hosts for expression of the encode peptide or polypeptide include, but are not limited to, E. coii. Suitable eukaryotic hosts include yeast such as 5. cerevisiae, other fongϊ, vertebrate cells, .invertebrate cells (e.g.. insect cells), plant cells, human cells, human tissue cells, and whole eukaryotic organisms, (e.g., a transgenic plant or a transgenic animal). Further, the vector nucleic acid can be used to generate a virus such as vaccinia or haeulovirus (far example using the Bac-to-Ba.eφ Bacuiovirus expression system (invitrogers Life Techno fogies, Carlsbad, CA)).

As noted above the disclosure includes vectors and genetic constructs suitable for production of a peptide of the disclosure or a peptide or polypeptide comprising such a peptide. Generally, the genetic construct also includes, in addition to tho encoding nucleic acid molecule, elements that allow expression, such as a promoter and regulatory sequences. The expression vectors may contain transcriptional control sequences that control transcriptional initiation, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well known to those in the art and may be functional in. but are not limited to, a bacterium, yeast, plant, or animal cell. The expression vector can also include a translation

regulatory sequence (e.g., an untranslated 5' sequence, an untranslated 3 ^! sequence, a poly A addition site, or an. internal ribosome entry site), a splicing sequence or splicing regulatory sequence, and a transcription termination sequence. The vector can be capable of autonomous replication or it can integrate into host DNA.

The disclosure also includes isolated host cells harboring one of the forgoing nucleic acid molecules and methods for producing a peptide by culturing such a cell and recovering the peptide or a precursor of the peptide. Recovery of the peptide or precursor may refer Io collecting the growth solution and need not involve additional steps of purification. Proteins of the present disclosure, however, can be purified using standard purification techniques, such as, but not limited to, affinUy chromatography, therrnaprecipitatioπ. immunoaffϊnity chromatography, ammonium sulfate precipitation, ion exchange chromatography, filtration, electrophoresis and hydrophobic interaction chromatography,

The peptides can be purified. Purified peptides are peptides separated from other proteins, lipids, and nucleic acids or from the compounds from which is it synthesized. The polypeptide can constitute at least 10, 20 _? 50 70, 80 or 95% by dry weight oi ^' the purified preparation,

In a twenty .first aspect, the disclosure features a method of increasing the level of cyche guatxxsine 3 '-monophosphate (cGMP) in an organ, tissue (e.g, the intestinal mucosa), or eel! (e.g., a cell bearing GC-A receptor) by administering a composition that includes a peptide of the disclosure.

In twenty second aspect, the disclosure features a method for treating a disorder ameliorated by increasing cGMP levels, the method comprising administering a pharmaceutical composition comprising, consisting essentially of or consisting of SEC} SD NO. 1 or a peptide or agonist of the disclosure and a pharmaceutically acceptable carrier.

in a twenty third aspect, the disclosure features a method for preparing a polypeptide of SEQ NO:1 or any of the other polypeptides described herein by: chemically synthesizing die polypeptide and at least partially purifying the synthesized polypeptide.

in a twenty fourth, the disclosure features a method for preparing a polypeptide of SKQ II ⁾ NO; 1 or any of the other polypeptides described herein by: providing a host cells (e.g., a bacterial or mammalian or insect cell) harboring a nucleic acid molecule encoding the polypeptide, eulturing the cell under conditions suitable hr expression of the polypeptide, and at least partially purifying the polypeptide from the cell or the culture media in which the cell is cultured.

In a twenty iϊfth aspect, the disclosure features a method for treating inflammation, including imiammation of the gastrointestinal tract, e.g.. inflammation associated with a gastrointestinal disorder or infection or some other disorder, the method comprising administering to a patient a pharmaceutical composition comprising a purified peptide comprising, consisting of or consisting essentially of polypeptide of S EQ ϊD NO; 1 or any of the other polypeptides described herein. In various embodiments the mfianiraation is associated with a gastrointestinal disorder, the inflammation is not associated with a gastrointestinal disorder.

in a twenty-sixth aspect, the disclosure features a method for treating hypertension The method comprises: administering to the patient a pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide or agonist of the disclosure and a pharmaceutically acceptable carrier. The composition can he administered in combination with another agent for treatment of hypertension,, for example, a diuretic, an ACE inhibitor, an angiotensin receptor blocker, a. beta-blocker, or a calcium channel blocker,

fn a twenty- seventh aspect, the disclosure features a method tor treating secondary hyperglycemias in connection with pancreatic diseases (chronic pancreatitis,

pancrea ^t ectomy., hemochromatosis) or endocrine diseases (acromegaly. Cushmg's syndrome, pheochromocytoma or hyperthyreαsϊsX drug-Induced hyperglycemias Cbenzothiadiazine saluretics, diazoxide or glucocorticoids), pathologic glucose ϊ.o!crancc, hyperglycemias, dyslipoproteinemias, adiposity, hyperlipoproteinemias and/or hypotensions is described. The method comprises; administering to the patient a pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide or agonist of the disclosure and a pharmaceutically acceptable carrier.

Also described are therapeutic methods employing any of the forgoing polypeptides (both with and without the proviso. The therapeutic methods include treating a disorder selected from the group consisting of: a gastrointestinal disorder, cystic fibrosis, congestive heart failure, benign prostatic hyperplasia, the method comprising administering a composition comprising any of the forgoing polypeptides (both with and without the proviso). The disorders that can be treated include: a gastrointestinal motility disorder, irritable bowel syndrome, chronic constipation, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonuleer dyspepsia, gasiroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, ulcerative colitis, arκl inflammatory bowel disease as well as other diseases and disorders described herein.

Also described are methods for producing any of the .forgoing polypeptides comprising providing a cell harboring a nucleic acid molecule encoding the polypeptide, euiϊuring the cell under conditions in which the peptide is expressed, and isolating the expressed peptide.

Also described arc methods for producing any of the forgoing polypeptides comprising chemically synthesizing the peptide and then purifying the synthesized peptide.

Also described arc pharmaceutical compositions comprising the forgoing polypeptide.

Also described are nucleic acid molecules encoding any of the forgoing polypeptides, vectors ⁽ e.g., expression vectors) containing such nucleic acid molecules and host cells harboring the nucleic acid molecules or vectors.

Certain of the polypeptides described herein have some homology to a natural Iy- oeeurriπg giumy ^j in or uroguanylin. Both guanylin and υroguanylin are commonly expressed as an immature prepropolypepiide thai is processed to yield the mature polypeptide. Thus, immature guanylin and uroguanvlin polypeptides generally include a so-called "pre sequence" followed by a "pro sequence" and then the mature polypeptide sequence, ^" The pre sequence is important for secretion of the polypeptides. The pro sequence is important for proper folding of the mature protein under ;u least some conditions.

As noted above, in mature guanylin or uroguairy-Hn and in active variants {hereof disulfide bonds are present between the first and third cysteines and between the second and fourth cysteines. The pro sequences of guanylin and uroguanylin are thought to be important for proper disulfide bond formation. Moreover, guarrylin and vsroguanylin are each thought to exist as an A-isomer and a E3 -isomer. POT each protein the A- and B-isomers have the same disulfide bond connectivity but differ in three -dimensional conformation. It is thought that only the B-isorner may lack some activities (see Lauber 2005 Protein and Peptide Letters 12: 153). The pro sequences might be important for formation of the active λ-Isomer. In addition, such sequences can protect the mature polypeptide from premature degradation in the body or stabilize a particular isomer of the polypeptide, in some cases, such sequences may influence oligomerkaikm. Accordingly, in some embodiments the polypeptides described herein are produced and or administered in a form that includes a pro sequence, a pre sequence or both a pre sequence and a pro sequence (a "prepro sequence ^1' } at their amino terminus. Thus, useful polypeptides can include a pre

- 6 S -

sequence, a pro sequence or a prepro sequence preceding (amino- terminal to) a GC-C receptor agonist polypeptide described herein, FIG. 4 depicts the pre sequence (SEQ

ID NOs: - ), pro sequence (SCQ ID NOs: - ) _s prepro sequence (SEQ ID

-NOs: - ), and mature sequence for a number guanylin and urαguanyim polypeptides as well a various combinations thereof (e.g., a polypeptide consisting of a pre sequence and a mature polypeptide}.

One OT mors; of a pre sequence, a pro sequence and a prepro .sequence can be present at the amino terminus of a GC-C receptor agonist polypeptide described herein. Thus, described herein arc polypeptides comprising, consisting of or consisting essentially of (from amino terminus to carboxy terminus) one or more of: a pre sequence (SEQ fϋ NOs: - ;pre sequences) and a pro sequence (SEQ ID NOs: _ - ;pro sequences) followed by a GC-C receptor agonist polypeptide described herein, e.g., mature human gυanylm or mature human uroguanyiin. Useful GC-C receptor polypeptides that can modified by the addition of pre. pro, and/or prepro sequences include, but are not limited to:

PGTCeCASλACTGC I'SEQ ID NO: )

P Cf FCE ICATA ACTGC (SEQ ID NO: }

PGTCEtCANAACTGC (SEQ ID NO: }

PGTCE- ICIAQAACTGC (SEQ ID NO; } PGTCEICARAACTGC (SEQ ID NO: )

FGTCEiCAEAACTGC (SEQ ID NO: )

PGl CHSCADAACTGC (SEQ ID NO: ) lO-fCEICλGAACTGC fSEQ ID NO: )

PGTCEICAAAACTGC (SEQ ID NO: ) PGTCEfCAMAACTGC (SEQ ID NO; )

PGTCEICAIAACTGC (SEQ ID NO: )

PGTCEICALAACTGC (SEQ ID NO: )

PGTCE 1C AVAACTGC (SEQ ID NO; )

PGTChϊCλHλACTGC (SEQ ID NO: )

!'GTCEGiCAYAACT(K: (SEQ ID NO: ) PGTCEiGCAYAACTGC (SEQ ID NO; ) PGTCEiCGAYAACTCC (SEQ ID NO: ) PGTCEICAGYAACTGC (SEQ ID NO; } PGTCE! ^' CλYGAACTGC (SBQ ϊD NO: } !'GTCEiCAYAGACTGC (SEQ ID NO: ) !'GTCEiCAYAAGCTGC (SEQ ID NO: ) PGTCmCAYAACGTGC (SEQ ID NO: ) PGTCKK ¹ AYAACTGCtC (SEQ ID NO: } PGTCAEiCAYAACTGC (SEQ ID NO; } FGTCE AICAYAACTGC (SEQ ID NO: ) PGTCE IACAYAACTGC (SEQ ID NO: ) PGTCE ϊCAAYAλCTGC (SEQ SD NO: ) PGTCE 1C AYAA ACTGC (SEQ ID NO: ) PGTCEICAYAACAATGC (SEQ ]D NO: } PGTCmCAYAACTλGC (SBQ LD NO; } PGTCπCAYA ACTGAC (SEQ IO NO: } PGTCAHICAAYAACTGC (SEQ ID NO: ) PGTCEAϊCAAYAACTGC (SEQ ID NO; ) and PGϊCHIACAAYAACTGC (SEQ ID NO; },

In soo^e cases it may be desirable to have a polypeptide that includes a pre sequence from a first guanylis or uroguanylin polypeptide and a pro sequence from a second gvianylin or uroguanyUn polypeptide. In other cases, the pre sequence and the pro sequence are from the same guanylin or uroguanylin polypeptide.

Useful polypeptides can include a naturally-occurring guanylin or urogua&ylin polypeptide in its. mature form, as a prepro polypeptide- (includes, from amino terminus to carboxy terminus, pre sequence, pro sequence and mature polypeptide), as & propolypeptide (includes, from amino terminus to carboxy terminus, pro sequence

and mature polypeptide) or as a prepolypeptide (includes, from amino terminus to carboxy terminus, pre sequence and mature polypeptide). FIG. 4 depicts these various guanylin or uroguanylin polypeptides,

hi some cases a polypeptide will be produced, e.g., reeombmaniiy, wiih a pre sequence and/or a. pro sequence, In certain cases the pre sequence and/or pro sequence is removed prior to administration of the polypeptide to a patient. In other cases the prεpropolypeptide, propolypcptide or the prepoϊypeptide is administered to the patient. The pre sequence and/or the pro sequence may stabilize the polypeptide or an active isomer thereof, facilitate efficient folding of the polypeptide or protect the polypeptide from degradation in the patient's body. Thus, pre sequences, pro sequences ursd/or proprosequences that do not significantly interfere with (5(74 ^' ? receptor agonist activity can be bcneSάal. In some cases the pre sequence and/or the prosequenee are removed by physiological processes after administration,

hi some eases useful polypeptides will include only a portion (e.g., 20, 15, 12, 1 1 , 10, 9, 8, 6, 5, 4 or fewer) of the amino acids of a pie sequence (SEQ ID NOs: - }, pro sequence (SBQ ID NOs: ^■■ ), prepro sequence (SEQ ID NOs: - ).

As can be seen in FIG. 4, pro sequences include Cys residues thai may form a disulfide bond. For example, many pro sequences include two Cys residues separated by 12 ammo acids. These Cys residues may form a disulfide bond. These Cys residues can be replaced by homocysteine, penicillamine, 3-mercaptopτoiine

(Koiodziej et ai. 1096 Lnt J Fcpi Protein Res 48:274); β, β- dirnethylcysfeme (Hunt et al. 1993 lnt J f ^J ept Protein Res 42:249) or diamine-propionic acid (Smith et al 1978 j Med Chem 21 : 1 17) to form alternative internal cross-links at the positions of the .normal disulfide bonds.

Metabolites of λsparaginc in some cases an asparaginc (Asn) within a polypeptide can he rnehibolizcd to have a differeru structure and the GC receptor agonist containing such a metabolite of Asn

may retain activity. Polypeptides where one or more λsn, e.g., one or more λsn within an embodiment of S EQ ϊD NO; I described herein are replaced by a metabolite of λsr; can be useful in the methods described herein and eaπ be present in a pharmaceutical composition thai optionally contains one or more additional active iπgreαi exits.

For example, one or more Asn within, a polypeptide and the peptide bond earboxy terminal thereto having the structure:

can repiatx'd by a group having a structure selected from:

Thus, the and the peptide bond earboxy terminal there to can be replaced by a cyclic imide:

Asp:

or ϊSGASΏ :

1 he λsp can be L-λsp or l. ⁾ -λsp. The isuλsπ cars be D-isoλsrt or L-isoAsα

(Considering the asparaginic only, one or more Asn having the structure;

is can be optional Iy replaced by a group having a structure selected from (a), (b) and

(C);

provided that an Asn at the earboxy terminus is not replaced by structure (a) or structure (c). When the Asn is at the earboxy terminus of the peptide, structure (a} cannot form. Since structure (c) is formed through structure {a}, structure (c) cannot be tbrnicd when the Asn is at the earboxy terminus.

The formation of the various metabolites of λsp is depicted below.

The details of one or more embodiments of the disclosure are set forth in (he accompanying description. All of the publications, patents and patent applications are hereby incorporated by reference.

DRAWINGS

FlG.1 depicts deletion variants of human guanyiin in which one, two, three or four amino acids are deleted The deleted amino acids are between Cys _;! and Cys _« as well as amino terminal to Cys _a ,

Fi ⁽ I 2 depicts insertion variants of human guanylin in which one, two, three or four amino acids are inserted. The inserted amino acids are between Cy^ and CyS _d as well as .amino terminal to Cys _a and carboxy terminal to CyS _1J .

FKl 3 depicts various polypeptides which include the amino acid sequence: Xaa ₅ Xaa? Xaa^ Cy$4 Xaa« Xaa _& Xaa-? Xaa^ Xaa^ Xaa: _(! Xaa _s j Cys _{i :} > Xaa^ Xaa^ Xaaj ₅ Xaak, (SEQ !.D NO:1 j wherein: Xaa _; is any amino acid or is missing: Xaa_» is any aπuno nάά or is missing; Xaa^ is any amino acid or is missing; Xaa^ is CHu; Xaa _< , is Tyr, 1 ^' rp. Phe or Leu; Xaa? is C'ys;

Xaa^ is any of the 20 naturally-υceαrring amino acids other than CX-'s or is missing; Xaa« ss any of the 20 naturally-occurring amino acids; Xaaio is Pro or GIy; Xaai ; is any of the 20 naturally-occurring amino acids; Xaa;? is Thr, VaI or GIy; Xaa^ is GIy or Ala; Xaa^ is Cys; and Xaaf6 is ^v of the 20 naturaily-oecurring amino acids or is missing.

F!G 4 is a table depicting the sequences of various gnaπylin and uroguanyiin polypeptides, including prc sequences, presequεnces, prepro sequences, mature sequences and combinations thereof.

DElAlLED DESCRIPTION

The peptides of the disclosure bind to the guanylatc cyclase (GCNC?) receptor, a key regulator of fluid and electrolyte balance in the intestine and kidney. When stimulated, this receptor, which is located on the apical membrane of the intestinal S epithelial surface, causes an increase in intestinal epithelial cyclic GMP {cGMP}.

This increase in eGMP is believed to cause a decrease in water and sodium absorption and aii increase in chloride and potassium ion secretion, leading to changes m intestinal fluid and electrolyte transport and increased intestinal motility. The intestinal GC-C receptor possesses an extracellular iigand binding region, a 10 transmembrane region, an intracellular protein kinase-like region and a cyclase catalytic domain. Proposed functions for the GC-C receptor are the fluid and electrolyte homeostasis, the regulation of epithelial cell proliferation and the induction of apoptosis (Shailhabhai 2002 Cmr OpIn DrugDis Deveϊ 5:261-268).

In addition to being expressed in gastrointestinal epithelial cells. GC-C is expressed in ^• ;*> extra -intestinal tissues including kidney, lung, pancreas, pituitary, adrenal developing, liver, bean and male txnά female reproductive tissues (reviewed in Vaandrager 2002 Moi Cell Biochem 230:73-83). This suggests that the GC-C receptor agonists can be used in the treatment of disorders outside the Gl tract, for example, congestive heart failure and benign prostatic hyperplasia.

20 Ghrehn, a peptide hormone secreted by the stomach, is a key regulator of appetite in humans. Ghrelin expression levels are regulated by fasting and by gastric emptying, (Kirn et a!., 2003, Neuroreprt 14: 1317-20; GualiHo et al, 2003, FEBS Letts 552: 105- 9). Thus, by increasing gastrointestinal motility, GC-C receptor agonists may also be used to regulate obesity.

25 In humans, the GC-C? receptor is activated by guanyhn (Gn) (U.S. Patent 5.06,097), isroguanyiin fUgn) (U.S. Patent 5, 140, 102} and lyraphogisarrylin (Forte et a!, 1999 Endocrinology \ 40: 1800 ^» 1806).

- ?6 -

Many gastrointestinal disorders, including IBS, are associated with abdominal or viscera! pain. Certain of the peptides of the disclosure include the analgesic or antinociceptive tags such as the earboxy-terminal sequence AspPhe immediately following a 1 rp, Tyr or Phe (i.e., a chymotrypsin cleavage site) or following Lys or 5 λrg {a trypsin cleavage site), Chymotrypsin in the intestinal tract will cleave such peptides immediately carboxy terminal to the Trp, Phe or Tyr residue, releasing the dipeptide. AspPhe. Thss dipeptide has been shown to have analgesic activity is animal models (Abdikkahi et ai. 2001 Ftmrfam Clin Pharmacol 15: I l 7-23; Mikfar et a) ! W7, 29:5^3-6; Edrnundson ct ai 1998 CHn Pharmacol liter 63:580-93), in this

W manner such peptides can treat both pain and inflammation. Other analgesic peptides can be present at the carhoxy terminus of the peptide (following a cleavage site) including: rødornoφhύvi. endo.morp3-rin~2, noeislatin, dalargin. lupron, ziconotide, and substance P. As described in greater detail below, various analgesic peptides and compounds can be covaienlly linked to or used in combination therapy with the

^■ )$ therapeutic peptides described herein.

In the human body an inactive form of chymotrypsin, ehymotrypsmogen is produced in the pancreas. When this inactive enzyme reaches the small intestine it i.s converted Lo active chymotrypsin by the excision of two di -peptides. Active chymotrypsin will cleave peptides at the peptide bond on the earboxy-terminai side of Trp, Tyr or Phe.

20 The presence of active ehyrrωtrypsm in the intestinal tract will lead to cleavage of certain of the peptides of the disclosure having an appropriately positioned chymotrypsin cleavage site. Certain of the peptides of the disclosure include a Trp, Tyr or Phe immediately followed by a carboxy-terminal analgesic peptide. It is expected that ehymotrypsin cleavage will release die analgesic peptide from peptide

25 oft.be disclosure having an appropriately positioned, chymotrypsiri cleavage site as the peptide passes through the intestinal tract.

Tripsinogen, like ehymotrypsin, is a serine protease that is produced in the pancreas and is present in the digestive tract. The active form, trypsin, will cleave peptides having a Lys or Arg. The presence of active trypsin in the intestinal tract will lead to

cleavage of certain of the peptides of the disclosure having an appropriately positioned trypsin cleavage site. It is expected that chymotrypsiii cleavage will release the analgesic peptide from peptide of the disclosure having an appropriately positioned trypsin cleavage site as the peptide passes through the intestinal tract.

5 In sonic cases, the peptides of the disclosure are produced as a prepro protein. The prepro protein can include any suitable prepro sequence, including but not limited to, for example, rαnatlLsalc llgawaalag gvtvqdgnfs fslesvkklk dfqepqeprv gkirofapip gepvvpilcs npntpedkp ickepnaqei Iqrleciaed (SEQ ID NO: ), mgeraasgli pgvavviill iqsiqsvyiq yqgfrvqbs mkklscileaq wapsprlqaq silpavchhp alpqdlqpvc asqeassifk tlrtia

!0 (SBQ ID NO: }. iriia (SEQ ID NO. }, rnπawllsvlc figalavlve gvtvqdgdls tplεsvkqlk hlrevqcpti mshkkfalri pkpvapdes qsafpeakp lcekpnacei Iqrleaiaqd (SBQ ID NO: }. and msgsqiwaav IMIvlqsaq gvyikyjhgfq vqlesvkkln deekqmsdp qqqksgilpd vcynpaipld kjpveasqea astlkalrti a (SEQ ID ^O: ) or a bacterial leader sequence such as: πϊkksilfiπsvlϋispfaqdakpvesskekitleskkcπiakksαksgpesmn . Where the peptide is

^■ :5 produced by a bacterial cell, e.g., £. coli, the forgoing leader sequence will be cleaved and the mature peptide will be efficiently secreted from the bacterial cell LLvS. Patent No. 5395,490 describes vectors, expression systems and methods for the efficient production of certain mature peptides having disulfide bonds in bacterial ceils and methods for achieving efficient secretion of such mature peptides. The vectors,

?.ϋ expression systems and methods described in U.S. Patent No. 5,395,490 cars be used to produce die polypeptides of the present disclosure.

The disclosure includes variant peptides that can include one, two. three, four, or five or more (e.g., 6, 7, S, 9, H), 11, 12, 13, 14, or ! 5) amino acid substitutions compared 25 to any of the peptides described above. The substitutionals) can be conservative or non-conservative. The πaturaHy-oecurring amino acids can be substituted by D- isomcrs of any amino acid, non-natural amino acids, natural and non-natural amino acid analogs, a_κi other groups. A conservative amino acid substitution results in the

- 7S -

alteration of an amino acid tor a Similar acting amino acid, or amino acid of like charge, poiaπty, or hydrophobieity. At some positions, even conservative amino acid substitutions can reduce the activity of the peptide. A conservative substitution can substitute a naturally-occurring amino acid for a non-natural! y-occurring amino acid. Among the naturally occurring ammo acid substitutions generally considered conservative are:

For Amino Acid Code Replace with anv of

Alanine Ala GIy, Cys, Ser

Argininc Arg Lys, His

Asparagine Asn Asp, GIu. GIn,

Aspartic Acid Asp Asn, GIu, Gin

Cysteine Cvs Met, Thr, Ser

(Histamine Gb Asn, GIu, Asp

Glutamic Acid GIu Asp, Asn. GIn

Glycine GIv Ala

I lisϊidine His Lys, Arg

Isoleiicme ϊle VaK Leu, Met

Leucine Leu VaI, He, Met

Lysine Lvs Arg, His

Methionine Met lie. Leu. Va!

Phenylalanine Phe IVr ₅ His, Trp

Proline Pro

Serine Ser Thr, Cys, Ala

Threonine Thr Ser, Met, VaI

Tryptophan Trp Phe, Tyr

Tyrosine Tyr Ph^ ffis

Valine Va! Leu, Oe, Met

in some circumstances it can be desirable to treat patients with a variant peptide that binds to and activates intestinal GC-C receptor, but is less active or more active than the Bon-vanarit form of the peptide. Reduced activity can arise from reduced affinity for the receptor or a reduced ability f.o activate the receptor once bound or reduced stability oS ^' frso peptide, increased activity can arise from increased affinity for the receptor or an increased ability to activate the receptor once bound or increased stability of the peptide.

In sonic peptides one or both members of one or both pairs of Cys residues which normally form a disulfide bond cart be replaced by homocysteine, penicillamine, 3- mereaptoproime (Kokxiziei ef a! i 996 Int J Pep! Protein Rex 48:274); (x [> dimethyleysteioe (Hunt et a!, 1993 int JPept Protein Res 42:2.49} or diarninopropiofiic acid { Smith et al . S 978 J Med Cheni 21 : 1 ! 7) to form alternative internal cross-links at the positions of the normal disulfide bonds.

Useful peptides can be produced either in bacteria including, without (imitation, E. colL or in other existing systems tor peptide or protein production (e.g.. Bacillus whlilis. baculovims expression systems using Drosophila S© cells, yeast or

AlarπeMous fungal expression .systems, mammalian cell expression systems), or they C3B be chemically synthesized.

If the pcpiide or variant peptide is to be produced in bacteria, e.g., E. cvlL the nucleic acid molecule encoding the peptide may also encode a leader sequence that permits the secretion of the mature peptide from the cell. Thus, the sequence encoding the peptide can include the pre seqwnee and the pro sequence of, for example, a naturally-occurring bacterial ST peptide. The secreted, mature peptide can be purified from the culture medium.

The sequence encoding a peptide of ^" the disclosure is can be inserted into ιx vector capable of delivering and maintaining the nucleic acid molecule in a bacteria! cell. The DNA molecule may be inserted into an autonomously replicating vector {suitable vectors include, for example, pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid may be a bacteria! or bacteriophage DNA such as bacteriophage lambda or M13 and derivatives thereof. Construction of a vector containing a nucleic acid described herem can he followed by transformation of a host cell such as a bacterium. Suitable bacteria! hosts include but are not limited to, /:.'. coli. B siώitih, Pstfiuiomofw:>, Salmonella. The genetic construct also includes, in addition to the

encoding nuclei e acid molecule, elements that allow expression, such as a promoter and regulatory sequences. The expression vectors may contain transcriptional control sequences that cαmroi transcriptional initiation, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well 5 known to those m the art. The expression vector cars also include a translation regulatory sequence (e.g., an untranslated 5' sequence, an untranslated 3' sequence, or an internal πbosome entry site). The vector can be capable of autonomous replication or it can integrate into host DNA to ensure stability during peptide production.

Hie protem coding sequence that includes a peptide of the disclosure can also be 0 iϊssed to a nucleic acid encoding a polypeptide affinity tag, e.g., glutathione S- transferase (GST), maltose E binding protein, protein A, FLAG tag. hexa-hishdine. rnyc tag or the iniluemia HA tag, in order to facilitate purification. The affinity tag or reporter fusion joins the reading frame of the peptide of interest to the reading frame of the geαe encoding the affinity tag such that a transnational fusion is generated. S Expression of the fusion gerse results in translation of a single polypeptide that includes both the peptide of interest and the affinity tag. In some instances where affinity tags are utilised. DNA sequence encoding a protease recognition site will be fused between the reading frames for the affinity tag and the peptide of interest.

Genetic constructs and methods suitable for production of immature and mature forms 0 of the peptides and variants of the disclosure in protein expression systems other than bacteria, and well known to those skilled in the art, can also be used to produce peptides in a biological system.

Mature peptides and variants thereof can be synthesized by the solid-phase method using an automated peptide synthesizer. For example, the peptide can be synthesized on Cyc(4~C.li> Bxl)~OCH ₂ ~4~(oxymetliyl)-phcnylacetaiτtidomcthy] resin using a double coupling program. Protecting groups must be used appropriately to create the correct disulfide bond pattern. For example, the following protecting groups can be used; i-butyioxycarhonyl {alpha- amino groups); acetamidomethyl (thiol groups of Cys

residues B and H); φmethylbenyl (thiol groups of Cys residues C and F); benzyl (y- carboxyl of glutamic acid and the hydroxy! group of threonine, if present), and hromobeπzyl {phenolic group of tyrosine, if present). Coupling is effected with symmetrical anhydride of t-bυtoxykarbonylaminø acids or hydroxybenzotriazolϋ ester (for asparaginc or glυlainine residues), and the peptide is deprotecteά and cleaved from the solid support in hydrogen fluoride, dimethyl sulfide, anisoie, and p- Oiioorcsol using 8/1/1/0,5 ratio (v/v/vλv) at D ⁰ C for 60 mm. After removal of hydrogen fluoride and dimethyl sulfide by reduced pressure and anisoic and p- thioeresoi by extraction with ethyl ether and ethyl acetate sequentially, crude peptides are extracted with a mixture of 0.5M sodium phosphate buffer, pR 8.0 and N,N- dimethyltonnamide using 1/1 ratio, v/v. Disulfide bonds between €ys residues can he formed using dimethyl sulfoxide (Tarn et al. (199!) j. Am, Chem. Soe, 1 13:6657-62). ^" [lie resulting peptide is the purified by reverse-phase chromatography. In some cases it may be necessary to first dissolve the peptide m 50% acetic acid in water before disulfide bond formation. Saturated iodine solution in glacial acetic acid is added { 1 ml iodine solution per 100 m! solution). After incubation at room temperature for 2 days in closed glass container, the solution is diluted fϊve-ibkl with deionixed water and extracted with ethyl ether four times for removal of unreacted iodine. After removal of the residual amount of ethyl ether by rotary evaporation the solution uf crude product is lyophilixed and purified by successive reverse-phase chromatography.

Peptides can also be synthesized by many other methods including solid phase synthesis using traditional FMOC protection (i.e., coupling with DCC-ROBt and depHUectioii vviih piperdine in DMF). Cys thiol groups can be trityi protected. Treatment unh TFA can be used for final deprotectiøπ of the peptide and release of the peptide from the solid-state resin. In many eases air oxidation is sufficient to achieve proper disulfide bond formation.

iDAJt-stjjIg.LQC-.C Receptor Binding and. Activity Assays

The ability of peptides, variant peptides and other compounds to bind So and activate the imestk-al GOC receptor can be tested using the TS4 human colon carcinoma cell line (American Type Culture Collection (Sethesda, Md.),

Bπeily, cells arc grown to eoo.fi ueney in 24- well culture plates with a 1 : 1 mixture of Ham's F 12, medium and Dυlbecco's modified Eagle's medium (DMEM), supplemented with 5% fetal calf serum and are used at between passages 54 and 60.

^■ ;0 Monolayers of T84 cells in 24-welI plates are washed twice with 1 tnl/weU DMEM. then incubated at 37°C for 10 min with 0.45 ml DMEM containing I niM isobutyimethyixanthine (IBMX), a cyclic nucleotide phosphodi esterase inhibitor. Test peptides (SOμl) are then added and incubated for 30 minutes at 37°C. The media Is aspirated and the reaction is terminated by the addition of ice cold 0.5 ml of OJN

15 IiCK The samples are held on ice for 20 minutes and then evaporated to dryness using a heat gun or vacuum eentritugation. The dried samples arc reauspended in ϋ.5ml of phosphate buffer provided in the Cayman Chemical Cyclic (JMI ⁵ £(A kit (Cayman Chemical Ann Arbor, Ml). Cyclic OMP is measured by BIA according Io procedures outlined in the Cayman Chemical Cyclic GMP EIA kit. 0

For the binding assay, T84 cell monolayers in 24-wεlf plates are washed twice with 1 ml of binding buffer (DMEM containing 0.05% bovine serum albumin and 25 niM Hf- PES, pi ! 7.2), then incubated for 30 min at 37 ^'3 C in the presence of mature radioactive^ y labeled E. coli ST peptide and the test material at various b concentrations. The cells are then washed 4 times with 1 ml of DMEM and solubilizeα with 0.5 ml/wel) IN NaOlL The level of radioactivity in the soTαhilizeα material is then determined using standard methods.

M^.DM |λfisi-Xiiiiiie§iina| jtraπM JiSIQ .assay

- R > -

in order to determine whether a test compound or a peptide, increases the rate of gastrointestinal transit, the test compound can he tested in the murine gastrointestinal transit (CHT) assay (Moon et al Infection ami Immunity 25: 127, 1979). hi this assay, charcoal, which can he readily visualized in the gastrointestinal tract is administered to mice after the administration of a test compound. The distance traveled by the charcoal is measured and expressed as a percentage of the total length of the colon.

Mice are tasted with free access to water tor 12 to 16 hours before the treatment with peptide or control buffer, ^' The peptides are orally administered at 1 μg/kg ■- !mg/kg of peptide in buffer (2OmM TrLs pH 7.5) seven minutes before being given an oral dose of 5% Activated Carbon (Aldrieh 242276-250G). Control mice arc administered buffer only before being given a dose of Activated Carbon. After LS minutes, the mice are sacrificed and their intestines from the stomach to the cecum are dissected. The total length of the intestine as well as the distance traveled from the stomach to the charcoal front is measured for each animal and the results arc expressed as the percent of the total length of the intestine traveled by the charcoal front. Results are reported as the average of 10 mice ± standard deviation. A comparison of the distance traveled by the charcoal between the mice treated with peptide versus the mice treated with vehicle alone is performed using a Student's t test and a statistically significant difference is considered for P< 0,0,5, Positive controls for this assay may include commercially available wild-type ST peptide (Sigma-Aϊdrich, St Louis, MO) and Zdnormøk a drug approved for IBS that is an agonist for the serotonin receptor 51TT4.

Similar assays can be performed in other rodents, for example, rats, hi addition, GlT assays can be performed and compared in wild-type versus rodents lacking the guanylate cyclase C receptor (GC-C KO), for example, using the GC-C KO mice described in Mann et ai 1997 Biochcm and Biophysical Research Communications 239:463.

Suckling ^" mouse model of intestinal secretion (SuMi assav

The peptides of the disclosure can be tested for their ability to increase intestinal secretion using a suckling mouse model of intestinal secretion. In this model a test compound is administered to suckling mice that, are between seven and nine days aid. After the mice are sacrificed, the gastrointestinal tract from the stomach to the cecum is dissected ("'guts"), ^" The remains ("carcass") as well as the guts are weighed and the ratio of guts to carcass weight is calculated. If the ratio is above 0.09, one can conclude that the test compound increases intestinal secretion. Controls for this assay may include wild-type ST peptide and Zeloorrn®,

Phsnylbenyoquinone-induced writhing ..model

The PBQ-iπchiccd writhing model can be used to assess pain control activity of ihc peptides and CiC-C receptor agonists of the disclosure. This model is described by Siegrøuuά et a). ( 1957 Proc. Soc. Hxp. Bio. Med. 95:729-73 !). Briefly, one hour after oral dosing with a test compound, e.g.. a peptide, morphine or vehicle, 0,02% phenyibenzoquioone (PBQ) solution f 12.5 raL/kg) is injected by intraperitoneal route into the mouse. The number of stretches and wrilhύigs are recorded from the 5* to the IO ^lh minute alter PBQ injection, and can also be counted between the 35 ^th ami 40 ^tf! minute and between the 60 ^l " and 6S ^lh minute to provide a kinetic assessment. ^' The results are expressed as the number of stretches and wnihiπgs (mean -t SBM) and the percentage of variation of the nociceptive threshold calculated from the mean value of ihe vehicle-treated group. The statistical significance of any differences between ttse treated groups and the control group is determined by a Dimnetfs test using the residual variance after a one-way analysis of variance fP< 0.05} using SigmaStat Software.

Colonic hyperalgesia animal models

Hypersensitivity to colorectal distension is a common feature in patients with IBS and may be responsible for Ihe major symptom of pain. Both inflammatory and non-

inflammatory animal models of visceral hyperalgesia to distension have been developed to investigate the effect of compounds on visceral pain in IBS,

Male Wi star rats (220-250 g) arc preroedicated with 0.5 mg/kg of acepromazme injected intraperitoneal Iy (IP) and anesthetized by intramuscular administration of 100 mg/kg of ketaniine. Pairs of nkhrome wire electrodes (60 cm in length and 80 μm in diameter) are .unplatted in the striated muscle of the abdomen, 2 cm laterally from the white line. The tree ends of electrodes are exteriorized on the back of the neck and protected by a plastic tube attached to the skin. Electromyographic (EMG) recordings arc started 5 days after surgery. Electrical activity of abdominal striated muscle is recorded with an electroencephalograph machine (Mini VlII, Alvar, Pads, France} using a short, time constant (0.03 sec.) to remove low-frequency signals (<3 Hz).

Ten days post surgical implantation, trinitrobenzenesulphonic acid (TN BS) is administered to .induce rectal inflammation. TNBS (80 nig kg" * in 0.3 ml 50 % cthanol) is administered intrarectal!}' through a silicone rubber catheter introduced at 3 cm from the anus under light diethyl -ether anesthesia, as described (Morteau et a!. 1994 Dig Ois Sci 39: 1239). Following TNBS administration, rats are placed in plastic tunnels where they arc severely limited in mobility for several days before colorectal distension (CRD), Experimental compound is administered one hour before CRD which is performed by insertion into the rectum, at 1 cm of the anus, a 4 cm long balloon made from a latex condom (Gue et al, 1997 Neurogasiroemerol Maiii. 9.271). The balloon is fixed on a rigid catheter taken from an emboiectomy probe (Fogarty). The catheter attached balloon is fixed at the base of die tail The balloon. connected to a barostat is inflated progressively by step of 15 mmHg, from 0 to 60 mraHg, each step of inflation lasting 5 rain. Evaluation of rectal sensitivity, as measured by KMG, is performed before ( 1-2 days) and 3 days following recta! instillation of TNBS.

The number of spike bursts that corresponds to abdominal contractions is determined per 5 rain periods. Statistical analysis of the number of abdominal contractions and evaluation of the dose-effects relationships is performed by a one way analysis of variance (ANOVA) followed by a post-hoc (Student or Duoπett tests) and regression 5 analysis for ED50 If appropriate.

H. _. ^P^:iMttce4Jrrl?erjdgrøiainodej

Male Wisiar Rats (200-250 g) are surgically implanted with nichrome wire electrodes as in the TNfSS model. Ten days post surgical implantation, partial restraint stress iα (PRS _. ), is performed as described by Williams et aϊ. ibr two hours ( Williams et al.

1988 Gastroenterology 64;61 1 ). Briefly, under light, anaesthesia with ethyl-ether, the ibreshoukiers, upρ«r ibrelimbs and thoracic trunk are wrapped in & confining harness of paper tape to restrict, bin not prevent body movements. Control sham-stress animals are anaesthetized but not wrapped. Thirty minutes before the end of the PRS

15 session, the animals are administered test-compound or vehicle. Thirty minutes to one hour after PRS completion, the CRD distension procedure is performed as described above ibr the TNBS mode! with barostat at pressures uϊ 15, 30, 45 and yOram Hg. Statistical analysis on the number of bursts is determined and analyzed as in the TNSS model above.

;?o S IL Water avoidance stress-induced hyperalgesia model

The effect of peptides/GC-C agonists of the disclosure on basal visceral nociception in a model of water avoidance stress-induced visceral hyperalgesia in adult male Wisiar rats can be tested. The stress involves confining rats to a platform surrounded by water for a period of 1 hour and then measuring their visceromotor

25 response to colonic distension using electromyography (EMG).

At least 7 days prior to stress measurements, animals are deeply anesthetized with pentobarbital sodium (45 trig/kg) and equipped with electrodes implanted into the external oblique musculature, just superior to the inguinal ligament. Electrode leads arc then tunneled suheytaneoυsly and externalized laterally for future access,

30 Following surgery, rats are housed in pairs and allowed to recover for at ltast 7 days.

On the day of the experiment animals are HgMIy anesthetized with halothane, and a Rubricated latex balloon (6 cm) is inserted ϊntra-anally into the descending colon. Animals are allowed to recover for 30 minutes, and colorectal distension (CRD) is initiated. The CRD procedure consists of graded intensities of phasic CRD (KX 20, 40, 60 mniHg; 20 s duration; 4 min intcr-stimuhis interval). Visceromotor response (VVlR) io C ¹ RL) is quantified by measuring EMG activity. To determine tlie effects of peptides/GC-C agonists of the disclosure on basal visceral nociception, a baseh ^' πe CRD is recorded, Animais are allowed 1 hour recovery and then the peptide/GC-C agonisi of the disclosure or vehicle is orally administered. At i hour following admirsistriuion of peptIde/GC~C agonist of the disclosure or vehicle CRD is repeated.

To determine the effect of peptides/GC-C agonists of the disclosure in a mode! of water avoidance stress-induced visceral hyperalgesia, a baseline CRD is recorded and then the animals were subjected to 1 hour of water avoidance stress. For water avoidance stress, the test apparatus consists of a PSexiglas tank with a block affixed to the center of the floor. The tank is filled with fresh room temperature water (25"C) to within i em of the top of the block. The animals are placed on the block for a period of 1 hour, ^' The sham water avoidance stress consists in placing the rats on the same piatibrm in a waterless container. A second CRD is performed at 24 hours post water avoidance stress. Following the second CRD, animals are allowed 1 hour recovery and then the peptide/GC-C agonist of the disclosure or vehicle is orally administered, At I hour following administration of peptide/GC-C agonist υf the disclosure or vehicle CRD is repeated. Mean +/- SEM is be determined and compared in the presence and absence of water avoidance stress conditions.

- m *

Kd determination and binding assays

To determine the affinity of peptides/GC-€ agonists of the disclosure lor GC- C receptors found in ral intestinal mucosa, a competition binding assay is performed using rat intestinal epithelial cells. Epithelial cells from the small intestine of rats are obtained as described by Kessler el al. (J. BhL Chem. 245; 5281-5288 ( 1970)). Briefly, animals arc sacrificed and their abdominal cavities exposed. The small intestine is rinsed with 300 ral ice cold saline or PBS. 10 em of the small intestine measured at 10 em from {he pylorus is removed and cut into 1 inch segments, intestinal mucosa is extruded from the intestine by gentle pressure between a piece of para film and a P-IOi)O pipette tip. Intestinal epithelial cells are placed in 2 ml 1*83 and pipetted up and down with a 5 mi pipette to make a suspension of cells. Protein concentration in the suspension is measured using the Bradford method (Aim!, βiυckcm. 72: 248-254 ( 1976)).

A competition binding assay is performed based on the method or ^' GiannelJa et al. {Am, ,/. Physiol. 245; G492-G498) between ['" ⁵ IJ labeled control peptide (e.g. wild-type guanyiirt, uroguanylin or ST peptide) and a peptide'6C-C agonist of the disclosure. The assay mixture contains: 0.5 ml of DfVlE with 20 rnM HEPES-KOH pϊl 7.0, ϋ.9 mg of the cell suspension listed above, 21.4 .rrauJ. [' ^"'5 Ij- labeled control. peptide (42.8 pM), and different concentrations of competitor peptide/GC-C agonist of the disclosure (0.01 to 1000 ,nM). The mixture is incubated at room temperature for 1 hour, and the reaction stopped by applying the mixture to GF/8 glass-fiber filters; (Whatman). The filters are washed with 5 ml ice-cold FBS and radioactivity is measured. KcI is determined. %B/Bo is the percentage of due ratio of radioactivity {rapped in each sample (B) compared to the radioactivity retained in a control sample with no cold competitor (Bo).

Similar competition binding assays are performed in intestinal epithelial cells from vvj Id-type and guaπylate cyclase C knockout (GC-C KO; Mann et al. 19^7 Biocheni ami Biophysical Research Communications 239:463} mice. Mouse intestinal epithelial ceils axe prepared identical to that above as for rat intestinal

- so -

epithelial ceils except the cells are homogenized with an Omni honwgemzer for 20 seconds on the maximum setting to make a suspension of cells. A competition binding assay is performed identical to thai described above between ^Ur 'l labeled peptiik/GC-C agonist of die disclosure and unlabeled peptide/GC-C agonist of the disclosure {competitor).

Pharmacokinetic property determination of peptides/GC-C agonists of the disclosure

Serum .samples are extracted (torn the whole blood of exposed {mice dosed orally or intravenously with peptides) of the disclosure) and control mice, then injected directly (I OmL) onto an in-line solid phase extraction (SPE) column (Waters Oasis \ ILB 25μnϊ column, 2.0 x tSram direci connect) without further processing. The sample on the SPE column is washed with a 5% methanol, 95% di l >O solution (2.1 mϊ../mm, 1.0 minute), then loaded onto an analytical column using a vah-e switch that places the SPB column in an inverted flow path onto the analytical column (Waters Xlerra MS CH Sμra IS column, 2.1 x 20πirn), The sample is eluied from the analytical column with a reverse phase gradient (Mobile Phase A: H) niM ammonium hydroxide in dlbϋ. Mobile Phase B: IO mM ammonium hydroxide in 80% acetcmitrik and 20% methanol; 20% B for the first 3 minutes then, ramping to 95% B over 4 πiirs. and holding for 2 min., all at a flow rate of 0.4 niL/min.). At 9. ! minutes, the gradient returns to the initial conditions of 20%B for 1 mm. Peptide is eluted from the analytical column and is detected b> triplc-quadrapole mass spectrometry (MRM, 764 { -i-2 charge state)> 182 ( ^• ? ^■ 1 charge state) Da; cone voltage ^:::; 30V; collision ^::; 20 eV; parent resolution ^:;; 2 Da at base peak; daughter resolution ^::; 2 Da at base peak). Instrument response is converted into concentration units by comparison with a standard curve using krκm-rs amounts of chemically synthesized peptide(s) prepared and injected in mouse plasma using the same procedure.

Similarly, pharmacokinetic properties are determined in rats using !.,CMS methodology. Rat plasma samples containing the peptide are extracted using a Waters Oasis K4AX % well solid phase extraction (SPE) plate. λ 200 uL volume of

rat plasma is mixed with 200 μL of " ¹ C ₉ , ^{i 5} N -labeled peptide in the well of a prepared SPE plate. The samples are drawn through the stationary phase with 15 mm Hg vacuum. All samples are rinsed with 200 μL of 2% ammonium hydroxide in water followed by 200 μL of 20% methanol in water. The samples are el u ted with 5 consecutive I OO μL volumes of 5/20/75 formic acid/water/rnethanoi and H)O μL 5/ i 5/80 formic acid/water/metnanol. The samples are dried under nitrogen and resuspeπded in 100 μL of 20% methanol in water. Samples are analyzed by a Waters Quatiro Micro mass spectrometer coupled to a Waters 1525 binary pump with a Waters 27?7 autosampler. A 40 μL volume of each sample is injected onto a Thermo

10 Hypersi! GOLD CIS column (2 JxSO mm, 5 urn). Peptide is doted by a gradient over 3 minutes with aeetomirile and water containing 0.05% trifluorαaeeϋc acid. The Quattro Micro mass spectrometer is run in multiple reaction monitoring (MRM) .mode using the mass transitions of, for example 764>182 or 682>1 36. Using this ^■ .methodology, peptide is dosed orally and by IV to rats at 10 rag/kg. Pharmacokinetic

15 properties including area under the curve and bioavailabilty are determined.

The stability uf peptides/GC-€ agonists of the disclosure in the presence of several 7.0 mammalian digestive enzymes is determined. Peptide/GC-C agonists of the disclosure arc exposed to a variety of in vitro conditions including digestive thymes and low ph environments designed, to simulate gastric fluid. Peptide/GC-C agonists of the disclosure are incubated with chymotrypsin, trypsin, pepsin, ammopeptkfnse, earboxypeptidase A. and simulated gastric fluid (sgf) at ph 1 ,0. Samples are collected 25 at 0, 3, and 24 h for all conditions except pepsin digestion and the SGI ⁷ . For the fatter two conditions, samples are obtained at 0, 1 , and 3 h. Negative control samples are prepared for initial and final time points, A separate, positive activity control is run in parallel for each condition. All samples are analyzed by LC/MS,

.Peptide GC-C agonists of the disclosure can be administered to mammals (e.g. humans) to determine the effect on bowel habits (including Bristol Stool Form. Scaly

score, stool frequency {number of stools per week), ease of passage and stool weigh!), Peptide/GC~C agonist is administered m a single dose or multiple doses (for example, once daily over a consecutive ? day period) and alterations in bowel habit are evaluated ( for each collected bowel movement), for example, prior to dose, during dosage (for multiple dosing), and postdose.

^" Che Bristol Stool Form Scale is:

I : Separate bard lumps, like nuts

2: Sis usage- shaped but lumpy 3: Like a sausage or snake but with cracks on its surface

4; Like a sausage or snake, smooth and soft

5: Soil blobs with clear-cut edges

6: Fluffy pieces with ragged edges, a mushy stool

?: Watery, no solid pieces The scale used to determine ease of passage is:

1. Manual disirøpaetion

2. Enema needed

3. Straining needed

4. Normal 5, Urgent without pain

6. Urgent with pain ?. incontinent

jM.nHxM.of .postoperative ileus, Female CD rats are used to test the effect of peptides/GC-C agonists of the disclosure on delayed transit induced by abdominal surgery and manual manipulation of the small intestine. Groups of at least .nine rats undergo abdominal surgery under isofiurane anesthesia. Surgery consists of laparotomy and 5 minutes of gentle manual intestinal massage. Following recovery from anesthesia, rats are dosed orally with either peptide/GC-C agonist (for example, 10 μg/kg) of the disclosure or vehicle (2OmM Tris) in a volume of ^' 3ϋϋμl. 1 hour after dosing, intestinal transit rate is

measured. Araraals are again dosed with 30θμl of the test article followed immediately by 500μl of a charcoal meal (10% charcoal, 10% gum arable in water). To calculate the distance of the small intestine traveled by the charcoal front, after 20 minutes;, the total length of the intestine as well as the distance traveled from the stomach to the charcoal front are measured for each animal.

Effect on cG MF levels and secretion in ligated .loops . rodent models

The effect of peptides/GC-C agonists of the disclosure on cGMP levels and secretion are studied by injecting peptides/GC-C agonists of the disclosure directly into an isolated loop in either wild-type or ClC-C KO mice. This is done by surgically Ii gating a loop in the small intestine of the mouse. The methodology for ligated loop formation is similar to that described in London et ai. ! 997 Am J Physio! p.G93- 105. The loop is roughly centered and is a length of 1 -3 cm. T he loops are injected with H)O μi of either SEQ I D N():3 (5μg) or vehicle (20 mM Tris, pH 7.5 or Krcbs Ringer, l OπiM Glucose, MEPES buffer (KRGH)), Following a recovery time of 90 minutes the loops are excised. Weights are recorded for each loop before and alter removal of the OλϊM contained therein.. The length of each loop is also recorded. A weight to iengtjh ratio (VWL) for each loop is calculated to determine the effects of the pcptide/GC-C agonist of the disclosure on secretion.. To deie.rffl.ine the effect of the peptide/GC-C agonist of the disclosure on cGMP activity, fluid from the loop is collected in ice-cold trichloracetic acid (TCA) and stored at -80 ^C C for use in an. assay to measure cGMP levels i.o the iluid. Intestinal fluid samples are TCA extracted, and cyclic GMP is measured by E)A according to procedures outlined in the Cayman Chemical Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, MI) to determine cyclic CJM I* levels in the intestinal fluid of the mouse in the presence of either peρtide/GC-C agonist of the disclosure or vehicle.

The effects of peptides/GC-C agonists of the disclosure on eGMP levels and secretion in ligated loops in female CD rats can also be determined using protocols similar to those described above. In the case of the rat, however four loops of intestine ars surgically ligated. The iϊrst three loops are distributed equally in the

small intestine and the fourth loop is located in colon, .Loops are 1 to 3 centimeters, and are injected with 200μL of either peptide/agonist of the disclosure (5μg) or vehicle (Kiebs Ringer, 1 OmM glucose, HEPES buffer (KRGH)).

The effect of peptides/GC-agonists of the disclosure on diuresis and natri uresis can be determined using methodology similar to thai described in WO06/001931 (examples 6 (p. 42} and 8 (p.45)). Briefly, the peptide/agonist of the disclosure { 1 SO- pmol) is inlused for 60 min into a group of 5 anesthetized rats. Given an estimated rat plasma volume of 10 mL, the infusion rate is approximately 3 pmoi/rnU ^' min, Blood pressure, urine production, and sodium excretion are monitored for approximately 40 minutes prior to the infusion, during the infusion, and for approximately 50 minutes after the infusion to measure the effect of the peptide/GC-C agonist on diuresis and natri uresis. For comparison, a control group of five rats is infused with regular saline, Urine and sodium excretion can be assessed. Dose response can also be determined. Peptide/GC-C agonist of the disclosure is infused intravenously into rats over 60 minutes. Urine is collected at 30 minute intervals up to 180 minutes after termination of pepiidε/GOC agonist miusion, and urine volume, sodium excretion, and potassium excretion art* determined for each collection interval. Blood pressure is monitored continuously. For each close a dose-response relationship for urine volume, sodium and potassium excretion can be determined. Plasma concentration of the peptide, G€- agonist is also determined before and after iv infusion,

Dl uresis Ex periroent :

Female Sprague-Dawley rats {> 170 g, 2-8 per group) arc given 3,OmL of iosotoπie saline peroral Iy, and then anesthetized with isoflurane /oxygen. Once an appropriate level of anesthesia has been achieved, a sterile polyurethaoe catheter {— ^' 16 cm, 0.6mm ID, 0.9mm OD) is inserted 1.5-2.0 cm into the urethra and secured using 1 - 2 drops of veterinary bond adhesive applied to urethra/catheter j miction. Rats are the?} dosed with either vehicle or test article via the intravenous or intraperitoneal route. Rats are then placed in appropriately sized rat restraint tubes, with the catheter protruding out

of the restrain! tube into a 10 mL graduated cylinder. Rats are allowed to regain consciousness, and the volume of urine excreted over a 1-5 hour duration is recorded periodically for each rat,

AdmjnLstratio.n.ot ^' ..p.eptides and GC-C receptor agonists

For {.refitment of gastrointestinal disorders, the peptides and agonists of the disclosure are preferably administered orally, e.g., as a tablet or cachet containing a predetermined amount of the active ingredient, pellet, gel, paste, syrup, bolus, electuary, slurry, sachet; capsule; powder; lyophiiixed powder; granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; as an oii-m-wafer liquid emulsion or a water-hi-oil liquid emulsion, via a liposomal formulation (see, e.g., EP 736299) or in some other form. Orally administered compositions can include hinders, lubricants, inert diluents, lubricating, surface active or dispersing agents, flavoring agents, and hmnectants. Orally administered formulations such as tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein. The peptides and agonists can be coadministered wife oilier agents used to treat gastrointestinal disorders including but not limited to the agents described herein. The peptides and agonists can also be administered by rectal suppository. For the _. treatment of disorders outside the gastrointestinal tract such as congestive heart failure and benign prostatic hypertrophy, peptides and agonists are preferably administered parenteral!}' or orally.

The peptides described herein can be administered alone or in combination with other agents. For example, die peptides can be administered together with an analgesic peptide or compound. The analgesic peptide or compound can be covalentϊy attached io a peptide described herein or it can be a separate agent that is administered together with or sequentially with a peptide described herein in a combination therapy.

Combination therapy can be achieved by administering two or more agents, e.g., a peptide described herein and an analgesic, peptide or compound, each of which is formulated and administered separately, or by administering two or more agents in a

„ Q -"; ..

single formulation. Other combinations are also encompassed by combination therapy. For example, two agents can be formulated together aud administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not he. for example, administration of a first agent (or combination of agents _. ? can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, the two or more agents can be administered within minutes of each other or within ], 2, 3. 6. 9, 12, 15, 18, or 24 hours of each other or within L 2, 3, 4, 5, 6, 7, S, 9, 10, 12. 14 days of each other or within 2, 3, 4, 5, (x 7, S, 9. or 10 weeks of each other. In some cases even longer intervals are possible. While in many eases it ;s desirable that the two or more agents used in a combination therapy be present in within, the patient ^' s body at the same time, this need not be so.

Combination therapy can also include two or more administrations of one or more of the agents used in the combination. For example, if agent X and agent Y are used in a combination, one could administer them sequentially in any combination one or more times, e.g., in the order X-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

Combination therapy can also include the administration of two or more agents via different routes or locations. For example, (a) one agent is administered orally and another agents is administered intravenously or (b) one agent is administered orally and another is administered locally, In each case, the agents can either simultaneously Oi' sequentially. Approximated dosages for some of the combination therapy agents described herein are found in the "BNF Recommended Dose" column of tables on pages 1 M 7 of WOO 1/76632 (the data in the tables being attributed to the March 200(J British National Formulary) and can also be found in other standard formularies and other drug prescribing directories. For some drugs, the customary preseαribeti dose for a.o indication will vary somewhat from country to country.

The agents, alone or in combination, can. be combined with any pharmaceutically acceptable carrier or medium. Thus, they can be combined with materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a

patient. The earners or mediums used can include solvents, dispersarus, coatings, absorption promoting agents, controlled release agents, and one or more inert exeipieots (which include starches, polyols, granulating agents, microcrysialline cellulose (e.g. celphere, €dphere beads®), diluents, lubricants, binders, disintegrating 5 agents, and the like), etc. !f desired, tablet dosages of the disclosed compositions may be coated by standard aqueous ur nonaqueous techniques.

Compositions of the present disclosure may also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, colorants, flavors, 0 dcsiccaπts, plasticizers, dyes, glidants, anti-adherents, anti-static agents, surfactants (wetting agents), antioxidants, iilm-coatmg agents, and the like. Any such optional ingredient must be compatible with the compound of the disclosure to insure the stability of the formulation. The composition may contain other additives as needed, including for exanple lactose, s glucose, fructose, galactose, trehalose, sucrose, maltose, raffinose, maltifol, mekzitose, stachyose, lactitol, paiatinite, starch, xylitol, πiatim ^' tol, myoinositol, and the like, and hydrates thereof, and amino acids, for example alanine, glycine and betainc. and peptides and proteins, for example albumen.

0 Examples of excipients for use as the pharmaceutically acceptable carriers and the pharmaceutically acceptable inert carriers and the aforementioned additional ingredients, include, but are not limited to binders, fillers, ciisintegrants, lubricants, anti -microbial agents, and coating agents such as:

5 BINDERS: com starch, potato starch, other starches, gelatin, natural and synthetic gurπs such as acacia, xanthaπ, sodium alginate, algmie acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, earboxymcthyl cellulose calcium, sodium carbαxyraefhyl cellulose), polyvinyl pyrrolidone (e.g., povidone, crospovidone, copovidone, etc), methyl D cellulose, Methocei, pre-gelatinized starch (e.g. , STARCH 1500® and STARCH 1500 LMφ, sold by (rolorcon, Ltd.), hydroxypropy! methyl cellulose, microerystaliine

. Q7 -

cellulose {e.g. AV1CEL™, such as, AVICEL-PIi-101™ _; ~l Q3 ^'m and -105™, sold by FMC Corporation, Marcus Hook, PA, USA), or mixtures thereof,

FiLiIiRS: tulc, calcium carbonate {e.g. , granules or powder), dibasic calcium 5 phosphate, tribasie calcium phosphate, calcium sulfate (e.g.,, granules or powder), .πύcroerystalline cellulose, powdered cellulose, dextrates, kaolin, mann.il.oL silicic acid, sorbitol, starch, pre- gelatinized starch, dextrose, fructose, honey, lactose anhydraie, lactose monohydraie, lactose and aspartame, lactose and cellulose, lactose find πiicroerystaliine CeHuIoSe, maltodextrin, maltose, mannitol, rnierowystalϊine :<) cellulose & guar gum, molasses, sucrose, or mixtures thereof,

DlSiNTEGRANTS: agar-agar, alginic acid, calcium carbonate, mieroerystallme cellulose, croscarraellose sodium, erospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre~gelatmized starch, clays, other 5 fslgms, other celluloses, gums (like geilars), low-substituted bydroxypropy! cellulose, or mixtures thereof,

LUBR ICANTS: calcium stearate, magnesium stearate, mineral oil. light mineral oil, glycerin, sorbitol, mamiitol, polyethylene glycol, other glycols, stearic acid, sodium 0 iaury! sulfate, sodium steary! fυmarate, vegetable based fatty acids lubricant, talc, hydrogenated vegetable oil {e. ,§ ^• ., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, con) oil and soybean oil), zinc separate, ethyl oieate. etliyi lauralc, agar, syloid silica gel (AEROSiI.. 200, W. R. Grace Co., Baltimore, MD USA), a coagulated aerosol of synthetic silica (Dcaassa Co., Piano, TX USA), a pyrogenic silicon dioxide 5 (CAB-O-SlL, Cabot Co., Boston, MA USA}, or mixtures thereof,

ANTI-CAKIN(J AGENTS; calcium silicate, magnesium siiieate, silicon dioxide, αfifoid&l silicon dioxide, talc, or mixtures thereof,

0 ANTIMICROBIAL AGKNTS: berualkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridiπium cliiodde, cresol,

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chlorobυtanoi, dchydi'oacetic acid, elhylparaben, methyfparabeπ., phenol, phenylethy! alcohol, pheπoxyethanol, phenyl mercuric acetate, phenyjrnercuric nitrate, potassium sorhate, propylparaben, sodium benzoate, sodium ddiydroacetate, sodium propionate, sorbic acid, tbimersoL thymo, or mixtures thereof, and

COATING AGFNTS: sodium carboxymethyl cellulose, cellulose acetate phthaiate, cϊhyiceOidose, gelatin, pharmaceutical glaze, hydroxypropyi. cellulose, hydroxy-propyl raethyicdJuiose (hypromeliese), hydroxypropyl methyl cellulose phthaiate, roethyleelluiose, polyethylene glycol, polyvinyl acetate phthaiate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalhne wax, gdlaπ gum, maltodextriπ, methaerySates, micro-crystalline cellulose and carrageeπaπ or mixtures iliercof.

The formulation can also include other excipients and categories thereof including but not limited to L-histidine, Pluronic®, Poioxajners (such as L-utrol® and Poloxamer 188), ascorbic acid, glutathione, permeability enhancers (e.g. lipids, sodium cbolatc, aeyieaππUnc, salicylates, mixoά bile sails, fatty acid micelles, chelators, fatty acid, surfactants, medium chain glycerides), protease inhibitors (e.g. soybean trypsin inhibitor, organic acids), pM lowering agents and absorption enhancers effective to promote bioavailability (including but riot limited UJ those described in IϊS6086918 and US59I 2014), creams and lotions (like maltodextrin and earrageenaπs); materiais ibr chewable tablets (like dextrose, fructose, lactose monohydratc, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, mieroery stall ine celhilose and guar gura, sorbitol crystalline); parenterals (like mannitol and povidone);, plasticizers (like dibutyl sebacate, plasticizers for coatings, pifi yvinylac etiUe phthaiate); powder lubricants (like glyceryl behena.e), soft gelatin capsules (like sorbitol special solution); spheres for coating (like sugar spheres); spheronization agents (like glyceryl behenate and rnierocrystaUme cellulose): susper-ding/gelling agents (like carrageeπan, gellan gum, niamiitol, mierocrystalline cellulose, povidone, sodium starch glycolate, xanthaπ gum); sweeteners (like aspartame, aspartame and lactose, dextrose, fructose, honey, maltodextrin, maltose, maomtoJ, molasses, sorbitol crystalline, sorbitol special solution, sucrose); wet

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granulation agents (like calcium carbonate, lactose anhydrous, lactose monohydrate, snaltodextrm, niarmitoi, microcrystalline cellulose, povidone, starch), caramel. carboxymethyiceilulose sodium, cherry cream flavor and cherry flavor, citric acid anhydrous, citric acid, confectioner's sugar, D&C Red No. 33, D&C Yellow ii\ 0 Aluminum Lake, disodium ©delate, ethyl alcohol 15%, F.D& C Yellow No. 6 aluminum lake, FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminum lake. FD&C Green No.3, FD&C Ret! No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C ¹ Ydlow No. 6 ₅ FD&C Yellow No.10, glycerol palm ^j tostearate, glyceryl monostcaratc, indigo carmine, lecithin, manitoi, methyl and propyl parabens, mono arairsonium giyeyrrhizmate, natural and artificial orange lluvor, pharmaceutical gbxc, poioxarπer 188, Folydextrose, polysorhate 20, polysorbaie 80, polyvidone, pregelatinized corn starch, pregelatinized starch, red iron oxide, sacciiarin sodium, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodiuni phosphate, strawberry flavor, synthetic black iron oxide, synthetic rtά iron oxide, titanium dioxide, and white wax,

Solid oral dosage forms .may optionally be treated with coating system^ (e.g. Opacify® fx film coating system, for example ϋpadry® blue (OY-LS-2092! ), Opadry® white (YS-2-7063), Opadry® white (YS-1-7040K and black ink (S-! -8106).

The agents, either in their free form or as a salt can be combined with a polymer such as poiylactic-glycoiøic acid (PLGA), poly-f lj-lactic-glycoiic-tartaric acid (P(F)LGl ^" ) (WO 01/12233), poiyglycolic acid (U.S. 3,773,919). polylactic acid (U.S. 4,767,628), ρo1y(«~caprolactone) and poly(aiky!ene oxide) (U.S. 20030068384) to create a sustained release formulation. Such formulations cars, be used to implants that release a peptide or another agent over a period of a lew days, a few weeks or several months depending on the polymer, the particle size of the polymer, and the size of the implant (see, e.g., U.S. 6,620,422), Other sustained release formulations and polymers for use in are described in EP 0 467 389 A2, WO 93/24150, U.S. 5,612 _; 052, WO 97/40085, WO 03/O758g?, WO 01/01964A2, U.S. 5,922,356, WO 94/!5558^ WO

02/074247 A2, WO 98/25642, U.S. 5,968,895. U.S. 6,180,608, U.S. 20030171296,

- KK) •

is chose?) from the group of. acetate, benzoate, citrate, fiimaraty, ωnbonate, dilorυphenoxyacetate, glycolatc palmoate, aspartate, metbancsuiphonatc, nialeatα pαrachiorophenoxyisobuiyrate, formate, lactate, succinate, sulphate, tartrate. cyelϋhex&ϊieearboxylate, bcxanoate, octanoatc, dccanoate, hexadeeanoale, δ octocleearioate, bcπzenesυlphonate, trimethoxyhenzoate, paratøhienesuiphonale, adumantanecarboxylαte, glycoxyiate, giutarnate, pyrrol idonecarboxylate, naphtha! enesulphonate, 1-glueosephosphate, nitrate, sulphite, dithionate and phosphate;), and phen&rrain; protein tyrosine phosphatase- 1 B (PTP-IB) inhibitors, such as A -401 , 674, KR 61639, iθ OC-060062, OC-83839, OC-297%2, MC52445, MC52453, LS (S I l 3? 15, mid those disclosed in WO99/S8S52 ! , WO99/58518, WO99/5S522, WO99/6143S, WO03/032916, WO03/032982, WO03/041729, WO03/0558S3, WO02/26707, WC)(}2/26743, JP2002 ! 1476S, and pharmaceutically acceptable salts and esters • hereof; is sulfonylureas such as acet.øhex amide (e.g. Dyrnelor, XlH Lilly), carbutamkie, chlorpropamide (e.g. Diabmeseφ), Pfizer), gliaroilide (Pfizer), gliciaxide (e.g. Diarncron, Sεrvier Canada Inc), glimepiride (e.g. disclosed in 1.1843797SS, such as λmαr>4 ^>v , Aveπtϊs), gUpentide, glipizide (e.g. Glucofrol or Glucotrol XL. Extended Release, Pfizer), gHqυidone, glisolamide. g1yburidc _' ^; g!ibcne}aimde (e.g. Mitταnase or

20 (llynase Piestab, Pharmacia & Upjolin and Diabeia, Aventis), tolaxarøicie (e.g.

Tolinasc), and tolbutamide (e.g. Orinase), and pharmaceutically acceptable salts and esters tbereo^

^■ meglitimdes such as repatgHnide (e.g. Pranidin®, Novo Nordisk), KλD1229

; PF/Kissci), and riateglinide (e.g. Stariix®, Novartis), and phamiacev-tically

25 αceeptable salts and esters thereof; u glycoside hydrolase inhibitors (or glucoside inhibitors) such as acarbose (e.g. Precose™, Bayer disclosed in US4904769), miglitol (such as GLYSET' ^' ", Pharmacia & Upjohn disclosed in US4639436), camiglibose (Methyl 6-deoxy-6-[(2R,3R,4R,5S)- 3,4,5-trihydroxy-2-{hydroxyniethyl)pipi;ridino3-aipha-D~gluc opyraτκjsidε, Marion

30 Merrell Dow), vogiibosc (T'akeda), adiposiπe, eniiglitate, pradiniiehi-Q, salbostatiπ., CKD-? i L MDL- 25,637, MDL-73,945, a d MOR 14, aM the αirapounds disclosed

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m US4062950, US4174439. US4254256, US4701559, 084639436, LJS5! 92772, US463476S, US51571 16, ϊJSS51HO7S, US5091418, US5217S77, US5 J 091 and

WOO I /4752 S Cpolyatnmes); u~amv1a.sc inhibitors such as lendamistat trestatin, and A I -3688, and the compounds 5 disclosed in US4451455, US4623714, and US4273765;

SGLϊ2 inhibtors including those disclosed in US64 I4126 and US6515137; an &P2 inhibitor such as disclosed in US6548529; insulin secreatagogues such as linogliride, A-4166, forskilin, dibutyrl cAMP, isobutyfmethylxanihine (IBMX), and pharmaceutically acceptable salts ami esters io thereof latiy acid oxidation inhibitors, such as clomoxir. and eiomojdr, and pharmaceutical!)' acceptable salts ami esters thereof,

λ2 antagonists, such as midaglizole, isaglidole, deriglidole, idazoxan, earoxan, and fkφaroxan, and phax"maceutical)y acceptable salts and esters thereof;

15 insulin and related compounds (e.g. insulin mimetics) such as biosa, LP- i 00»

S'Ovarapki insuHn dtiemir, insulin Hspro, insulin giargine, insulin zinc suspension (Sente and ultrakπtc), Lys-Pro insulin, GLP-ϊ ( 1-36} amide, GLP-I (73-7) {insulintropin, disclosed in US5614492), LY-3ϊ5902 (Lilly), OLP-I (7-36J-NHZ), AL-401 (Autoimmune), certain compositions as disclosed in US4579730.

20 US4S4y4ϋ5, US4%3526, US5642868, US5763396, USSS24638. US5843866,

US6153632, US6I 91 105, and WO 85/05029, and primaie, rodent, or rabbii insulin including biologically active variants thereof including allelic variants, more preferably hmπan insulin available in recombinant form (sources of human insulin include pharniaceutiealiy acceptable and sterile formulations such as those available

?.$ from EIi Lilly {Indianapolis, hid. 46285} as I-lumulin ^' " (Imniars insulin rDNA origin), also see the THH PHYSICIAN'S DESK REFERENCE, 55.sυp.th Ed. (2001 ) Medical Economics, Thomson Healthcare (disclosing other suitable human insulins); non-thiazolidinediones such as JT-SOi and targϋtazar (GW-2570/GI- 262579). and p)>anyiaeeαticaiiy acceptable salts and esters thereof;

3C PPAExϊ/γ dual agonists such as AII-HO39242 (Azti-azeneca), GW-409544 (Glaxo- Welkorøe), BVT-142, CLX-0940, GW-1536, GW-1929, OW-2433, KRP-297

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(Kyorin Merck; 5-[(2,4-DiOXO ihiazolidinyDraethyl] ϊnethoxy-N-[|4~

(triiluorosneihyl )phesτyi j m«thyl]bens ^j .amide), L-7%449, LR-DO ₅ MK-076?

(Merek/KyorijvBanyu), SB 219994. muragiitazar (8MS), tesaglitxar (Aslrazeneca), ivglitazar 0TT-5U1 ) and thosϋ disclosed in WO99/ 16758, WO99/193 13, WO9W20614, WO99/38850, WOOO/23415. WO00/234I 7, WOOO/23445,

WOOG/50414, WOO I /00579, WOO 1/79150, WO02/062799, WOG3/004458,

WO03/016265, WO03/018010, WO03/03348 K WO03/033450, WO03/033453,

WO03/G43S85, WO 031053976, U.S. application Ser. No. 09/664,598, filed Sep. 18.

2000, Murakami ei ai. Diabetes 47, i 841-1847 ( 1998), aud pharmaceutically acceptably salts and esters thereof; other insulin seBsitizing drugs;

VPAC'2 receptor agonists;

GLK modulators, such as thost- disclosed in WO03/015774; retinoid modulators such as those disclosed in WO03/000249; GSK 3β/GSl< 3 inhibitors such as 4-[2-{2-bfθmophenyl )-4-(4-πuorophenyl-lH-

5iT5id;izol-5- yi ipyrkiioc and those compounds disclosed in WO03/024447,

WO03/037869. WO03/037877, WO03/037891, WO03/068773, EPl 295884,

KPI 295885. and the like; glycogen phosphoryϊase (MG i..?a) inhibitors such as CT-36S.296, CP-316.819, BAYR.3401, and compounds disclosed in WO0I/943U0, WO02/2053G,

WO03/037864, and pharmaceutically acceptable salts or esters thereof;

ATP consumption promoters such as those disclosed in WOU3/007990;

TR.B3 inhibitors; vaniiloid receptor Sigands such as those disclosed in WO03/049702; hypoglycemic agents such as those disclosed in WO03- 015781 and WOG3/Q4G1 14; glycogen kinase 3 inhibitors such as those disclosed in WO03/035663 agents such as those disclosed in WO99/51225, US20O30134890, WO01/247S6. and

WO03/05%7ϋ; insulin-responsive DNA binding protein- 1 (IRDBP-I) as disclosed in WOCB/057827, iind the like: adenosine A2 antagonists such as those disclosed m WO03/035639, WO03/035640,

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arκl the like;

PFλRδ agonists such as GW 501516, GW 590735, arid compounds disclosed in JP 10237049 and WO02/14291 ; dipeptidyl peptidase IV (DP-IV) inhibitors, such as isoleucine thisxolidide. NVP- DPP728λ i 1 -[ [[2-[ ^' (5-cyajiopyridin-2-yl}ajnino]ctlvvi]araino]acctyl]-2-cyano-( S)- pyrroiidine, disclosed by Hughes et al. Biochemistry, 38(36), 1 1597-1 1603, 1999), P32/98, NVP-LA F-237, P329S, TSL225 (tryptophyl- 1,2,3, 4-tetrahydro-isoquinoiine- 3-earboxy!ie acid, disclosed by Yavmπia et al, Bioorg, & Med. Cheat Lett. S (1998) 1 537- 1 540). valine pyrrotidide. TMC-2A/2B/2C, CD-26 inhibitors, FE99901 i, P<tf 10/K364, VIP 0177, DPP4, SDZ 274-444, 2-cyanopyrroiUiides and 4- cytuiispyrrolidides as disclosed by Ashworth et ai, Bioorg, & Med, Chem, Lett.. VoI 6, No. 22, pp 1 163-1 166 and 2745-2748 (1 ¹ W(S) ,and the compounds disclosed in LJS6395767, US65732S7, US6395767 (compounds disclosed include BMS-4771 IS, BMS--47I2I ! and BMS 538,305), WO99/3850L WO99/46272, WO99/67279, WO99/6727S, WO99/6143 i WO03/00449S, WO03-O04496, EV \ 258476.

WO02/0S3128, WO02/062764, WO03/000250, WO03/002530, WO03/00253 I . VVOU3/002553, WO03/002593, WO03/000180, and WO03/00018 ! ; GLP-! agonists such as exendm-3 and exendin-4 (including the 39 aa peptide synthetic exeτκiin-4 called Exenatide®). and compounds disclosed in US2003087821 and NZ 504256, pharmaceutically acceptable salts and esters thereof; peptides including amlintide arid Symlin® (pramiintidc acetate); and giycokinase activators such as those disclosed m US2002103 199 (fused Ketcroaroτrs8tk compounds) and WO02/48106 (i&oindolin-i-one-aubstituted propfonani idc compounds).

The peptides and agonists described herein useful in the treatment of obesity can be administered as a cofherapy with electrostimulation (US2004O01520!

The peptides and agonists described herein can be used in combination therapy with agents that activate soluble giianylate cyclase, for example those described in US20G4G 192680.

The peptides ami agonists described herein can be used in combination iherapy with a phosphodiesterase inhibitor, FDE inhibitors are those compounds which slow the degradation of cyclic AMP (cAMP) and/or cyclic GMF (cGMP) by inhibition of the phosphodiesterases, which can lead to a relative increase in the intracellular concentration of cAMF and/or cGMP. Possible PDE inhibitors are primarily those substances which are to be numbered among the class consisting of the PDID inhibitors, the class consisting of the PDE4 inhibitors and/or the class consisting of the PDE5 inhibitors, in particular those substances which can be designated as mixed types of PDE3/4 inhibitors or as mixed types of PD E 3. '4/5 inhibitors. By way of example, those PDE inhibitors may be mentioned such as arc described and/or claimed in the following patent applications and patents: DE1470341, D1ϊ2108438, DIϊ2! 23328, DE230S339, DE2305575. DE2315B01 , DG240290S, DE2413935, DE2451417, DE2459090, DE2646469, DE272748L DH2825048. DB283716 K DE2845220, DE2847621 , DB2934747, DE3021 792, DH3O38166, DE3044568, EP00071S, EP0008408, EP0010759, EP0059948, EP0075436. EP0096517, EPOl 12987, EPOi 16948, EP0150937, EP0ϊ58380, EP0I 61632, EP016ϊ91 S, EP0167 I21, £1*0199127, HP0220044, EP0247725, HP0258191 , EP0272910, EP0272914, EP0294647, EPO3OO726, EP0335386, HP0357788, EPC389282, EP0406958, EP0426J 80, EP0428302, EPO43581 L BP0470805, HPO4822O8, EP0490823, EPO5O6194, EP051 1865, EP0527] 1 7 ₅ EP0626939, EP0664289, EP0671389, EP0685474, EP06B5475, EϊP0685479, JP92234389, JP94329652, JP95010875. U.S. PaL Noa. 4,963,561, 5,141 ,93I ₅ WCX ⁾ I i 7991 , WO9200968, WO921296K WO9307 I46, WO9315044, WO9315045, W()93 18024, WO9319068, WO93 19720, WO9319747, WO93 19749. WO9319751 , WO93255R WO9402465, WO9406423, WO94I2461 , WO9420455, WO9422852, WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045. WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681 , WO95ϊ7392, WO95I7399, WO9519362, WO9522520, WO95243S1 , WO9527692, WO9528926, WO953S28 L WO9535282, WO9600218, WO960ϊ 825, WO960254K WO961 1917, DE3142982,

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DEi 1 16676, DE2162096, EP0293063, EP0463756, EP0482208. EP0579496, IiP066?34S USό,331,543, US20050004222 (including those disclosed in formulas 1- XIII and paragraphs 37-39, 85-0545 and 557-577} and WO9307124, EPOi 63965, S- PO3935OO, 1^*0510562, EP0553174. WO9501338 mά WO%()3399. POE5 inhibitors which may be mentioned by way of example are RX-RA -69, SCH-51 $66 _S KT-734. vesnarinone, zaprinast, SKP-96231, ER-21355, BE/GP-385, NM-702 and sildenafil (Viagra©). PDE4 inhibitors which may be mentioned by way of example are RO-20-1724. MEM 1414 (RJ533/R I5OO; Pharmacia Roche), DENBUFYLLiNE, ROLIPRAM, OXAGRELATE, NmiAQUAZO ^' NE. Y-590, DH -6471, SKF-94120, MOTAPTZON E, LIXAZ1NONE, INDOLlDAN, OLPRINONE, AlIZORAM, KS- 506-G, DIFAMfYIIJNE, BMY-433S1 , ATIZORAM, AROFYLLϊNE, FILAMINAST, POB-093, UCB-2%46, CDP-Ui). SK.F-I 07g()6, PICLAMILAST, RS-I7597, RS ^» 25344-000, SB-207499, TiBENELAST, SB-210667, SB-21 1572, SB- 2i 1600, SB~212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL. ANAORHL)DE, IBUDILAST, AMRiNONE, PϊMOBENDAN, CiLOSTAZOL, OUAZiNONE and N-(3,5-dichloropyrid~4~yl}~3-cyclopropyh ⁱ neihoxy4- dilluororactKoxybcπϋainide. PDE3 inhibitors which may be menUoiied by way of example are SULMAZOLE, AMPIZONE, C SLOSTAM IDE, CARB AZER AN, PIROXIMONE, IMAZODAN, CI-930, SIGUAZODAN, ADIBENDAN, SATϊϊRϊNONϊϊ, SKF-95654, SI)Z-M JCS-492, 349-U-85, EMORADAN, EMD-

53998, EMD-57033, NSP-306, NSP-307, REV1ZϊNONE, NM-702, WIN-62SS2 and W IN-6329 i , ENOXlMONE and MILRINONE. PDE3/4 inhibitors which may b€ mentioned by way of example are BENAFENTiUNE, TREQU1NSIN, ORG-30029, ZARDAVERINE, L-686398, SDZ-iSQ-844, ORG-2024ϊ , EMD-54622, and "\ OLAFEHTRINE. Other PDE inhibitors include: dlomilasl, pentoxifylline, rotlumiJasL ιad;i!aiil(Cialisφ), theophylline, and vai-drøaϋi(Levitra®), zaprinast (PDE5 specific).

^' lite peptides and agonists described herein can be used in combination therapy (for example, in order to decrea.se or inhibit uterine contractions) with a tocolytic agent

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inekidmg but not limited to beta-adrenergic agents, magnesium sulfate, prostaglandin inhibitors, and calcium channel blockers.

The peptides and agonists of thy disclosure can be used in combination therapy with aji aoti-neorslastie agents including but not limited to alkvlatins atients, c-pipodophyllotβxiπs, nitrosoureas, antimetabolites, vinea alkaloids, anlhracyclme antibiotics, nitrogen mustard agents, and the like. Particular anti-neoplasiie agents may include tamoxifen, taxol, etoposidc and 5-ftuυrou.raeiL The peptides and agonists of the. disclosure can be used in combination therapy (for example as in a ehemoiherapeutic composition) with an antiviral and monoclonal antibody therapies.

The peptides and agonists of the disclosure can he used in combination therapy UOr example, in prevention/treatment of congestive heart failure or another method described herein) with the partial agonist of the nociceptin receptor ORLI described by Oooicy et aL (The Journal of Pharmacology and Experimental Therapeutics, 283 (2); 735-741, 1997). The agonist is a hexapepiiάe having the amino acid sequence Ae- HYY (RK) (WI) (RK)-NlC ("the Dooley peptide"), where the brackets show allowable variation of amino acid residue. Thus Dooley peptide can include but are not limited to KYYRWR, RYYRWR, KWRYYR, RYYRWK, RYYRWX (all-D amiri acids), RYYRIK, RYYRlR. RYYKIK, RYYKIR, RYYKWR, RYYIiWK, RYYRWR., RYYRWIv, RYYRIK, RYYlCWR, RYYKWK, RYY R W K and KYYRVVK, wherein the amino acid residues are in the L-førm unless otherwise specified. The peptides and agonists of the disclosure can also be used in combination therapy with peptide conjugate mαdifieations of the Dooley peptide described in WO0198324. The peptides and agonists of the disclosure can also be used in combination therapy (for example in the prevention and/or treatment of ! BS and associated bloating) with nerve-acting agents sych as lidoeaine, iopiramatε, mexiifine, and gahaperitin as described in US20060205678.

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A number of disorders might be treated with GC-C receptor agonists mά agents that increase cG.MP levels including the peptides and agonists of the disclosure,

S The peptides and agonists of the disclosure can be used alone or in combination therapy for the treatment or prevention of congestive heart failure. Such agents can be used in combination with natriuretic peptides (e.g., atrial natriuretic peptide, brain natriuretic peptide or C~iype natriuretic peptide], a diuretic, or an inhibitor of angiof εnsi π converting enzyme, i π

The peptides and agonists of the disclosure can be used alone or in combination therapy for the treatment or prevention of benign, prostatic hyperplasia (BPH). Such agents can be used in combination with one or more agents for treatment of BPH, for example, a 5-aipha reductase inhibitor (e.g., finasteride) or an alpha adrenergic

15 inhibitor (e.g., doxaxosiπe).

The peptides and agonists of the disclosure can be used alone or in combination therapy for the treatment, prevention or reduction of visceral pain associated with a gastrointestinal disorder or pain associated with another disorder.

20

The peptides and agonists of the disclosure can be used alone or in combination therapy for the treatment or prevention of obesii.y-rdat.ed disorders (e.g. disorders that are associated with, caused by, or result from obesity). Examples of obesity-related disorders include overeating and bulimia, hypertension, diabetes, elevated plasma

25 insulin concentrations and insulin resistance, dyslipidemias, hyperhpidemia, endometrial breast, prostate and colon cancer, osteoarthritis, obstructive sleep apnea, cholelithiasis, gallstones, heart disease, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome,

30 FroliUch's syndrome, GH-deficient subjects, normal variant short stature. Turner's

„ ^■ ?{)« ..

syndrome, and oilier pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat- tree mass, e.g., children w ^ή h acute lymphoblastic leukemia. The agents of the disclosure may be used to reduce or control body weight {or fat) or to prevent and/or treat obesity or 'JtIiCf appetite related disorders related to the excess consumption of food, ethanof and other appetizing substances. The agents may be used to modulate lipid metabolism, reduce body fat (e.g. via increasing fat utilization) or reduce (or suppress) appetite (e.g. via inducing satiety). Further examples of obesity-related disorders are metabolic syndrome, also known as syndrome X, Insulin resistance syndrome, sexual and reproductive dysfunction, such as infertility, hypogonadism in Busies m\ά hirsutism in females, gastrointestinal motility disorders, such as obesity-related gastroesophageal reflex., respiratory disorders, such as obesity-hypoventilation syndrome {Pickwickian syndrome), cardiovascular disorders, inflammation, such as systemic inflammation of the vasculature, arteπoseierosis, hypercholesterolemia, hyperuricemia, knver back pain, gallbladder disease, gout, and kidney cancer. The agents of the present disclosure are also useful for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy,

^' The peptides and agonists of the disclosure can be used alone or in combination therapy for the treatment, or prevention of gastrointestinal related disorders including: chronic intestinal pseudo-obstruction (Qgiivie's syndrome), colonic pseudoobstruction, Crohn's disease, dyspepsia (including functional dyspepsia or ^■ lonuleer dyspepsia), dυodenogasirie reflux, functional bowel disorder, functional gastrointestinal disorders, functional heartburn, gastroesophageal reflux disease (GHRD). gastrointestinal motility disorders, gastroparesis (e.g. idopatiiic gasiroparesisK hypertrophic pyloric stenosis. Inflammatory bowel disease, irritable bowel syndrome (IBS), post-operative ileus, and uleeratK'e colitis. The peptides and agonists of the disclosure can be used aioπe or in combination therapy to patient suffering from or susceptible to G! disorders relating to damage to the GI tract stemming from impact or surgical intervention. The peptides and agonists of the disclosure can be used alone or in combination therapy to patients at risk for or having

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particular diseases associated with hypomotility (e.g. colonic inertia) or stasis in the Gl tract. For example, diabetic neuropathy, anorexia nervosa, and achlorhydria are frequently accompanied by gastric hypαmotϋity. Damage to the Gl tract following surgical intervention, for instance, can result in substantial gastric stasis. The peptides ami agonists of the disclosure can be administered alone or in combination therapy to patients susceptible to or having a Gϊ disorder associated with diabetes (e.g. diabetic gasirnpathy). The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent and/or treat GI disorders characterized by at least one of nausea, vomiting, heartburn, postprandial discomfort, diarrhea, constipation, indigestion or related symptoms. The peptides and agonists of the disclosure can be used alone or i.n combination therapy to prevent and/or treat G! disorders associated with at least one of diabetes, anorexia nervosa, bulimia, achlorhydna, achalasia, anal Bssure, haemorrhoids. Irritable bowel syndrome, intestinal pseudoobstruction, scleroderma and gastrointestinal damage,

The peptides and agonists of the disclosure can be used to prevent and/or treat constipation. Constipation can be used to describe bowel patterns which include one or more of hard, small, infrequent stools; the sensation of difficulty in passing stool, specifically excessive or ineffectual straining; the sensation of incomplete evacuation. Constipation has also been described as the passage of stool less than a certain number (e.g, 3} of times per week. A number of conditions can be associated with constipation. Constipation can be associated with numerous disorders and conditions. For example, constipation can be ( 1 ) associated with the use of a therapeutic agent (e.g. antihypertensives, anticonvulsants, antispasmodics, analgesics, anticholinergics, antidepressants, antipsychotics, cation-containing agents, anticonvulsants, ganglion blockers, vinca alkaloids}; (2) associated with a muscular, neuropathic, metabolic or endocrine disorder (including but not limited to myotonic dystrophy, dcnrsamyositis, systemic sclerosis., scleredema, amyloidosis (neurologic or muscular), ischemia, tumor of the central nervous system, autonomic neuropathy, Cbagas disease, cystic fibrosis, diabetes mcHitus, Hirschsprung disease, hyperthyroidism, hypocalcaemia, hypothyroidism. Multiple Sclerosis, neurofibromatosis, Parkinson's disease, and

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spinal cord lesions (for example, related to sacral nerve damage related to trauma or a tumor or the enteric nervous system)); (3) post-surgical constipation (postoperative ileus); (4) associated with a structural colon alteration (for example that associated with Neoplasm, stricture, volvulus, anorectal, inflammation, prolapse, rectoede, or 5 fissure); (5) associated with the a gastrointestinal disorder; (6) associated with a .systemic illness or disorder (for example, electrolyte abnormalities, thyroid disease, diabetes radlitus, panhypopituitarism, λddison's disease, pheochroiisocytorna, uremia, porphyria); (?) chronic constipation; (8) associated with the use ofanaigesic drugs (e.g. opioid induced constipation); {9} associated with megacolon; and { 10) idiopathic

K) constipation, (functional constipation). Functional constipation can be associated with normal transit, slow transit (e.g. one or fewer bowel movements per week) and pelvic Ii OUT dyssynergia. Pelvic Door dyssynergia is considered a disorder of the rectum and anus although these patients also have abnormal contractions throughout the colon. Patients with pelvic floor dyssynergia have abnormal colonic pressure waves prior to

15 defecation and present with symptoms that may include a sensation of incomplete evacuation, excessive straining, a need .for digital disirøpaeticm, perianal heaviness, λ ^• ϊiά tenesmus. Constipation can be associated with bloating and abdominal pain. The peptides and agonists of the disclosure can be used to prevent and/or treat low stool frequency or poor stool consistency.

20

The peptides and agonists of the disclosure can be used to treat decreased intestinal motility, slow digestion or slow stomach emptying. The peptides and agonists can be used to relieve one or more symptoms of IBS (bloating, pain, constipation), GERD (acid reilux imo the esophagus), duodenogaslrie reflux, functional dyspepsia, or 25 gastroparesis (nausea, vomiting, bloating, delayed gastric emptying) and other disorders described herein. The peptides and agonists of the disclosure ears be used to treat flatulence.

The peptides and agonists of the disclosure can be used to increase intestinal motility. 30 slow colonic transit, and to prevent and/or treat gastrointestinal imrnotiiity and other conditions calling for laxative or stool softener therapy. Gastrointestinal inimotility

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can include constipation, and also includes delayed oral cecal transit tims. irregular Taxation, and oilier related gastrointestinal motility disfunction including impaction, impaction is a condition where a large mass of dry, hard stool develops in the rectum, often due io chronic constipation. Thus mass may be so hard that it cannot be excreted, ^" The subjects aiϊeeieα by constipation or gastrointestinal imraotilily can be refractory to laxative therapy and/or stool softener therapy.

The peptides and agonists of the disclosure can be used for the treatment or prevention of cancer, prc-cancerous growths, or metastatic growths. For example, they can be used for the prevention or treatment of: eolorectal/ioeal metastasized colorectal cancer, intestinal polyps, gastrointestinal tract cancer, Sung cancer, cancer or pre-eaneemus growths or metastatic growths of epithelial cells, polyps, breast, colorectal lung, ovarian, pancreatic, prostatic, renal, stomach, bladder, fiver, esophageal and testicular carcinoma, carcinoma (e.g., basal cell, basosquarnous, Brown-Pear ce, ductal carcinoma, Ehrlich. tumor, fCrebs, Merkd cell, small or non- small ceil king, oat cell, papillary, bronchiolar, squamous cell transitional cell. (Walker), leukemia (e.g., B-cell, 1-cclL MTLV. acute or chronic lymphocytic, mast cell, myeloid), histiocytoma, histiocytosis, ^" Modgkin's disease, noπ-Hodgkm's ivrophoma, plasmacytoma, reticuioendotheliosjs, adenoma, adenocarcinoma, adenolϊbroirsa, adenolyrnphoma, ameloblastoma, angiokeratoma, angiolymphαid hyperplasia with eosinophilic*, sclerosing angioma, angiomatosis, apudorøa, brαπchionia, malignant carcinoid syndrome, carcinoid heart disease, carcinosarcoma, cementoma, ehoiangioma, cholesteatoma, chondrosarcoma, chondroblastoma, ehondiosareoniu, chordoma, choristonia, craniopharvngioma, chrondrorna, cylindroma, cystadenoearcinoma, cystadenonia, cystosarconia phyilodes. dysgenninorøa, ependymoma, lϊwing sarcoma, fibroma, itbrosarconia, giant ceil tumor, ganglioneuroma, glioblastoma, glornangioma, granulosa cell tumor, gyrtandrobiaatoma, hamartoma, hemangioendothelioma, hemangioma, hemangio- pericytoma, hernangiosarcoma. hepatoma, islet cell tumor, Kaposi sarcoma, leiomyoma, leiomyosarcoma, ieukosarcoma, Leydig cell tumor, lipoma, iiposarcoma, lymphangioma, lymphangiomyoma, lympbaαgiosarcoma, mcduiloblasioma,

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meningioma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, aeurilernmoma, neuroma, neuroblastoma, neuroepithelioma, neurofibroma, neurofibromatosis, odontoma, osteoma, osteosarcoma, papilloma, paraganglioma, paraganglioma, Doochromallin, piπeaJoraa, rhabdomyoma, rhabdomyosarcoma, Sertoli eel! tumor, teratoma, ttaeca cell tumor, and other diseases in which cells have become dysplastie, immortalized, or transformed.

The peptides and agonists of the disclosure can be used tor the treatment or prevention of: Familial Adenomatous Polyposis (FAP) (autosomal dominant syndrome) (hat precedes colon cancer, hereditary nonpolyposis colorectal cancer (HNPCC), and inherited autosomal dominant syndrome.

For treatment or prevention of cancer, precancerous growths and metastatic, growths, the peptides and agonists of the disclosure can be used in combination therapy with radiation Uϊ chemotherapy utic agents, an .inhibitor of a cGMP-depeπdeπt phosphodiesterase or a selective cycIooxygenase-2 inhibitor, A number of selective eyelooxygenase-2 inhibitors are described in US20010024664, U.S. PaL No. 5,380,738, U.S. Pat. No, 5344,991, U.S. Pat No, 5,393,790, U-S, Pat, No. 5,434J7B ₅ U.S. Pat, N( _S . 5,474,995. U.S. Pat. No. 5310,368, WO02/062369, WO 96/06840, WO 96/033&S. WO 96/03387, WO 96/19469, WO 96/25405, WC) 95/15316, WO

94/15932, WO 94/27980, WO 95/00501 , WO 94/13635, WO 94/20480, and WO 94/26731 , the disclosures of which are herein incorporated by reference. [Pyrazol-1 ~ vi ]bt'ϊizenesu!fonamides have also been described as inhibitors of cyclooxygenase-2.

The peptides and agonists of the disclosure can be used in the treatment or prevention of inflammation. Thus, they can be used alone or in combination with an inhibitor of cGMP-dependent phosphodiesterase or a selective cyclooxvgenαse-2 inhibitor for treatment of: organ inflammation, IBD (e.g. Crohn's disease, ulcerative colitis), asthma, nephritis, hepatitis, pancreatitis, bronchitis, cystic fibrosis, ischemic bowel diseases, intestinal miiarnmations/allergies, eoetiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, and other inflammatory disorders. The peptides and

- ? S 3 -

agonists of the disclosure can be used alone or in combination therapy in the treatment or prevention of gastrointestinal tract inflammation (e.g. inflammation associated with a gastrointestinal disorder, gastrointestinal tract infection, or another disorder}. They can bt- used done or in combination therapy with phenoxyaikyearboxylic acid derivatives lor the treatment of interstitial cystitis, irritable bowel syndrome, ulcerative colitis, and other inflammatory conditions, as mentioned in US200S0239902AL

The peptides and agonists of the disclosure can also be used to treat or prevent insuiin-relaied disorders, for example: U diabetes meliitus, hyperglycemia, obesity, disorders associated with disturbances in glucose or electrolyte transport, and insulin secretion in cells, or endocrine disorders. They can be also used in insulin resistance treatment and post-surgical and non-post surgery decrease in insulin responsiveness.

The peptides and agonists of the disclosure can be used to prevent and/or treat pulmonary and respiratory related disorders, including, inhalation, ventilation and mucus secretion disorders, pulmonary hypertension, chronic obstruction of vessels uii d airways, aeiite respiratory failure, and irreversible obstructions of vessels and bronchi. One may administer an agent of the disclosure for treating broncHospasra, tor inducing bronchodilaiion, ibr treating chronic obstructive pulmonary disease ( ^■ including chronic bronchitis with normal airflow), for treating asthma (including bronchial asthma, intrinsic asthma, extrinsic asthma, acute asthma, chronic or inveterate asthma (e.g. late asthma and airways hyper-responsiveness), dust-induced asthma, allergen-induced asthma, viral-induced asthma, cold-induced asthma, pollution-induced asthma and exercise-induced asthma) and ibr treating rhinitis (including acute-, allergic, hatrophie rhinitis or chronic rhinitis (such as rhinitis caseosa, hypertrophic rhinitis, rhinitis puαiienta, rhinitis sicca), rhinitis medicamentosa, membranous rhinitis (including croupous, fibrinous and pseudomembranous rhinitis), scrofulous rhinitis, perennial allergic rhinitis, seasonal rhinitis (including rhinitis nervosa (hay fever) and vasomotor rhinitis). The peptides of the disclosure may also he useful in the treatment of dry eye disease and chronic

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sinusitis. The peptides of the disclosure may also be used to prevent and/or treat disorders characterized by acute pulmonary vasoconstriction such as may result from pneumonia, traumatic injury, aspiration or inhalation injury, fat embolism in the long, acidosis inflammation of the lung, adult respiratory distress syndrome, acute D pulmonary edema, acute mountain sickness, post-cardiac surgery, acute pulmonary hypertension, persistent pulmonary hypertension of the newborn, perinatal aspiration syndrome, hyaline membrane disease, acute pulmonary thromboembolism, herapin- prot amine reactions, sepsis, status asthmaticus or hypoxia (including iatrogenic hypoxia) and oilier tonus of reversible pulmonary vasoconstriction. Such pulmonary 9 disorders also are also characterized by inflammation of the lung including those associated with the migration mtα the lung of nonresident cell types including the various leucocyte subclasses. Also included in. the respiratory disorders contemplated ure: bullous disease, cough, chronic cough associated with inflammation or iatrogenic induced, airway constriction, pigeon fancier's disease, eosinophilic bronchitis. s asthmatic bronchitis, chronic bronchitis with airway obstruction (chronic obstructive bronchitis), eosinophilic lung disease, emphysema, fanner's lung, allergic eye diseases f including allergic conjunctivitis, venial conjunctivitis, venial keratoconjunctivitis, and giant papillary conjunctivitis), idiopathic pulmonary fibrosis, cystic fibrosis, diffuse pan bronchiolitis and other diseases which are characterized by inflammation 0 of t.be king and/or excess mucosal secretion. Other physiological events which are contemplated to be prevented, treated or controlled include platelet activation hi the lung, chronic inflammatory diseases of the lung which result in interstitial fibrosis, such us interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoid arthritis, or other autoimmune conditions), chronic 5 obstructive pu'rπonary disease (COFD)Csuch as irreversible COPD), chronic sinusitis, fibroid iung, hypersensitivity lung diseases, hypersensitivity pneumonitis, idiopathic interstitial pneumonia, .nasal congestion, nasal polyposis, and otitis media.

[lie peptides and agonists of the disclosure can he used alone or in eombitherapy to Q prevent or treat: retinopathy, nephropathy, diabetic angiopathy, and edema formation

^■ - 21 S -

The peptides and agonists of the disclosure can he used alone or in combiiherapy io present or treat neuϊϋiogical disorders, for example, headache, tensson-type headache, migraines, anxiety, stress, cognitive disorders, cerebral ischemia, brain trauma. movement disorders, aggression, psychosis, seizures, panic attacks, hysteria, sleep disorders, depression, schizoaffective disorders, sleep apnea, attention deficit syndromes, memory loss, dementia, memory and learning disorders as discussed in Moπeada and HIggs 1995 FASEB J. 9:1319-1330; Severina 1998 Biochemistry 63:794; Ue t* ai. 2000 P-NAS 97: 10763-10768; Hobbs 1997 TIPS 18:484-491 ; Murad I $94 Adv, Pharmacol 26: 1 -335; and Denainger et al, 199 ^f > Biochim. Biophys. Acta 1411 :334-350 and narcolepsy. They may also be used as & sedative.

The peptides and deteetably peptides and agonists of the disclosure can be used as markers to identity, detect, stage, or diagnosis diseases and conditions of small intestine, including, without limitation: Crohn's disease, colitis, inflammatory bowel disease, tumors, benign tumors, such as benign stromal tumors, adenoma, angioma, adenomatous (pedunculated and sessile) polyps, malignant, carcinoid tumors, endocrine eeil tumors, lymphoma, adenocarcinoma, ibregut, midgut, and hindgυt carcinoma, gastroinstestinal stroma! tumor (GIST), such as leiomyoma, cellular leiomyoma, leiomyoblastoma, mid leiomyosarcoma, gastrointestinal autonomic nerve tumor, malabsorption syndromes, celiac diseases, diverticulosis, Meckel's diverticulum, colonic diverticula, megacolon, Hirschsprung's disease, irritable bowel syndrome, mesenteric ischemia, ischemic colitis, colorectal cancer, colonic polyposis, polyp syndrome, intestinal adenocarcinoma. Liddle syndrome, Brody myopathy, infantile convulsions, and ehoreoatiieiosis

The peptides and agonists of the disclosure can be conjugated to another molecule (e.g., Li diagnostic or therapeutic molecule) to target cells bearing the GC-C- receptor, e.g., cystic fibrosis lesions and specific cells lining the intestinal tract. Thus, they can he used to target radioactive moieties or therapeutic moieties (active moieties like a radionuclide, an enzyme, a fluorescent labd. a metal chelating group, a ehemilυmiπescent label, a bioiumineseent label a chemoiherapeutϊc. a toxin, an

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inactive prodrug, a radiosensitizirig agent, a photodyπamie agent) to the intestine to aid in imaging and diagnosing or treating colorectal/metastasized or local colorectal cancer. In addition, they can be used to deliver antssense molecules or nucleic acid molecules (like normal copies of the p53 tumor suppressor gene) to the intestinal tract. The peptides and agonists of the disclosure can also be used to increase the number of GC-C ^' molecules on the surface of a cell, hi some embodiments the cell is a metastasized colorectal cancer cell. In one embodiment the peptide or agonist of the disclosure is therapeutically conjugated to a second agent. In certain embodiments, •he second agent can be radioactive or radiostabie. In certain embodiments the second agent cars be selected from the group consisting of a compound that causes cell death, u compound that inhibits cell division, a compound that induces cell differentiation, a ohemotherapeυtie, a toxin and a radiosensitking agent, In certain embodiments the second agent can be selected from the group consisting of; methotrexate, doxorubicin, daunombiciru cytosinarabinoside, etoposide, 5-4 fiuorouracii, roelphaiaπ, chlorambucil, eis-platio, vindesine, mitomycin, bleomycin, purothionin. nmwniomycin, M-benzotjuimme derivatives, trenimαn. ricin, riein A chain, Pseudomonas exotoxin, diphtheria toxin, Clostridium perfrfngens phospholipase C, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, cobra venom factor, gekmirs, saporin, modeccin, viseiirmn. volkensirj, αitroimidazole, and misonidaznle. In certain embodiments the second agent can be a cytoxic agenϊ selected from the group consisting of cemadotin, a derivative of ccrnadoiirk <x derivative of hemiasierlin, esperamiein C, neocarajKsstatin, maytansinoid D.M ϊ , 7-chJoromethyl-10,n niethylenedioκy~camptothecin, rliizoxin, and the haiichondrin B analog, ER-086526.

The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent and^br treat inner ear disorders, e.g., to prevent and/or treat Meniere's disease (including symptoms thereof such as vertigo, hearing loss, tinnitus, sensation of ^" fullness in the ear), MaI do debarquement syndrome, otitis externa, otitis media, otorrhea, acute mastoiditis, otosclerosis, otic pain, otic bleeding, otic inflammation, Lerrnoyez's syndrome, vestibular neuronitis, benign paroxysmal

- 2 ! 7 -

positional vertigo (BPPV), herpes zoster oticus, Ramsay Hunt's syndrome, herpes, labyrinthitis, purulent labyrinthitis, perilymph fistulas, presbycusis, ototoxicity { including drug-induced ototoxicity), neuronal as (including acoustic neuromas), aerotitis media, infectious myringitis, bullous myringitis, squamous cell carcinoma, basal cc)j carcinoma, pre-cancerous otic conditions, nonchromaffin paragangliomas, cheraodectornas, glomus jugulare tumors, glomus tympanicum iuniors, perichondritis, uuval eczematαid dermatitis, malignant externa! otitis, subperichondriai hematoma, ecruiriinomas, impacted cerumen, sebaceous cysts, osteomas, keloids, otalgia, tinnitus, tympanic membrane infection, tympanitis, otic furuncles, petrositis, conductive and sensorineural hearing loss, epidural abscess, lateral .sinus thrombosis, subdural empyema, otitic hydrocephalus, Dandy's syndrome, bullous myringitis, diffuse external otitis, foreign bodies, keratosis obturans, otic neoplasm, otomycosis, trauma, acute barotitis media, acute eustachian tube obstruction, postsurgical otalgia, cholesteatoma, infections related to an otic surgical procedure, and complications associated with any of said disorders, The peptides and agonists of the disclosure can be used alone or in combination therapy to maintain fluid homeostasis in the inner ear, neuronitis (including viral neuronitis), ganglionitis, geniculate

The peptides and agonists of tJhe disclosure can be used alone or in combination therapy to prevent and/or treat disorders associated with fluid and sodium retention, e.g., diseases of the electrolyte- water/ electrolyte transport system within the kidney, «ut and urogenital system, congestive heart failure, hypertension, iiypolension, salt dependent forms of high blood pressure, hepatic edema, and liver cirrhosis, In addition they can be used to facilitate diuresis or control intestinal fluid ^' The peptides and agonists of the disclosure can also be used to treat disorders where there is abnormal proliferation of epithelial cells within the kidney (e.g. as in the case of renal cancer).

The peptides and agonists of the disclosure can be used alone or in combination therapy ι.o prevent and/or treat kidney disease. "Kidney disease" includes renal failure {including acute renal failure), renal insufficiency, nephrotic edema,

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glomerulonephritis, pyelonephritis, kidney failure, chronic renal failure, nephritis, nephrosis, azotemia, uremia, immune renal disease, acute nephritic syndrome, rapidly progressive nephritic syndrome, nephrotic syndrome, Berger's Disease, chronic πephriiic/prøtemυπe syndrome, tubulointerstital disease, nephrotoxic disorders, renal infarction, atheroerobolic renal disease, renal cortical necrosis, malignant πcpbroaπgiosclerosis, renal vein thrombosis, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, Barter's Syndrome, Liddle's Syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, hereditary nephritis, and nail-patella syndrome, along with any disease or disorder that relates to the renal system and related disorders, as well as symptoms indicative of, or related to, renal or kidney disease and related disorders

The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent or treat polycystic kidney disease. Polycystic kidney disease" "PK.D" (also called "polycystic renal disease ¹⁵ ) refers to a group of disorders characterised by a large number of cysts distributed throughout dramatically enlarged kidneys. The resultant cyst development leads to impairment of kidney function and can eventually cause kidney failure. "PKD" specifically includes autosomal dominant polycystic kidney disease (ADFKD) and recessive autosomal recessive polycystic kidney disease (ARPKD), in ail stages of development, regardless of the underlying cause.

The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent and/or treat disorders associated with bicarbonate secretion, e.g., Cystic Fibrosis.

The peptides and agonists of the disclosure can be used alone or in combination therapy ' to prevent and/or treat disorders assoeiated with bile secretion. In addition, they cars be used to facilitate or control chloride and bile fluid secretion in the gall bladder.

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The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent and/or treat disorders associated with liver cell regeneration. This may include administration of the peptides and agonists to liver transplant recipients and to patients with drug or alcohol induced- Ii vei damage. Furthermore, the peptides and agonists may be useful to treat liver damage as in the case of viral mediated hepatitis. The peptides and agonists of the disclosure may be used alone or in combination to prevent and/or treat liver abscess, liver cancer (either primary or metastatic), cirrhosis (such as cirrhosis caused by the alcohol consumption or primary biliary cirrhosis), amebic liver abscess, autoimmune hepatitis, biliary atresia, coccidioidomycosis disseminated, δ agent (hepatitis 5), hemochromatosis, hepatitis a. hepatitis b, hepatitis c, or any other acute, subacute, fulminant or chronic hepatitis of vjrai, metabolic or toxic etiology, hepatocellular carcinoma, pyogenic liver abscess, R eye's syndrome, sclerosing cholangitis, Wilson's disease, drug induced hepatotoxicity, or fulminant or acute liver failure. The peptides and agonists may be used in stimulating hepatic regeneration after surgical hepateetomy.

The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent and/or, treat myocardial infraction, coronary artery disease, nitrate- induced tolerance, nitrate tolerance, diastolic dysfunction, angina pectoris, stable, unstable and variant (Prinzmetal) angina, atherosclerosis, thrombosis, endothelial dysfunction, cardiac edema, stroke, conditions of reduced blood vessel patency, eg., fx>stpercutaneous transluminal coronary angioplasty {posi-PTCA}, and peripheral vascular disease,

The peptides and agonists of the disclosure can be used alone or in combination therapy io prevent and/or treat glaucoma.

The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent arκl/or treat immunodeficiency.

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The peptides and agonists of the disclosure can be used alone or in combination iherapy Io prevent and/or treat bladder outlet obstruction and incontinence.

The peptides and agonists of the disclosure can be used alone or in. combination therapy to prevent and/or treat male (e.g. erectile dysftmctioα) or female sexual dysfunction, dysmenorrhea, endometriosis, polycystic ovary syndrome, vagina! dryness, uterine pain, or pelvic pain. These peptides and agonists of the disclosure can be utilized a^ tocolytic agents that decrease or arrust uterine contractions. The peptides and agonists of the disclosure can be used to prevent/treat premaiirre/preterm labor. Premature or preterm labor can be associated with, for example, an iHness/disorder/corsdi.iϋπ of the mother (such as pre-edampsia, high blood pressure Oϊ diabetes, abnormal shape or size of the uterus, weak or short cervix, hormone imbalance, vaginal infection that spreads to the uterus, abnormalities of the placenta, such as placenta previa, and excessive amniotic fluid}, premature rupture of the amniotic membranes ("water breaks ^' "), large fetus, and more than one fetus. The peptides or agonists of the disclosure can be used to prevent uterine rupture. The peptides or agonists of the disclosure can be used treat rapid uterine contractions (for example, associated with placental abruption wherein the placental abruption is associated with hypertension, diabetes, a multiply pregnancy, an unusually large amount of amniotic fluid, numerous previous deliveries, or advanced maternal age (e.g. >40 years old), in certain embodiments they can be used in combination with a phosphodiesterase inhibitor. The peptides and agonists of the disclosure can be used alone or in combination therapy to prevent and/or treat infertility, for example, male infertility <me to poor .sperm quality, decreased sperm motility or low sperm count.

The peptides miά agonists of the disclosure can be used alone or in combination therapy to prevent and/or treat osteopenia disorders {bone loss disorders), "Bone loss disorders" include conditions and diseases wherein the inhibition of bone loss and/or the promotion of bone formation is desirable. Among such eorκlitiorLs and diseases are osteoporosis, osteomyelitis, Paget's disease (osteitis deformans}, periodontitis, hypercalcemia, osteonecrosis, osteosarcoma, osteolyic metastases, familial expansile

osteolysis, prosthetic loosening, periprostatic osteolysis, bone loss attendant rheumatoid arthritis, ami cleidocranial dysplasia (CCD). Osteoporosis includes primary osteoporosis, endocrine osteoporosis (hyperthyroidism, hyperparaihyroidisirs , Cushing's syndrome, and acromegaly), hereditary and congenita] forms of osteoporosis (osteogenesis imperfecta, homocystinuria, Menkes' syndrome, and RiIe- Oay syndrome) and osteoporosis due to immobilization of extremitiesosteomyeiitis, or an infectious lesion in bone leading to bone loss. The peptides and agonists can be used alone or in combination therapy to stimulating bone regeneration. The bone regeneration may be following reconstruction of bone defects in eraπio-maxiilofaeiai surgery, or ibl lowing an implant into bone, for example a dental implant, bone supporting implant or prosthesis. The bone regeneration may also be following a bone fracture.

The peptides and agonists of the disclosure maybe used alone or in combination therapy (for example, with otheτ agents that increase cGMP) to prevent or treat disorders related to an alteration in cGiVIP including, but not limited to Alzheimer's disease, psoriasis, skin necrosis, scarring, fibrosis, baldness. Kawasaki ^' s Disease, nutcracker oesophagus (US20050245544), septic shock, NSAlEHnduced gastric- disease or disorder, ischemic renal disease or disorder, peptic ulcer, sickle cell anemia, epilepsy, arid a neuroinfianimatory disease or disorder (tor example as described in WO05 ! 05765 s.

Treatment of the side-effeets of opioid administration

GGC receptor agonists, e.g., GCC receptor agonist polypeptides described herein. may useful in r.he trca.rπent of one or more side effects of opioid administration, e.g., opioid induced constipation, nausea and/or vomiting. hi the case of constipation, the GCC" receptor agonist polypeptide can be administered at a dosage to induce taxation within a desired time {e.g., within 15 minutes, 30 minutes, 1 hoar, 2 hours, 3 hours, 4 hoists, 5 hours. 6 hours, 7 hours, 8 hours, 9 hours, H) hours, 12 hours, ! S hours or 24 hours).

The the OCX ^' . ^' receptor agonist polypeptide can be administered to maintain regular bowel movements in a patient who is a chronic opioid use? (e.g., a terminally-ill patient). The administration can be via any convenient route (e.g., sublingual, parenteral, intravenous, subcutaneous),

Thus, the polypeptides described herein can be administered Io a patient that is taking i*ne or more of the following opioids: Acetorphine, Acetyldihydroeodeine, Acetyiiϊtorphone, AlføitaniL AHylprodπie. Anilcridiπe, Bαnidone. Benzylmoφhϊπe, Bezitram ide, Buprenorphine, Butoφhanol, CaiieMaαi l/Carfentanyl, Clonitaxene, Codeine, Oodeine-N-Qxide, Codeinoπe, Cyeiaxocine, Cvdoφhaa Desomoφhme, Dextromorarciide, Dextropropoxyphenc, Dezocinc, Diacetyldihydromorpiiirse, Diamoφhme/Diacetyimoφbine (Heroin), Dicthylthiarabutene, Di fenoxin, .Diliydxocodeine, Dib.ydujcodeinone Enol Acetate, Dihydroeioφhine, Dihydrobocodeinc, Diliydromorphine. Dimethylthiambutene, Diphenoxylate, Dipropanoyhrioφhines Drobetaboi Ethyiketocyclazociiie, Ethyl morphine,

F.tonita/.eπc, Eioφhine, Fenta yl l lydrocodone, Slydromoφhone, isorøethadoπe, Ketobemidonc, Laudanum, Lefctarninc, I _^ evallorphaπ, Levo-AIphaceiylmethadoi { 1..λAM), Li'vorncihoφhan, Levoφhanol, Loperairύde, Meptazinoi, Metazυcinc, Methadcaie, Monoaceiylπsoηphine, Moφhine, Morphine-6-Giucuronide, Morphine-N- Oxide, Moφbinonc. MPFP (I -Methyl 4-Phcnyl 4-Propionoxypiperidine), Myorphine, Naibuphme/Nalbufine, N icocodeine, N icodieodeine, Nicoraoφhϊne, Norcodeiπe, Ohmtrfcπtunyi, Oxycodone, Oxymorpbonc, Pentazocine, PEPAi-* {l-Pheuetbyl-4- P!κ ^s nyi-4-Piperidinol Acetate { lister)}. Pethidine (Meperidine), Phenadoxone, Pheϊiazc-cme, Phenoperidine, Pholcodeine, Pimiαodϊne. Piritramide, Prodiπe, Pixψiram, Propoxyphene, Racemeihoφhan, RεmifcnUini], Sufentanil, Thebsine, ^' Thidientanil/rhiofentanyl, Tilidiπe, and Tramadol The peptide can be coadministered with or co-formulated with any of the preceedϊng peptides.

Where the GCC receptor agonist :s αv-ibrnrulaied with an opioid the composition may further include one or more other active ingredients thai may be conventionally employed in analgesic and/or eough-coki- antitussive combination products. Such

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conventional ingredients include, for example, aspirin, acetaminophen, phenylpropanolamine, phenylephrine, chlorpheniramine, caffeine, and'or guaifenesin. Typical or conventional ingredients that may be included in the opioid component are described, tor example, in the Physicians' Desk Reference, 1999, the disclosures of which are hereby incorporated herein by reference, in their entirety.

in addition, the composition may further include one or more compounds that may be designed to enhance the analgesic potency of the opioid and/or to reduce analgesic tolerance development. Such compounds include, for example, dextromethorphan or other NMDA antagonists (Mao, M J. ct aL Pain 1996, 67, 361). L-364.718 and other CCK antagonists (Dourish, C. T. et al., Eur J Pharmacol 1988, 147, 469), NOS inhibitors (Bliargava, H. N. et a!.. Neuropeptides 1996, 30, 219), PKC inhibitors (Bilsky, Ir,. J. et aL J Pharmacol Kxp Ther 1996, 277, 484), and dynorphin antagonists or aniisera (Nichols, M. L. et al. Pain 1997, 69, 317). The disclosures of each of the foregoing documents are hereby incorporated herein by reference, in their entireties.

The combination products, such as pharmaceutical compositions comprising opioids in combination with a CJCC agonist may be in any dosage form, such as those described heroin, and can also be administered in various ways, as described herein. In a preferred embodiment, the combination products of the disclosure are foπxmiaied together, in a single dosage form (that is, combined together in one capsule, tablet, powder, or liquid, etc.). When the combination products are noi formulated together in a single dosage form, the opioid compounds and the CJCC agonists may be administered at the same time {that is, together), or in any order. When not administered at the same time, preferably the administration of an opioid and a GCC agonist occurs less than about one hour apart, less than about 30 minutes apart, less than about I 5 minutes apart, and less than about 5 minutes apart. Administration of the combination of an opioid and a GCC agonist can be, for example, oral, although other routes of administration, as described above, are contemplated to be within the scope of the present disclosure. Although it is the opioids and GCC agonists may KuIi be administered in the same fashion (thai is, for example, both orally), if desired, they

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may each be -administered in different fashions (that is, for example, one component of the combination product may be administered orally, and another component may be administered intravenously). The dosage of the combination products of the disclosure may vary depending upon various factors such as the pharmacodynamic characteristics of the particular agent and .its mode and route of administration, the age, health and weigh! of the recipient, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, and the effect desired.

Although the proper dosage of the combination products of this disclosure will be readily ascertainable by one skilled in the art, by way of general guidance, where an u jpk*iά α sits pounds is combined with a GCC" agonist, for example, typically a daily

A .in, sage may rarsge from about (3.01 to about 100 milligrams, Q. I \o about IO sϊϊiiiigϊams of the opioid, 15 to about 2.00 milligrams, 1 , 2, 3. 4, 5, 6, 7, 8, 9, IO milligrams of opioid per kilogram of patient body weight. The opioid-OCC agonist combination product can include, for example, from 1 to 30 μg, 1 to 40 μg, I to 50 μg, 1 to 100 μg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg. 1 to 500 μg. I to 600 μg, 1 to 70Q μg. ! to 800 μg. 1 to 900 μg, 1 to 1000 μg. 10 to 30 μg, 10 to 40 μg, 10 to 50 μg, 10 to J OU μg, 10 to 200 μg, 10 to 300 μg _r 10 to 400 μg, 10 to 500 μg. 10 to 600 μg, 10 to 700 μg, 10 to 800 μg, 10 to 900 μg _? 10 to 1000 μg, 100 to 200 μg, 100 to 300 μg, 100 to 400 μg, 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to 800 μg, 100 to 900 μg, 100 to 1000 μg, 100 to 1250 μg, 100 to 1500 μg, 100 to 1750 μg.. 100 to 2000 μg. 100 to 2250 μg. ! 00 to 2500 μg, 100 to 2750 μg, I Oi ⁾ to 3000 μg. 200 to 300 μg, 200 to •400 μg, 200 io 500 μg, 200 to 600 μg _s 200 to 700 μg, 200 in 800 μg, 200 k> 900 μg, 2(M) U) i 000 μg, 200 to 1250 μg, 200 to 1.500 μg, 200 to 1750 μg, 200 to 2000 μ.g, 200 to 2250 μg, 200 ω 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 to 6(K) μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg, 300 to ! 250 μg, 300 to 1500 μg, 300 to 1750 μg, 300 to 2000 μg, 300 to 2250 μg. 300 to 2500 μg, 300 to 2750 μg. 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg. 40O U) 700 μg, 400 to 800 μg. 400 to 900 μg. 400 to 1000 μg, 400 to 1250 μg- 400 Io 1500 μg, 400 to 1750 μg. 400 to 2000 μg. 400 io 2250 μg, 400 to 250O μg, 400 io 2750 μg, 400 to 3000 μg, 500 to 600 μg. 500 to 700 μg. 500 to SOO μg. 500 to 900 μg, 500 to 1000

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μg. 500 to 1250 μg, 500 to 1500 μg. 5(){} to 1750 μg. 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 to 800 μg, 600 to 900 μg, 600 to 1000 μg, 600 to 1250 μg, 600 to 1500 μg, 600 Io ! 750 μg, 600 to 2000 μg. 600 to 2250 μg, 600 to 2500 μg, 600 to 2750 μg, 600 to 30(Ki μg, 700 to 800 μg, 5 ?()(} to 9(50 μg, 700 Io 1000 μg, 700 Io 1250 μg, 700 to 1500 μg, 700 to 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg. 700 to 2750 μg, 700 Io 3000 μg, 800 to 900 μg, 800 to 1000 μg. SOO io 1250 μg, SOO to 1500 μg, 800 to ! 750 μg, 800 in 2000 μg. 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 io 3000 μg, 900 to 1000 μg, 900 to 1250 μg, 900 to 1500 μg, 900 to 1750 μg, 900 to 2000 μg. 900 to 2250 μg, 900 o to 2500 μg, 900 to 2750 μg, 900 io 3000 μg, 10(30 to 1250 μg, 1000 to 1500 μg, ! 000 Ui 1750 μg. 1000 to 2000 μg, 1000 to 2250 μg. 1000 to 2500 μg, ! 000 io 2750 μg, 1000 io 3000 μg. 2 io 500 μg. 50 to 500 μg, 3 io 100 μg. 5 io 20 μg, 5 io 100 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg. 60 μg, 70 μg, 75 μg, SO μg, 90 μg, 100 μg, 150 μg. 200 ug, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg. 500 μg, 550 μg, 600 μg, 650 μg, 700 r> μg, 750 μg, KOO μg. 850 μg. 900 μg. 950 μg, 1000 μg, 1050 μg, 1100 μg, 1150 μg, 1200 μg, 1250 μg, BOO μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg. 1600 μg. 1650 μg, 1700 μg, i 750 μg. 1800 μg, 1850 μg, 1900 μg, 1950 μg. 2000 μg, 2050 μg, 2100 μg, 21 50 μg, 2200 μg. 2250 μg, 2300 μg, 2350 μg, 2400 μg. 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2S(K) μg, 2850 μg. 2900 μg, 2950 μg, 0 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg. 4250 μg, 4500 μg, 4750 μg, 5000 μg uFa GCC a <.tsro ^v nist described herein.

When provided as a single dosage form, the potential exists for a chemical interaction between the combined active ingredients (for example, an opioid and a GCC agonist). For this reason, the preferred dosage forms of the combination products of this 5 disclosure are formulated such that although the active ingredients are combined in a Single dosage form, the physical contact between the active ingredients is minimized (that is, reduced}.

in order to minimize contact, one embodiment of this disclosure where the product is D orsiiy administered provides for a combination product wherein one active ingredient

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is enteric coated. By enteric coating one or more of the active ingredients, it is possible not only to minimize the contact between the combined active ingredients, but also, it is possible to control the release of one of these components in the μastrt 'intestinal tract such that one of these components is not released in the stomach but rather is released in the intestines. Another embodiment of this disclosure where oral administration is desired provides for a combination product wherein one of the active ingredients is coated with a sustained-release materia! which effects a siislaiaed-relea.se throughout the gastrointestinal tract nnά also serves to minimize physical contact between the combined active ingredients. Furthermore., the sustained- released component can be additionally enteric coated such that the release of this component occurs only in the intestine. Still another approach would involve the formulation of a combination product in which the one component is coated wtih a sustained and/or enteric release polymer, and the other component is also coated with a polymer such as a low-viscosity grade of hydroxypropyi methyl cellulose (HPMC) or other appropriate materials as known in the art, in order to further separate the active components. ^" The polymer coating serves to form an additional barrier to interaction with the other component.

Dosage forms of the combination products include those wherein one active ύmrediem is enteric coated can be in the form of tablets such that the enteric coated component and the other active ingredient are blended together and then compressed into a tablet or such that the enteric coated component is compressed into one tablet layer and the other active ingredient is compressed into an additional saver. Optionally, in order to further separate the two layers, one or more placebo layers may be present such that the placebo layer is between the layers of active ingredients, in addition, dosage forms of the present disclosure can be in the form of capsules wherein one active ingredient is compressed into a tablet or in the form of a plurality uf rmerϋtahlets, panicles, granules or non-perils, which are then enteric coated. These enteric coated niierotablets, particles, granules or non-perils are then placed into a capsule or compressed into a capsule along with a granulation of the other active ingredient.

These as well as other ways of minimizing contact between the components of combination products of the present disclosure, whether administered in a single dosage form or administered in separate forms but at the same time by the same manner, will be readily apparent to those skilled in the art in light of the present disclosure.

Peptides as iimnuttβgens

The peptides of the disclosure can be used as immunogens to create antibodies lor immunoassays. The peptides of the disclosure can be used as immunogens to treat arid/or prevent one or more disease symptoms associated with traveler's diarrhea and for vaccination against pathogens, including but not limited to enterotoxigenic E.coii (IVTECj. They may also be used in vaccines which also comprise interleukisi 18 and cither saponin adjuvant or CpG adjuvant for example as described in WO05039634 and WO0503%30. The methods described in US2004GI 46534, US4220584, US42U539L US51821O9, US461B049, US454593 L US48S6663, US4758655, WQ08402700, FR2525592, and FR2532850 can be similarly used to create immunogens eoroprisiusi the peptides of the disclosure. US6043057, US5S34246, US5268276, and BP36S81V, speeitlealiy describe an expression system containing CTB (cholera toxin Beta subumt) fused to an ST-Hke peptide under a foreign promoter for use as a vaccine. The nucleic acids that encode the peptides of the disclosure may be used as genetic vaccines as described in US20050260605 &nά WOOl 480! 8. The nucleic acid molecules may also be used for the manufacture of a functional ribonucleic acid, wherein the functional ribonucleic acid is selected from the group comprising ribozymes, antisen.se nucleic acids and si RNiA (as described in WOOS 103073),

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U.S. 200201 76841, U, S- 5,672,659, U.S. 5,893,985, U.S. 5,134, 122. U.S. SJ92.74I, U.S. 5,102,74 L U.S. 4,668,506. U.S. 4,713,244. U.S. 5,445,832 U.S. 4,93 i , 279, U.S. 5.980,945, WO 02/058672, WO 9726015, WO 97/04744, and. US200200I9446. In such sustained release formulaiions mieropartieles (Delie arse Blanco- Prieto 2005 5 Molecule 10:65-80) of peptide are combined with mieropartieϊes of polymer. One or more sustained release implants can be placed in the large intestine, the small intestine or both. U.S. 6J)I 1,01 i and WO 94/06452 describe a sustained release formulation providing either polyethylene glycols (i.e. PEG 300 and PEG 400) or tπaceim. WO 03/053401 describes a formulation which may both enhance bioavailability and 0 provide controlled releascof the agent within the GI tract. Additional controlled release formulations are described in WO 02/38129, EP 326 151, U.S. 5,236,704, WO 02/3039S ₅ WO 98/13029; U.S. 20030064105, U.S. 200301384S8A1, U.S. 20030216307λ L U.S. 6,667,060, WO 01/49249, WO 01/4931 1. WO 05/49249, WO IU/49M h and U.S. 5,877,224,

The agents ears be administered, e.g., by intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, topical, sublingual, intraarticular (in the joints), intradermal, buccal, ophthalmic (including intraocular), intranasal? (Including using a cannula), intraspinaliy, intrathecal^ or by other routes. 0 The agents can be administered orally, e.g., as a tablet or cachet containing a predetermined amount of the active ingredient, gel, pellet, paste, syrup, bolus, electuary, slurry, capsule, powder, lyophilized powder, granules, sachet, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, as an oil-m-water liquid emulsion or a water-in-oil liquid emulsion, via a mieellar ibrnialation (see, e.g. WO 5 97/1 1682} via a liposomal formulation (see, e.g., EP 736299,WO 99/59550 and WO 97/13500). via formulations described in WO 03/094886, via bilosonie (bile-salt based vesicular system), via a dendrimer, or in some other form. Orally administered compositions can include binders, lubricants, inert diluents, lubricating, surface active oτ dispersing agents, Savoring agents, and humeetants. Orally administered C; formulations such as tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active

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ingredient therein. The agents can also be administered transdermal!}' (i.e. via reservoir-type or matrix-type patches, microneedles, thermal poraiioπ, hypodermic needles, iontophoresis, electroporation, ultrasound or other forms of sonophoresis. jet mjeefiorh or a combination oi ^' any of the preceding methods (Prausrήtz ei ai. 2004, Nature Reviews Drug Discovery 3:1 15-124)}. The agents can be administered using high- velocity transdermal particle injection techniques using the hydrogeJ particle S ^' uπmiiatkm described in U.S. 20020061336. Additional particle formulations are described in WO 00/45792, WO 00/53160, and WO 02/19989. λ.n example of a transdermal formulation containing piaster and the absorption promoter dimethylssosorbide can be found in WO 89/04179. WO 96/1 ! 705 provides formulations suitable for transdermal adminisitratioπ. The agents can be administered in the form a suppository or by oilier vaginal or rectal means. The agents can be administered in a transmembrane formulation as described in WO 90/07923. The agents can be admmistcd non-iπvasively via the dehydrated part icicles described in U.S. 6,485,706. The agent can be administered in an enteric-coated drug formulation as described io WO 02/49621 , The agents can be administered intranassaty using the formulation described in U.S. 5,179,079. Formulations suitable for parenteral injection are described in WO 00/62759. The agents cars be administered using the casein formulation described in U, S. 20030206939 and WO 00/061 OS. The agents can be administered using the particulate formulations described in U.S. 20020034536.

The agents, alone or in combination with other suitable components, can he administered by pulmonary route utilizing several techniques including but not limited to intratracheal instillation (delivery of solution into the lungs by syringe), intratracheal delivery of liposomes, insufflation (administration of powder formulation by syringe or any other similar device into the lungs) and aerosol inhalation. Aerosols (e.g., jet or ultrasonic nebulizers, metcjred-dose inhalers (MD Is), ami dry-powder inhalers (DPIs)) can also be used in intranasal applications. Aerosol formulations are stable dispersions or suspensions of solid material and liquid droplets m a gaseous medium mύ can be placed into pressurized acceptable propeilants. such

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as hydrofjuroalkanes (I-iFAs, i.e. HFA-134a and HFA-2.2.7, or a mixture- {hereof}, diehlorodilluorornεthane (or other chlorofluocarbcm propellants such as a mixture of PropeHants I i ₅ 12, and/or i 14}, propane, nitrogen, and the like. Pulmonary formulations may include permeation enhancers such as fatty acids, saccharides, 5 chelating agents, enzyme inhibitors (e.g., protease inhibitors), adjuvants (e.g., glycocholate, sαrfactin, span 85, and naiamostat), preservatives (e.g., benxalkoniuro chloride or chlorobutanoϊ), aid ethanoϊ (normally up to 5% but possibly up to 20'Hs, by weight), Bthanol is commonly included in aerosol compositions as it cars improve the iiiπction of the metering vaϊve and in some cases also improve the stability of the

^• to dispersion. Pulmonary formulations may also include surfactants which include but are not limited to bile salts and those described in U.S. 6,524.557 and references therein. The surfactants described in. U.S. 6,524.557, e.g., a C8-C16 fatty acid salt, a bile salt, a phospholipid, or alky! saccaridc are advantageous in that some of them also reportedly enhance absorption of the peptide in the formulation. Also suitable in the

15 disclosure are dry powder formulations comprising a therapeutically effective amount of active compound blended with an appropriate carrier and adapted for use in connection with a dry-powder inhaler. Absorption enhancers which can be added to dry powder formulations of the present disclosure include those described in U.S. 6,632,456, WO 02/080884 describes new methods for the surface modification of

20 powders. Aerosol formulations may include U.S. 5,230,884, U.S. 532,499, WO 01 7/8694, WO 01/78696, U.S. 2003019437, U. S. 20030165436, and WO 96/4 ⁽ 3089 (which includes vegetable oil). Sustained release formulations suitable for inhalation are described in U.S. 200100364Sl Al ₅ 20G30232019A1 , and U.S. 20040018243 A l as well as m WO 01/13891 , WO 02/067902, WO 03/072080, and WO 03/079885.

25 Pulmonary formulations containing microparticϊes are described in WO 03/015750, U.S. 20O3O0OS013, and WO 00/00! 76. Pulmonary formulations containing stable glassy state powder are described in U.S. 20020141945 and U.S. 6309,67 L Other aerosol formulations are desribed in EP 1338272Ai WC) 90/097S i , U. S. 5,348,730, U.S. 6,436,367, WO 9!/04OU , and U.S. 6,294,153 mά U.S. 6,290.987 describes a so liposomal based formulation that can be administered via aerosol o.r other means. Powder formulations for inhalation are described in iλS, 20030053960 and WC ⁾

0 i/6034 i . 1 he agents can be administered mtranasaliy as described in U.S. 20010038824.

The agents can be incorporated into microemulsions, which generally are ihermodyiiarakaϋy stable, .isotrαpically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an inleriacia! film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). For the preparation ofnήcroemuϊsions, surfactant (emuisifϊer), co- Burfaciant (co-cmuisifier), an oil phase and a water phase are necessary. Suitable surfactants include any surfactants thai are useful In the preparation of emulsions, e.g., esimfsi tiers that are typically used in the preparation of creams. The co-surfactant (or "co-cmulsifer") is generally selected from the group of poiygiyceroi derivatives, glycerol derivatives and fatty alcohols. Preferred ernuksifier/co-ernuisifkτ combinations are generally although not necessarily selected from the group consisting of: glyceryl rnonøstearate and polyoxyethylene stearate; polyethylene glycol and ethylene glycol pamritostearate; and capriUc and capric triglycerides and ϋSeoyl lϊiacrogolglycefides. The water phase includes not only water but also, typically, buffers, glucose, propylene glycol polyethylene glycols, preferably lower molecular weight polyethylene glycols (e.g., PEG 300 and PEG 400), and/or glycerol, and the like, while the oil phase will generally comprise, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono- άi~ and triglycerides, mono- and di-esters of PEG (e.g., oleoyl maeragol giycericks), etc.

The agents of the disclosure can be incorporated into phaπuaceuticaily-accepiabie πanopartick, nanosphcrc, and nanocapsule formulations (Ddk and Blanco-Prieu* 2005 Molecule 10:65-80). Nartocapsules can generally entrap ccmipcainds in a stable and reproducible way {Henry-Mieheliand et ai., i 987; Quintanar-Guerrero ct ai., 199H; Douglas ei aL, 1987). To avoid side effects due to intracellular polymeric overloading, isltrailne particles (sized around 0.1 μrn) can be designed using polymers able to be degraded in vis'o (e.g. biodegradable polyaikyi-eyaπoacrylate rumopaiticSes). Such particles arc described in the prior art (Couvreur et ai, 19S0;

104 -

19SS; zur Muhkπ et aL 1998; Zaxnbaux et al. 1998; Pinto-AIphaπcJry et aL 1995 and U.S. Pat. No. 5,145,684).

The agents of the disclosure can be ibrrnulaied with pH sensitive materials which may s include those described in WO04041195 (including the sea! and enteric coating described therein) and pH-sensitive coatings that achieve delivery in the colon including those described in US4910021 and WO9001329, US4910021 describes using a pH- sensitive material to coat a capsule. WO9001329 describes using pH- sensitive coatings on beads coinaining acid, where the acid in the bead cove prolongs 0 dissolution of the pϊ-ksemiiive coating, ϋ. S. Patent No. 5,175, 003 discloses a dual mechanism polymer mixture composed of pH-sensHive enteric materials and IiLm- forming plastieizers capable of conferring permeability to the enteric material, for use in drug-deli very systems; a matrix pellet composed of a dual mechanism polymer mixture permeated with a drug and sometimes eos-ering a pharmaceutically neutral 5 nucleus; a membrane-coated pellet comprising a matrix pellet coated with a dual mechanism polymer rasxtirre envelope of the same or different composition; and a pharmaceutical dosage form containing matrix pellets. The matrix pellet releases acid- soluble drugs by diffusion in acid pH and by disintegration at pH levels of nominally about 5.0 or higher. The agents of the disclosure may be formulated in the pi! 0 triggered targeted control release systems described in WO04052339, The agents of ^* Hie disclosure may be formulated according to the methodology described in any of VVO03105812 (extruded Iiyrdratable polymers); WO0243767 {enzyme ekavahϊe membrane transiocators); WO03007913 and WO03086297 (mucoadhesive systems); WO02072075 (bilayer laminated formulation comprising pH lowering agent and b absorption enhancer); WO04064769 (amidaied peptides); WO05063156 (solid lipid suspension with pseudotropie ami/or thixotropic properties upon melting); WO03035029 and WO03035041 (erodible, gastric retentive dosage forms); US5007790 and US5972389 (sustained release dosage forms}; WO041 1271 ! (oral extended release compositions); WO05027878, WO02072033, and WO02072034 0 (delayed release compositions with natural oτ synthetic gum); WO05030182

(controOed release formulations with nn ascending rate of release); WG05048998

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(microencapsulation system); US Patent 5.952, 314 (biopolymer); US5108758 (glassy amylose matrix delivery); US 5840860 (modified starch based delivery), JPl 0324642 (delivery system comprising ehitosan and gastric resistant materia! such as wheat gliadm or zein); 11S5866619 and US636S629 (saccharide containing polymer); US S 553 1 152 (describes a drug delivery system containing a water soluble core (Ca pectinate or other water-insoluble polymers) and outer coat which bursts (eg hydrophobic polyraer-Eudragπt)); US 6234464; US 6403330 (coating with polymer containing casein and high mcthυxy pectin; WO0174175 (Maiilarti reaction product); WO05063206 (solubility increasing formulation); WO04019872 (transferring fusion io proteins). The agents of the disclosure may be formulated using gastrointestinal retention system technology (GIRBS; Merrion Pharmaceuticals). GIRES comprises a comroiled-release dosage form inside an inflatable pouch, which is placed in a drug capsule for oral administration. Upon dissolution of the capsule, a gas-generating system inflates the pouch m the stomach where it is retained for 16-24 hours, all the

15 time releasing agents of the disclosure.

The agents of thy disclosure can be formulated in an osmotic device including the ones disclosed in US4503030, US5609590 and US5358502. IJS4593030 discloses an osmotic device for dispensing a drug to certain pH regions of the gastrointestinal tract.

:>0 More particularly, the disclosure relates to an osmotic device comprising a wail formed of u semipermeable pH sensitive composition that surrounds a compartment containing a drug, with a passageway through the wall connecting the exterior of the device with the compartment. The device delivers the drug at a controlled rate in the region of the gastrointestinal tract having a pH of less than 3.5, and the device self-

25 dcstructs and releases all its drug in the region of the gastrointestinal tract having a pfl greater than 3,5, thereby providing total availability for drug absorption, Li. S. Patent No ss. 5,609, 590 and 5, 358,502 disclose an osmotic bursting device for dispensing a beneficial agent to an aqueous environment. The device comprises a beneficial agent and osrnugeot surrounded at least in part by a. semi-permeable membrane. The

30 beneficial agent may also function as the osmagent. The semi-permeable membrane is permeable to water and substantially impermeable to the beneficial agent and

- 1 lib ~

osmagenL A trigger means is attached Io the semi -permeable membrane (e. g. , joins two capsule halves). ^' The trigger means is activated by a pH of from 3 io 9 and triggers the eventual, but sudden, delivery of the beneficial agent. These devices enable the pB~triggered release of the beneficial agent core as a bolus by osmotic bursting.

The agents of the diseiosure may be formulated based on the disclosure described hi ϋ. S. Patent No. 5,316, 774 which discloses a composition for the controlled release of an active substance comprising a polymeric particle matrix, where each particle defines a network of internal pores. The active substance is entrapped within the pore network together with a blocking agent having physical and chemical characteristics selected κ> modify the release rate of the active substance from, the interna] pore network. In one embodiment, drugs may be selectively delivered to the intestines using an enteric materia! as the blocking agent, The enteric- material remains intact in th$ stomach but degrades under the pH conditions of the intestines, in another embodiment, the sustained release formulation employs a blocking agent, which remains stable under the expected conditions of the environment to which the active .substance is to be released. The use of pH-sensitive materials alone to achieve site- specific delivery is difficult because of leaking of die beneficial agent prior to the rekasu site or desired delivery time and it is difficult to achieve long time lags before release oft.be active ingredient after exposure to high pH (because of rapid dissolution or degradation of the pH-sensitive materials).

The agents may also be formulated in a hybrid system which combines pH -sensitive materials and osmotic delivery systems. These hybrid devices provide delayed initiation of sustained-release of the beneficial agent. In one device a pH-sensitive matrix or coating dissolves releasing osmotic devices that provide sustained release of the beneficial agent see U, S. Patent Nos. 4,578, 075, 4,681 , 583, and 4,851 , 23 L A second device consists of a semipermeable coating made of a polymer blend of an .insoluble and a pH -sensitive materia!. As the pH increases, the permeability of the coating increases, increasing the rate of release of beneficial agent see U. S. Patent Nos, 4,096, 238,4, 503,030, 4, 522, 625, and 4,587, 1 17.

- 107 -

The agents of [he disclosure may bef formulated in terpoluraers according to U. S, Patent Nt). 5,484, 610 which discloses terpolymers which are sensitive to pH and smpeiatute which are useful carriers for conducting bioactive agents through the gastric juices of the stomach in a protected form. The terpolymers swell at the higher 'Miysioiogk pH of the intestinal tract causing release of the bioactive agents into the ntestine. The terpolymers are linear and are made up of 35 to 99 wt % of a emperature sensitive component, which imparts to the terpolynier LCST (lower critical solution temperature) properties below body temperatures, 1 to 30 wt % of a P-H sensitive component having a pK.a in the range of from 2 to 8 which functions hrough ionization or deionization of carboxylic acid groups to prevent the bioactive :igent from being lost at low pH but allows bioactive agent release at physiological pH >f about IA and a hydrophobic component which stabilizes the LCSl ^" below body temperatures and compensates for bioact.Ye agent effects on the terpolymers. The lerpolyrners provide for sale bioactive agent loading, a simple procedure tor dosage orm Fabrication and the terpolymer functions as a protective earner in the acidic environment of the stomach and also protects the bioactive agents from digestive enzymes until the bioactive agent is released in the intestinal tract,

The agents of the disclosure may be formulated in pH sensitive polymers according to those described in U. S. Patent No. 6.103, 865. U. S. Patent No. 6.103, S65 discloses pH-sensiiive polymers containing sulfonamide groups, which can be changed in physical properties, such as swellahϋity and solubility, depending on pB and which can be applied for a dπig-deiivery system, bio-material, sensor, and the like, and a preparation method therefore. The pϊi-sensitive polymers are prepared by introduction of sulfonamide groups, various in pK.a, to hydrophiiic groups of polymers either through coupling to the hydrøphiϋe groups of polymers, such as aeryiamkie, N, N- dmiethyiaeryϊamide, acrylic acid, N-isopropylacrylamide and the like or copolymerizaiion with other polymerizable monomers. These pH- sensitive polymers may have a structure of linear polymer, grafted copolymer, hydrogef or jnteφcneiTatmg network polymer.

- 50S -

The agents of the disclosure may be formukύed according U. S. Patent No, 5. 656, 292 which discloses a composition for pH dependent or pi-! regulated controlled release of active ingredients especially drugs. The eornposiϋoo consists of a eornpactable mixture of the active ingredient and starch molecules substituted with acetate and dicarboxylaie residues. The preferred dicarhoxylate acid is succinate. The average substitution degree of the acetate residue is at least 1 and 0. 2-1. 2 for the dicarboxyiate residue. The starch molecules can have the acetate and diearboxylate residues attached to the same starch molecule backbone or attached to separate starch molecule backbones. The present disclosure also discloses methods for preparing said starch acetate die&rboxylates by traosesterification or mixing of starch acetates and starch dicarboxylates respectively.

The agents of the disclosure may be formulated according to the methods described in U. S- Patent Nos. 5,554, 147,5, 788, 687, and 6,306. 422 which disclose a method for the controlled release of a biologically active agent wherein the agent is released from ^• ά hydrophobic. pH-sensitive polymer matrix. The polymer matrix .swells when the environment reaches pϊ-i S.5, releasing the active agent. A polymer of hydrophobic and weakly acidic comorsorBcrs is disclosed for use in the controlled release system. Also disclosed is a specific embodiment in which the controlled release system may be used. The pH -sensitive polymer is coated onto a latex catheter used in ureteral catheterization. A ureteral catheter coated with a pH-seπsitive polymer having an antibiotic or urease inhibitor trapped within its matrix will release the active agent when exposed to high pi! urine.

The agents can be administered using COLAL® colonic drag delivery technology (U.S. Patent No. 6,534,549} BTG International, Ltd,.; Alizyme, pie; Cambridge, UK } in which small pellets containing the agents are coated with ethyl cellulose and a specific form of amylase. This coating prevents drug release in the stomach and small intestine. When the pellets reach the colon the amyiose in the coating is broken down by bacterial enzymes and the agent is released.

- 109 -

The agents of the disclosure may be formulated in/with bioadhesive polymers according Io US Patent No. 6365, 187. Bioadhesive polymers in the form of, or as a coating on, microcapsules containing drags or bioactive substances which may serve for therapeutic, or diagnostic purposes in diseases of the gastrointestinal tract, are described in US6365187. The polymeric microspheres all have a bioadhesive force of at temi 11 mN/cπr (1 10 Tvi/n^} Techniques lor the fabrication of bioadhesive microspheres, as well as a method for measuring bioadhesive forces between microspheres and selected segments of the gastrointestinal tract in vitro are also described. ThLs quantitative method provides a means to establish a correlation between the chemical nature, the surface morphology and the dimensions of drug- loaded microspheres on one hand and bioadhesive forces on the other, allowing the screening of the most promising materials {tore a relatively large group of natural and synthetic polymers which, from theoretical consideration, should be used for making bioadhesive microspheres. Solutions of medicament in buffered saline and similar vehicles are commonly employed to generate an aerosol in a nebulizer. Simple nebulizers operate on Bernoulli's principle and employ a stream of air or oxygen to generate the spray panicles. More complex nebulizers employ ultrasound to create the spray particles. Both types are well known in the art and are described in .standard textbooks of pharmacy such as Sprowls' American Pharmacy and Remington's The Science and Practice of Pharmacy. Other devices for generating aerosols employ compressed gases, usually hydrofluorocarbons and chlororluorocarbons, which are mixed with the medicament and any necessary exeipients in a pressurized container, these devices are likewise described in standard textbooks such as Sprowls and Remington..

^" The agents can be a free acid or base, or a pharmacologically acceptable salt thereof. Solids can be dissolved or dispersed immediately prior to administration or earlier. In some circumstances the preparations include a preservative to prevent the growth of microorganisms. The pharmaceutical 'forms suitable for injection cars include sterile aqueous or organic solutions or dispersions which include, e.g., water, an alcohol, an organic solvent an oil or other solvent or dispersani (e.g., glycerol, propylene glycol,

- HO -

polyethylene glycol, and vegetable oils). The fomiuhnions may contain antioxidants, buffers, hacteriostais, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubiiizers, thickening agents, stabilisers, and preservatives. Pharmaceutical agents can be sterilized by lifter sterilization or by other suitable means. The agent can be fused to immunoglobulins or albumitu albumin variants or fragments thereof, or incorporated into a liposome to improve half-life. Thus the peptides described herein maybe fused directly or via a peptide linker, water soluble polymer, or prodrug linker to albumin or a.n analog, fragment, or derivative thereof. Generally, the albumin proteins that are part of the fusion proteins of the present disclosure may bε derived from albumin cloned from any species, including human. Human serum albumin (HSA) consists υf a single non-glyeosylated polypeptide chain of 585 amino acids with a formula molecular weight of 66,500, The amino acid sequence of human HSA is known [See Meiouπ, ev al, (1975} FEBS Letters 58:136; Behrens, et al. U975) Fed. Proc. 34:591 ; Lawn, ei aL (1981 ) Nucleic Acids Research 9:6102-61 14; Minghetti, et al. (1986) j. Biol. Chem 261 :6747. each of which arc incorporated by reference herein}. A variety of polymorphic variants as well as anal ugs and fragments of albumin have been described. [See Weitkarnp, et aL (1973} Ann. Hum. Genet, 37:219], For example, in EP 322,094, various shorter forms of llSA. Some of these fragments of HSA are disclosed, including HSA(I- 373), HSAπ-388), HSA( I-389), HSA(I -369), and HSA( i -419) and fragments beiween I -369 and I -419. EP 399,666 discloses albumin fragments that include HSA(ϊ-! 77) and HSA(I -200) and fragments between HSA(M 77) and HSA( 1-200). Methods related to albumin fusion proteins can be found in US 7,056,7OK US 0,994,857, US h if r>; /7j?at ft , uspU; . gov/netac gi/siph-

bOhtip^'/oatft.Lϊspto.tiov/Tvcl ^' acg.i/ ^' nph-' l ^> ar^^Sect J ^« PlX) )&Sect2=HITOFF<^^PALL&p ^« l&u^%2Fiietahtml%2J ^; PTQ ⁰ X Fsrchnιnn.hm^r^&fK}&Fθ0&sl-6946S 34.PN.&OS-PN/6946134&RS-PN/ I]26,946J.?4. US ll^/^criiiusirto.goy/neta^gim^

- J H -

.lOhπ)yj/H^lJlijspi,o.goy/netao.ui/nph-

hO hit p './/pat i ^' t u ^pto . &oy/netac gi/n.ph-

Fsryki?mdiMi4M&^^

1x26,905,688 and the related priority documents and references cited therein. The agent CM) also be conjugated to polyythyient- glycol {PEG} chains. Methods for pegykuoo and additional formulations containing PEG-conj urates (i.e PEG-hased

15 hydrogels, IMvX?) modified liposomes) can be found in Harris and Chess, Nature

Reviews Drug Discovery 2; 214-221 and the references therein. Peptides can also be modified with alky! groups (e.g., Cl -€20 straight or branched alky) groups}; fatty acid radicals; and combinations of PEG, alky] groups and fatty acid radicals (see U.S. Patent 6,309,633; SoUero et a!., 2(JOI. Innovations in Pharmaceutical Technology 106-

20 11 Q). The agent can be administered via a nanocochleatc or coehieate delivery vehicle (Bio Delivery Sciences International). The agents can be delivered ttaπsmueosally (i.e. across a mucosal surface such as the vagina, eye or nose) using ibiTJUil&tions such as thai described in U.S. 5,204JI)S. The agents can be formulated sn microcapsules as described in WO 88/0! 165, The agent can be administered intra-

?5 orally using the formulations described in U.S. 20020055496. WO 00/47203, and If. S. 6,495, 120. The agent can be delivered using nanoerraiiskm formulations described in WO 01 /91728A2.

- \ \2 ~

In general one can provide for controlled release of the agents described herein through the use of a wide variety of polymeric carriers and controlled release systems 5 including erodibJe and non-erodibJe matrices, osmotic control devices, various reservoir devices, enteric coatings and multiparticulate control devices.

Matrix devices are a common device for controlling the release of various agents. In such devices, the agents described herein are generally present as a dispersion within

10 the polymer matrix, and are typically formed by the compression of a polymer/drug mixture or by dissolution or melting. The dosage release properties of these devices may be dependent upon the solubility of the agent in the polymer matrix or, in the case of porous matrices, the solubility in the sink solution within the pore network, and the tortuosity of the network. In one instance, when utilizing an credible

^■ ;» polymeric matrix, the matrix imbibes water and forms an aqueous-swollen gel that entraps the agent. The matrix then gradually erodes, swells, disintegrates or dissolves in the GI tract, thereby controlling release of one or more of the agents described herein. In non- credible devices, the agent is released by diffusion through an inert matrix.

20

Ag€nts described herein can be incorporated into an credible or non-erodible polymeric matrix controlled release device. By an credible matrix is meant aqueous- erodible or wafer-swdlabie or aqueous-soluble in the sense of being either credible or swciiable or dissolvable in pure water or requiring the presence of an acid or base to

25 ionsze the polymeric matrix sufficiently to cause erosion or dissolution. When contacted with the aqueous environment of use, the credible polymeric matrix imbibes water and forms an aqueous-swollen gel or matrix that entraps the agent described herein. The aqueous-swollen matrix gradually erodes, swells, disintegrates or dissolves in the environment of use, thereby controlling the release of a compound

30 described herein to the environment of use.

The credible polymeric matrix into which an agent, described herein can he incorporated .may generally be described as a set of exeipients that are mixed with the agent following its formation that, when contacted with the aqueous environment of use imbibes water and tonus a water-swollen gel or matrix that entraps the drug form. Drug release may occur by a variety of mechanisms, for example, ihe matrix may disintegrate or dissolve from around particles or granules of the agent or the agent may dissolve in the imbibed aqueous solution and diffuse from the tablet, beads or granules of the device. One ingredient of this water-swollen matrix is the water- sweHablc, credible, or soluble polymer, which may generally be described as an osmopoiynier, hydroge! or waier-swdlabk* polymer. Such polymers may be linear, branched u? erosslinkcd. The polymers may be homopolymcrs or copolymers. In certain embodiments, they may be synthetic polymers derived from vinyl, acrylate, meihacrylaie, ureihane, ester and oxide monomers, hi other embodiments, they can be derivatives of naturally occurring polymers such as polysaccharides (e.g. ehitin, chitosan, dexiran and puϋulan; gum agar, gom arable, gitm karaya, locust bean gum, gum tragaeanih, earrageerians, gum ghatti, guar gum, xanthan gum and selerogluean), starches (e.g. dextrin and maitodexiriπ), hydrophilic colloids (e.g. pectin), phosphatides (e.g. lecithin), alginates (e.g. ammonium alginate, sodium, potassium or calcium alginate, propylene glycol alginate), gelatin, collagen, and cdlulosics, Ceflulosics are cellulose polymer that has been modified by reaction of at least a portion of the hydroxy] groups on the saccharide repeat units with a compound to t ^' brni an ester-linked or an ether-linked subsrituent. For example, the eeKulosio ethyl cellulose has an ether linked ethyl substkuent attached to the saccharide repeat unit. while the ceilnlosic cellulose acetate lias an ester linked acetate substitucnt. hi certain embodiments, the eelluJosics for the credible matrix comprises aqueous-soluble and acjueous-emdibie celiuiosics can include, for example, ethyl cellulose (HC), methylethy! cellulose (MBC), carboxymethyl cellulose (CMC), CV! EC, hydroxyetbyl cellulose (HHC), hydroxypropyl cellulose (HPC) ₅ cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate bυtyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, IiPMCAS, hydroxypropyi uicthy! cellulose acetate trimeUitatc (HPMCAT), and ethylhydroxy cthy 1 cellulose

- ] !4 -

{HHi ^' iC}. In certain embodiments, the ceilulosics comprises various grades of low viscosity (MW less than or equal to 50,000 dalions, for example, the Dow Methocel " ^* scries ES, Kl SLV, E50LV and KlOOLY) and high viscosity (MW greater than 5(1000 rfaltons, for example, E4MCR, ElOMCR, K4M, KI 5 M and KiOOM and (he s Methoccf" K series ^' ) HPMC. Other commercially available types of HPMC? .include the Shin Etsu Metolose 90SM series.

The choice of matrix material can have a large effect on the maximum drug concentration attained by the device as well as the maintenance of a high drug co.ncentrat.ion. ^' The matrix material can be a concentration-enhancing polymer, for ^■ 10 example, as described in WO05/011634.

Other materials useful as the erodibie matrix material include, but are not limited to, pullulan, polyvinyl pyrrolidonc, polyvinyl aieohol, polyvinyl acetate, glycerol tatty acid esters, polyacrylamide, polyacrylic acid, copolymers of ethacryiic acid or :5 πicthacryhc acid (EUDRAGSIO, Rohm America, inc., Piscataway, New jersey) and other acrylic acid derivatives such as homopolymers and copolymers of hutyi methaerylate, methyl niethacryiate, ethy Irnefhacrylate, ethyl acryiate, (2- dimethylarninoethyl) snethaeryiaie, and (trimethylarninoetliyi) methacrylate chloride.

20 The erodibie matrix polymer may contain a wide variety of the same types of additives and exc.ipien.ts known in the pharmaceutical arts, including osmopolymers, osrnagem, uoJubility-e-nhancing or-retarditig agents and excipients that promote stability or processing of the device. Alternatively, the agents of the present disclosure may be administered by or

25 incorporated into a non-erodible matrix device. In such devices, an agent described herein is disiri bated in an inert matrix, ^' The agent is released by diffusion through the inert matrix. Examples of materials suitable for the inert .matrix include insoiubie plasties (e.g methyl aerylate-mefhyl methaerylate copolymers, polyvinyl chloride, polyethylene), hydropbilic polymers (e.g. ethyl cellulose, cellulose acetate,

30 crosslinked polyviπylpyiTolidoπe (also known as crospovidoπe)), and fatty compounds (e.g. caraauba wax, microcrystalϋne wax, and triglycerides). Such devices

- 3 15 -

arc described further in Remington: The Science and Practice of Pharmacy, 20th cdirion (2000),

Matrix controlled release devices maybe prepared by blending an agent described herein and other exeipieπts together, and then forming the blend into a tablet, caplet, pill, or other device formed by compressive forces, Such compressed devices may be formed using any of a wide variety of presses used in the fabrication of pharmaceutical devices. Examples include single-punch presses, rotary tablet, presses, and multilayer rotary tablet presses, all well known in the art. See for example, Remington: The Science nnό Practice of Pharmacy; 20th Edition, 2000. The compressed device may be of any shape, including round, oval, oblong, cylindrical, or triangular. The upper and lower surfaces of the compressed device may be flat, round, concave, or convex.

In certain embodiments, when formed by compression, the device has a strength of at least 5 KJionoods (Kp)ZCm ² (for example, at least 7 Kp/cra"). Strength is the fracture force, also known a a the tablet hardness required to fracture a tablet formed from the materials, divided by the maximum cross-sectional area of the tablet normal to that Jbret ¹ . The fracture force may be measured using a Sehleuniger Tablet Hardness Tester, Model 61). The compression force required to achieve this strength will depend on the size of the tablet, but generally will be greater than about 5 kP/cm\ friability is a well-know measure of a device's resistance Io surface abrasion that measures weight loss in percentage after subjecting the device to a standardized agnation procedure. Friability values of from 0.8 to 1.0% are regarded as constituting the upper limit of acceptability. Devices having a strength of greater than 5 kP/cixf generally are very robust, having a friability of less than 0. 5%. Other methods for forming matrix conirolied-reiease devices axe well known in {he- pharmaceutical arts. See for example. Remington; The Science and Practice of Pharmacy, 20th Edition, 2000.

As noted above, die agents described he.re.in may also be incorporated into an osmotic control device. Such devices generally include a core containing one or more agents

- ! 16 -

as described herein and a water permeable, non-dissolving arκl non- eroding coating surrounding the core which controls the influx of water into the core from an aqueous environment ((f use so as to cause drug release by extrusion of some or all of the core to the environment of use. In certain embodiments, the coating is polymeric, aqueous-permeable, and has at least one delivery port The core of the osmotic device optionally includes an osmotic agent which acts to imbibe water from thy surrounding environment via such a semipermeable membrane. The osmotic agent contained in the core of this device may be an aqueous -swel table hydro philie polymer or it may be an osniogen, also known as an osmageαt. Pressure is generated within the device which forces l.he agent(s) out of the device via an orifice (of a size designed to mini mixe solute di Jϊusion while preventing the build-up of a hydrostatic pressure head).

Osmotic agents create a driving force for transport of water from the environment of use mto the core of the device. Osmotic agents include but are not limited to vvater- swdl&ble hydrophilie polymers, and osmogens (or osmagens). Thus, the core may include wilt er-swellab Ie hydrophiiie polymers, both ionic and notύonic, often referred to as osmopolymers and hydrogels. Hie amount of water-swellable hydrophobic polymers present in the core may range from about 5 to about 80 wt% (including for example. J O to 50 wi%). Nonlimiting examples of core materials include hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene nxide (PEO), polyethylene glycol (PECJ), polypropylene glycol (PPG), poly (2- hydfoxyeihvi πietbacrylate), poly (acrylic) acid, poly (methacrylie) acid, potyvinyipyjToiidone (PVP) and erosslinked PVP, polyvinyl alcohol (PVAK FV'A/PVP copolymers and PVA/PVP copolymers with hydrophobic monomers such as methyl rncthaerylate, vinyl acetate, and the like, hydrophilic polyurethanes containing large PEC) blocks, sodium croscaππeilαse, carrageenaπ, hydroxycthyl cellulose (HECt'), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymcthyl cellulose (CMC) and carboxyethyi cellulose (CBC), sodium alginate, polycarbophii, gelatin, xanthan gum, and sodium starch glycol at. Other materials includu hydrogels comprising interpenetrating networks of polymers that ■nay be formed by addition or by condensation polymerization, the components of

- i 17 ^■ •

which may comprise hydrophilic and hydrophobic monomers such as those just mentioned. Water-swell able hydrophilic polymers include but are not limited to PKO. PEG, PV ⁷ P, sodium eroseamiellose, Mi ¹¹ MC, sodium starch glvcoiate. polyaerylie acid itnd cross! inked versions or mixtures thereof.

5

The core may also include an osmogen (or osmagent). The amount of osrπogers present in the core may range from about 2 to about 70 wt% (including, for example, from 10 to 50 wt%). Typical classes of suitable osmogens are water-soluble organic acids, sails and sugars that are capable of imbibing water to thereby effect an osmotic

ΉJ pressure gradient across the barrier of the surrounding coating. Typical useful osmogens include but are not limited to magnesium sulfate, magnesium chloride, euieium chloride, sodium chloride, lithium chloride, potassium sulfate, sodium carbonate, .sodium sulfite, lithium sulfate, potassium chloride, sodium sulfate, rmmnitol, xyϋtoi, urea, sorbitol, inositol, raffinose. sucrose, glucose, fructose, lactose,

^■ ;s citric acid, succinic acid, tartaric acid, and mixtures thereof, hi certain embodiments, the osmogen is glucose, lactose, sucrose, maπnitøl, xylite!, sodium chloride, including combinations thereof.

The core trury include a wide variety of additives and excipiesits that enhance the perlbmianci' oi ^' thc dosage form or that promote stability, tabieting or processing,

20 Such additives and exeipicnts include tabieting aids, surfactants, water- soluble polymers, pH modifiers, fillers, binders, pigments, disintegranis, antioxidants, lubrkams and ilavorants. lNonlimiting examples of additives and exeipienis include but are not iiπiited to those described elsewhere herein as well as microcrystalHne cellulose, metallic salts of acids (e.g. aluminum stearate, calcium stearate, magnesium

25 stearate, sodium stearate. zinc stearate), pH control agents (e.g. buffers, organic acids, organic acid salts, organic and inorganic bases), tatty acids, hydrocarbons and iatty alcohols (e.g. stearic aαd, palmitic acid, liquid paraffin, stearyl alcohol, and palmUoi), tatty acid esters (e.g. glyceryl (nrøno-aad S-) stearates, triglycerides, glyceryl ϊpaimiticsicaric) ester, sorbitaii esters (e.g. sorbitaπ monostearate, saccharose

30 moiiusteanue, saccharose monopalrnitatc, sodium sleary! furaaratcj, polyoxyethyiene sorbuaα esters}, surfactants (e.g. alky! sulfates (e.g. sodium iauryi sulfate, magnesium

i-ϊury] sulfate), polymers (eg, polyethylene glycols, polyoxyethylene glycols, polyoxyeihylene, poiyoxypropylene ethers, including copolymers thereof), polytetrafluoroothyiene), and inorganic materials (e.g. talc, calcium phosphate), cycIotlcK-trins, sugars (e.g. lactose, xylitoi), sodium starch glycolate). Noπhmitmg examples of disimegrants are sodium starch glycolate (e. g., Explotab ^'** CLV,

(raicrocrystalline cellulose fe. g., Avice! ^1M )> microcrystalliαe silicilkd cellulose (e.g., FroSolv ^"" ), croscarmellose sodium (e. g., Ac-Di-SoI ^'** ). When the agent described herein is a solid amorphous dispersion formed by a solvent process, such additives may be added directly to the spray-drying solution when forming aα agent described hcrein/i'oncentπition-enhancing polymer dispersion such that the additive is dissolved or suspended in the solution as a slurry. Alternatively, such additives may be added following the spray-drying process to aid in forming the final controlled release device.

A ooniiniiting example of an osmotic device consists of one or more drug layers containing an agent described herein, such as a solid amorphous drug/polyπiεr dispersion, and a swelkr layer that comprises a water-sweliahle polymer, with a coating surrounding the drug [aver and swcller layer. Each layer may contain other cxcipients such as tableting aids, υsmagents. surfactants, water-soluble polymers and water-sweliahie polymers.

Such osmotic delivery devices may be fabricated in various geometries including bilayer (wherein the core comprises a drug layer and a swdler layer adjacent to each other), trilayer (wherein the core comprises a swell er layer sandwiched between two drug layers) and concentric (wherein the core comprises a central swcJIcr ageni surrounded hy the drug layer). The coating of such a tablet comprises a membrane permeable to water but substantially impermeable to drug and ex ci pi en Ls contained within. The coating contains one or more exit passageways or porls in coπmiunicaiiorj with the drug-cofttainmg layer(s) for delivering the drug agent. The drug-containing kyer(s) of the core contains the drug agent (including optional øsmagents and

1 19 -

hydrops iiie water-soluble polymers), while the swelier layer consists of an expandable hydrogel, with or without additional osmotic agents.

When placed in an aqueous medium, the tablet imbibes water through the membrane, causing the agent to form a dispensable aqueous agent, and causing the hydrogel layer to expand axxi push against the drag-containing agent forcing the agent out of the exit passageway. The agent can swell, aiding in forcing the drug out of ihe passageway. Drug can be delivered from this type of delivery system either dissolved or dispersed in the agent that is expelled from the exit passageway.

The rate of drug delivery is controlled by such factors as the permeability and thickness of the coaling, the osmotic pressure of the drug-containing layer, Ihe degree of. hydrophiiicily υf the hydrogel layer, arid the surface area of the device. Those skilled in the art. will appreciate that increasing the thickness of the coating wiii reduce the release rate, while any of the following will increase the release rate: increasing the permeability of the coating; increasing the hydrophilieity of the hydrogel layer; increasing the osmotic pressure of the drug-containing layer; or increasing the device's surface area.

Other materials useful in forming the drug-containing agent in addition to the agent described herein itself, include HPMC, PEO and PVP and other pharmaceutically acceptable carriers, In addition, osmagents such as sugars or salts, including but not limited to sucrose, lactose, xylitol, mannitol, or sodium chloride, may be added. Materials which are useful for forming the hydrogel layer include sodium CMC, PEO (e.g. polymers having an average molecular weight from about 5,000,000 to about

7,5OiU)OO daitons), poly (acrylic acid), sodium (poiyacrylate), sodium crosearrrsellose, sodium starch glycolat, PVP, crosslmked PVP, and oilier high molecular weight hydrophilic materials.

i n the case of a bilayer geometry, ihe delivery pori(s) or exit passagewa y(s ) may be located on the side of the tablet containing the drug agent or may be on both sides of

- i 20 -

the tablet or even on the edge of the tablet so as to connect both the drug layer and the swell er layer with the exterior of the device. The exit passageway(s) may be produced by mechanical means or by laser drilling, or by creating a diftϊcislt-to-coat region on the tablet by use of special tooling during tablet compression or by other means.

The osmotic device can also be made with a homogeneous core surrounded by a semipermeable membrane coating, as in US3845770. The agent described herein can he incorporated into a (.ablet core and a semipermeable membrane coating can be applied via conventional tablet-coating techniques such us using a pan coaler. A drug delivery passageway can then be formed in this coating by drilling a hole in the coating, either by use of a laser or mechanical means. Alternatively, the passageway may be formed by rupturing a portion of the coating or by creating a region on the tablet that ss difficult to coat, as described above. In one embodiment, an osmotic device comprises: (a) a single-layer compressed core comprising: (i) an agent described herein, (ii) a hydroxyethylcel1ulo.se, and (iii) an osmagent wherein the hydroxyothyledlulose is present in the core from about 2,0% to about 35% by weight and the osmageot is present from about 15% to about 70% by weight; (b) a water- permeable layer surrounding the core; and (c) at least one passageway within the water- permeable layer (b) tor delivering the drug Io a fluid environment surrounding the tablet In certain embodiments, the device is shaped such that the surface area to 'volume ratio (of a water-swollen tablet.) is greater than 0.6 ram ^{' 1} (including, for example, greater than 1.0 mrn ^"! ). The passageway connecting the core with the fluid environment can be situated along the tablet band area. In certain embodiments, the shape is an oblong shape where the ratio of the tablet tooling axes, i.e., the major and minor axes which define the shape of the tablet, are between 1.3 and 3 (including, for example, between 1.5 and 2,5). In one embodiment, the combination of the agent described herein and the osiπagent have an average ductility from about 100 to about 200 M pa. an average tensile strength from about 0.8 to about 2.0 Mpa, and an average brittle fracture index less than about 0.2, The single-layer core may optionally include

a disirstegraπt, a bioavailability enhancing additive, and/or a pharmaceutically acceptable exdpient, carrier or diluent.

in certain embodiments, entrainment of particles of agents described herein in the extruding fluid during operation of such osmotic device is desirable. For the particles to be well entrained, the agent drug form is dispersed in the fluid before the particles have an opportunity to settle in the tablet core. One means of accomplishing this is by adding a disintegram that serves to break up ihe compressed core into its particulate components. NimEmiimg examples of standard disirstegrants include materials such as sodium starch giycoiate Ct-. g. , Explotab ^" CLV), microcrystallme cellulose Ce. g., A vice! ^{'" 1} }, rrueroerystdlme siliciiied cellulose (e. g., ProSolv ^" ) and erosearroεllose sodium (e. g., Ac-Oi-Sol' ^" ), and other disintegrants known to those skilled in the art. Depending upon the particular formulation, some disirsiegrarits work belter than others. Several disintegrants tend to form gds as they swell with water, thus hindering drug delivery from the device. Non-gelling, non-swelling disintegrants provide a snore rapid dispersion of the drug panicles within the core as water enters the core. In certain embodiments, non-gelling, non-swelling disinlegrants are resins, for example, Um-exchange resins. In one embodiment, the resin is Ambcrlite' ^"' IRP 88 (available tVom Rohm and Haas, Philadelphia, PA _. ). When used, the disintegrant is present in amounts ranging from about 50-74% of the core agent.

Water- soluble polymers are added to keep particles of the agent suspended inside the device before they can be delivered through the pa$sageway(s) (e.g., an orifice). High viscosity polymers are useful in preventing settling. However, the polymer in combination with the agent is extruded through the passageway(s) under relatively low pressures. A\ a given extrusion pressure, the extrusion rate typically sknv^ with increased viscosity. Certain polymers in combination with particles of the agent described herein form high viscosity solutions with water but are still capable of being extruded from the tablets with a relatively low force. In contrast, polymers having a low weight-average, molecular weight {< about 3OOJ3OO) do not form sufficiently viscous solutions inside ϊht* tablet core to allow complete delivery due to particle

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settling. Settling of the particles is a problem when such devices are prepared with no polymer added, which leads to poor drug delivery unless the table! is constantly agitated to keep the particles from settling inside the core. Settling is also problematic when she particles are large aτκl/or of high density such that the rate of settling increases.

hi certain embodiments, the water-soluble polymers for such osmotic devices do not interact with the drug. In certain, embodiments the water-soluble polymer is a non- ionic polymer, A noiil uniting example of a non-ionic polymer forming solutions iiaving a high viscosity yet still extrudable at low pressures is Natπxso! ^'* 250H (high molecular weight hydroxyetbyleeiliilose, available from Hercules Incorporated, Aquaion Division, Wilmington, DFi; MW equal to about I million daltons and a degree of polymerization equal to about 3,700). Narrosol 25OJH ^'** provides effective drug delivery at concentrations as low as about 3% by weight of the core when eornbsneu with an osmagent, Natrosol 25OH ^" ' NF is a high-viscosity grade nonionJc cellulose other that is soluble in hot or cold water. The viscosity of a 1% solution of Natrosol 250M using a Brookfield LVT (30 rpm) at 25°C is between about 1 , 501) and about 2,500 cps- in. certain embodiments, hydrøxyethyieelMose polymers for use in these monolayer osmotic tablets have a weight-average, molecular weight froπα about 300,000 to about L 5 million. The hydroxyethylcellυlose polymer is typically present in the core in an amount from about 2.0% to about 35% by weight,

Another example of an osmotic device is an osmotic capsule. The capsule shell or portion of the capsule shell can be semipermeable. The capsule can be filled either by a powder or liquid consisting of an agent described herein, excipients that imbibe water to provide osmotic potential, and/or a water -sweHabk polymer, or optional iy solubiliziϊig exeipiems. The capsule core can also be made such that it has a hi layer or multilayer agent analogous to the bifayer, trilaycr or concentric geometries described above.

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Another class of osmotic device useful in this disclosure comprises coated swellabie tablets, for example, as described in EF37S404. Coated swellabie tablets comprise a tablet core comprising an agent described herein and a swelling material, preferably a hydrophihc polymer, coated with a membrane, which contains holes, or pores through winch, in the aqueous use environment, the hydrophihc polymer can extrude and carry out the agent. Alternatively, the membrane may contain polymeric or low molecular weight water-soluble porosigens. Porosigens dissolve in the aqueous use environment, providing pores through which the hydrophihc polymer and agent may extrude. Examples of porosigens are water-soluble polymers such as HPMC, PEG, and low molecular weight compounds such as glycerol, sucrose, glucose, and sodium chloride, hi addition, pores may be formed in the coating by drilling holes in the coating using a laser or other mechanical means. In this class of osmotic devices, the membrane material may comprise any film-forming polymer, including polymers which are water permeable or impermeable, providing that, the membrane deposited on the tablet core is porous or contains water-soluble porosigens or possesses a macroscopic hole for water ingress and drug release. Embodiments of this class of sustained release devices may also be multi layered, as described, for example, in EP37S404.

When an agent described herein is a liquid or oil. such as a lipid vehicle formulation, lor example as described in W O05/01 1634, the osmotic controlled -release device may comprise a soft-gel or gelatin capsule formed with a composite wall, and comprising the liquid formulation where the wall comprises a barrier layer formed over the external surface of the capsule, an expandable layer formed over the barrier layer, and a semipermeable layer formed over the expandable layer. A delivery port connects the liquid formulation with the aqueous use environment. Such devices are described, for example, it) US6419952. US6342249. US5324280, US4672850, US4627S50, US4203440. and US3995631 . The osmotic controlled release devices of the present disclosure can also comprise a coaling, in certain embodiments, the osmotic controlled release device coating exhibits one or more of the following features: is water-permeable, has at least one

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port for the delivery of drug, and is non-dissolving and non-eroding during release of lhe drug formulation, such that drag is substantially entirely delivered through the delivery pori(s) or pores as opposed to delivery primarily via permeation through the coating material itself Delivery ports include any passageway, opening or pore whether made mechanically, by laser drilling, by pore formation either during the coating process or in situ during use or by rupture during use. in certain embodiments, the coating is present in an amount ranging from about 5 to 30 wt% (including, for example, 10 l.o 20 wι%) relative to the core weight.

One form m coating is a semipermeable polymeric membrane thai has the pori(s) formed therein either prior to or during use. Thickness of such a polymeric membrane may vary between about 20 and 800 μm (including, for example, between about 100 to 500 μm}. The diameter of the delivery port (s) may generally range in size from 0. i to 300Q μm or greater (including, for example, from about 50 to 3000 μm in diameter). Such pori(s) may be formed post-coating by mechanical or laser drilling or maybe formed in situ by mpture of the coatings; such rupture may be controlled by intentionally incorporating a relatively small weak portion into the coating. Delivery ports may also be formed in sttu by erosion of a plug of water-soluble materia! or by rupture of a thinner portion of the coating over an indentation in the eore. In addition, delivery pons may be formed during coating, as in the ease of asymmetric membrane coatings of the type disclosed in lj S5612050 and US5698220. The delivery port may he formed /« .ήlu by rupture of the coating, lor example, when a collection of beads that may be of essentially identical or of a variable agent are used. Drug is primarily released from such beads following rapture of the coating and, following rupture, such release may be gradual or relatively sudden. When the collection of beads has a variable agent, the agent may be chosen such that the beads rupture at various times following administration, resulting in the overall release of drug being sustained for a desired duration.

Coatings may be dense, mieroporous or asymmetric, having a denser region supported by a thick porous region such as those disclosed in US5612059 and US5698220.

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When the coaling is dense the coating can be composed of a water-permeable material. When the coating is porous, it may be composed of either a water-permeable or a water-imperτneable materia!. When the coating is composed of a porous vvaier- irapermeabie materia], water permeates through the pores of the coating as cither a Ikjiiid or a vapor. Noniimiimg examples of osmotic devices that utilize dense coatings include L ^' S ^' 3995631 and US3S45770. Such dense coatings are permeable to the external fluid such as water and may be composed of any of the materials mentioned in these patents as well as other water- permeable polymers known in the art The membranes may also be porous as disclosed, for example, m US5634005 and US5458887 or even be formed from water-resistant polymers. IJSS 120548 describes another suitable process lor forming coatings from a mixture of a water-insoluble polymer and a kachable water-soluble additive. The porous membranes nitty also be formed by the addition of pore-formers as disclosed in US4612008. in addition, vapor-penneable coatings may even be formed from extreme! y hydrophobic materials such as polyethylene or polyvinyl ide-ne difluorid that, when dense, are essentially vvater-impetffieable, as long as siϊch coatings are porous. Materials useful in forming the coating include but are not limited to various grades of acrylic, vinyls, ethers, polyamuies. polyesters and cellukisie derivatives that are water-permeable and water- insoluble at physiologically relevant pHs, or are susceptible to being rendered water- iπsolublε by chemical alteration such as by crosslinkrag. Nonlirniting examples of suitable polymers (or crosslinked versions) useful in forming the coating include plastiei/.ed, usiplasticized and reinforced cellulose acetate (CA), cellulose diaceiate, cellulose triacetate, CA propionate, cellulose nitrate, cellulose acetate butyrate (CAB), CA ethyl carbamate. CAP, CA methyl carbamate, CA succinate, cellulose acetate tπmeliitau* (CAT). CA dimethylarainoacetate, CA ethyl carbonate, CA chloroaeetate. CA ethyl oxalate, CA methyl sulfonate, CA butyl sulfonate, CA p-toluene sulfonate, agar acetate, am y lose triacetate, beta gluc&π acetate, beta glucan triacetate, acetaldehyde dimethyl acetate, triacetate of locust bears gum, hydroxiated ethyleue- vinylaccURC KC, PEG, PPG, PEG/PPG copolymers, PVP, I-IBC, HPC, CMC, CMEC, HPMC, I f PMCP, HPMCAS, HPMCAT, poly (acrylic) acids tmά esters and poly-

•• 126 -

Craethseryik) acids and esters and copolymers thereof, starch, dextran, dextrin, ehitosan, collagen, gelatin, polyalkenes. polyethers, polysulfones, polyethersuifortes, polystyrenes, polyvinyl halides, polyvinyl esters and ethers, natural waxes and synthetic waxes. In various embodiments, the coating agent comprises a cdiulosic polymer, m particular cellulose ethers, cellulose esters and cellulose ester-ethers, i.e., cellulosie derivatives having a .mixture of ester and ether substituents, the coating materials arc made ox derived from poly (acrylic) acids and esters, poiy fmet ^' haeryiie) acids and esters, and copolymers thereof, the coating agent comprises cellulose acetate, the coating comprises a ceilulosic polymer and PE(J, the coating comprises cellulose acetate and PEG.

Coating is conducted in conventional fashion, typically by dissolving or suspending the coating materia! in a solvent and then coating by dipping, spray coating or by pan- coating, in eenasn embodiments, the coating solution contains 5 to 15 χvt.% polymer. ^' lypieai solvents useful with the ceϊlυlosie polymers mentioned above include hut are not limited to acetone, methyl acetate, ethyl acetate, isopropy! acetate, n-butyl acetate, methyl isobυtyi ketone, methyl propyl ketone, ethylene glycol rnoπoethyi ether, ethylene glycol raanoethyl acetate, methylene diehloride, ethylene diehioride, propylene dichloride, nitroethane, nitropropaπe, tetrachloroethane, 1 ,4~dioxane, tetrahydroibran, dϊglymε, water, and mixtures thereof. Pore- formers and non- solvents (such as water, glycerol and ethanol) or plastiekers (such as diethyl phthalate) may also be ackkd in any amount as long as the polymer remains soluble at the spray temperature. Pore-formers and their use in fabricating coatings are described, for example, in US5612059. Coatings may also be hydrophobic micropores layers wherein the pores are substantially filled with a gas and are not wetted by the aqueous medium but are permeable to water vapor, as disclosed, for example, in IJS579S 1 19. Such hydrophobic but water-vapor permeable coatings are typically composed of hydrophobic polymers such as polyalkenes, polyacrylic acid derivatives, polyethers, polysuHVsnes, polyethersulfoncs, polystyrenes, polyvinyl haiides, polyvinyl esters and ethers, natural waxes and synthetic waxes. Hydrophobic microporous coating materials include but are not limited to polystyrene, poiysuitbπes, polyeihersuffones,

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polyethylene, polypropylene, polyvinyl chloride, polyvniylidene tlucride and [jo 3 ytelra 11 ϋoroeihyleno. Such hydrophobic coatings can be made by known phase aversion methods using any of vapor-quench, liquid quench, thermal processes, caching soluble material from the coatina or bv sintering: coatiiia particles. In thermal nroeesses, a solution of polymer in a latent solvent is brought to liquid-liquid phase eparatioα in a cooling step. When evaporation of the solvent is not prevented, the ^■ esulting π-enibrasie will typically be porous. Such coating processes may be wndueied by the processes disclosed, for example, in US4247408. US4490431 and JS4744906. Osmotic controlled-release devices may be prepared using procedures known in the pharmaceutical arts. See for example. Remington: The Science arid Practice of Pharmacy, 20th Edition, 2000.

As further noted, above, the agents described herein may be provided in the form of micropartieulaies, generally ranging in size from about lOμrn to about 2mm (including, for example, from about l OOμm to lmm in diameter). Such multiparticulates may be packaged, tor example, in a capsule such as a gelatin capsule or a capsule formed from an aqueous-soluble polymer such as HPMCAS, HPMC or starch; dosed m a suspension or slurry in a liquid ; or they may be formed into a tablet, c-iplet, or pill by compression or other processes known in the art. Such multiparticulates may be made by any known process, such as wet- and dry- granulation processes, extrusion/spheronization, roller-compaciion, melt-congealing, or by spray-coating seed cores. For example, in wet-and dry- granulation processes, the agent described herein and optional exeipients may be granulated to form jnuhiparticuiates of the desired size. Other exeipients, such as a binder (e. g., rmerocrystalline cellulose), may be blended with the agent to aid in processing and forming the multiparticulates. In the case of wet granulation, a binder such as rrncTocrystaiHne cellulose may be included in the granulation fluid to aid in forming a suitable multiparticulate. See, for example, Remington : The Science and Practice of Pharmacy, 20"£difion, 2000. in any case, the resulting particles may themselves constitute the therapeutic composition or they may be coated by various Hlm-forming

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materials such as enteric polymers or water-sweHable or water-soluble polymers, or they may be combined with other excipients or vehicles to aid in dosing to patients. Suitable pharmaceutical compositions in accordance with the disclosure will generally include sn arnαum of the active corapound(s) with an acceptable pharmaceutical diiucnt or exeipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences { 18th Edition, Mack Publishing Company. 1995).

Kite

The agents described herein and combination therapy agents can be packaged as a. kit that includes single or multiple doses of two or more agents, each packaged or formulated individually, or single or multiple doses of two or more agents packaged cr formulated in combination. Thus, one or more agents can be present in first container, and the kit can optionally include one or more agents in a second container, ^" foe container or containers are placed within a package, and the package can optionally include administration or dosage instructions. A kit can include additional components such as syringes or other means for administering the agents as well as diluents or other means for formulation,

Thus, the kits can comprise: a) a. pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier, vehicle or diluent; and fa) a container or packaging. The kits may optionally comprise inslaictions describing a method of using the pharmaceutical compositions in one or more of the methods described herein (e.g. gastrointestinal motility disorders, chronic intestinal pseudoobstruction, colonic pseudo-obstruction, Crohn's disease, diiodenogasiric reilux, dyspepsia, functional dyspepsia, nonuicer dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GERD), gastroparcsis, irritable bowel syndrome, post-operative ileus, ulcerative colitis, chronic constipation, and disorders and conditions associated with constipation (e.g. constipation associated with use of opiate pain killers, post-surgical constipation, and

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constipation associated with neuropathic disorders as well as other conditions and disorders described herein). The kit may optionally comprise a second pharmaceutical composition comprising one or more additional agents Including but not limited to those including analgesic peptides and compounds, a phosphodiesterase inhibitor, an 5 agent used to treat gastrointestinal and other disorders (including those described herein), an agent used to treat, constipation, an antidiarrheai agent, an insulin or related compound (including those described herein), an anti-hypertensive agent, an agent useful in the treatment of respiratory and other disorders, an anti-obesity agent, an anti-diabetic agents, an agent that activates soluble guanylate cyclase and a 10 pharmaceutically acceptable carrier, vehicle or diluent. The pharmaceutical composition comprising the compound described herein and the second pharmaceutical composition contained in the kit may be optionally combined in the same pharmaceutical composition.

15 A kit includes a container or packaging for containing the pharmaceutical compositions and may also include divided containers such as a divided bottle or a divided foil packet. The container ean be, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a "refill" ⁵ of tablets tor placement into a different container), or a blister pack with individual doses for

;?o pressing out of ihe pack according to a therapeutic schedule, it is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle which is in turn, contained within a box.

25 An example of a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely υscd for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The

30 recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be

• no-

packed. Next the tablets or capsules are placed in the recesses accordingly and the -sheet of relatively stiff materia! is scaled against the plastic foil at the face of the toll which is opposite itom the direction in which the recesses were farmed, As a result. she tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in die sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

St maybe desirable to provide a written memory aid containing information and/or instructions i ^' or the physician, pharmacist or subject regarding when the medication is to be taken. A "daily dose" can be a single tablet or capsule or several tablets or capsules to be taken on a given day. When the kit contains separate compositions, a daily dose of one or more compositions of the kit can consist of one tablet or capsule while a daily dose of another one or more compositions of the kit can consist of several tablets or capsules. A kit can take the form of a dispenser designed to dispense the daily doses one at a time in the order of their intended use. The dispenser can be equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a πieehanieal counter which indicates the number of daily doses that have been dispensed. Another example of sueb a memory-aid is a battery- powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when, the next dose is to be taken.

Methods to increase chemical and/or physical stability of the agents the described herein are found in U.S. 6,541 ,606, U.S. 6,068,850, U.S. 6,124,261 , U.S. 5.904,935, and WO 00/15224, U.S. 20030069182 (via the additon of nicotinamide), U.S. 20030I75230AL U.S. 2003017523OA1 , U.S. 30030175239AI , U.S. 20020045582, U.S. 20010031726, WO 02/26248, WO 03/014304, WO 98/00152λI, WO

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98/00157λ L WO 90/12029, WO 00/04880, and WO 91/04743. WO 97/04796 and the references Cited therein.

Methods w increase bioavailability of the agents described herein are found in U.S. 6,008.187, U.S. 5,424,289, U.S. 20030198619, WO 90-OI 329, WO 01 /49268, WO 00/32172, and WO 02/064166. Glycyrrhizinate can also be used as an absorption enhancer (sec, e.g., EP397447}. WO 03/004062 discusses Ulex europacus 1 (UEAl) and UEAl mimetics which may be used to iarget the agents of the disclosure to the Gl tract. The bioavailability of the agents described herein can also be inerased by addition of oral bioavailahility-enhanemg agents such as those described in IJ. S. 6,818,615 including but not limited to: cyclosporins (including cyclosporins A through Z ss defined in Table 1 of U.S. 6,818,615}, for example, cyclosporin A (cyclosporin), cyclosporin F, cyclosporin D, dihydro cyclosporin A, di hydro cyclosporin €, acetyl cyclosporin A, PSC-833, {Me- Ik'-4}-cycfosporin (SDZ-NiK-! BI I ) (both ik>τri Sandoz Pharmaceutical Corp.), and related oligopeptides produced by species in the genus Topycladium); antifungals including but not limited to ketocoπazoie; cardiovascular drag including but not limited to MS-209 (BASF), amiodarone, nifedipine, reserpine, quinidine, nicardipine, ethacrynic acid, propafenone, reserpine, arniloride; anti-migraine natural products including but not limited to ergot alkaloids; antibiotics including but not limited to ceibperazoπe, tetracycline, diioroquine, fosfoπiycin; antiparasitics including bin not limited to ivermectin; rnuiϋ-drug resistance rcvcrscrs including but not limited to VX-? IO and VX-853 (Vertex Pharmaceutical Incorporated); tyrosine kinase inhibitors; including but not limited to genisteiα and related isoflavonoids, quercetin; protein kinase C mhibtiors iαciuding but not limited to calpliostin; apoptosis inducers including bin not limited to ceramides; and agents active against, endorphin receptors including but not limited to morphine, morphine congeners, other opioids and opioid antagonists including (but not limited to) naloxone, naltrexone ami nalrrsefcne).

Ilie agents described herein can be fused to a modified version of the blood serum protein transferrin. U.S. 2003022120L U.S. 20040023334, U.S. 20030226155, WO

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Oφ'020454, and WO 04/019872 discuss the manufacture miά use of transferrin fusion proteins. Transferrin fusion proteins may improve circulatory halflrfe and efficacy, decrease undesirable side effects and allow reduced dosage.

The peptides and agonists of the disclosure can be recombinant! y expressed in bacteria. Bacteria expressing the peptide or agonists cars be administered orally, rectal Jy, inucosaily or in via some other mode of administration including but not limited to those described herein. Bacterial hosts suitable for such administration include hut are not limited Io certain Lactohaciena (e.g. Lacfocøccus iaciis, ! άtciobacillm piantarum, Lact. rhamnosus and Lact. paracasei ssp. Paraca.sie and other species found in normal human flora (Ahroe el al. Journal of Applied Microbiology 1998 ϊ5:S8}), certain Streptococcus sp. (e.g. S. gordonii), and certain 8. :;tώfiJLs strains (including pSM539 described in Porno et al. BMC Biotechnology 2004 4:27), The polypeptides imά agonists described herein can be administered using the Heiiobacter baaed preparation methods described in WO06/015445. Bacteria expressing the peptides/agonists described herein may comprise DNA encoding the peptide/agoπist on one or vαore bacterial chromosomes and/or may comprise DNA encoding the peptide/agonist on one or more extrøohrørnosornal dements.

Dosage

The dose range for adult humans is generally from 0,005 mg to i() gMay orally, tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the disclosure which is effective at such dosage or as a multiple of the same, lor instance, units eontaining S mg to S(Ki jng, usually around 10 mg to 200 mg. The precise amount of compound administered to a patient will be the responsibility of the attendant physic? art. However, the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity,

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λ dosage usύt (e.g. an oral dosage unit) can include from, for example. I io 30 μg, 1 to 40 μg. 1 -0 50 μg, 1 to 100 μg, 1 to 200 μg. 1 to 300 μg. 1 to 400 μg, I to 500 μg, 1 to 600 μg, I to 700 μg- 1 to 800 μg, 1 to 900 μg. 1 to 1000 μg. 10 to 30 μg. 10 to 40 μg. K) iu 50 μg, 10 io KK) μg, 10 to 200 μg. 10 Io 300 μg, 10 to 400 μg, 10 to 500 μg, 10 to 600 μg, i 0 to 700 μg, 10 to 800 μg, i 0 to 900 μg, 10 to 1000 μg, 100 to 200 μg, 300 to 300 μg. I (M) to 400 μg, 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to BOO μg, 100 to 900 μg, 100 to 1000 μg. ! 00 to 1250 μg, 100 to 1500 μg, 100 to 1750 μg. 100 to 2000 μg. 100 to 2250 μg, 100 to 2500 μg. i 00 to 2750 μg, K)O to 3000 μy, 200 to 300 μg, 200 to 401) μg, 200 to 500 μg. 200 to 600 μg, 200 Jo 700 μg, 20C? to 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg. 200 to 1500 μg, 200 to 1750 μg, 200 to 2000 μg. 200 to 2250 μg, 200 to 2500 μg. 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 io 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to i 000 μg, 300 Io 1250 μg, 300 to 1500 μg, 300 to I 750 μg, 300 io 2000 μg, 300 to 2250 μg, 300 to 2500 μg, 300 to 2750 μg. 300 to 3000 μg, 400 to 500 μg. 400 to 600 μg, 400 to 700 μg, 400 to 800 μg. 400 to 900 μg, 400 to 1000 μg. 400 to 1250 μg, 400 to 1500 μg, 4(K) to 1750 μg, 400 to 2000 μg, 400 to 2250 μg. 400 io 2500 μg, 400 to 2750 μg, 400 to 3000 μg. 500 to 600 μg. 500 to 700 μg. 500 to 800 μg. 500 to 900 μg, 500 to 1000 μg, 500 to 1250 μg, 500 to J 500 μg, 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 to 800 μg, 60(J to 900 μg. 600 to ! 000 μg, 600 to 1250 μg, 600 to 1500 μg, 600 io 1750 μg, 600 to 2000 μg, 600 to 2250 μg. 600 to 2500 μg, 600 to 2750 μg, 600 to 3000 μg, 700 to 800 μg. 700 to 900 μg, 700 to 1000 μg, 700 to 1250 μg, 700 to 1500 μg, 700 to 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 70(J to 2750 μg, 700 io 3«K)0 μg, 800 to 900 μg, 800 io 1000 μg, 800 to 1250 μg _; 800 to 1500 μg.. SOO to 1750 μg, 800 So 2000 μg, 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 to 3000 μg, 900 to I OC)O μg, 900 io 1250 μg, 900 to 1500 μg, 900 to \ 750 μg, 900 to 2000 μg. 900 Lo 2250 μg. 900 to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, ! QGO to 1250 μg. 10(50 to 1500 μg, 1000 to 1750 μg. 1000 to 2000 μg, 1000 to 2250 μg, 1000 to 2500 μg, 1000 to 2750 μg. 1000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg. 5 to 100 μg, 50 μg, 100 μg. I 50 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg. 500 μg, 550 μg, 600 μg, 650 μg, 700 μg. 750 μg, 800 μg, 850 μg, 900 μg.

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950 μg, 1000 μg, 1050 μg, 1100 μg, Il50 μg, 1200 μg, 1250 μg, 1300 μg.1350 μg,

HOO μg, 450 μg, 1500 μg, 1550 μg, 1600 μg. 1650 μg, 1700 μg, 1750 μg, 1 SOO μg, 1 850 UJ>. 900 μg, 1950 μg, 2000 μg, 2050 μg. 2100 μg, 2150 μg, 2200 μg, 2250 μg.

2300 μg, 2350 Ug ₁ 2400 μg. 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg. 2750 μg, 2800 μg, 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg. 4250 μg. 4500 μg, 4750 μg, 5000 μg of a peptide or GC-C agonist described herein, in certain embodiments the dosage unit and daily dose are equivalent, In various embodiments, the dosage unit is administered with food at anytime of the day, without food at anytime of the day, with food after an overnight fast (e.g. with

10 breakfast), at. bedtime after a low fat snack, in various embodiments, the dosage umi. is administered once a day, twice a day. three times a day, four times a day, rive times a day, six times a day. The dosage unit can optionally comprise other agents.

A dosage unit (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg, 1 IS to 40 μg, 1 to 50 μg, I to 100 μg, 1 to 200 μg. I to 300 μg. 1 to 400 μg, 1 to 500 μg, 1 to 600 μg, I to 700 μg, 1 to 800 μg, 1 to 9(10 μg, 1 to 1000 μg, 10 to 30 μg, I O to 40 μg, 10 to 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg, 10 to 400 μg, 10 to 500 μg, U) to 600 μg, 10 to 700 μg, 10 to 800 μg, 10 to 900 μg, 10 to 1000 μg _? JOO to 200 μg, 100 to 300 μg, 100 to 400 μg, J 00 io 500 μg. IiK) to 600 μg. 100 to TOO μg, 100 to 800 w xg. S(H) to 900 μg.100 to 1000 fig, 100 to 1250 μg, 100 to 1500 μg, !00 to 1750 μg.

100 to 2000 μg.100 to 2250 μg, 100 to 2500 μg, 100 to 2750 μg, !00 io 3000 μg, 200 to 300 μg, 200 to 400 μg, 200 to 500 μg,.200 to 600 μg, 200 io 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 Lo 1750 μg, 200 to 2000 μg, 200 to 2250 μg, 200 to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to

25 400 μg, 300 to SOO μg, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg, 300 to 1250 μg, 300 to 1500 μg.300 to 1750 μg, 300 to 2000 μg, 300 to 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg, 400 to 700 μg, 400 to 800 μg, 400 to 900 μg, 400 to 1000 μg, 400 Lo 1250 μg. 400 to 1500 μg.400 to 1750 μg, 400 to 2000 μg.400 to 2250 μg, 400 to 2500 μg, 400

SC to 2750 ug, 4(Ks to 3000 μg, 500 to 600 μg, 500 to 700 μg, 500 to SOO μg, 500 to 900 μg, 500 to 1000 μg, 500 to 1250 μg, 500 to 1500 ug, 500 to 1750 μg, 500 to 2(KK) μg.

500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg, 500 Io 3000 μg. 600 to 700 μg, 600 Lo 800 μg. 600 to 900 μg, 600 to 1000 μg. 600 to 1250 μg. 600 to 1500 μg, 600 to ! 750 μg, 6(K) to 2000 μg. 600 to 2250 μg, 600 to 2500 μg, 600 to 2750 μg, 600 to 3000 μg, 700 to 800 μg, 700 to 900 μg, 700 to 1000 ug, 700 to 1250 μg, 700 to 1500 μg, 700 to ! 750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 io 2750 μg, 700 to 3000 μg. 800 to 900 μg, 800 to 1000 μg, 800 to 1250 μg, SOO to 1500 μg, 800 to 1750 μg. 800 to 2000 μg., 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 to 3(KK) μg, 900 to 1000 μg, 900 to 1250 μg, 900 to 1500 μg. 900 to 1750 μg, 900 to 200« μg, 900 to 2250 μg, 900 to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, 1000 to 1250 μg, 1000 to 1500 μg, 1000 to 1750 μg. 1000 to 2000 μg, 1000 to 2250 μg, 1000 to 2500 μg, 100(5 to 2750 μg, 1000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, S to 20 μg, 5 to 100 μg. 50 μg, 100 μg, I 50 μg, 200 μg, 250 μg, 300 μg, 350 μg, 4(M) μg, 450 μg. 500 μg, 550 μg, 600 μg, 650 μg. 700 μg, 750 μg, 800 μg, 850 Ug ₅ 900 μg, 950 μg, 1000 μg, 3050 μg, 1 100 μg, H 50 μg, 1200 μg, 1250 μg, BOO μg _; 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg, 1600 μg, 1650 μg, 1 700 μg, 1.750 μg, 1800 μg, 1850 μg, 1900 μg, 1950 μg.2000 μg, 2050 μg, 2100 μg, 2150 μg, 2200 μg, 2250 μg. 2300 μg, 2350 μg, 2400 μg, 2450 μg, 2500 μg. 2550 μg. 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2800 μg. 2850 μg, 2900 μg, 2950 μg.3000 μg. 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and from 50 τ.ng io 650 mg (e.g. 50 røg, 100 mg, 150 nig, 200 rag, 250 mg, 300 nig. 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg) of Modulon® (tritoebutine sϊutleatul.

A dosage unit (e.g. an oral dosage unit) can include, for example, from ! to 30 μg, I so 4ϋ μg, 1 io 5(5 μg, 1 io 10(5 μg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg, I to 500 μg, ! io 600 μg, } io 700 μg, 1 to 800 μg, 1 to 900 μg, I to 1000 μg, 10 to 3(5 μg, 10 to 40 μg. 10 to 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg. 1(5 io 400 μg, 10 to 500 μg, ! (J to 600 μg, 10 to 700 μg, ! 0 to 800 μg, 10 to 900 μg, 10 to 1000 μg, S 00 to 200 μg, 100 to 300 μg, 100 to 400 μg, 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to 800 μg, 100 to 900 μg, 100 to 1000 μg, 100 to 1250 μg, 100 to 1500 μg, 100 to 1750 μg, i 00 to 2000 μg, i 00 to 2250 μg, 100 to 2500 μg, 100 to 2750 μg, 100 to 3000 μg, 200

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lo 300 μg, 20(1 so 400 μg. 200' to 500 μg, 200 to 600 μg, 200 io 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 to 1750 μg, 200 to 2000 μg, 200 to 2250 μg, 2J)O to 2500 μg, 200 to 2750 μg. 200 io 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 to|όOO μg, 300 to 700 μg, 300 to 800 μg. 300 to 900 μg, 300 to I Oi)O μg, 300 io ! 250 fig, 300 to 1500 μg, 300 to ! 750 μg. 300 to 2000 μg _: 300 to 2250 μg, 300 to 2500 μg, 3J)0 to 2750 μg. 300 io 3000 μg. 400 to 500 μg, 400 to 600 μg, 400 to 700 μg, 400 to|SOO μg, 400 to 900 μg, 400 io 1000 μg. 400 to 1250 μg. 400 to ! 500 μg, 400 to 1750 jig, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400 to 2750 μg. 400 to 3000 μg, 500 to 600 μg, 500 to 700 μg. 500 to 800 μg. 500 to 900 μg, 500 to 1000 μg, 500 to ! 250 μg, 500 to 1 500 μg. 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg. 500 to 2500 μg. 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 to BOO μg. 600 to 000 μg, 600ItO 1000 μg, 600 to 1250 μg. 600 to 1500 μg, 600 to

^• 4000 μg, 4250 μg, 4500 μg, 41750 μg, 5000 μg of a peptide or agonist described herein and from 1 mg io 80 nig (e.g. 1 nig, 5 mg, 10 mg, 15 mg, 20 rag, 25 rng, 30

• 13'

nig, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 nig- 65 nig, 70 nig, 75 rag, SO rag) of Propufsidφ (cisapride).

λ dosage unit (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg. I lo 40 μg. ) tυ 50 μg, 1 to 100 μg, 1 to 200 μg, I to 300 μg, I to 400 μg, 1 to 500 μg, I to 600 μg, 1 Jo 700 μg, 1 Io 800 μg, 1 to 900 μg, 1 to 1000 μg, 10 to 30 μg. 10 to 40 μg, 10 to 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg, 10 Io 400 μg, 10 to 500 μg, 10 to 600 μg. 10 to 700 μg, 10 to SOO μg, 10 to 900 μg, 10 to 1000 μg, 100 Io 200 μg, 100 to 300 μg, 100 to 400 μg. 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to 800 μg. 100 to 900 μg, 100 to 1000 μg, 100 to ϊ 250 μg, 100 to 1500 μg, 100 to i 750 μg, 100 to 2000 μg, 100 to 2250 μg, 100 to 2500 μg, 100 to 2750 μg, 100 to 3000 μg. 200 to 300 μg, 200 to 400 μg, 200 Io 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to 800 μg, 200 to VOO μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg. 200 to 1750 μg, 200 to 2000 μg, 200 to 2250 μg, 200 to 2500 μg. 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg. 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg. 30(5 lo 1250 fig, 300 to 1500 μg, 300 to 1750 μg, 300 in 2000 μg, 300 lo 2250 μg, 300 to 2500 μg, 300 to 2750 μg. 300 io 3000 μg, 400 to SOO μg, 4 ⁽ K) to όOO μg, 400 to 700 μg, 400 to 800 μg, 400 to 900 μg. 400 ω 1000 μg, 400 to 1250 μg, 400 to 15(K) μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400 to 2750 μg, 400 to 3000 μg, 500 to 600 μg, 500 to 700 μg, 500 Io 800 μg. 500 ω 900 μg, 500 to 1000 μg, 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg. 500 Io 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 io 800 μg, 600 to 900 μg, 600 to 1000 μg, 600 to 1250 μg, 600 to 1500 μg, 600 to 1750 μg, 600 to 2000 μg, 600 to 2250 μg. 600 io 2500 μg, 600 lo 2750 μg, 600 lo 3000 μg, 700 to 800 μg, 700 to 900 μg, 700 to 1000 μg, 700 to 1250 μg. 700 io 1500 μg, 7(K) to 1750 μg, 700 to 2000 μg, 700 to 2250 μg. 700 to 2500 μg, 700 to 2750 μg, 700 to 3000 μg. 800 io 900 μg, 800 Io 1000 μg. 800 to 1250 μg, BOO to 1500 μg, SOO io 1750 μg, 800 to 2000 μg, 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 to 3000 μg, 900 to 1000 μg, 900 to 1250 μg, 900 to 1500 μg, 900 to t 750 μg, 900 to 2000 μg, 900 to 2250 μg, 900 io 2500 μg, 900 to 2750 μg, 900 to 3000 μg, 1000 to ; 250 μg, ! 000 H> 150(5 _μg . ! 000 to 1750 μg, 1000 to 2000 μg, 1000 to 2250 μg, 1000

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to 2500 μg, i 000 io 2750 μg. 1000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg _τ 5 to 20 μg, S io 100 μg, 50 μg, 100 μg, ISO μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg. 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, SOO μg. 850 μg. 900 μg, 950 μg, 1000 μg. 1050 μg, 1 100 μg- 1 150 μg _; 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg, 1800 μg, 1850 μg, 1900 μg, 1950 μg, 2000 μg. 2050 μg, 2100 μg, 2150 μg, 2200 μg, 2250 μg, 2300 μg, 2350 μg, 2400 μg. 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg. 2700 μg, 2750 μg, 28'JU μg, 2850 μg, 2900 μg, 2950 μ.g, 3000 μg, 3250 μg, 3500 μg. 3750 μg. 4000 μg, 4250 μg. 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and from 10 mg to 600 rag (e.g. 10 mg, 20 mg, 30 mg, 40 nig, 50 mg, (>0 mg, 70 mg. 80 mg, 90 mg, 100 mg, 1 10 mg, 120 mg, 130 mg, 140 mg, 150 mg, i 60 mg, 200 nig, 250 mg, 300 rag, 350mg, 400 mg, 450 mg, 500 nig, 550 mg, 600 rag) of BcotytS/Beπlylϋ]® (dleidcnnine }.

λ dosage unit (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg, 1 to 40 μg, ! Co 50 μg, ! to 100 μg. 1 io 200 μg, I to 300 μg, 1 to 400 μg, I io 500 μg, I io 600 μg, I to 700 μg, 1 io 800 μg, 1 Io 900 μg, 1 io 1000 μg, 10 to 30 μg., 10 to 40 μg, 10 50 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg, 10 to 400 μg, ! 0 io 500 μg, 10 to 600 μg, 10 io 700 μg, 10 Io 800 μg, 10 to 900 μg, K) to 1000 μg, 100 to 200 μg, ! 00 to 300 μg, 100 to 400 μg, 1 OC* to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 io 800 μg, 100 Io 000 μg, IC)O Io 1000 μg. 100 to 1250 μg, 100 to S 500 μg, 100 Io ) 750 μg, 100 to 2001) μg, 100 to 2250 μg, 100 io 2500 μg, 100 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 200 io 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 70(5 μg, 200 to 800 μg, 200 io 900 μ.g, 200 to 1000 μg, 200 to 1250 μg. 200 to 1500 μg, 200 io ! 750 μg, 200 to 2000 μg, 200 to 2250 μg.200 Io 2500 μg. 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg. 300 io 500 μg, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg, 300 to 1250 μg, 300 to 1500 μg, 300 to 1750 μg, 300 to 2000 μg, 300 io 2250 μg. 300 io 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg. 400 to 600 μg, 400 io 700 μg, 400 to 800 μg, 400 to 900 μg, 400 to 1000 μg. 400 to 1250 μg, 400 to 1500 μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400 to 2750 μg, 400 to 3000 μg, .500 Io 6(K) μ.g, 500 to 700 μg, 500 to 800 μg, 500 to 900

- ϊ >9 -

μg, 500 iυ 1000 μg, 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg, 500 to 2000 μg. 500 to 2250 μg. 500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 Io 800 μg, 600 to 900 μg, 600 to 1000 μg. 600 Io 1250 μg, 600 to 1500 μg, 600 to 1750 μg. 600 to 2000 μg. 600 Io 2250 μg, 600 to 2500 μg, 600 to 2750 μg, 600 to 3000 μg, 700 to 800 μg. 700 to 900 μg, 700 to 1000 μg, 700 to 1250 μg, 700 to 1500 μg. 7(M) to 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg. 700 to 3000 μg, 800 to 900 μg, 800 to 1000 μg, 800 to 1250 μg, SOO to 1500 μg, 800 to 1750 μg, 800 to 2000 μg. 800 to 2250 μg, SOO to 2500 μg, SOO to 2750 μg. SOO to 3000 μg, 900 to 1000 μg, QOO to ! 250 μg, 900 to 1500 μg, 900 to 1750 μg, 900 to 2000 μg, 900 Io 2250 μg, 9(XHo 2500 μg, 900 to 2750 μg. 900 io 3000 μg, 1000 to 1250 μg. 1000 to ϊ 500 μg. 1000 to 1750 μg. 1000 to 2000 μg, 1000 to 2250 μg, 1000 in 2500 μg. U)OO to 2750 μg, 3000 to 3000 μg, 2 to 500 μg, 50 to 500 μg. 3 to 100 μg. 5 Io 20 μg, 5 io 100 μg, 50 μgj, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1050 μg, UDO μg, 1 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg. 1600 μg, 1650 μg, 1700 μg. 1750 μg, 1800 μg, 1850 μg, 1900 μg, ! 950 μg, 2000 μg, 2050 μg, 2100 μg, 2! 50 μg, 2200 μg, 2250 μg, 2300 μg, 2350 μg, 2400 μg, 2450 μg, 2500 μg, 2550 μg. 2600 μg, 2650 μg, 2700 μg. 2750 μ.g, 2800 μg. 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg. 3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and from 1 mg to 25 mg (e.g.1 ing, 2 nig, 3 rag, 4 mg, 5 mg. 6 ing, 7 mg, 8 mg, <> mg, 10 rng, 1 1 mg, \2 rag, 13 mg, 14 mg, 15 nig, 16 mg, 17 mg, ϊB mg, 19 mg, 20 mg, 21 mg, 22 nig, 2,3 mg, 24 ing, 25 rag) of Questran® (cholestyramine).

A dosage unit (e.g. an oral dosage unit) can include, for example, from ! to 30 μg, 1 to 40 μg, 1 to 50 μg. i to 100 μg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg, I io 500 μg, 1 to 600 μg. 1 io 700 μg, 1 to 800 μg, 1 to 900 μg, 1 to 1000 μg, 10 to 30 μg, 10 to 40 μg, 10 to 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg, 10 to 400 μg, l O to SGO μg. 10 to 600 μg, 10 \o 700 μg, 10 to 800 μg, 10 to 900 μg. 10 to 1000 μg, 101 ⁾ to 200 μg. 100 to 300 ug, 100 to 400 μg, 100 Co 500 μg. 100 to 600 μg, 100 Lo 700 μg. 100 io 800 μg, 100 to 900 μg, 100 to 10(10 μg, 100 to 1250 μg, 100 to 1500 μg. 100 to 1750 μg,

- 140 -

100 to 2000 μg, 100 to 2250 μg, 100 to 2500 μg, 100 to 2750 μg, 100 to 3000 μg.200 to 500 μg, 200 to 400 μg.200 ^: to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to 800 μg, 200 to 90(S μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 io 1750 μg, 200 io 2000 μg, 200 to 2250 μg, 200 to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg.300 Io 500 μg, 300 io;600 μg, 300 to 700 μg, 300 to 800 μg.300 to 900 μg, WJ io 1000 us, 300 to 1250 ik 300 to 1500 μg, 300 to 1750 μt», 300 to 2000 ug, 300 io 2250 μg, 300 to 2500 μg, 300 io 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg, 400 to 700 μg, 400 to=800 μg, 400 tu 900 μg, 400 to 1000 μg, 400 to 1250 μg, 400 io !500 μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 io 2500 μg, 400 to 2750 μg, 4(K) to 3000 μg, 500 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg, 500 to 1000 μg, 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg.500 io 2000 μg, 500 io 225(5 μg, 500 to 2500 μg.500 io 2750 μg, 500 to 3000 μg.600 to 700 μg, 600 to 800 μg, 600 to 900 μg.60(Ho 1000 fig.600 to 1250 μg, 600 to 1500 μg, 600 to 1750 μg, 600 Eo 2000 μg, (H)O:to 2250 μg, 600 to 25(K) μg.600 to 2750 μg, 600 to 3000 μg, 700 tυ SOO μg, 700 to 900 μg, 700 to 1000 μg.700 to 1250 μg.700 to 1500 μg.700 to 1750 μg, 700 to 20D0 μg, 700 to 2250 μg, 700 Io 2500 μg, 700 io 2750 μg, 700 to 3000 μg, 800 t.o 900 μg.800 to 1000 μg, 800 Io .1250 μg, 800 to 1500 μg, 800 to 1750 μg, 800 to 2000 μg, 800 to 2250 μg, 800 to 2500 μg, S0O to 2750 μg, 800 to 3000 μg..900 io KM)O μg, 900-to 1250 μg, 900 to 1500 μg, 900 to 1750 μg.900 to 2000 μg.900 to 2250 μg, 900 to 2500 μg, 900 io 2750 μg, 900 to 3000 μg, i000 to 1250 μg. iOOOto 1500 μg, 1000 to 1750 μg.1000 to 2000 μg, 1000 io 2250 μg.1000 to 2500 μg, 1000 to 2750 μg, _. 1000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, S to 20 μg, 5 to .100 μg, 50 μg, 100 μg.150 μg.200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg.700 μg, 750 μg, 800 μg, 850 μg, SH)O μg, 950 μg.1000 μg, 1050 μg, 1100 μg, 1150 μg, 1200 μg, 1250 μg. BOO μg.1350 μg, 1400 μg, 1450 μg, 1500 μg.1550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg, 1800 μg. 1850 μg, 1900 μg, 1950 μg, 2D00 μg.2050 μg, 2100 μg, 2150 μg, 2200 μg, 2250 μg. 2300 μg, 2350 μg, 2400 μg, 2450 μg.2500 μg.2550 μg.2600 μg.2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg, 2=900 μg.2950 μg.3000 μg, 3250 μg.3500 μg, 3750 μg. -K)OO μ.g, 4250 μg, 4500 μg, 4750 μg, 5000 μg ofa peptide or agonist described herein and from 100 nig to 3000 mg (e.g.100 mg, 200 mg, 300 mg, 400 mg, 500 nig,

^■ 14! -

600 mg, 025 mg. 700 mg, 800 rag, 900 ing, 1000 mg, 1250 mg, 1300 rag, 1400 mg, 1500 nig, 1600 mg, ! 700 mg, 1800 mg, 1875 nig, 1900 mg, 2000 mg, 2100 mg, 2200 nig, 2300 mg, 2400 mg. 2500 mg,} of Equal ac?rn©/pibercoπ ^> !> {Calcium Puiyearbophil).

λ diisage unit (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg, 1 Sc 40 μg. 1 So 50 μg. I to 1 (K) μg. 1 to 200 μg, 1 Io 300 μg, 1 to 400 μg, 1 to 500 μg, 1 Iu 6(10 μg, I to 700 μg, 1 to 800 μg, 1 io 900 μg, ! to 1000 μg, 10 Io 30 μg, 10 Lo 40 μg. 10 to 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg, 10 to 400 μg. 10 to 500 μg, K) to t>00 μg. I O to 700 μg, I O to 800 μg, 10 io 900 μg, 10 U) 1000 μg, 100 to 200 μg, 100 to 3CKl μg, 100 to 400 μg, 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to 800 μg, 100 to 900 μg, 100 to 1000 μg. 100 to 1250 μg, 100 to 1500 μg. ϊ 00 to 1750 μg, 100 to 2000 μg, 100 to 2250 μg. 100 to 2500 μg, 100 to 2750 μg, 100 to 3 ⁽ KH ⁾ μg, 200 U. 300 μg. 200 to 400 μg, 200 to 500 μg, 200 to 600 μg. 200 io 700 μg. 200 to 800 μg, 200 to 000 μg, 200 to 1000 μg, 200 to 1250 μg. 200 to i 500 μg, 200 to 1750 μg. 200 to 2000 μg, 200 Jo 2250 μg. 200 to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 Hs 600 μg, 300 to 700 μg, 300 to 800 μg. 300 to 900 μg, 300 So 1000 μg, 300 to 1250 μg, 300 to 1500 μg, 300 to 1750 μg. 300 to 2000 μg, 300 us 2250 μg, 300 to 2500 μg, 300 to 2750 μg- 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg. 400 ui 700 μg. 400 io 800 μg, 400 to 900 μg, 400 to 1000 μg, 400 to 1250 μg, 400 to 15(M) μg. 400 to 1750 μg. 400 to 2000 μg.400 to 2250 μg, 400 to 2500 μg. 400 to 2750 μg, 400 io 3000 μg, 500 io 600 μg, 500 to 700 μg. 500 io 800 μg. 500 to 900 μg, 500 to 1000 μg, 500 to 1250 μg. 500 to 1500 μg. 500 io 1750 μg _: 500 to 2000 μg, 500 to 2250 μg. 500 io 2500 μg, 500 to 2750 μg. 500 to 3000 μg. 600 to 700 μg, 600 to 800 μg, 600 to 900 μg, 6(K) to 1000 μg, 600 to 1250 μg, 600 to I 500 μg, 6 ⁽ 50 to 1750 μg, 600 io 2000 μg. 600 to 2250 μg, 600 to 2500 μg, 600 to 2750 μg, 600 to .1000 μg, 700 to SOO μg. 700 to %»0 μg, 700 io 1000 μg, 700 to 1250 μg, 700 to 1500 μg. ^" 700 tu ! 750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg. 700 to 3(KKi μg, 800 to 900 μg, 800 to 1000 μg. 800 to 1250 μg. 800 to 1500 μg, 800 to 1750 μg, SOO to 2000 μg. 800 to 2250 μg, 800 to 2500 μg. 800 to 2750 μg, 800 to 3000 μg, 900 to i OOO μg, V(KJ to 1250 μg. 900 to 1500 μg, 900 io I 750 μg. 900 to

- 342 -

2000 μg, 900 Io 2250 μg, 900 to 2500 μg, 900 to 2750 μg _t 900 to 30Of) μg, 1000 io 1250 μg, 1000 to 1500 μg. 1000 to 1750 μg, 1000 to 2000 μg, 1000 to 2250 μg, 1000 !o 2500 μg, 1000 to 2750 μg, 1000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg, 5 to 100 μg. 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg. 500 μg. 550 μg, SOO μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, H ⁾ OO μg, 1050 μg. 1 100 μg, 3 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 ug, !4 ^{ ;0 μg. ! 450 μg, 15ϋ0 μg, 1550 μg, USOO μg, 1650 μg, 1700 μg, 1750 μg, 1S00 μg, 1850 μg. 1900 μg. 1950 μg, 2000 μg. 2050 μg, 2H)O μg, 2150 μg, 2200 μg. 2250 μg. 2300 μg, 2350 μg. 2400 μg. 2450 μg, 2500 μg. 2550 μg, 2600 μg, 2650 μg _; 2700 μg, 2750 μg, 2800 μg. 2850 μg. 2900 μg, 2950 μg. 3000 μg, 3250 μg, 3500 μg. 3750 μg, 4000 μg, 4250 μg, 4500 μg. 4750 μg, 5000 μg of a peptide or agonist described herein and from 1 mg to 20 nig (e.g. 1 nig, 2 nig, 2.5 nig, 3 rag, 4 mg, 5 mg, 6 mg, 7 nig, 7.5 mg, 8 mg, 9 mg, 10 mg, 1 1 mg, 12 rag, 12,5 mg, 13 mg, 14 mg. mg, 17,5 mg, 18 mg, 19 mg, 20 mg} of darifenacin (Enables®).

λ dosage unit (e.g. an oral dosage unit) can include, lhr example, from i to 30 μg, 1 to 40 μg, I to 50 μg, 3 io 100 μg. 1 to 200 μg, 3 to 300 μg. 1 to 400 μg- I Io 500 μg, I to 600 μg, I to 700 μg, 1 to 81)0 μg, 1 to 900 μg, 1 Io 1000 μg, 10 to 30 μg, 10 to 40 μg, 10 to 50 μg, ! 0 to 100 μg, 10 to 200 μg. 10 to 300 μg, 10 to 400 μg, 10 to 500 μg, 10 to 600 μg, 10 to 700 μg, 10 to SOO μg, 10 to %0 μg, 10 to 1000 μg, 100 to 200 μg, ! 00 to 300 μg, 100 to 400 μg, i 00 to 500 μg, 100 to 600 μg _s 100 to 700 μg. 100 to KOO μg, 100 to 9Of) μg. 100 to 1000 μg, 100 to 1250 μg, 100 to 1500 μg, 100 to 1750 μg, HK ⁾ Lo 2000 μg. 100 to 2250 μg, 100 to 2500 μg. 100 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 200 to 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 to ! 750 μg, 200 to 2000 μg, 200 to 2250 μg, 200 to 2500 μg, 200 to 2750 μg. 200 Io 3000 μg, 300 to 400 μg. 300 to 500 μg, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg, 300 to 1250 μg, 300 to 1500 μg, 300 to 1750 μg, 300 to 2000 μg. 300 Io 2250 μg. 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg. 400 to 700 μg, 400 U) 800 μg, 400 Jo 900 μg, 400 to 1000 μg, 400 to 1250 μg. 400 to i 500 μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400

- 143 -

to 2750 μg, 400 to 3000 μg. 500 to 600 μg. 500 Io 700 μg. 500 to 800 μg. 500 to 900 μg. 500 Io 1000 μg. 500 to 1250 μg, 500 to 1500 μg, 500 w 1750 μg, 500 to 2000 μg, 500 to 2250 μg, 500 Io 2500 jig, 500 to 2750 μg, 500 to 3000 μg. 600 to 700 μg, 600 Lo mi) μg, 600 to 900 μg, 600ito 1000 μg, 600 to 1250 μg, 600 to 1500 μg _; 600 to ! 750 μg, 600 to 2000 μg, 600 :to 2250 μg, 600 to 2500 μg, 600 io 2750 μg, 600 to 3000 μg, 700 to 800 μg, 700 to 900 μg, 700 to 1000 μg, 700 to 1250 μg, 700 to 1500 μg, 700 to 1 750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg, 700 to 3000 μg. SOO to 900 μg, 800 to 1000 μg, 800 to 1250 μg, 800 to 1500 μg, 800 lo ! 750 μg. 800 to 2000 μg, 8D0 to 2250 μg, 800 to 2500 μg, SOO to 2750 μg, SOO to 3(K)O μg, 900 to KK)O μg, 900.to 1250 μg, 900 to 15 ⁽ 50 μg, 900 to 1750 μg, 900 to 2000 μg, 900 to 2250 μg, 90Oj tυ 2500 μg, 900 to 2750 μg, 900 to 3000 μg. 1000 to 1250 μg, 1000 to 1500 μg, 1000 to 1750 μg, 1000 to 200(S μg, S 000 io 2250 μg, ! 000 ?o 2500 μg. 1000 to 2750 μg, 51000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg, 5 to 100 μg, 50 μg: 100 μg. 150 μg, 200 μg. 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, SOO μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg. 850 μg, 900 μg. 950 μg, 1000 μg, 1050 μg, 1 100 μg, 1 150 μg, 1200 μg, 1250 μg, 1300 μg. 1350 μg, 1400 μg _s 1450 μg, 1500 μg. 1550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg, 1 S0O μg. 1850 μg, 1900 μg. 1950 μg, 2000 μg, 2050 μg. 2100 μg, 2150 μg, 2200 μg, 2250 μg, 2300 μg, 2350 μg, 2400 μg, 2450 μg, 2500 μg, 2550 μg, 2600 μg. 2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg, ψSQ μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein imά irom S mg to 250 mg (e.g. 1 ing, 2 nig, 3 mg, 4 mg, 5 mg. 6 nig, 7 rng, 8 nig, 9 mg, 10 nig, 20 ro». 30 mg, 40 rag, 50 mg, 60 mg, 70 mg, SO rag, 90 mg, 100 mg, HO πsg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg} of Ondansetron HCi (Zofran#).

A dosage unit (e.g an oral dosage umt) can include, tor example, from 1 to 30 μg, 1 io 40 μg, ! Io 50 μg, 1 to 100 ^g. 1 to 200 μg. 1 to 300 μg. 1 to 400 μg, I to 500 μg, 1 to 600 μg, 1 to 700 μg, I to 800 μg, I to 900 μg. 1 to 1000 μg. 10 to 30 μg, 10 to 40 μg, 10 to 50 μg. 10 to 100 μg] 10 to 200 μg, 10 to 300 μg, 10 to 400 μg. 10 to 500 μg. 10 to 600 μg, I O to 700 μg, J O to 800 μg. 10 to 900 μg, 10 to 1000 μg, 100 to 200 μg,

- 144 -

100 to 300 μg, 100 to 400 μg| ϊ 00 io 500 μg. 100 to 6(K) μg, 100 to 700 μg, 100 to 800 μg, KH) to 900 μg, KM) to 1000 μg, 100 to 1250 μg, 100 to 1500 μg, 100 to 1750 μg, H)O to 2000 μg. 100 in 2250 μg, 100 to 2500 μg, 100 to 2750 μg, 100 io 3000 μg, 200 to 300 μg, 200 to 400 μg, 200 to 500 μg, 200 to 600 μg. 200 to 700 μg, 200 io 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 Io 1250 μg, 200 to 1 500 μg, 200 to 1750 μg, 200 to 2000 μg, 200 to 2250 μg, 200 to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μ.g, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg, 300 to 1250 jig, 300 to 1500 μg, 300 to 1750 μg, 300 to 2000 μg, 300 U) 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 io 3000 μg, 400 io 500 μg, 400 to 600 μg. 400 to 700 μg, 400 id 800 μg, 400 to 900 μg, 400 to 1000 μg, 400 to 1250 μg, 40(5 to 1500 μg. 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 io 2500 μg, 400 to 2750 ug, 400 to 3000 μg, 500 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg, 500 Io 1000 μg- 500 io 1250 μg, 500 io ϊ500 μg, 300 to 1750 μg. 5(M) to 2(KK) μg. 500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 io 800 μg. 600 to 000 μg, 600 to 10DC* μg, 600 to 1250 μg, 600 to 1500 μg, 600 to 1750 μg. 600 to 2000 μg, 600 to 2250 μg, 600 to 2500 μg, 600 to 2750 μg, 600 to 1(H)O μg, 700 io 800 μg, 700 jo 900 μg. 700 to 1000 μg. 700 to 1250 μg, 700 to 1500 μg, 700 to 1750 μg, 700 io 2000 μg, 700 to 2250 μg, 700 to 2500 μg. 700 io 2750 μg, 700 to 3000 μg, 800 to 900 μg, 800 to 1000 μg. 800 Io 1250 μg, 800 to 1500 μg, 800 Lo I 750 μg. 800 to 2000 μg. 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 to 3000 μg, QOO to 1000 μg, 900 to 1250 μg, 900 to 1500 μg, 900 to 1750 μg. 900 io 2000 μg. 900 io 2250 μg, 9OQ to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, 1000 to 1250 μg, 1000 to 1500 μg. HK)O io 1750 μg, 1000 to 2000 μg, ! 000 to 2250 μg. K ⁾ OO to 2500 μg. 10(50 to 2750 μg,;!000 to 3000 μg, 2 lo 500 μg, 50 to 500 μg, 3 io H)O μg, 5 to 20 ug, 5 to 100 μg, 50 μg _; 100 μg, 150 μg, 200 μg, 250 μg. 300 μg. 350 μg, 400 μg, 450 μg, 500 μg. 550 μg, 600 μg. 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg. 950 μg, 1000 μg, 1050 μg, 1 K)O μg, 1 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg. 1450 μg, 1500 μg, ϊ550 μg, 1600 μg, 1650 μg, 1700 μg. 1750 μg. 1800 μg, 1850 μg, 1900 μg., 1050 μg, 2000 μg, 2050 μg, 2100 μg, 2150 μg.2200 μg. 2250 μg, 2300 μg, 2350 μg. 2400 μg, 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μ ug, 3750 μg.

- 145 -

4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μ.g of a peptide or agonist described herein and from I rag to 3000 mg (e.g. 1 nig, 2 mg, 3 mg, 4 nig, 5 rag, 6.mg, 7 rag, S mg, 9 mg, JO mg, 20 ing, 30 mg, 40 mg, 50 mg, 60 rag, 70 mg, 80 mg, 90 rag, ! 00 mg, 200 rag, 250 mg _s 300 mg, 350 ing, 400 mg, 450 rag, 500 mg, 750 mg, 1000 mg, 1250 nig, 1500 rag, I 750 mgj 2000 mg, 2250 rag, 2500 rag, 2750 mg, 3900 mg) of Cimctrαpiurø {Alginorφ}, i

A ciosagc unit (e.g. an oral dosage unit) can include, ibr example, from 1 to 30 μg, 1 !.o 40 μg. ! to 50 μg. I to l OOiμg, 1 to 200 μg, 1 to 300 μg. 1 to 400 μg, I So 500 μg, 1 to 600 μg, 1 to 700 μg, 1 to 800 μg, 1 to 900 μg, 1 to 1000 μg, 10 to 30 μg, 10 to 40 ug, 10 to 50 μg, 10 to 100 μgj 10 to 200 μg, ! 0 to 300 μg, ! 0 to 400 μg. 10 to 500 μg. ! 0 to 600 μg, ! 0 to 700 μg. I i) to 800 μg, ! 0 to 900 μg, 10 to 1000 μg, ! 00 io 200 μg, 100 to 300 μg. 100 to 400 μgj 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to 80C μg, t OO io 900 μg, JOO io 1000 μg, 100 to 12S0 μg, 100 to 1500 μg, 100 to 1750 μg _s 100 to 2000 μg, I (K) to 2250 μg, 100 io 2500 μg, 100 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 200 to 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 Io 800 μg, 200 to MOO μg. 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 to 1750 μg. 200 to 2000 μg, 200 to 2250 μg. 300 to 2500 μg, 200 to 2750 μg, 200 So 3000 μg, 300 io 400 μg, 300 to 500 μg. 300 iό 600 μg, 300 to 700 μg, 300 io 800 μg, 300 to 900 μg, 300 to i 000 μg, 300 to J 250 μg, 300 io 1500 μg. 300 to 1750 μg. 300 to 2000 μg. 300 to 2250 μg, 300 to 2500 μg, 300 to 2750 μg. 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg, 400 io 700 μg, 400 id 800 μg, 400 to 900 μg, 400 to 1000 μg, 400 to 1250 μg, 400 to 1500 μg, 400 to ! 750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400 Io 2750 μg, 400 to 3000 μg. SOO to 600 μg, 500 to 700 μg. 500 to SOO μg. 500 io '-K)O μg, 500 to ! 000 μg, 500 to 1250 μg, 500 to ! 500 μg, 500 to 175ϋ μg, 500 IG 2000 μg, SfK) to 2250 μg, 500 to 2500 §ig, 500 to 2750 μg, 500 io 3000 μg, 600 to 70(3 μg, 600 io 800 μg, 600 io 900 μg, 600 to 1000 μg, 600 to 1250 μg, 600 to 1500 μg, 600 to 1750 μg, 600 to 2000 μg, 60Q to 2250 μg, 600 to 2500 μg. 600 io 2750 μg, 600 to 3000 μg, 700 to 800 μg, 700 fo ^00 μg, 700 iϋ 1000 μg, 700 to 1250 μg. 700 to 1500 μg, 700 io 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg, 700 to 3(KKs μg. 800 to 900 μg, 800 io 1000 μg, 800 to 1250 μg, 800 H) 1500 μg, 800

: . 146 -

to ! 750 μg, 800 to 2(K)O μg. TOO to 2250 μg. 800 Io 2500 μg, BOO to 2750 μg, 800 to 3000 μg, 900 to 1000 μg, 900 to 1250 μg, 900 to 1500 μg, 900 to 1750 μg, 900 to 2000 μg, 900 to 2250 μg, 900 to 2500 μg, 900 Io 2750 μg, 900 to 3000 μg, ! 000 to 1250 μg, 1000 to 1500 μg, KK)O to 1750 μg, 1000 to 2000 μg, 1000 to 2250 μg, 1000 io 2500 μg, 1000 to 2750 μg,!1000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 Io 100 μg, 5 to 20 μg. 5 to 100 μg, 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 ug, 450 μg, SOO μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg. 850 μg, 900 μg, 950 μg, 1000 μg. 1050 μg, I IjOO μg, U 50 μg, 1200 μg, 1250 μg. 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 11550 μg, 1600 μg, 1650 μg. 1.700 μg. 1750 μg, I SOO μg, 1 S50 μg. 1900 μg, 1950 μg, 2000 μg, 2050 μg, 2 ! 00 μg. 2150 μg. 2200 μg, 2250 μg, 2300 μg. 2350 μg, 2400 μg, 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg, 2900 μg, 2950 μg. 3000 μg, 3250 μg, 3500 μg. 3750 μg. 4000 μg, 4250 μg, 4500 μg, 4750 μg. 5000 μg uf a peptide or agonist described herein and from 1 mg to 1000 mg (e.g. 1 mg, 5 mg, 10 rag, 15 mg, 20 mg, 25 rag, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg _; 250 mg, 300 mg, 350 nig, 400 mg, 450 mg, SO© mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 rag, 900 rag, 950 mg, 1000 nig) of Dolasetron (Anzemel®),

A dosage um ^' i (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg. i to 40 μg, ] to 50 μg. 1 to iOOjμg, 1 to 200 μg, 1 to 300 μg, i to 400 μg, 1 Io 500 μg, 1 io 600 μg. 1 to 700 μg. 1 to 800 μg, 1 to 900 μg, 1 to 1000 μg, 10 to 30 μg, 10 to 40 μg. 10 io 50 μg. 10 to 100 μg| 10 to 200 μg, 10 to 300 μg, 10 to 400 μg, 10 to 500 μg. 10 to 600 μg, 10 io 700 μg, 10 to 800 μg, 10 to 900 μg, 10 to 1000 μg, 100 to 200 μg. 100 $o 300 μg, 100 to 400 μgj 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 to 800 μg, 100 tι> ⁴ SOO Ug, 100 to 1000 μg, 100 to 1250 μg, I GO to ϊ SOO μg. 100 to 1750 μg, 100 to 2000 μg, 100 io 2250 jjg, 100 io 2500 μg, 100 to 2750 μg, i 00 to 3000 μg, 200 io 300 μg, 200 to 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to SOO μg, 200 to 9(K) μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg. 200 to 1750 μg, 200 to 2000 μg, 200 to 2250 μg. 201} to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 ug, 300 to KKK) μg, 300 to J 250 jig, 300 io 1500 μg, 300 to 1750 μg, 300 to 2000 μg, 300 i - 14? -

in 2250 μg. 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg. 400 to 600 μg. 4(K) to 700 μg- 4(50 id 800 μg. 400 to 900 μg, 400 to 1000 μg. 4(K) to 1250 μg. 400 to ! 500 μg, 400 to 1750 jig, 400 to 2000 μg. 400 to 2250 μg, 400 to 2500 μg, 400 to 2750 μg, 4(K? to 3000 μg, 500 io 600 μg, 500 Io 700 μg, 500 to SOO μg, 500 to 900 μg, 500 to 1000 μg, 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 jig, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg. 600 io 800 μg, 600 to 900 μg, 6OQ to 1000 μg, 600 to 1250 μg, 600 io S 500 μg. 600 to ! 750 μg. 60(5 Lo 2000 μg. 600 to 2250 μg. 600 io 2500 μg. 600 to 2750 μg. 600 to 3000 μg. 700 to 800 μg, 700 io 900 μg, 700 to 1000 μg, 700 to 1250 μg, 700 to 1500 μg, 700 to 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg. 700 to 3000 μg, 800 to 900 μg, 800 to 5000 μg, 800 to \ 250 μg. 800 to 1500 μg, SOO in 1 750 μg, 800 to 2000 μg, $00 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 to 3(KK) μg. 90(5 io i 000 μg, 900 to 1250 μg. 900 to 1500 μg, 900 to 1750 μg, 900 to 2000 μg. 900 to 2250 μg. 90Q to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, ! 000 to 1250 μg, 1000 to 1500 μg, i (K)O to 1750 μg. K)(K) to 2000 μg, 1000 Io 2250 μg, 1000 to 2500 μg, 1000 to 2750 μgJlOOO to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg, 5 to 100 μg, 50 ug, 100 μg. 1 50 μg, 200 μg, 250 μg, 300 μg, 350 μg. 4OU μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg. 750 μg, 800 μg, 850 μg, 900 μg, 950 μg. ] 000 μg, 1050 μg, ϊ 1:00 μg, 1 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg. 1450 μg, 1500 μg, 1:550 μg, 1600 μg. 1650 μg, 1700 μg, 1750 μg, 1800 μg, 1850 μg, 1900 μg, 1950 μg, 3000 μg, 2050 μg, 2100 μg, 2150 μg, 2200 μg, 2250 μg, 2300 μg, 2350 μg, 2400 μg.2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2800 μg. 2850 μg, 1900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg. 4000 μg, -1250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and from I mg to I 80 tng (eg. 1 mg, 2 nig, 3 mg, 4 mg, 5 mg, 6 mg, 7 røg, 8 mg, 9 ϊog, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 1 10 mg, 120 mg, 130 mg, 140 mg, ^' ) 50 mg, 160 mg, 170 ing, 1 SO mg) of Zelnomv$> (iegaserod).

A dosage unit (e.g. sm oral dosage unit) can include, for example, from 1 to 30 μg, 1 to 40 μg, 1 to 50 μg, 1 to I GGlμg, S to 200 μg, I to 300 μg, 1 to 400 μg, 1 io 500 μg, 1

^; .. !4S --

in 60 ⁽ 3 μg, I iυ 700 μg, 1 to 800 μg. J to 900 μg. I to 1000 μg, 10 to .30 μg, 10 to 40 μg, K ⁾ Jo 50 μg, 10 to 100 μi 10 to 200 μg, 10 to 300 μg, 10 to 400 μg, 10 to 500 μg, 10 it) 6 ⁽ K ⁾ μg, 10 to 700 μg. IK) to 800 μg. 10 to 900 μg, 10 to 1000 μg, 100 to 200 μg, H ⁾ O lø 3 ⁽ K ⁾ μg, H ⁾ O Io 400 μg. 100 Io 500 μg, 100 Io 600 μg. 100 to ?00 μg. 100 to 800 μg, 100 to 900 μg, 100 to I 000 μg, 100 to 1250 μg, 100 to 1500 μg, 300 to 1750 μg, 100 to 2000 Mg, 100 to 2250 iμg, ! 00 to 2500 μg, ! 00 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 200 to 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to 800 μg. 200 Io 900 μg, 200 Io 1000 μ|g, 200 to 1250 μg, 200 to 1500 μg, 200 to i 750 μg, 200 to 2 ⁽ K ⁾ O μg. 200 to 2250 μg, 200 Io 2500 μg, 200 io 2750 μg. 200 io 3000 μg, 300 to

10 400 μg, 300 to 500 μg, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg. 300 to ! 250 μ.g, 300 to ! 500 μg. 300 to 1750 μg, 300 to 2000 μg, 300 to 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg, 400 to 7(K) μg, 400 to 800 μg, 400 Io 900 μg, 400 io 1000 μg. 400 to 1250 μg, 400 io 1500 μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400

15 to 2750 μg, 400 to 3000 μg, 500 to 600 μg, 500 to 700 μg, 500 to S(K) μg, 500 to 900 ug, 500 to 1000 μg, 500 to 1250 μg, 500 to ! 500 μg, 500 to 1750 μg, 500 io 2000 μg, 500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg. 600 to 700 μg, 600 to 800 μg. 600 to 900 μg, 600 to 1000 μg, 60(3 to 1250 μg, 600 to 1500 μg, 600 to J 750 μg, 600 to 2000 μg, 600 to 2250 μg. 600 to 2500 μg, 600 to 2750 μg, 600 to

^' -J0 3000 μg. 700 io 800 μg, 700 to 900 μg, 700 Io 1000 μg, 700 to 1250 μg, 700 to 1500 μg, 700 to 1750 μg, 700 to 2Q00 μg, 700 to 2250 μg, 700 to 2500 μg. 700 to 2750 μg, 700 to 3000 μg. BOO io 900 μg, 800 Io 1000 μg, 800 Io 1250 μg, 800 to 1500 μg, SOO to 1750 μg, 800 to 2000 μg ₅ 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 to 3000 μg, 900 to 1000 μg, 900 to ϊ250 μg, 900 to 1500 μg, 900 to 1 750 μg. 900 to

9 ⁵ ^ 2000 μg, 900 to 2250 μg, 900 to 2500 μg, 900 to 2750 μg, 900 to 3000 μg. LO(M) to 1250 μg, 1000 to 1500 μg, 1000 to 1750 μg, 1000 to 2000 μg. 1000 to 2250 μg, 1000

000 to 3000 μg, 2 to 500 μg, 50 to 500 μg. 3 to 100 μg, 5 io 20 μg, 5 to 100 μg. 50 μg. 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 Mg. 450 μg, SS)O μg, 550 μg. 600 μg, 650 μg. 700 μg, 750 μg. 800 μg. 850 μg, 900 μg,

30 950 μg, 1000 μg. 1050 μg _: 1 1iOO μg, 1 150 μg. 1200 μg. 1250 μg, 1300 μg, 1350 ug.

1400 μg, 1450 μg. 1500 μg, Ij 550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg, 1800 μg, - 149 -

I SSO μg, 1900 μg, 1950 μg, 2000 μg, 2050 μg, 2] 00 μg, 2150 μg, 2200 μg. 2250 μg, 2300 μg. 2350 μg, 2400 μg, 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg, 2750 μg. 2800 μg, 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg. 4250 μg. 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and from ) μg to 500 μg (e.g. 1 μg, 5 μg, 10 μg. 50 μg, 75 μg, 100 μg, 125 μg, 150 μg. 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg. 350 fig, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 300 μg) oπ..evsm$t ^! {hyoscyamiϊiε! sulfate).

A dosage unit (e.g. an orai dosage unit) can include, for example, from 1 to 30 μg, I to 40 μg, I to 50 μg, 1 to lOOlμg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg. 1 Io 500 μg, 1 to 600 μg, 1 to 700 μg, 1 to 800 μg, 1 to 900 μg, I to 1000 μg, 10 Io 30 μg, 10 LO 40 μg, 10 to 50 μg, 10 to 100 μgj K) to 200 μg. 10 to 300 μg. 10 io 400 μg, 10 to 500 μg, 10 to 600 μg, 10 to 700 μg, 10 to SOO μg, 10 to 900 μg, 10 to 1000 μg, 100 to 200 μg, 100 to 300 μg. 100 to 400 μgj 100 io 500 μg, 100 io 600 μ.g, 100 to 700 μg, 100 to 800 μg, 100 to 900 μg, 100 to 10(30 μg, 100 to 1250 μg, 100 to 1500 μg, 100 to 1750 μg, LOO to 2000 μg, tOO to 2250 μg _? 100 to 2500 μg, ^" 100 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 200 to 400 μg, 200 to 500 μg.200 to 600 μg, 200 to 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg. 200 to 1500 μg, 200 to 1750 μg, 200 Uf 2000 μg, 200 io 2250 μg, 200 to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg. 300 Id 600 μg, 300 to 700 μg, 300 to 800 μg, 300 Io 900 μg, 300 to 1000 μg, 300 to 1250 μg, 300 to 1500 μg, 300 to ! 750 μg, 300 U) 2000 μg, 300 to 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg, 400 to 700 μg, 400 to 800 μg, 400 to 900 μg. 400 to 1000 μg, 400 to 1250 μg. 400 to 1500 μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg. 400 to 2750 μg, 400 to 3000 μg, 5:00 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg. 500 to H500 μg, 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg, 500 to 2000 μg. 500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 to SOO μg, 600 to 900 μg, 60(1 to i 000 μg, 600 io 1250 μg, 600 to ! 500 μg, 600 to !7SO μg, 600 to 2000 μg, 600 to 2250 μg. 600 to 2500 μg, 600 to 2750 μg, 600 to 3000 μg. 700 io 800 μg, 700 jo 900 μg, 700 io 1000 μg, 700 to 1250 μg. 7(H) io 1500 μg, 700 to 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg,

I - 1 so -

700 to 3000 μg.800 tυ 900 μg, 800 Io 1000 μg, Wi) to 1250 μg, 800 to 1500 μg, 800 io 1750 μg, 800 to 2000 μg, 800 to 2250 μg, SOO to 2500 μg, 800 io 2750 μg, SO XOMo 3000 μg.900 to 1000 μg, 900 to 1250 μg, 900 U) I500 μg, 900 io 1750 μg, 900 tto< 2000 μg, 900 to 2250 μg, 90(1 Io 2500 μg, 900 io 2750 μg, 900 to 3000 μg, 1OCKJ to 1250 μg, 1000 to 1500 μg, 1000 io 1750 μg, 1000 to 2000 μg, KK)O to 2250 μg, 1000 to 2500 μg. HHK) io 2750 μg,|ϊ000 io 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg, 5 to 100 μg, 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg.400 μg, 450 μg, 500 μg, 550 μg.600 μg.650 μg, 700 μg, 750 μg, SOO μg, 850 μg.900 μg, 950 μg.1000 μg, 1050 μg, 1 i|θθ μg, 1150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg.1450 μg, 1500 μg, 11550 μg, 1600 μg, 1650 μg, 1700 μg.1750 μg, 1800 μg, 1850 μg, 1900 μg.1950 μg, 3000 μg, 2050 μg, 2100 μg, 2150 μg, 2200 μg, 2250 μg, 2300 μg, 2350 μg.2400 μg, 2450 μg, 2500 μg.2550 μg, 2600 μg, 2650 μg.2700 μg, 2750 μg.2800 μg, 2850 μg, 2900 μg, 2950 μg, 3000 μg, 32S0 μg, 3500 μg.3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg ofa peptide or agonist described herein and from 50 mg io 50«:) mg (e.g.50 rag, 60 mg, 70 mg _f 80 rug, 90 mg, i00 røg, 125 mg, 150 mg, 175 mg, 200 mg, 225 rag, 250 mg, 275 mg, 300mg, 325 nig, 350 SBg, 375 mg.400 nig, 425 mg, 450 mg.500røg) ofDicetdφ (pmaveriinrs bromide).

A dosage unit (e.g. an oral dosage unit _. ) can include, for example, from 1 to 30 μg, 1 ,o 40 μg, I io 50 μg, 1 to lθθ| μg _? 1 to 200 μg, ! to 300 μg, i Ea 400 μg, 1 to 500 μg, t :o 600 μg, 1 to 700 μg, 1 to SpO μg, 1 Io 900 μg, 1 to 1000 μg, 10 to 30 μg, 10 to 40 ig, i 0 to 50 μg, 10 to 100 μ.gl 10 to 200 μg, 10 to 300 μg, 10 Io 400 μg, 10 to 500 μg, 0 to 600 μg, 10 io 700 μg, 10 to 800 μg, 10 to 900 μg, IO Io 1000 μg, 100 Io 200 μg, 00 Io 3 DO μg, 100 to 4(K) μgi 100 to 500 μg. 100 to 600 μg, 100 to 700 μg, 100 to SOO ;g, 100 to 900 μg, 100 to 1000 μg, 100 to 1250 μg, 100 to 1500 μg, 100 io 1750 μg, OO to 2000 μg, 100 to 2250 μg. 100 to 2500 μg, 100 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 21)0 io 400 μg, 200 to 500 μg, 200 to 600 μg. 200 io 700 μg, 200 to SOO μg, 200 to 000 μg, 200 io 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 io 1750 μg, 200 io 2000 μg, 200 to 2250 μg. 200 to 2500 μg, 200 to 2750 μg _s 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg. 300 W 600 μg, 300 to 700 μg. 300 to SOO μg, 300 io 900 μg. 300 to iOOO μg, 300 to 1250 μg, 300 to 1500 μg, 300 to 1 750 μg, 300 io 2000 μg, 300 i - 151 -

to 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 Io 3000 μg. 400 io 500 μg, 400 to 600 μg. 400 to 700 μg, 400 iϋ 800 μg, 400 to 900 μg, 400 to K)0(i _μg , 400 io 1250 μg, 400 Io 1500 μg, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg. 400 to 2750 μg, 400 to 3000 μg, 5:00 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg. 500 to 1000 μg, 500 io J 250 μg, 500 io 1500 μg, 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 jig, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 to 800 μg, 600 ω 900 μg, 600 to 1000 μg, 600 to 1250 μg, 600 io ! 500 μg, 600 to 1750 μg, 60(5 Io 2000 μg, 600 io 2250 μg, 600 to 2500 μg, 600 to 2750 μg, 600 io 3000 μg, 700 to 800 μg, 700 io 900 μg, 700 to ϊ000 μg, 700 to 1250 μg. 700 io 1500 μg, 700 to 175(1 μg. 700 to 2000 μg. 700 io 2250 μg, 700 to 2500 μg, 700 to 2750 μg, 700 to 3000 μg, 800 to 900 μg, SOO to ! 000 μg, 800 to 1250 μg, 800 to 1500 μg, 800 to 3 750 μg, 800 io 2000 μg, 800 to 2250 μg, 800 io 2500 μg, SOO to 2750 μg, 800 io 3000 μg, 900 to ! UOO μg, 900 to 1250 μg, 900 to 1500 μg, 900 to 1750 μg, 900 to 20(Hl μg. 900 Lo 2250 μg. 900 to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, ! 000 io 1250 μg, 1000 io 1500 μg. 1000 to 1750 μg. i 000 to 2000 μg, 1 (K)O to 2250 μg, 1000 to 2500 μg, 1000 to 2750 μg J 1000 in 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg, 5 io 100 μg. 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg. 750 μg, 800 μg, 850 μg, 900 μg, 950 μg. i OOO μg. 1050 μg, I tjOO μg, 1 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg. 1450 μg, 1500 μg, liS50 μg, 1600 μg, 1650 μg. 1700 μg, 1750 μg, 1800 μg, S 850 μg, 1900 μg. 1950 μg, 2000 μg, 2050 μg, 2100 μg, 2150 μg, 2200 μg. 2250 μg, 2300 μg, 2350 μg, 2400 μg, 2450 μg, 2500 μg, 2550 μg, 2600 μg, 2650 μg.2700 μg, 2750 μg, 2800 μg, 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and tro.ro 50 mg lo 500 mg (e.g. 50 nig, 75 mg, 100 mg, 125 mg, 135 Big, 150 mg, 175 mg, 200 rng, 225 mg, 250 nig, 275 mg, 300 mg, 325 mg, 350 mg _: 375 mg, 400 mg, 425 mg, 450 mg. 47$ Dig, 500 mg) of roeheverme (DUSPATA IM, DUSPATAIJN®, COLOFAt Mil®, COLOTALt=I.

λ dosage unit (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg, 1 to 40 μg, 1 to 50 μg, 1 to lOOiμg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg. i to 500 μg, i i - 152 -

to 600 μg, ! to 700 μg, ! to SpO μg, I to 900 μg, I to 1000 μg, 10 to 30 μg, 10 to 40 μg, t O to SO μg, 1 O tO 100 μg 10 to 200 μg, 10 to 300 μg. O tO 400 μg, 10 to 500 μg.

0 to 600 μg, 10 io 700 μg, Ip to 800 μg, 10 to 900 μg, 10 to 1000 μg, 100 to 200 μg.

100 to 300 μg, 100 to 400 μg 100 to 500 μg, 100 to 600 μg, 100 to 700 μg. 100 Io 800 μg. 100 io 900 μg. 100 to I OQO μg. 100 to 1250 μg, 100 to 1500 μg. 100 to 1750 μg, i (JO to 2000 μg. 100 to 2250 jig, 100 to 2500 μg, 100 to 2750 μg, ! 00 to 3000 μg, 200 to 300 μg, 200 to 400 μg, 20(1 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μ^, 200 to 1250 μg, 200 to 1500 μg, 200 to 1750 μg. 200 to 2000 μg, 200 to 2250 μg, ioO to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 fcj 600 μg, 300 to 700 μg, 300 to SOO μg, 300 Lo 900 μg. 300 to 1000 μg, 300 to 1250 μg, 300 to 1500 μg, 300 to 1750 μg, 300 to 2000 μg, 300 to 2250 μg, 300 io 2500 μg. 2J00 to 2750 μg. 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg. 400 to 700 μg, 400 tcj 800 μg, 400 to 900 μg, 4(M) to 1000 μg, 400 Io 1250 μg, 400 to ! 5(K) ug, 400 to 1750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400 to 2750 μg.. 400 to 3000 μg, i00 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg, 500 to 1000 μg. 500 to 12J50 μg, 500 to 1500 μg, 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 μg, 500 to 2750 μg. 500 to 3000 μg. 600 to 700 μg, 600 la SOO μg, 600 to 900 μg, 6ϋ(j to H)OO μg. 600 to 1250 μg. 600 to 1500 μg, 600 to 1750 μg. 600 to 2000 μg. 6CKJJ io 2250 μg, 600 to 2500 μg, 600 to 2750 μg. 600 to 3000 μg, 700 to 800 μg, 700 Io 900 μg, 700 to 1000 μg, 700 to 1250 μg, 700 to 1500 μg, 700 io 1750 μg, 700 to 2CJ00 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg, 700 io 3000 μg, 800 to 900 μg. 800 to 1000 μg, SOO to 1250 μg, 800 io 15Qd μg, 800 to ! 750 μ«. 800 to 2000 IUJ. 800 to 2250 μe, 800 to 2500 us, SOO to 2750 μj>. SOO to 3000 μg, 900 to 1000 μg, 90Cj to i 250 μg, 9(30 io 1500 μg, 900 to 1750 μg, 900 to 2000 μg. 90(J to 2250 μg. 9i)U to 2500 μg. 900 to 2750 μg, 900 to 3000 μg. ) 000 to 1250 μg, 1000 to 1500 μg, 1000 to 1750 μg, 1000 to 2000 μg, KK)O to 2250 μg, 1000 io 2500 μg, 1000 to 2750 μgj l OOO to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg. 5 io H)O μg. 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg. 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, φθ μg, 650 μg. 700 μg, 750 μg, 800 μg, 850 μg, 900 μg. 950 μg, 1000 μg. 1050 μg. 1 ljOO μg, 1150 μg, 1200 μg, 1250 μg. 1300 μg, 1350 μg, 1400 μg. 1450 μg, 1500 μg, 11550 μg, 1600 μg, 1650 μg. 1 700 μg, 1750 μg, 1800 μg,

! - 153 -

! 850 μg, 1900 μg. 1950 μg, 2000 μg, 20S0 μg, 2100 μg, 2150 μg, 2200 μg. 2250 μg, 2300 μg, 2350 μg, 2400 μg, 3450 μg, 2500 μg, 2550 μg, 2600 μg.2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg, φ)00 μg, 2950 μg. 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described hheerreeiinn aanndd ffrroomm 11 raragg ttoo 112200 ppiigg ((ee..gg.. 11 raragg,, 22..55 mmgg,, 55 mmgg,. 7.5 nig, 10 nig, 12.5 mg. 15 mg, 20 mg, 25 mg, 30 mgj 40 rag, 50 mg, 6 >00 mmgg,, 7 ,0.., _i m _St& g,, , 80 mg, 90 mg, 100 mg. 1 10 mg, 120 mg) of Propantliilme bromide (Pro-Banthmeφ).

λ dosage unit (e.g. an oral dφage unit) can include, tor example, from 1 to 30 μg, 1 to 40 μg, 1 to 50 μg, I to K?θjμg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg. I to 500 μg, I to 600 μg, 1 to 700 μg, 1 to 800 μg, 1 to 900 ug, 1 to 1000 μg. ! 0 to 30 μg, 10 to 40 μg, 10 to 50 μg, 10 to LOO μgj 10 to 200 μg, 10 to 300 μg. 10 lo 400 μg. 10 to 500 μg, ! 0 to 600 μg, 10 to 700 μg, J j ⁾ to 800 μg, 10 Io 900 μg, i 0 to 1000 μg, 100 to 200 μg, 1 GQ to 3(K) μg. 100 to 400 μgj 100 to 500 μg, 100 to 600 μg, ! 00 to 700 μg, 100 to 800 μg, 100 to 900 μg, 100 to 1000 μg, 100 to 1250 μg, 100 to 1500 μg, 100 to 1750 fig, ! 00 to 2000 μg, 100 to 2250 jig, 100 to 2500 μg, 100 to 2750 μg, 100 to 30(K) μg, 200 lo 300 μg, 200 to 400 μg, 20Cf to 500 μg.200 to 600 μg. 200 to 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μji, 200 to 1250 μg, 200 to 1500 μg. 200 to 1750 μg, 200 U* 2000 μg. 200 Io 2250 μg, 2|00 to 2500 μg, 200 to 2750 μg. 200 to 3000 μg, 30(5 to 400 μg. 300 to 500 μg. 300 id 600 μg, 300 to 700 μg, 300 to SOO μg, 300 to 000 μg, 300 to KK)O μg, 300 to 1250 fig, 300 to 1500 μg. 300 to 1750 μg, 300 to 2000 μg, 300 to 2250 μg, 300 to 2500 μg, 3:00 io 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg. 400 to 700 μg, 400 tcj 800 μg, 400 to 900 μg. 400 to 1000 μg, 400 to 1250 μg. 400 to 1500 μg, 400 to 1 750 jig, 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg. 400 to 2750 [jg, 400 to 3000 μg, $}0 to 600 μg, SOO to 700 μg, 500 to 800 μg, 500 io 900 μg. 500 to 1Ot)O μg. 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg. 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 Jig, 500 to 2750 μg, 500 to 3000 μg, 600 to 700 μg, 600 to 800 μg. 600 to 900 μg, 600 to 1000 μg, 600 Lo 1250 μg, 600 to 1500 μg, 600 to 1750 μg, 600 to 2000 μg, 60(j to 2250 μg, 600 Io 2500 μg. 600 to 2750 μg, 600 to 3000 μg. 700 to 800 μg, 700 jo 900 μg. 700 to 1000 μg, 700 to 1250 μg, 700 to 1500 μg, 700 io 1750 ug, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μg,

\ - 154 -

700 to 3000 μg, 800 to 900 μg, SOO to 1000 μg, 800 to 1250 μg, 800 to 1500 μg. HtK) in 1750 μg. 800 to 2000 μg, φθ to 2250 μg, SOU to 2500 μg, 800 to 2750 μg, 800 to 3000 μg,. 900 Io ) 000 μg, 9O0J to 1250 μg, 900 to i 500 μg, 9Qi) to 1750 μg. 90!) to 2000 μg. 900 Io 2250 μg, 900; to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, 1000 Io 1350 μg, 1000 to 1500 μg, 1 €100 to 1750 μg, J 000 to 2000 μg, 1000 to 2250 μg, 1000 to 2500 μg, 1000 to 2750 μg.i lOOO to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to H)O μg ₅ 5 to 20 μg, 5 to 100 μg. 50 μgl 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 000 μg, 650 μg, 700 μg _? 750 μg, 800 μg, 850 μg, 900 μg, 950 μg. K)OO μg, 1050 μg, 1 1JOO μg, 1 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1J550 μg. 1600 μg, 1650 μg, 1700 μg, 1750 μg. 1800 μg, 1850 μg, 1900 μg, 1950 μg, 2JOO0 μg. 2050 μg, 2100 μg, 2150 μg. 2200 μg, 2250 μg, 2300 M.g, 2350 μg, 2400 μg. 2J450 μg, 2500 μg, 2550 μg. 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg. φoQ μg. 2950 μg, 3000 μg. 3250 μg, 3500 μg, 3750 μg. 4000 μg.4250 μg, 4500 μg, 4|750 μg, 5000 μg of a peptide or agonist described herein and from 100 μg to 5000 μg (e.g. 100 μg, 200 μg, 300 μg _; 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, |000 μg, 1250 μg, 1500 μg, 1750 μg, 2000 μg, 2250 μg, 2500 μg, 2750 ug, 3000 μg. J500 μg, 4000 μg, 4500 μg, 5000 μg} of GranisetroB (Kytri ! ^• #). |

A dosage unit (e.g. an oral ddsage unit) can include, for example, irora 1 to 30 μg, 1 io 40 μg, 1 to 50 μg, ϊ io lOOjμg, 1 to 200 μg. 1 to 300 μg. 1 to 400 μg. 1 to 500 μg. 1 in 600 μg, \ to 700 μg, 1 to 8j)0 μg, 1 to 900 μg, I to 1000 μg, 10 to 30 μg, IO io 40 μg. 10 to 50 μg, 10 to 100 μgj 10 to 200 μg. 10 to 300 μg, 10 to 400 ug, 10 to 500 μg. H) to 600 μg, 10 to 700 μg, 10 to 800 μg, 10 to 900 μg, H) to 1(300 μg, 100 to 200 μg, ! 00 to 300 μg- 100 io 400 μgj 100 to 500 μg, 100 to 600 μg, 100 to 700 μg. 100 to 800 μg. 100 to 000 μg, 100 to J OCJO μg, KK) to 1250 μg. 100 to 1500 μg, 100 to 1 750 μg. H)O to 2000 μg, 100 to 2250 |ϊg, 100 to 2500 μg, 100 to 2750 μg, 100 to 3000 μg. 200 to 300 μg, 200 to 400 μg, 200 io 500 μg, 200 to 600 μg, 200 to 700 μg, 200 to 800 μg, 200 to 900 μg, 200 to 1000 μg. 2(K) to 1250 μg, 200 to 1500 μg, 200 to i 750 μg, 200 so 2000 μg, 200 to 2250 μg, 2 ¹ OO to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to ■■100 μg. 300 IO 500 μg, 300 id 600 μg, 300 to 700 μg, 300 to 800 μg, 300 to 900 μg,

; - 155 -

300 to !000 μg, 300 to 125(J jig, 300 to 1500 μg, 300 to 1750 μg, 300 to 2000 μg, 3(K) iv! 2250 μg.300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 to 600 μg, 400 to 700 μg.400 lϋ 800 μg.400 to 900 μg, 400 to 1000 μg, 400 to 1250 μg, 400 to 1500 μg, 400 Io 1750 jig, 400 Io 2000 μg, 400 to 2250 μg, 400 io 2500 μg, 400 to 2750 μg.400 to 3000 μg, 500 lo 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg, 500 io 1000 μg, 500 to 1250 μg, 500 to 1500 μg, 500 to 1750 μg, 5(K) to 2000 μg. 500 to 2250 μg.500 to 2500 μg, 500 to 2750 μg, 500 to 3000 μg, 600 io 700 μg, 600 io 800 μg, 600 io 900 μg, 600 to 1000 μg, 600 to 1250 μg.600to !500 μg, 600 io 1750 μg, 600 to 2000 μg, 600 to 2250 μg, 600 Io 2500 μg _; 600 to 2750 μg.60(5 to 3000 μg.700 to HOO μg.700 jo 900 μg, 700 to 1000 μg, 700 to 1250 μg.700 to i500 μg, 700 io 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 U) 2500 μg, 700 to 2750 μg. 700 to 3000 μg, 800 to 900 μg, 800 to 1000 μg, 800 to 1250 μg.800 to 1500 μg, 800 \o ) 750 μg.800 tυ 2000 μg, 800 to 2250 μg, SOO to 2500 μg, 800 to 2750 μg, 800 to

1850 μg, 1900 μg, 1950 μg, 2000 μg, 2050 μg, 2100 μg, 2150 μg, 2200 μg. 2250 μg, 2300 ue. 2350 ua, 2400 uu_ 2450 μ«. 2500 μ«, 2550 uii, 2600 us>. 2650 μ«, 2700 UCL, 2750 μg, 2800 μg, 2850 μg.2|900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg, 4500 μg, 4750 μg, 5000 μg of a peptide or agonist described herein and from 50 μg to 300p μg (e.g. 50 μg, 100 μg, 200 μg. 300 μg, 400 μg, 500 Mg, 600 μg, 700 μg, 800 μg, #0 μg _; 1000 μg, 1250 μg, 1500 μg, 1750 μg, 2000 μg, 2250 μg, 2500 μg, 2750 μg, 3000 μg) of Lotronex φ (aiosetron hydrochloride),

A dosage unit (e.g, an oral dφage unit) can include, for example, from i to 30 μg. 1 50 40 μg, 1 to 50 μg, 1 to HK)J μg, 1 to 200 μg, 1 to 300 μg. 1 So 400 μg, 1 to 500 μg. 1 i - 156-

to 600 μg, 1 to 700 μg _} I io 8f)0 μg, 1 Io 900 μg, 1 to 1000 μg, 10 to 30 μg, 10 to 40 μg, \ 0 to 50 μg., 10 to 100 ugi 10 to 200 μg, 10 to 30(J μg, 1 C) to 40« μg, 10 to 500 μg. ] 0 to 600 μg, ! 0 to 700 μg, 1 j) to SOO μg, i 0 to 900 μg, ! 0 to 1000 fig, 100 to 200 μg, 100 Io 300 μg, !00 to 400 μg| HH) to 50(1 μg. 100 to 600 μg, 100 to 700 μg, 100 to SO(J μg, tOO to 900 μg, 100 to I OQO μg, 100 to 1250 μg, i 00 io 1500 μg, H ⁾ O to 1750 μg, 100 it. 2000 μg, KK) to 2250 μg, 100 to 2500 μg, 100 to 2750 μg, 100 io 3000 μg, 200 u> 300 μg. 200 to 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 io 800 μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg. 200 to 1750 μg, 200 to 2000 μg, 200 to 2250 μg, i00 to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg, 300 to 500 μg, 300 to! 600 μg, 300 to 700 μg. 300 to 800 μg, 300 to 900 μg, 300 to 1000 μg, 300 to J 250 jig, 300 to 1500 μg, 3(K) io 1750 μg, 300 to 2000 μg, 300 in 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 io 500 μg, 400 to 600 μg, 400 to 700 μg, 400 IcI 800 μg, 400 to 900 μg, 400 to 1000 μg, 400 to 1250 μ.g. 400 to 1500 μg, 400 to 1750 jig, 400 to 2000 μg, 400 to 2250 μg. 400 to 2500 μg, 400 to 2750 μg, 400 to 3000 μg, 5:00 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg _s 500 to 1000 μg. 500 io 12J5O μg, 500 to 1500 μg, 500 to 1750 μg, 500 io 2000 μg. 500 to 2250 ug, 500 to 2500 jig, 500 to 2750 μg, 500 to 3000 μg, 600 io 700 μg, 600 m 800 μg. 600 to 900 μg, 6OQ to 1000 μg, 600 to 1250 μg, 600 to 1500 μg, 600 to 1750 μg, 600 to 2000 μg, 60CJ to 2250 μg, 600 to 2500 μg, 600 to 2750 μg. 600 to 300(5 _μg . ?0Q to HOO μg, 700 jo 900 μg, 700 to i 000 μg. 700 to 1250 μg. 700 to 1500 μg, 700 to 1750 μg. 700 to 2(K)O μg, 700 to 2250 μg, 700 to 2500 μg. 700 to 275Q μg, 700 to 3000 μg, 800 to 900 μj>. 800 to 1000 μg, 800 to 1250 μg, 800 to 1500 μg, 800 to 1750 μg, 800 to 2000 μg, φθ to 2250 μg, 800 to 2500 μg. 800 to 2750 μg, 800 to 3000 μg. 900 io 1000 μg. 900 io 1250 μg, 900 to 1500 μg, 900 to ! 750 μg, 900 Io 2000 μg, 900 Io 2250 μg. 90C| to 2500 μg, 900 to 2750 μg, 900 to 3000 μg, 1000 to S 250 μg, 1000 to 1500 μg, lφθ to 1750 μg, 1000 to 2000 μg, KKK) to 2250 μg. 1000 to 2500 μg, 1000 to 2750 μgJlOOO io 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to ! 00 μg, 5 io 20 μg. 5 io 100 μg, 50 μg, 100 μg _: 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg. 450 μg. 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, HK)O μg, 1050 μg, 1 lloO ug, ^" 1 150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1J550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg. 1800 μg, i - [57 -

1850 μg, 1900 μg, 1950 μg. 2D00 μg, 2050 μg, 2100 μg, 2150 μg. 2200 μg, 2250 μg, 2300 μg, 2350 μg, 24(H) μg.2^50 μg, 2500 μg, 2550 μg, 2600 μg. 2650 μg- 2700 μg, 2750 μg, 2800 μg, 2850 μg. 2p00 μg. 2950 μg, 3000 μg. 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg. 4500 μg. 4|?50 μg, 5000 μg of a peptide or agonist described herein and from 10 rng to 600 rag (eg, .10 rng, 20 mg, 30 røg, 40 mg, 50 mg, 60 mg, 70 Rig, SO mg, 90 mg, 100 nig, 325 mg. 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 350 rng. 400 mg, 450 mg, SOQ rng, 55(5 rag, 600 mg) of Xilaxan® {rifaximin}.

A <iosage unit (e.g. an oral dosage unit) can include, for example, from 1 to 30 μg, 1 Io 40 μg, I to 50 μg, 1 to 100 |μg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg, 1 to 500 μg. ) U) 600 μg, 1 to 700 μg, 1 to 800 μg, 1 to 900 μg, 1 to 1000 μg, 10 to 30 μg. 10 to 40 μg, 10 to 50 μg, 10 to ! 00 μgj ! 0 to 200 μg, 10 to 300 μg, 10 to 400 μg, 10 to 500 μg, 10 to 600 μg, !0 to 700 μg, f t to 800 μg, 10 to 900 μg, 10 to KH)O μg, 100 to 200 μg, 100 to 300 μg, 100 to 400 μgj 100 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 io 800 μg, 100 to 900 μg, 100 to 100D μg, 100 to 1250 μg. S OO io 1500 μg, 100 to 1750 μg, 100 io 2000 μg, 100 to 2250 μg, 100 to 2500 μg, 100 to 2750 μg, 100 to 3000 μg, 200 to 300 μg, 200 to 400 μg, 2C)θi to 500 μg, 200 to 600 μg, 200 to 700 μg. 2(K) to 800 μg, 200 to 900 μg. 200 to 1000 μg. 200 io 1250 μg, 200 fo 1500 μg, 200 to 1750 μg. 200 to 2000 μg. 200 to 2250 μg, 2PO to 2500 μg, 200 to 2750 μg, 200 to 3000 μg, 300 to 400 μg. 300 io 500 μg, 300 io! 600 μg, 300 to 700 μg. 300 to 800 μg, 300 to 900 fig, 300 to KK)O μg. 300 to 1250 |jιg, 300 to 1500 μg, 300 to ] 750 μg. 300 to 2000 μg, 300 to 2250 μg, 300 to 2500 μg. 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 io 600 μg. 400 to 700 μg. 400 to! 800 μg. 400 Io 900 μg, 400 to 1000 μg. 400 to 1250 μg, 400 to 1500 μg. 4Ot) to 1750 jig. 400 to 2000 μg, 400 to 2250 μg, 400 to 2500 μg, 400 io 2750 μg. 400 to 3000 μg, 5|00 to 600 μg, 500 to 700 μg, 500 to 800 μg, 500 to 900 μg, 500 to 1000 μg, 500 LO 12|50 μg. 500 io 1500 μg, 500 to 1750 μg, 500 to 2000 μg, 500 to 2250 μg, 500 to 2500 ijtg, 500 to 2750 μg, 500 io 3000 μg, 600 to 700 μg, 600 to 800 μg, 600 to 900 μg, 6ϋU to LOGO μg. 600 to 1250 μg, 600 to 1500 μg, 600 to 1750 μg, 600 to 2000 μg. 6GOj to 2250 μg, 600 to 2500 μg, 600 to 2750 μg. 600 to 3000 μg. 700 io 80t) μg, 700 tjo 900 μg, 700 to i 000 μg, 700 to ! 250 μg, 700 to 1500 μg, 700 to \ 750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg. 700 to 2750 μ.g, i - i 58 ^■■

700 to 3(KK) μg, 800 to 900 μg, SOO to 1000 μg, 800 to 1250 μg, 800 to 1500 μg, 800 !o 1750 μg.800 to 2000 μg, 800 to 2250 μg, 800 to 2500 μg, 800 to 2750 μg, 800 io 3000 μg, 900 lϋ 1000 μg.90()|io 1250 μg, 900 to 1500 μg, 900 to !750 μg, 900 to 2000 μg, 900 to 2250 μg, 9(M)IlO 2500 μg, 900 to 2750 μg, 900 to 3000 μg, 1000 to 1250 μg, 1000 to 1500 μg, 1000 to 1750 μg, 1000 to 2000 μg, 1000 to 2250 μg.1000 to 2500 μg, 1000 to 2750 μg. il000 to 3000 μg, 2 to 500 μg, 50 to 500 μg, 3 to 100 μg, 5 to 20 μg, 5 to 100 μg, 50 μgl 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 5(Ki μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1050 μg, HOO μg, 1150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg, 1800 μg, 1850 μg.1900 μg, 1950 μg, 2000 μg, 2050 μg, 2100 μg, 2150 μg, 2200 μg, 2250 μg, 2300 μ.g, 2350 μg, 2400 μg.2450 μg.2500 μg, 2550 μg, 2600 μg, 2650 μg, 2700 μg, 2750 μg, 2800 μg, 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μg, 4000 μg, 4250 μg.4500 μg, 4750 μg, 50(K) μg ofa polypeptide oragonist described herein and from 10 mgto 600 ing (eg, 10 ing, 20 mg, 30 mg, 40 mg.50 mg, 60 rag, 70 mg, 80 mg, 90 mg, 100 mg, 120 mg, 140 mg, 160 mg, 1 SO mg, 200 mg, 220 mg, 240 mg, 260 rag, 280 mg, 30(1 mg, 320 rag, 340 mg, 360 mg, 380 mg, 400 mg, 420 rag, 440 mg, 460 mg, 480 mgi, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg) of furoseiπide {Lasix}, j i

A dosage unit (e.g. an oral, initraveaous or intramuscular dosage unit) can include, for example, frøm 1 to 30 μg, 1 to 40 μg, ! to 50 μg. 1 to 100 μg, 1 to 200 μg, 1 to 300 μg, 1 to 400 μg, I to 500 μg, j to 600 μg, 1 to 700 μg, 1 to 800 μg. I to 900 μg, 1 to 1000 μg, 10 to 30 μg, 10 to 4φ μg, 10 to 50 μg, 10 to 100 μg, 10 to 200 μg, 10 to 300 μg, 10 to 400 μg, 10 to 500 μji, 10 to 600 μg, 10 io 700 μg. 10 to 800 μg, i 0 to 000 μι>, 10 to 1000 μg, KSO in 20(1 μg. 100 to 300 μg, 100 to 400 μg, 1.00 to 500 μg, 100 to 600 μg, 100 to 700 μg, 100 id 800 μg, 100 to 900 μg, 100 to 1000 μg, 100 to 1250 μg, 100 to I SOO μg, KK) UJ ! 75O μg, 100 to 2000 μg. 100 to 2250 μg, 100 to 2500 μg. I QO iυ 2750 μg, 100 to 3000 μg. 300 to 300 μg, 200 to 400 μg, 200 to 500 μg, 200 to 600 μg, 200 to 700 μg, 200 io SOO μg, 200 to 900 μg, 200 to 1000 μg, 200 to 1250 μg, 200 to 1500 μg, 200 io 1750 μg, 200 to 2000 μg, 200 to 2250 μg, 200 to 2500 μg, 200 to

! - 159 -

275(S μg, 200 to 3000 μg. 30θjio 400 μg, 300 to 500 μg, 300 to 600 μg, 300 to 700 μg, 300 to 800 μg. 300 to 900 μgJ3Q0 Io H)OO μg, 300 to 1250 μg, 300 to 1500 μg, 300 to \ 750 μg, 300 to 2000 μg, 300jto 2250 μg, 300 to 2500 μg, 300 to 2750 μg, 300 to 3000 μg, 400 to 500 μg, 400 tb 600 μg ₅ 400 to 700 μg, 40(5 io 800 μg, 400 to 900 μg, a 400 io 1000 μg, 400 to 1250 i|g _; 400 to ! 500 μg, 400 to i 750 μg, 400 to 2000 μg, 400 to 2250 μg, 400 io 2500 μg, 4Jrø to 2750 μg, 400 to 3000 μg, 500 to 600 μg, 500 to 700 μg. 500 to 800 μg, 500 to! 900 μg. 500 to 1000 μg, SOO to 1250 μg, 500 to 15(K) μg, 500 io 1750 fig, 500 to 20130 μg, 500 to 2250 μg, 500 to 25(X) μg, 500 to 2750 μg, 500 to 3CK)O μg, 600 to 700 μg, 600 to 800 μg, 600 to 900 μg. 600 to 1000 μg, 600 to C 1250 μg, 600 to 5500 μg, 6θθ|to ! 750 μg, 600 Io 2000 μg, 600 to 2250 μg, 600 to 2500 μg, 600 to 2750 μg. 60θ|to 3000 μg, 700 to 800 μg, 700 Io 900 μg, 700 to 1000 μg, 700 to 1250 μg, 700 to ISDO μg, 700 to 1750 μg, 700 to 2000 μg, 700 to 2250 μg, 700 to 2500 μg, 700 to 2750 μ|.g. 700 to 3000 μg, 800 to 900 μg ₅ SOO to 1000 μg, 800 to 1250 μg, 800 to ! 500 μg, SpO to 1750 μg, 800 to 2000 μg, 800 to 2250 μg, SOO to 5 250(1 μg, 800 to 2750 μg, 80θito 3000 μg, 900 to iOOϋ μg, 900 to 1250 μg, 900 to 1500 μg, 900 to 1750 μg, 90θito 20Oi) μg. 900 to 2250 μg, 900 to 2500 μg, 900 to 2750 μg, 900 to 3000 μg. 1000 to i 250 μg, 1000 to 1500 μg. i OiK) to 1750 μg, 1000 to 2000 μg, SOOO to 2250 μg, 1000 to 2500 μg, 1000 to 2750 μg, 1000 to 3000 μg. 2 to 500 μg, 50 Io 500 μ.g, 3 to iOij) μg, 5 to 20 μg, 5 to 100 μg, 50 μg. 100 μg, ! 50 μg, 200 0 μg, 250 μg, 300 μg, 350 μg. 400 μg, 450 μg, 500 μg, 550 μg. 600 μg. 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 jjig. 950 μg, 1000 μg, 1050 μg, 1 100 μg, 1 150 μg, 1200 μg, 1250 μg. 1300 μg. 1350 μg, 1400 μg, 1450 μg. 1500 μg, 1550 μg, 1600 μg, 1650 μg, 1 700 μg. 1750 μg, 1800 μjg, 1850 μg. 1900 μg, 1950 μg, 2000 μg. 2050 μg, 2100 μg. 2150 μg, 2200 μg _: 2250 μig, 2300 μg, 2350 μg, 2400 μg. 2450 μg. 2500 μg, 2550 6 μg, 2600 μg, 2650 μg, 2700 μjg, 2750 μg, 2800 μg _; 2850 μg, 2900 μg, 2950 μg, 3000 μg, 3250 μg, 3500 μg, 3750 μig, 4000 μg. 4250 μg, 4500 μg, 4750 μg, 5000 μg of a polypeptide or agonist described herein and from 0.2 mg to 10 røg (e.g. 0,2 nig, 0.4 mg, 0.5 mg, 0.75 mg, 1 nig, !j.5 mg, 2 mg, 2.5 mg, 3 ing, 3.5 mg. 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 rag, 6.5 mg. 7 mg. j-5 rag, S mg, 8.5 mg, 9 mg, 9,5 mg, H ⁾ mg) of 0 bumcianide (Bimiex®), i

- 160 -

The precise amount of each of the two or more active ingredients in a dosage unit will depend on the desired dosagcjof each component. Thus, it can be useful to create a dosage unit that will, when administered according to a particular dosage schedule (e.g., a dosage schedule specifying a certain number of units and a particular timing for administration), deliver tlip same dosage of each component as would be administered if the patient was being treated wife only a single component, in other circumstances, it might be desirable to create a dosage unit that will deliver a dosage of one or more components that is less than that which would be administered if the patient was being treated only with a single component. Finally, it might be desirable to create a dosage unit that will deliver a dosage of one or more components that is greater than that which would be administered if the patient was being treated only with a single component. This pharmaceutical composition can include additional ingredients including but not limited to the exeipients described herein. In certain embodiments, one or more therapeutic agents of the dosage unit may exist in an extended or control release fdrmuialion and additional therapeutic agents may not exist in extended release formulation. For example, a peptide or agonist described herein may exist in a controlled release formulation or extended release formulation in die same dosage unit with another agent that may or may not be m either a controlled release or extended release foirmuiation. Thus, in certain embodiments, it may be desirable to provide for the immediate release of one or more of the agents described herein, and the controlled release of one or more other agents.

In cert a i vi embodiments the dosage unit and daily dose are equivalent. In certain embodiments the dosage unit land the daily dose are not equivalent. In various embodiments, the dosage unit is administered twenty minutes prior to food consumption, twenty minutes! after food consumption, with food at anytime of the day, without food at anytime of the day, with food after an overnight fast (c.y. with breakfast), at bedtime after a low fat snack. In various embodiments, the dosage unit is administered once a day, tvyiee a day, three times a day, four times a day, five times a day, six times a day.

^■■ S 6 I ^■■

When two or more active ingredients are combined in single dosage form, chemical interactions between ihe aetivje ingredients may occur. For example, acidic and basic active ingredients can react with each other and acidic active ingredients can facilitate the degradation of acid labile substances, Thus, in certain dosage forms, acidic and basic substances can be physically separated as two distinct or isolated layers in a compressed tablet, or in ihe cφre and shell of a press-coated tablet. Additional agents that are compatible with aeidijc- as well as basic substances, have ihe flexibility of being placed in cither layer, ijπ certain multiple layer compositions at least one active ingredient can be enteπe-coatjed. In certain embodiments thereof at leasi one active ingredient cun be presented in a controlled release form. In certain embodiments where a combination of three lor more active substances arc used, they can be presented as physically isoϊaiφd segments of a compressed mutlilayer tablet, which can be optionally film coated]

The therapeutic combinations described herein can be formulated as a tablet or capsule comprising a plurality of beads, granules, or pellets, AIi active ingredients including the vitamins of the combination axe formulated into granules or beads or ^■ pellets thai are further coated jwith a protective coat, an enteric coat, or a film coat to avoid the possible chemical interactions. Granulation and costing of granules or beads is done using techniques weϋiknown to a person skilled in the art. At least one active ingredient can present in a controlled release form. Finally these coated granules or beads are filled into hard gelajliπ. capsules or compressed to form tablets.

^' he therapeutic combinations; described herein can be formulated as a capsule composing microtablets or rranitahlets of all active ingredients. Microtablets of the idividual agents can be prep bred using well known pharmaceutical procedures of ablet making like direct compression, dry granulation or wet granulation. Individual rdcrotahlets can be filled inUj hard gelatin capsules, A final dosage form may comprise one or more microtablets of each individual component. The mieroiablets mav be film coated or enterid coated.

- ! 62 -

^' Flic tberapeuUe combinations! described herein can be formulated as a capsule comprising one or more mierφtablets and powder, or one or more rnieroiablets and granules or beads. In order to!avoid interactions between drugs, some active ingredients of a said combination can be formulated as raicrotahieis and the others S filled into capsules as a powder, granules, or beads. The mieroiablets may be film coated or enteric coated. At lbast one active ingredient can be presented in controlled release form. !

^" The therapeutic combinations! described herein can be fomiiilated wherein the active 0 ingredients are distributed in the inner and caitεr phase of tablets, In an attempt to divide chemically incompatible components of proposed combination, few interacting components are converted in granules or beads using well known pharmaceutical procedures in prior art. The prepared granules or beads (inner phase) are then mixed with outer phase comprising fie remaining active ingredients and at least one ■5 pharmaceutically acceptable <jxoipient. The mixture thus comprising inner and outer phase is compressed into tablets or molded into tablets. The granules or beads can be controlled release or immediate release beads or granules, and can further be coated using an enteric polymer in aft aqueous or non-aqueous system, using methods and materials that are known in the art, 0 !

The therapeutic combination^ described herein can he formulated as single dosage unit comprising suitable buffi-ring agent. All powdered ingredients of said combination are mixed and a suitable quantity of one or more buffering agents is added to the blend to minimise possible interactions. 5 !

The agents described herein, alone or in combination, can he combined with any pharmaceutically acceptable earner or medium. Thus, they can be combined with materials feat do not produeeian adverse, allergic or otherwise unwanted reaction when administered to a patiedt. The carriers or mediums used can include solvents, 0 dsspersants, coatings, absorption promoting agents, controlled release agents, and one or more inert exάpients (which include starches, polyois, granulating agents,

! - 163 -

microerystalflne cellulose, diluents, lubricants, binders, disintegrating agents, and the like), etc. If desired, tablet dosages of the disclosed compositions may be coated by standard aqueous or nonaqueous techniques.

S Analgesic Agents in eombithόrapv

The peptides and ago.ni.sts described herein can be used, in combination therapy with an analgesic agent, e.g., an analgesic compound or an analgesic peptide. These peptides and compounds can be administered with the peptides of the disclosure (simultaneously or sequentially). They can also be optionally covalently linked or iα attached to an agent described herein to create therapeutic conjugates. Among the useiiii analgesic agents are: Oa channel blockers, 5BT receptor antagonists (for example 5HT3, 5HT4 and 5HTl receptor antagonists), opioid receptor agonists (loperamide, feάotozine, and fentaπyi), NKl receptor antagonists, CCK receptor agonists (e.g., Joxigfumide), NKI receptor antagonists, NK3 receptor antagonists,

15 norepincphrine-serotonjn reuptake inhibitors (NSRI), vanilloid and caiiuahanokl receptor agonists, and sialorphin. Analgesics agents in the various classes axe described in the literature. i

Among the useful analgesic peptides are sialorphin-related peptides, including those

20 comprising the ammo acid sequence QHNPR (SEQ ID NO; ), including: VQHNFR (SEQ ID NO: ); VRQHNFR (SEQ ID NO: ); VRGQHNFR (SEQ ID NO: ); YRGPQHNPR (SEQ ID NOJ }; VRGPRQHNPR (SEQ ID NO: j; VRGPRRQBNPR (SEQ 3D M); }; and RQHNPR (SEQ ID NO: ), Siaiorphin- relaied peptides bind to neprilysin and inhibit .πeprilysin-niediated breakdown of

25 substance P and Met-enkephalin. Thus, compounds or peptides that are inhibitors of •leprilysin are uselisl analgesic agents which can be administered with the peptides of the disclosure in a co-therapy or linked to the peptides of the disclosure, e.g., by a eiivalent bond. Sialophin am! related peptides are described in U.S. Patent 6,589,750; U.S. 20030078200 Al; and WO 02/051435 A2.

30

^■ 164 -

Opioid receptor antagonists and agonists can be administered with the peptides of the disclosure in co-therapy or linked to the agent of the disclosure, e.g., by a covafent bond. For example, opioid receptor antagonists such as naloxone, naltrexone, methyl naloxone, naimefene. cypridilne, beta funaHrexamine, naloxonazine, πaltriπdole, and jjor-biπaliorphimine are thought to be useful in the treatment of IBS, it can be useful to formulate opioid antagonists of this type is a delayed and sustained release formulation such thai initial release of the antagonist ss in the mid to distal small intestine ami/or ascending colon. Such antagonists are described rn WQ 01 /321 SO A2, Enkephalin peniapeptkie (HOE825; T>τ~D-Lys-Gly~Phe-L-hornoserine) is an agonist ol ^' fhc Biu and delta opioid receptors and is thought to be useful for increasing intestinal motility {Eur J. Pharm. 219:445, 1992), and this peptide can be used in conjunction with the peptides of the disclosure. Also useful is trimebutine which is thought to bind to rnu/deita/kappa opioid receptors and activate release of raotilin and modulate the release of gastrin, vasoactive intestinal peptide, gastrin and glucagons. Kappa opioid receptor agonists such as fedotozine, asimadoiine, and ketocvc ^' iazocinc, and compounds described in WO(BWOSl and WOOS/007626 can be used with or linked to the polypeptides described herein. Sn addition, mo opioid receptor agonists such as morphine, diphenyloxylate, frakefai'nide {H-ϊ>τ-D~Ala-Phe{F)-Pbc-NH2; WO 01/019849 Ai ) and loperamide can be used.

Tyr-Arg {kyotorphiri} is a dipiepiide that acts by stimulating the release of met - enkephalins to elicit an analgesic effect (J. Biol. Ch em 262:8165, 19S7), Kyotorphin can be used with or linked to the peptides of the disclosure.

C hfoniograniπ -derived peptide (CgA 47-66; see, e.g., Ghia et al. 2004 Regulatory Peptides 1 19:199} can be used with or linked to the peptides of the disclosure.

CCK. receptor agonists such as oacruiem from amphibians and other species are useful analgesic agents that can be used with or linked to the peptides of the disclosure. i

- 165 -

Coootoxm peptides represent !a large class of analgesic peptides that act at voltage gated Ca channels, NMDA receptors or nicotinic receptors. These peptides can be used with or linked to the peptides of the disclosure.

Peptide analogs of ihymulin (FR Application 2830451 ) can have analgesic activity and can be used with or linked to the peptides of the disclosure.

CXIv (C"€K.a ox CCKb) recepfør antagonists, including loxiglurnide and dexloxigiuniide (the R-isomer of loxiglumide) (WO 88/05774} can have analgesic activity and can be used with ^: or linked to the peptides of the disclosure.

Other useful analgesic agents, include S-HT4 agonists such as tegaseroά (Zehiorm®), mosapridc. metoclopraniidc, iacopride, cisapride, renzapride, benzimidazolone derivatives such as BIMIJ 1 aid BiMU 8, and lirexapride. Such agonists are described in: EP 132 H42 Al, WO U3/0S3432AL EP 505322 A L EP 505322 BL US 5,510,353, EP 507672 AL EP 507672 Bl, and US 5,273,983.

Calcium channel blockers such as zieonotide and related compounds described iα, for ωsample, EF625162B1 , US 5364,842, US 5M7λ54, US 5,824,645, US 5,859.186. US 5,094,305, US 6,087,091, US 6,136,786, WO 93/13128 AL EP 1336409 AL EP S35126 A L EP 835126 BL US 5,795,864. US 5,891 ,849, US 6,054,429, WO 97/0 i 351 A 1 , can be used with or linked to the peptides of the disclosure.

Various antagonists of the NK -I . NK-2, and NK-3 reeepfors (for a review see Giardina et a!. 2003 Drugs 6: 758) can be can be used with or linked to the peptides of trie dbelosure.

NK ! recepLor antagonists such as: aprepUaπt (Merck & Co Iπc), vofopitaiu, eziopitant (Pfizer, foe), R-673 {Hoffinann-La Roche Ltd), SR-48968 (Sanolϊ Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo Smith Kline), TAK-637 ( ^' Fakeda/ Abbot), SR- 14033, and related compounds described in, for example. EP i - ) 66 -

S73753 Al , US 20010006972 Al , I)S 2003010941 ? Ai. WO 01/52844 AL cars be used with or linked to the peptides of the disclosure.

NK1-2 receptor artiagonists such as ncpadutant (Mcnarinϊ Ricerche SpA), saredutani (Samtfi-Synthdabo), GW597599 (Glaxo Smith Kline). SR- 144190 (Sanofi-

Synthelabo) and UK-290795 (Pfizer Inc) can be used with or linked to the peptides of the disclosure.

NK3 receptor antagonists such as σsanetant (SR-14280! ; Sanoiϊ-Syruhdaho), SSR- 241 586. tainetaitt arid related compounds described in, for example, WC) 02/094! 87 λ2, HP 876347 Al, WO 97/21680 AU US 6,277,862. WO 98/1 1090, WO 95/2841 SJ, WO 07/ 19927, and Boden et a!. U Med Chenu 39:16A4-75, 1^%} can be used with or imked lo the peptides oi ^' tlie disclosure,

Norcpmephrinc-serotoπin reuptake inhibitors fNSRf) such as røilnaciρraτi and related compounds described in WO 0.V077897 Al can be used with or linked to the peptides of the disclosure.

VaniSloid receptor antagonists such as arvanil and related eompouds described in WO 01 ^;I 64212 Al can be used with or linked to the peptides of the disclosure.

1 he analgesic peptides and compounds can be administered with the peptides and agonists of the disclosure (simultaneously or sequentially). The analgesic agents can also be eovaieniiy linked to the peptides and agonists of the disclosure to create therapeutic conjugates. Where the analgesic is a peptide and is covalently linked to an agent described herein ihe resulting peptide may also include at bast one trypsin cleavage site. When present vnthin the peptide, the analgesic peptide may be preceded b>Mif M is at the carboxy terminus) or followed by (if it. Is at the amino terminus) a trypsin cleavage site that allows release of the analgesic peptide.

- 167 -

In <kldnhτ, ro Malorphuw dated peptides, analgesic peptides include AspPhe, endomorphisv L ondomoφhm-2, tioeisuhn, dalaigin, ϊupron, and substance S ^s

C Dsabcks, ObCMt) ami n»theijϊisυrdcrs

Phαϊ macv'uucal compositions eompnsing at least fv,o of 1 ) an agent *h,u shoiulatcs ' - ^■ jo production of eλMP (eg „ giueagort-liiα* peptide 1 (GLP-I )). 2} sα\ agent that the ikvradatϊOB oi a cvclsc nucleotide (c y , a pKssphodtesteusc πilubuor), and 3) a ocptjdc or agonist of the disdosuro tor treating diabetes and obcMiy.

1C Such JTϊ connection %itb paucrcafic disejses (chππuc pancreatitis paiieieaseetoπn, ^'s emt>cbromau>sis) or ondoerme diseases {auomegah, Cushmg ^' s s>τ3dπ^mc, obcoch omiie>tøma or hyperthyretisis). drug-uiducul hsperglx ceniiαs ^lueo^e la tjietauev. hypcrglycerrias. dyshpυprot anemias. adipt)sUy,

I he nifabiior can be specific foi a particular (c,s/ . utυup IH OJ Gioup phosphodiesterase; snhibuoi , sαch as jMpvHO«iJi". cnprotX lliπus and or !BMX. Speejik ρiθhp1k ^« d? esterase

?λ nhibifors \\ hιc.i mhib^ group Hl pliosphodiestciascs Jc ⁽ J MF- inhibited phospiiAh^teuscs). including intiolidduc {L Y1951 1 ^* > K ci!osUπ ^> κie ^OFC 56^s ^> ), !i \a/inwo ( RS S2S5λ), \ -590, mwodarie (C1914). SKF 9-120, qua/mono. K M I i0, tjiosia/olc, hemorandane (RWJ 2286"), sigua/odαne (isKivh 94 8361 milrinone (WIN 47203), enmsmcme (MDL 7iM3 κ

Z5 r'HTsobendar.e (J^D-CG 1 155, Vt«. ^'! l- I 54, satennone t BPF S634), sdma^ole (λR I V.5), I I)-I ύ 2 12, moiapizone. piroxπnone, and Kl i 18233 can be useful. In addition, phosphodiesterase inbibnors whielϊ inhibit group IV phospLodiesteia^es iC VVIP-speeuii, phosphodiesterases), such as rohpiam ?!K O2 ^' 'ϊ 1 ; pyrmlidone), :i)udazoitcim ne (Rt) 204724), eta/olate (SQ 65442), denbuiylhnc (BRI 30892 K

'όθ lCϊf>3 i '> ^? , and RP ^" 340I can be used

It^

Other Kgems fur Iλse i^LCombithcπiEi

AJ.-o wjtmr the disclosure are pharmaceutical compositions eompπ _^ mg a peptide os ;,«omsts- o. ^' ώj d ⁱ sclosure and a second therapeutic agent 1 he .second therapex.*se . ^« enι cm be jdmniMered to treat any condition for w hich it ¹ S use nil uicsuduig

^■■ > ^judiUo/is tLn ate \λ* eurtMdeieJ to be the primary indication for tredtmen. v\ 4h the second ιbe> apeuiK, t.gent 1 he second therapeutic agent can be adminisslutcd s.tnalκinot>usl) or _^ «,ueotially i'he second theiapcuti _* . ageni can he ctn ^lentiy li'iκed ^t 1 i>e peptide;* »jnd αgorπst;* of Ac disclosure to create α iherapeαuc eonju^αie W hen the seeom. theiαpeutic agent '$ another peptide a linker uicluuuig t io^e i ) uc^vπbed urc5n may he used bctvλ een the peptide of the disclosure and ή-c second ^* jc;apeu ^* ;e peptide

}- sanip.es or addmoπaϊ therapeutic agents to treat ga^πυmte.sunai and other JiH>rdet> melude

15 age: I ⁺ ^ to neat constipation {c g.. a elilondc channel aetis atoi su^h ^ the bicxbc iaU> ac\L _^ ubipioston*. (iormerK know n as SFl-021 1 , Sucanipo Ph irπtaeeuheais, tne , Bethe&da, M l)), a laκatt\c (eg a hulk-forming laxatn c (e.g. nonstaich .xih ^iieehαπdes, i\^k>nel Cablet (poh,earbophil calcium). Plantαgo O ^χ ata>? ¹ , Iλjuaiaet.n^ K'aleruin PoKcubophsDV fiber fc g MBhR ⁽ XJN^ (Caietum

20 PolveϊjabopKil). λπ i ^nurje kixame, a stmiυknt Saxatn e (such as Jiphorλ lnclh .me* _\ c g bisac* M-JsI ;, anthiaquinoncs (e.g ctscaia &eαnc K and sutfactant ]ax.rj\e- ^; (v ^x L cantor of , diHTfeatcs), an emoU.ent luhπtabπg ageut (sαvh a^ minora! oiL ^SX CCγΉC i'\ά dui'u\ites). \huύ a\ s Buantree Lαboratoπet., Branitzec \i \K de\io\:glumme I crest I αin .'-atOE 'e.s. also kno^ n as CR 2*)\ 7 RotL'.pharm tRotm Repeater

?-> i aboraurnitn ^pA i), saime laxatn es, cnenus, suρpt>.sUoπes. ααd CR . ^" "00 (RottaphaπiJ ^ Rotta Resetreii Lahoπitoiuini SpA),

<κ AI i edueiiψ agents sucn d^ pioion pump inhibitoi a (c g , oir epra<-.o!e t Pr lose. '$) esotnepiti/oic ( KexiuuvK;). Luisopia/ole (Prevaeid^). panti)praλ'ic; Pro:ont\λ ) and 30 MbepλL- ojj i \ciplicxø)> ixnά f li.>tamiπe ϊ I2-reeeptoi antagur.\->t (abo kr.im u as H2

^■ Tceptor blockers m.iiidiug cimeudine, ranitidine, lainotidme and ;iι/αtιdino),

- 169 -

prokmetic agents including itopride. octreotide, bethaneeooL mctoclopramide (Regiamφ), domperidone (Motϋnim®), erythromycin (a.nd derivatives thereof) or ci sapri dc { propuIsklφ ^*5 ) ;

Prokinetieiπ polypeptides horaojogs, variants anil chimeras thereof including thυse described in US 7,052,674 which can be used with or linked io the polypeptides described herein;

pro-niodlϋy agents such as the vasostatin-dcrived peptide, chromogranin A (4-16) (.see, e.g., Gbia el al. 2004 Regulatory Peptides 121:31 } or røotiliπ agonists (e.g., GM- 61 1 or rnitemcinal iumarale) or noeicepiin/Orphariin FQ receptor modulators {US200S016QO! ?);

other peptides which can bind to and/or activate GC-C including those described in ϋS2O05O2870f;7;

complete or partial 5HT (e.g. SHTI, 5Hη, 5HT3, 5HT4) receptor agonisls or antagonists (ineiuding 5HTiA antagonists (e.g. AGi-001 f AG! therapeutics). 5HT2B antagonist (e.g. PGN 1091 arid FGNl 164 (Phaπnagene Laboratories Limited}, and SUVA rcccpior agonists (such as tcgascrod (ZELNORM®). prucalopride. nκ>sapridc. metoclsfpramido, zacopride, cisapride, renzapride, benxiπiidazoloac derivatives such as BlMU J and BlMU 8. and iirexapride). Such agonists'modulatos arc deseribcd in: KP 1321 142 AI , WO 03/053432 A K EP 505322 Al, EP 505322 Bl , US 5,510353, HP 507672 Al, EP 507672 Bl , US 5 _; 273,983, and IJS 6,951,867); 5HT3 receptor r;g.ι>uist.« such us MK.O-733; and 5MI3 receptor antagonists sueh as DDP-225 (MCI- 225, DyiKigt'π Pharmaeeutieals. Inc.), eilansetron fCairaactm€'k alosctron (■..ulroiicx©), Ondansetron IiOl (2otranβ>), Dolasetrυn {ANZHMRT-®}, paicmosetron { Aloxi(E>), Granisetroπ (fCytol®), YM060(raτπosetron; Asteϋas Pharrna Inc.;

- ! 70 -

rainosetroπ. «iay be given as a daily dose of 0.002 to 0.02 mg as described in BPO 1588707) and ATI-7GGG (λryx Therapeutics, Santa Clara CA);

muscarinic receptor agonists;

luiti-iπfiammatory agents;

antispasmodics Including but not limited to anticholinergic drugs (like dicyclomine (e.g. COUϊΏCX®. Foπrmlex®, Lornine®, ProtyloKD, Viscerol®, BentyKS, Bentyloi®), hyoscyamine (e.g. IB-Slat®, Nulev®, Levsiπ®, Levbϊdάk Lcvsiπcλ Timecapsφ, Levsin/SL®, Anaspaz®, A-Spas S/L®, Cysiospazφ. { ^■ 'ystospaz-M ^' i\ Dαnnamar®, Colidrυps Liquid Pediatric®, Gastroscd'®, .Hyco HlixirφO, MVOSOMB ⁾ , lϊyospazφ, Hyosyne®, Losamine®, Medispazφ, Ncosolφ, Spacol®, Spasdeiφ, Symax®, Symax SI,®}, Doπnatal (e.g. Donnatal Extentabs®}, ciidinium (e.g. Quarzan, in combination with Librium ^::: LibraxK methanthdine (e.g. Baritbine), Mepensr.otate (e.g. Canti!}. homatropine (e.g. hycodan. fioniapin), Propanthdinc bromide (e.g. Pro-Banthine), Glycopyrrolate (e.g. Robinul®, Robino! I ^' orteS}- scopolamine (e.g. Traiisderai-ScopQi), Transderm-V®}, hyαsine-N- buiyibroniide (e.g. Buseopan©}, Pirenzepine (e.g. Gastrozepin®} Propantheline Bromide (e.g. Fropanthei®}, dicycloverine {e.g. Merbeπtyl®) , gfyeopyrronium broπude (e.g. GiycopyrroiaLe®} ₅ hyoscine bydrobroraide , hyoscine mcmobromide . methantheiiniuro, ; ^: uid octatropine); peppermint oil; and direct smooth muscle relaxants like cimetrυpiuπi btoraidt, mebeverine (DUSPATAL®, DUSPATALIN®, COLOFA(^ MR®. COLOTAL®). otiionium bromide (oeiiloniurn), pinaverium (e.g. DiceCel® (pinavcrium bromide; Solvay S. A.)), Spasfon® (hydrated phloroglucino! and triτnethylρhloroglucinol)and trimebυliae (including t ^ή mcbutine niakate (Modulon®);

antidepressants, including but not limited to tbose listed herein, as well as tricyclic antidepressants like amtiriptyiine (Elavil®), desipramine (Norpramin®), irøipramiπc

- ϊ ? ! ^■ -

(To Irani 1®), amoxapϊne (Asendin®), nortriptyline; the selective serotonin reuptake inhibitors (SSRFs _. i like paroxetine (Paxil©), fluoxetine (Prozac®-}, sertraline (Zoloft®), and citralopram (Celexaφ); and others like doxepin (Smequaπ®} and ! ra/.odone (Desyrel®);

centrally-acting analgesic agents such as opioid receptor agonists, opioid receptor antagonists {e.g., naltrexone);

agents for the treatment of Inflammatory bowel disease;

agents for she treatment of Crohn's disease and/or ulcerative colitis (e.g., alequel (Bnzo Siochem, Inc.; Farmingsale, NY), the anti-inflaπunatory peptide RDP5S (Genzymc, inc.; Cambridge, MA), and TRAFIC El-EN ^VM (ChemoCentryx, Inc.; San Carlos, CA);

agents that treat gastrointestinal or visceral pain;

agents that increase eGMP levels (as described in IJS2004012! 994) like adrenergic receptor antagonists, dopamine receptor agonists and PDE (phosphodiesterase) inhibitors including hut not limited to those disclosed herein;

purgatives that draw tluids to the intestine (e.g., ViSϊCQL®, a combination of. sodium phosphate monobasic monohydrate and sodium phosphate dibasic anhydπite);

Corticotropin Releasing Factor (CRF) receptor antagonists (including NBϊ-3404). (Neurocrine Biosciences. San Diego, CA), CR1-S9-41 , astressin, R 121919 (Janssen Pharmaceutica), CPl 54,526, NBi-27914, Antalarmin, DMP696 (Bristol-Myers Squibb! CP-3 I 63U (Piizcr, fee,}, SB723620 (GSK), GWS76008 (Neurocrine/Glaxo Smith Kline), ONO-2333Ms (Ono Pharmaceuticals), TS-041 (Janssen), AAG561 (Novariis) and those disclosed in US 5,063,245, US 5,861 ,398, US2004Q224964,

US20&40! 98726, U S20040176400, US20040171607, US200401 ϊ0815, IJS20040006066, and US20050209253);

gkicagon-ϋke peptides (glp- !} and analogues thereof (including excixl»i~4 and GTP- 5 010 (Gastroiech Pharma A)) and inhibitors of DFP-IV (DPP-IV mediates the inactivatioH of glp- 1 )ι

i-allsop&m, enantioπiericaϊly-pure K-toπsoparti, and pharmaceuticaify-aeceptable salts thereof (US 20040229867);

10 tricyclic anti-depressants of the dibenzothiazepine type including but not limited to Dextøfisopara® (Vela Pharmaceuticals), tianeptine (StablαnOi ¹ ) and other agents described i v? US 6,683,072;

16 (E)-4 { i,3bis(cyc!ohexyltxiethyi)-L2,34,-tc ^> trahydro-2,6~diono~9H-puήn-S-yl}ciniiamic acid nortacthylenc glycol methyl ether ester and related compounds described in WO 02/067942;

the prohiofic PROBACTRLXOϊ ⁾ (The BioBalan.ee Corporation; New York, NY) which 20 contains microorganisms useful in the treatment of gastrointestinal disorders;

ant idiarrhcii! drugs induding but not limited to loperamide { Imodium, Pepto Diarrhea), diphenoxylate with atropine {Lomotil, Lomoeot), cholestyramine (Questran, (^hoiybar). atropine (C.-o-Phenotrope. DiarsαL Diphenoxylate, Loieπe, 2.5 [..ogen, L«nox, Vi-Atro, atropine sulfate injectioϊi} and Xifaxan® (rifaximin; Salix PliarniaeeuticiiLs Ltd), TZF-201 ( ^" Traπzyme P harm a inc.), the neuronal acetylcholine receptor (nA€hR) blocker AG[-0()4 (AGI therapeutics), and bismuth subsalicylate (Pepto-bisraol);

30 anxiolytic drugs including but not limited toAtivan (lorazepani), alprazolam

[ ^' Xanax®), chlordiazcpoxide/clidiniura (ϋbriumφ), Libras®), clonazepam

- ϊ?3 -

(KlonopύvS), clorazepate (Tranxene®), diazepam (Valium®), estazolam (ProSom®), fksrazepam (Balrnane®). oxazepam (Serax®}, prazepam (Centrax®), teinaxepam (ResiotHly}, triazolam (Halciort®;

5 Bedelk® (Mσntmoriilomte beideϋitic; ϊpsen Ltd), Solvay SLV332 (ArQuIe Inc.), YKF (SK Ph&rrπa), Asiraadoline (lioga Phamuieeuiieals/Merek), AGϊ-003 (AG! Therapeutics};

neurokinin antagonists including those described in U 820060040950;

potassium channel modulators including those described in US?,002,015;

the serotonin modulator AZD7371 (AstraZeneea PIc);

:5 M 3 muscarinic receptor antagonists such as daπieπaem (Enabkx; Novaxlis ACJ and /arnitcnitcin (P fixer);

herbal ami natural therapies including but not liniit€d to acidophilus, chamomile tea, L-venhig primrose oil, fennel seeds, wormwood, comfrey, and compounds of Bao-Ji- 20 Wan (magfiOioL, honokioϊ, imperatorie. and isoimpcratorin) as m LJS6923992; and

Cv)røpositiθiis comprising lysine and an anti-stress agent for the treatment of irritable bowel syndrome as described in EPOl 550443.

The peptides and agonists described herein can be used in. combination

£5 therapy with insulin and related compounds including primate, rodent, or rabbit insulin including biologically active variants thereof including allelic variants, more preferably human insulin available in recombinant form, Sources of human insulin include pharmaceutically acceptable and sterile formulations such as those available from Eh Lilly (Indianapolis, Ind. 46285) as r-Jumu!in ^{l ivi} (human insulin rDNA origin).

30 See die THE PHYSICIAN'S DBSK REFERENCE, 55.sup.th Ed. {2001 } Medical Economics, Thomson Healthcare (disclosing other suitable human insulins). The

^■■ 174 -

peptides and agonists described herein can also be used in combination therapy with agents thai can boost, insulin effects or levels of a subject upon administration, e.g. glipizide and/or rosigliiaxone. The peptides and agcmistsdescribed herein can be used in combit ^' herapy with SYM LIN® (praraliπtide acetate) and Exenatide® (synthetic exendin-4; a 39 aa peptide).

The peptides and agonists described herein can also be used in combination therapy with agents (e.g., Entereg ^!>/1 (alvimopan; forrneτi> called adolor/ ADL, S- 2698), eonivsptan and related agents describe in US 6,645,959} used for the treatment of postoperative ileus and other disorders.

The peptides and agonists described herein can be used in. combination therap> with an anti-hypertensive agent including but not limited to; ! I } diuretics, such as thiazides, including chlorthalidone, ehlorthiaϊdde, dichlorophenamide, hydroflumethiazide, indaparnide, polythw.ide, and hydrochlorothiazide; loop diuretics, such as bumetanide, ethacrynic acid, furosemide, wnύ torserrhde; potassium sparing agents, such as amiloride, and mamiercae; carbonic anhydrasc inhibitors, osmotics(such as glycerin) and aldosterone antagonists, sue Ii as spirunol&ctDne, epirsnone. and the like; (2) beta-adrenergic blockers such as acebutolol, atenolol, betaxoloi, bevantoioL. bisoproIoL bopindoloi. earteoloL can-cdiloK celiproloL esmoloK indenobl, nietaproloL nadolol, nebivolol. penbutolol, pindolol, propanoic!, sotak>l, tertatoIoL tilisoloi, and timolol, and the like; ;3) calcium channel blockers such as amlodipine, araaiclipine, azeinidipine, hαrnidipine, benidipine, bepridil, einakiipine, eievidipine, diϊtiaxem, eforsidrpine, ielodipine. galioparnil, ssradipiπc, lacidipisie. ieniifdipiπe, iercanidipine, nicardipine, nifedipine, niivadipine, nimodepine. nisoldipine, nitrendipine, manklipine, pranidipinSv and verapamil, and the like; (4) angiotensin converting enzyme (ACB) inhibitors such as benazepril; captopπl; ceranapril: cilazapril: delapril; eπaiapril; enalopril; fosinopril; iraidaprii; lisinopril;

losksopril; moexipril; quinapril;. quiiiapriiat; ramiprii; peπndoprii; psriπdropril; quaoipnl; spirapril; tenocaprit; trandolapril and zofenoprii, and the like; (S) neutral eπdopeptklase inhibitors such as omapairilat, ca.dax.atri! and ecadotril, fosidotrih sarapatriJat, AVε7688, ER4O3O, and the like; (6) endothelin antagonists such as tezoseπtan, A308165, and YM62899, and the hke;

(?) vasodilators such as hydralazine, clonidine, minoxidil; and aicotinyl alcohol, arid the like;

(8) angiotensin SI receptor antagonists such as aprosaπan, candesart&n, eprosartan, irbesartan, iosarttui, oiπiesartan, pratosarian. tasosartan. telmisailan, vaisartan, a«d hXP-3137. FI682SK, and RNH6270, and the Hke;

(9) tt/j) adrenergic blockers such as nipradiloL arotinoiol and amosulalol, and the like;

{10} alpha 1 blockers, such as terazosin, urapidil, prazosin, tamsulosm, bunaxosm, tπmazosin, doxazosin, naftopidii, indoramin, WHP 164, and XENOiO, and the like;

{1 1} alpha 2 agonists such as iofexidine, tiamcnidme, rnoxonsdinc. nlmenidme and guanoben/, and the like;

(12) aldosterone inhibitors, and the like: and

{ 13 ) angiopυietm-2-biπdiπg agents such as those disclosed hi VVO03.030833.

Specific anti-hypertensive agents that can be used in eonibmaiioπ with oeptidcs and a *g»o^ nists described herein include, but are not lirπiiyd to;

diuretics, such as thiazides {e.g., chlorthalidone, eyclothiazide (CAS RN 2259-96-3), chlorothiazide (CAS RN 72956-09-3, which maybe prepared as disclosed in

US2809194), diehlorophenarrade, hydroflumethiazide, indapamide, poiythiazide, bendrofitαncthazide, methyclothaϋide, pol>thiazide. triehSormethaxide, chlorthalidone, indapamide, metolazoπe, quinethazone, alihiazide {CAS RN S5S8-16- 9, which may be prepared as disclosed in British Patent No. 902.658), benztlnazide (CAS RN 91-33-8, which may be prepared as disclosed in US3 108097), buthiazide (which may be prepared as disclosed in British Patent Nos. 861,3(S?), and

- 176 -

hydrochlorothiazide), loop diuretics (e.g. bimiεiaπkie, ethaerymc add. furoseraide, and loraxemide), potassium sparing agents (e.g. arniloride, and triamterene (CAS Number 396-0 !-O)), and aldosterone antagonists (e.g. spironolactone (CAS Number 52-01-7), epirenone, and the like); ^-adrenergic blockers such as Arniodaroπe (Coniaroo.e, Paeeronε), bunolol hydrochloride (CAS RN 3 ϊ 969-05-8, Parke-Oavis), aoebutolol (±N-|3 ^» Acetyi-4-[2-hydroxy-3-[( l melhylethyl)arainojpropoxyjphenyl]- buianamide, or (±)-3'-λθ£;tyi-4'~[2-hydroxy -3-(isopropySammo) propoxy] buiyramlide), aeebutoiol hydrochloride (e.g. Sectral®, Wyeth-Ayersi), alprenoloi hydrochloride (CAS RN 13707-88-5 see Netherlands Patent Application No. 6,605,692), atenolol (e.g. Tenormin®, AstraZeneca), carteoioi hydrochloride (e.g. Cmxolφ Fϋmtab®, Abbott), CeUproloi hydrochloπde (CAS RN 57470-78-7, also sec in US4G34009), cetamolol hydrochloride (CAS RN 77590-05-5, see also US405%22.k iabetaloi hydrochloride (e,g. Normodyne®, Scheriagj, csmolαl jivdrochlϋride (e.g. Brevibloc® .Baxter), levobetaxolol hydrochloride (e.g. Betaxon™ Ophthalmic Suspension, Alcon), ϊevobυnolol hydrochloride (e.g. Bstagan®

Liquitliniφ with C CAPS ¹ Compliance Cap, Allergasi), nadolol (e.g. Nadolol, Mylan), prautoU)! (CAS RN 6673-35-4, see also US340S387), propranolol hydrochloride (CAS RN 318-98-9), sotalol hydrochloride (e.g. Betapace AP^Berlex), timolol (2- Propaπol,l-i(] ,1 -diiτieihyJethyl)amino]-3-[[4-4(4-moφho]inyl)- l,2,5-thiadia>:ol-3- yijoxyj-, hemihydπite. (S)-, CAS RN 9I524-I6-2), timolol oialeate (S}-!-[(! ₅ i- dimeihyiethyi} aminoj-3-[[4- (4-morphoHnyl)-] ,2,5-thi.adiazol ~3- yl] oxy]-2-pjOpaun)] (Z}-2-buteocdioLitc { 1 : 1 ) sail, CAS RN 26921-17-5), bisoprolol (2-Propanoi, 1 -[4-[[2- I l-mt;thy!cthoxy)t:thoxy]-inetliy!]phenoxyl]-3-[{ l-ϊτieth- yletby!}amino!--, (±), CAS RN 66722-44-9), bisoprolol fumarate {s«ch as (±)-l-|4-[[2-(l -Metiwietboxy) ethoxy ]mcth yl ]pheπoxy]-3-[( I -metbylethy 1 }amino]-2-propanol t E) -2-butenedioate (2: 1 } (salt,;, e.g., Zebeta™ , Lederle Consumer), ncbivalol (2B- l~Benzϋpyran--2- methanol, aa -{ irn.inobis(.methy!cnc)]bis[6-flt,κ>ro-3 ,4-dihydro-, CAS RN 99200-09-6 see also U.S. Pat. No, 4,654362), cicϊoprolol hydrochloride, such 2-frαpaπol, l -[4- 12-(cyciopπ ⁾ pyiτnethoxy)ethoxy]pheτit>xy]~3~[ 1 -me-thyiethyi }aiπino]~ , hydrochloride, A.A.S. RN 63686-79-3), dexprυpranolol hydrochloride (2-PropanoU -[ 1-methylethy)-

iiiiiino]~3-{l~naphthαlenyloxy)-hydrochloride (CAS RN 13071-1 1 -9), diaeetoloi hydrochloride {Acef amide, N-[3-acctyl-4-[2-h>'droxy-3-({ 1 -methyl- cthyl)aτϊiiiio]proρoxy] [phenyl]-, monohydrochloride CAS RN 69796-04-9), dilevalol hydrochloride (Ben/amide, 2-hydroxy~5-[ i -hydτoxy-2~[ i -methy 1-3- 5 pheny}propyl)affiiiκ>]cthyl}-, monohydrochloride, CAS RN 75659-08-4), exaproiol hydrochloride (2-Propanol, l -(2-eyclohexylphenαxy)~3-[(l -methylethyijamino]-, hydrochloride CAS RN 59333-90-3), flestoloS sulfate (Benzoic acid, 2-fluro-,3-{[2~ [amiπocarb(.>nyl)aminoj- -dimethyieihyϊjamiiw] -2 -hydros ypropyl ester, (±>- sulfate ( 1 :1 ) (salt), CAS RN 88844-73-9; metalol hydrochloride (MethanesuMonaniide, N- [4- 0 I \ -hydru?ty-2-(metJ)ylamJno)propyl]phenyVj-, monohydrochloride CAS RN 7701 ^» 65- 7), πieinprolol 2~Prf>panoL l-(4-{2-me{hoxyethyi)p1ienoxy]- ^' 3-[] ~-Tiethylethy!)amiπo]-; CAS RN 37350-58-6), rrseioprolol tartrate (such as 2-Prapanoi, l -[4~(2- incth(jχyethyI)ρhenoxy]-3 -[( i -methylethyljamino] -, e.g., Lopressor® , Novariis), pamatolol sulfate (Carbamie acid, [2-[4'[24iyc!roxy-3-[(l- 5 iTR"thy1ethyl)araaiojpK>poxyl]phenyl]-cihyl]~ _? methyl ester, (±) sulfate (sa.lt) (2: 1 ). (^AS RN 59954-01-7), penbutoio! sulfate {2-Propanol, l~(2-eyc!opt*rit>1phenoxy)-3- ! I J -dimcihyk- thyhaiϊiino] 1 , (Ss-, suiføie {2: 1} (salt), CAS RN 38363-32-S), pry ctoiøl ( Aeet amide, N-( 4~| 2-h ydroxy-3-[ ( 1 -methylethy] )ammo]-propox y jphenyH-, CAS RN 6673-35-4;) tiprcnoloi hydrochloride (Propanol, !-[{ ! -methylethy [)amiiiu]- G 3-{2-(mctiαy]thio)-phenoxyi-, hydrochloride, (±), CAS RN 39832-43-4), tolamoϊol (Benzaniide, 4-[2-[[2-hydroxy-3-(2-τπet ^" hylphenoxy)~piOpy!]araiιαoicthoxyrj-, CAS RN 38103-61-6), bopindolo!, indenolol, pindolol, propanolol, tertatαlol, and tilisolol, and the like; calcium channel blockers such as besylale sali of amlodipine (such as 3- eihy]-5-Tnethyl-2-(2-aininoethoxymethyl)-4-(2-chlorophciiyI) ~] ,4-dih>'dro-6-inethyi- 5 3.5-pyτidiαedicarboxylate benzetiesuiphonate, e.g., NorvascCφ. Plizεr), clentiazeni iτja]eatt; (l ,5-Benzothiazepin-4(5I-0-one, 3-{acetyioxy)-8-chloro-5-[2~ (dir-iethylamiiiojethyl]-2,3-dihydro-2-(4-methoxyphenyl}-(2S -cis)-, (Z)-2- butenedioate ! I: I }, see also US4567195), isracHpine (3,5-Pyridinediearboxylic acid, 4~ {4-beιv/ofurazanyl)-l ,4-dihydro-2,6-dhneihyϊ-, methyl 1-τncthylethyi ester, 5r)-4(4- 0 ben?,o iu razanyl)- 1.4-<.lihydro-2 _; 6-diτneihyl-3,5-pyridinαiicarboxyiate. sec aiao

US4466972); niraodipine {such as is isopropyi (2- methoxyethyl) L 4- dihydro -2,6-

- 178 -

dimethyl -4- ( ^" 3-THtropbenyl) -3,5- pyridine - dicarboxyiale, e.g. Nimotυp®, Bayer), fdodipine (such as ethyl methyl 4-{23-dich!orophenyi)-i,4-dihydro-2 _! 6-dimeihyl- 3,5-pyτklijiedicarboxylaie- , e.g. Piendil® Ex tended- Release, AstraZeneca LP), iiiivadipine {3.5-Pyridinedicarboxylic acid, 2~cyano-1 ,4-dihydro-6-mefhyl~4~(3~ nitrophenyI)-3-me-thyl 5~( 1 -niethylethyi) ester, also see US3799934), nifedipine (such as 3,5-pyridmedicarbo ^' x.yiie acid,i,4-dihydro-2,6-dimethyi-4-{2-nitτoplienyl)-, dimethyl eslcr. e.g., Procardia XL® Extended Release Tablets, Pfizer), dilliazem hydrochloride (such as 1 ,5~Ben5f.othia/.epin~4(5I-i )-tme,3-(aeetyloxyy 5f 2- (dimethyIarøJno}ethy!J-2,-3-dihydro-2(4-mcthoxyp3ieτiyl)- _I πioπohydroehloride, (-f-)- ess., e.g., 1 iiizacφ, Forest), verapamil hydrochloride (such as benzeneaceironitrik, (alpha)-[[3-[[2-{3,4-dimcthoxyphenyl) ethyl lraelhy!aniino]propy[ ^' j-3 ,4-dimεthox y- {aipha)-{l-τnelhyk*thyl) hydrochloride, e.g., ϊsoptin® SR, Knoll Labs), teiudipine hydrochloride (3,5-Pyridinedicarboxylic acid, 2-[{dimethylamino)metbyl ]4-{2-[ ^' { 1 E)- J-( IJ -dimethylethoxy)-3-oxo-l -propenyl]phenyl]-l ,4-dibydro-6-melhy)-, diethyl esler, monohydrochloride) CAS RN 108700-03-4), belibsdii (Phosphonic acid, [2-{2- phcnoxyethyl)-] ,3-propane- diyijbis-, letrabuiy! ester CAS RN 103486-79-9), fostedil (Phosphonic acid, [[4-(2-beiizoibiazolyl}pheπyi]πiet1iyS]-, diethyl ester CAS RN 75889-62-2), ararήdipme, azelnidipine, barnklipine. benidipine, beprklii, cinaidipme, elcvidipϊnc, efonidipinc, gallopamil, iacidipme, lemiidipirse, Jercanidiptne, rnonatepii rαaleate (I -Piperazinebiuanamide, N-(O ₅ I l-dihydrodibcnzo(h,e)tliiεpin-l ! -yi)4-(4- fmorophenyl)-, {;!)-, jZ)-2-buteτκ*dioate (i :1) (±)-N-(6, ! 1-Dihydrodiberizo{b,e)thiep~ in- 1 1 ~yl)-4-{ρ-{liioroρhenyl)-l -piperazinebutyramide maleate { 1 : 1} CAS RN 132046- 06-1 ), nicardipine, msoldipine. nitrendipine, raaαidipme, pranidipine, and the like; T-chaniid calcium antagonists such as mibefradϋ; angiotensin converting enxyiiie (ACE) inhibitors such as benazepril, benazepril hydrochloride {such as 3-[[ ! -

(clhcuyearboτiy! κ>-pheπyl-(JS)~pr<.>pyi]aτniπo]-2,3.4,5-tet.rab )'dro-2-oxo~i M -1 -(3S)- benxaxepine-l -acetic acid monohydrochloride, e.g., Lotreϊ®, Novartis), captopril (such as l -f (2S)-3-mercapto-2-metbyipropionyVj-L-prolJne, e.g., Captopril, Myian, CAS RN 62571 -86-2 and others disclosed in US40468S9), eeranapril (and others disclosed in US4452790), eetapril (alaeepril, Dainippon disclosed in Eur. Tlierap.

^■■ 1 19 -

Res. 39:671 (1986); 40:543 (1986)). cilazapril (Hofϋnan-LaRoche) disclosed in J. Cardjovasc. Pharmacol. 939 ( 1987), indafaprii (deiapril hydrochloride (211- 1 ,2,4- Bcnzothiadiazme-T-sυlfonamide, 3~bicycio[2.2.I]hept-5-en-2~yl-ό~chioro-3.4- dihvdro-, IJ -dioxide CAS RN 2259-96-3); disclosed in US43S5O5 S ), enalapril (and others disclosed in US4374829), enalopiii, etialoprilat, fosinopril, ((such as [.-proline, 4-cydohexyi-I -[[[ ^' 2 -methyl- 1 -(I -oxopropoxy) prαpoxy](4-phenyLbυtyi) pliθsρhiny]]acetylj-, sodium salt, trans—, e.g.. Monopήl, Bristol-Myers Squibb and others disclosed in US4168267), fosinopril sodium (L.-Proli-ie, 4-cyck>hexyi-ϊ-[[{R)~ [C I S)-2 -methyl- 1 ~{ 1 -t>x- opropoxy)propox), vmklapril, indolaphi (Schenng. disclosed in j. Cafdiovasc. Pharmacol 5:643, 655 (3983)), lismopril (Merck), losinopril niot'xipril, nioexipril hydrochloride (3-fsoqιππoIinecarboxyIic acid, 2-[{2S)-2-[ [(1S)- i-(ethoxycarbonyl)-3φheωyipropy{]aτϊjino]-l-oxopropyl]-l _! - 2 _: ,3 _> 4-tetraliydro-6,7- dhncihoxy-, monohydrocliloride, (3S}~ CAS .RN 82586-52-5), quinapril, quinapπlai, ramipril (Hoechssl) disclosed in EP 79022 and Curr. Ther, Res. 40:74 (19S6) _: perindoprii erbummc (such as 2S,3aS,7aS-l~[(S)-N-[(S)-1 -

(^arboxybutyi jalanyrihcxahydro^-indolinecarboxylic acid, 1 -ethyl ester, compound with lert-butyhimme (1 :1), e.g.. Accon®, Soivay), pcrindopril (Servier, disciosed in iϊur. i. din. Pharmacol. 31 :519 (1987)}, quanipril (disclosed in US4344949), spiraprii (Schering, disclosed in Acta. Pharmacol, Toxicol. 59 (Supp. 5): 173 (1986)}, tenocapriL trandolapril, zoieπopril {and others disclosed in US4316906), rcndapri! (ieαtiapril, disclosed in Clin. Exp. Pharmacol. Physiol. 10: 131 (1983)}, pivopriL YS980, tcprmidc {Bradykiisin potentiator BPP9a CAS UN 351 15-60-7). BRL 36 ₅ 378 (Smith Kline Beecham. see EP80822 and BP60668), MC-83S (Chugai. see CA. 102:72588V and Jap, J. Pharmacol 40:373 ( 1986), CGS 14824 (Ciba-Geigy, 3-([l - cUχtχyca*bonyi-3-phenyH ! S)-ρropyJ]amino)-2,3 ,4.5-tetrahydro~3-ox- o~ I -( ^' 3S)- hrøzazopiise- 1 acetic acid HCl, see U.K.. Patent No. 2103614K COS 16.617 (Ciba- Geigy, 3(S}-[[( 1 S)-5-amino-1 -carboxypentyljamino |-2,3,4 _r 5-tetrahydro--2-oxo- 1 H-I- benzazepffie-I-ethaτjoic acid, see US4473575), Ru 44570 (Hoeehsf, see Anjaeimitteifoϊschung 34: 1254 (1985)), R 31-2201 (Hofϊhian-LaRoehε see FEES Lett. 165:20! (1984)), C1.925 {Pharmacologist 26:243, 266 ( 1984)}, WY-44221 (Wyeth, sye J. Mud. Chern, 26:394 ( 1983)), and those disclosed in US2003QG6922

- 180 -

(paragraph 28), US4337201 , US443297! (phosphonamidates); neutral endopcptida.se inhibitors such as omapatrihl (VaπJev®}, (XJS 3044O, cadoxatrϊl and ecadotrij, iasidotn. (also known as aladotril or alatriopril), sampatrilat, mixanpril, and gcsmopatrϋat, AVE7688, ER4O3O, and those disclosed in US5362727, US5366973, US5225401 , US47228iO. US52235ϊ6, US4749688, IJS5552397, US5504080, LJS5612359, US5525723, EP0599444, EP0481522, EP0599444, EP0S9S610, JϊP0534363, HP534396, EPS34492, EP0629627; cndotheLsn aiit agonists such as tczosentan, A308165, and YM62899, mά the like; vasodilators such as hydralazine (apresolinc), clonidine fclonidme hydrochloride (IH- ia)ida/.ai~2-ajninc, N-(2,6-dichIorophenyl)4 _> 5~dihydro-, monohydraehloride CAS RN 420S-9i -8), catapres, minoxidil (loniten), nicotmyl alcohol (roniacol), diitiazem hydrochloride (such as I,5-Benzothiazepm-4(5H)-one3-(acetvloxy)-5[2~ (dimethyIaramo)cthy!j-2,-3-dihydro-2(4-m ^ς thoxyphcnyl)-, Trionohydrochic'ridc, (+i- cis, e.g.. TiazacOI), Forest), isosorbide dinitratc (such as l ,4:3,6-diarihydro~D-g]ιιcitol ^' 2,5-dmitraie e.g., Isordil© Titradosc®, Wyeth-Ayers!.}. sosorbidc τnonoτπtraic (such as l ,4:3,ό-dianhydro-D--glucito- 1,5-nitratc, an organic mwatc, e.g., Isπio®, Wyeth- Ayerst}, nitroglycerin (such as 2.3 propanetriol trinitrate, e.g., Nit.ross.at® Parke- Davis), verapamil hydrochloride (such as benzcneacetonitrile, (±)-(alpha}[3-{[2~(3,4 dimethox>phe«yl)ciJiy!]mcthyl<uTurio)propyi3-3 _> 4-dirnethoxy-{alpha)- (1-methyletliyl) hydrochloride, e g.. Covera HS® Extended-Refeasc, Searle), chromonar (which iπay be prepared as disclosed in LJS3282938), cfonitate (Annalcn 1870 155), droprenilamine (which may be prepared as disclosed m DE252L 1 13 _. K Hdofiazine (which may be prepared as disclosed in ^" US3267104); prenylamine (which may be prepared as disclosed in US3152173), propatyl nitrate (which .maybe prepared as disclosed in French Patent No. 1 , 103 λ i 3), mioflaxine hydrochloride ( ! -

PspcrazisieaceJamide, 3-(a£mnocarbony1) _J? ~[4.4-bis(4~f1uoropheny])butyi]-N ^r -(2.6~ d ^j chloix>ρhenyl)~, dihydrochioride CAS RN S3S^8--67--3). mixidine (Bcn/cαecthunamint;, 3,4-dimethoxy~N-(! -raetby!-2-pym>1idin>'!idene}- Pyrrolidine, ^-[{3,4~diniethoxyphenethyi)imiiio]-l-methyl-l-Methyl-2-r(3, 4- dimetlioxyphcnethyllimmo ^' jpyrrolidine CAS RN 27737-3S-8), molsidomine ( 1,2,3- OxadiazcHum, 5-[(elhoxyc8rbony])amino)-3-(4~moφhoHny!)-, inner salt CAS RN

257 H-SO-O), isosorbide mononitrate (D-Glueitol, l,4;3,6-dianJhydιt.κ S-nitrate CAS RN 16051 -77-7), eryfhrityl tetπmitrate (K2,3,4-ButanGietrol tctranittate, (2R,3S)~rel- CAS R N ^" 7297-25-8), c!onitrate( ! ,2-Propanedio!, 3-chtora-, dimirate (7Cl, SCl 9Cl) CAS RN 2612-33-1 ), dipyridamole Bhaπol, 2,2',2",2 ⁱⁿ -[f4.S-di-i - δ piρeridiαylpyrimido[S,4-tljpyriϊr!idine-2.6-ciiyl)diϊiil ri!o|tetrak.is- CAS RN 58-32-2), πicorarujii (CλS RN 65141-46-0 3-}, pyridinecarboxamide (N-[2-(nnrooxy)elbylj- Nisoldipine3,5~Pyridmodicarboxylic acid, 1.4-dihydro~2,6-dimethyi-4-(2- nitropliersyi}-, methyl 2-raethyipropyl ester CAS RN 63675-72-9), τπfcdipine3,5- Pyridincdicarboxyiic acid, l,4-dihydro-2,6-dimethy[-4-(2-dfrophenyl)-, dimethyl 0 ester CAS RN 21829-25-4), perhexiline πmleate (Pipeτidi.ne, 2-(2,2- dicyclohcxylethyl)-, (2Z)-2-butcnedioatc (1 :1) CAS RN 6724-53-4), oxprenolϋi hydrochloride (2 -Propanol, 1 ■•[{ ! »methylethyl)arairiθ]-3-[2-(2-propenyloxy)pbcnoxy |- . hydrochloride CAS RN 6452-73-9), pentrinitrol ( 1 ,3-Propanedioi, 2,2- his[(nii.rooxy)methyi]-, mononitrate (ester) CAS RN 1607-17-6), verapamil metJiylamino]prapyi]-

3,4-diiiiedlθxy-tt-(l--methylethyϊ)- CAS RN 52-53-9) and the like; angiotensin II receptor antagonists such as, aprosartaπ, zolasartan, olmesartaπ, pratosartan, F1682SK, RN116270, candesartan f 1 M-Ben/.imidazole-7-cai-boxyiic acid, 2-etboxy-l-[[2'-(i H~ TL'trazoi-5-yl)[l ,r-biphenyl]4-yl3methylj- CAS RN i 394Sl -59-7), candesattan 0 cilexetil {(^/-)-!-(cycb3ii;xylearbonyloxy)ethy!~2-ethoxy-l-[[2'-(H-!- tetra2;ol-5- y])biphcnyl-4-yi]-lH-beii7imidazole caxboxylaie, CAS RN 145040-37-5, US57O31 10 and US5196444), eprosartan (3-f l-4-carboxypheny1methyl)-2-n-biityf-imJdttzo!-5-yJ}~ {2-thienyimeihyi) prøpenoie acid, US5185351 and US5650650), irbesatian (2-n- hmyi~3- i] ^" _2'-(lh-tetrazoi-5->-])biphenyl-4-yl]methyi]l,3-diaxa/.sp iR){ ^' 4,4)non-l-ei>4- 5 one, US5270317 and US5352788), loaartan (2-N-butyl-4-chiort>5-hydn>xysnethyi-i- i (2'-(lϊ-l-telrazol-5-yl)bipheny!-4-yf)-metiiy! jimtdazole, potassium salt, US513^069, US5153197 and US5128355), tasosartaB (5.8-dihydro-2,4-dimeihyi-8-[(2 ^! -(IH- tctrazol-5-yl}| ^' Ll'-bipheiiyrj4-yl)methyl]~pyrido[2 _! 3-d]pyraπidin-7{6H}-onej US5149699), iehnssartan {4'-[( 1 ,4-dirøelhyl-2'-proρyi-{2,6 ^< -bi-IB-benzimidazol}-1'~

30 _> ])j-j l, r-bJphcrιyl]-2-carboxyϊic acid, CAS RN 14470! -48-4, US5591762),

>πilfasarta.;i, abitcsartan, valsartan (Diovanφ (Novattis), (S)- ^' N-vateryi~N-|[2 ^< -(lH-

- ! 82 ^■•

tcS.razo1-5-yI)bipheny]-4-yI)metliy1] valine, US5399578K EXP-3137 (2-N-butyl-4- di]oro4-[{2'-{iϊ-f-tetrazol-5-yl)biphenyi-4-y1)-mcthy]]irai da2θle-5"Cai-bθλy1ic acid, USSBSOό^ USS iSS^T and USSnSSSSλ S-fa'-Oetrazoi-S-yO-i .r-biphen-φ yl)mcihyi"5,7-dimethyi~2-cthy ^r I-3H'imidazϋ| ^" 4 _J 5-b]pyridiiic, 4'[2-ethyl~4~methy1~6~ {5 _> 6,7,S~{etrahyc1roimidazo[l,2-a]pyiidin-2-yr|-ben2imidazol-l- yrj-i'nethy]]-l J "- biphenyϊ]-2~ carboxylic acid, 2~hutyl-ό~(1 -methoxy-I -τnethylethyl)~2-[2 ⁽ -}S H~letrazoi- 5-yl)bJphcπyi-4-ylmcthyljgumai ⁱ ;oliτi-4(3H)-onc, 3-P'-earboxybiphenyI-4~y1)mctbyI]- 2-cyclopϊOpyi- 7 -methyl- 3H-imidazo[4,5-b]pyτidiπe. 2-butyl-4-chloro-] -[(2 ¹ -tetrazoϊ- 5-yl)biphenyM-yi)methyi jimidazole-earboxylic acid, 2-butyϊ-4-chioro-ϊ -[[2'-( ϊ U- -bipheπyl j-4-y! jmethyl]- 1 acid- 1 -

(ethox>'carbonyi-oxy)ethyl ester potassium salt, dipotassium 2-bLuyI-4-6nethylthio)"]- J [2-f [[{propyl amino)carboαy! )ammo]-sull ^" bnyrj(l , r-biphenyl)-4-yl Jπiethy]]-1 H- imidazok-5-carboxyiatc, iτicthy!-2-|[4-buty[-2-niethyl-6-oxo-5-[[2'-( I ii-tetrazol-5- y[)-i 1 ,1 "-biphεny! j-4-y] }methy!]-I-(6H)-pyrimidinyi]methyI]-3-thioρhencarboxy!ate, 5-f (3,5-dibutyi- Mi-1 ,2 ,4-triazol-ϊ -y!)methyi j-2-[2-( 1 M-fetrfjzol-5~ylpheπy) })pyridiπc, ό-bu ty 1 -2-(2- pheny I ethyl)-5 [ [2 ' -{ Ui-tcirazoi -5 -y!}[ 1 , 1 ' -bipheny I J-4- mcthyl]pyrimidiii-4-{3H)-t>ne D,L lysine salt, 5-τnelhyl-7~n~proρy!~8-[[2 ^> -(l E- ietrazo3-5-yl }biphenyt~4~yl]methy!]~[ 1.2,4]-triazob[ 1 ,5-c]pyrιmidiπ-2(3H)-one, 2,7- dicthy!-54[2 ^s -(5-tdrazoly}biphenyl-4~yl3methyl]-5H-pyra2θk>[l ,5~bj[l ,2 _! 4]triazoie potassium salt, 2-j ^" 2-butyl-4,5-dihydro-4-oxo-3-| ^" 2 '-( 1 li-teirazoi~5-y!)-4- hiphenylmethy i j-3 H-imidazoi [4,5~cjpyridine-5-y Imethy! [benzoic acid, ethyl ester, potassium salt, 3 -methoxy-2,6-dimethyl-4-{ ^' [ 2 ^* ( I H-ietra^oI-5-yl)- 1 , 1 '-biphenyi~4~ vrimeLhoxyipyridine, 2~et,hoxy-] -f[2'-(5-oκo-2,5-dihydro-} ,2,4-(.)xadiϊtzoϊ-3- y I)b iplieπ yi -4- yi jroeihyi)-1 ϊϊ-bcnzimidazo!e-7-carbo.\yϋc. acid, 1 -[N-(2'-{ I H-teirazol- 5-y 1 }bipbenyI-4-yi-mcthyl)-N-valeroly!amiπomethyl)cydoρcntane- 1 -carbo.x ylic acid, 7-methyl-2n-propy!-3-f[2'l B~tetra/.ol-5-yl)biphenyl-4~yi]mcthyi]~3ϊ-l-ύiύdazo(4,5- όjpyridine, 2-[5-[(2-etbyi-5,7-dimcthyl-3H-unidazo[4,5-b]pyridine-3-yl)m e{hy]]-2~ qumolinyfjsodiuiii benzoate, 2"buiyi-6-ch1oro-4-hydroxymetliyl-5--methyl-3-iT2 ^'i -(1I-l- ietrazol--5~yi )biphenyl~4~yTjmκthyTjpyridiϊie, 2-[[[2~butyl- 1 -[{4- carboxyphenyl)raethyl]« I H-imidaz«!-5-yl]metbyV|aτnino)henxoic acid ietrazol-5- yi)biphenyl-4-yljmethyrjpyrimidin-6-oαe, 4{S)-j ^' 4-(c^rboxymetliy1)phακ>xy]-N-[2(R)-

- 1 S3 -

j4-{2-sυlfobeτiϊ;i*iτύdo)imidazo!-] -yl]ocianoγrj-L-proline, l-{2,ό-dimethy]phenyI}-4~ butyl- i ,3-dihydro-3 -[[δ-[2-{ 1 iI-tctrazol-5-yI)phenyS]-3-p>τidinyi jmethy! J-2H- i«mteøl~2-oπ.e, 5.S-ethano-5,8-dimethyI-2-n-pa)pyI-5,6,7,8-tetrahydro- ! -[JTC I i-ϊ- ictfaz.{) ^] -5-y!)bipheny ^] -4-yijmeihy ^] ]-l H,4H-1 J,4a,Sa-tctrazacyclopcntanap1ithaleric~^~ 5 one, 4~[ 1 -[T -( i ,23 _} 44ctrazol-5-yl)biphen-4-yl)methySaniino]-5,6 _I 7 ₅ 8-tetrahydiO-2- trify ^j quim^oline, 2-(2-chIorobenzoyl)imino-5-ethyl-3-[2'-( !H-teirazoie-5- yl)biphen.vJ-4-y]}mcthyi-],3,4-t.hiadiazolme, 2-[5-ethyl-3~[2-(IH-ietrazo1e~5- yl}biphen>i--4-y ^] jiτ3cthyl-],3,4-thia7.oiin<>2~ylidene]aτnijπocarbon yϊ~l - cyclopeϊttencarboxyiic acid dipotassium salt, and 2-butyl-4-[N-røeihyi-N~{3~

10 rnethyIcrotonoyl)aminoj-l-([2 ^> -(l H-tetrazol-5-yl}biphcnyl-4-yl]methyl]- lI-l-imid/.oi«- S-carboxylic acid ! -ethoxycarbonyioxyethyl ester, those disclosed in patent publications E-P475206, IϊP497I SO, EP539086, EP539713, EP535463, EP53S465, i;P542059, EP49712L EP535420, EP407342, EP4I 5886JϊP4243 I7, EP435827, FP433983, EP475898, EP490820, EP528762, EP324377, EP323841 , EP420237,

^■ 15 fP500297, EP426021 , EP48G204, EP429257, EP430709, EP434249, EP446062, HP505954, EP5242 I 7, EP514197, EP514198, EP5I4193, EPS 14192. EP4S0566, EP468372, EP485929, EP503162, E.PS3305S, HP467207 EP39973 I , EP399732, EP412848, EP4532 I0, EP456442, EP470794, EP470795, EP495626. EP495627, EP499414, EP499416, EP499415. EP51 1791 , EP5I6392, EP520723. EP520724, 0 EP539066. EP438869, EP505893, EP53O702, EP400B35, EP400974, EP4OIO3O, EP407102, EP4I 1766, EP409332, EP412594, EP419048, EP480659, EP48ϊ614, EP490587, EP467715, EP479479, EP502725, EP503838, EP505098, EP5051 1 1 ϊ:.P5i 3,979 EP507394, EP5 JO812, EP51 1 767, EPS 12675, EP512676, EP51287O, EP5I 7357, EP537937. EP534706, EP527534, EP540356, EP461040, EP540039.

2S EP46536S, EP498723, EP49S722, EP498721, EP515265, EP5037JJ5, EP50I 892, f:P5! 983ϊ . EP532410, EP498361 , EP432737, EP504S8S, EP5O8393. EP508445, EP403159, IϊP403158, EP4252) L EP427463, EP437103, EP48I448. EP488S32, EP50I 269, EP50O4O9, EP5404O0, EP005528, EP028834, EP028833, EP411507, EP42592L EP430300, EP434038, EP442473, EP443568, EP4458ϊ I , EP459136,

30 BP483683, EP518033, EP520423, EP531876, EP531 S74, EP3923I 7, EP468470, BP470343, EP502314, EP529253, EP543263, EP540209, EP449699, EP465323,

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EP52176S, EP4ϊ 5594, WO92/ f 4468, WO93/08171, WO93/08169, WO91/00277, WO9i /00281. WO9ϊ ^/ 14367, WO92/00067, WO92/00977, WO92/20342, WO93/04045, WO93/04046, WO91/15206, WO92/ I47 I4, WO92/G9600, WO92/ 16552. WO93/05025, WO93/03018, WO9ϊ/07404, WO92/02508, W092/OS53, WO9 J/19697, WO91/ 11909, WO9I/12001 , WO91/1 1999, WO9I/ϊ5209, WO91/15479, WO92/20687, WO92/20662, WO92/20661 , WO93/01 17 ^" , WO91/14679, WO91/13063, WO9?Jl 3564, WO9I/I7I48, WO9J /1 SS88, WO91/19715, WO92/02257, WO92/04335, WO92/05161 , W()92/07?52, WO92/ 15577, WO93/03033, WO91/16313, WO92/00068, WO92/02510, WO92/09278, WO9210179, WO92/10 i 80, WO92/10186, WO92/I0181, WO92/I0097, WO92/10183, WO92/10182, WOy2/I0l87, WO92/10184, WO92/I01 S8, WO92/KH80, WO92/10185, WO92/20651 , VVO93/03722, Wθy3/06S28, WO93/03040, WO92/192 1 1 , WO92/22533, WO92/06081 , WO92/05784, WO93/00341 , WO92/04343. WO92/04059, US5 UWJ.77, US5187168, US5149699, US5185340, US4880804, US5ϊ38069, US491 6I29. US5153 ϊ97, USS173494. US51379O6, US5155126, US5I 40CB7, US513^902. US5157026, US5053329, US5132216, US5057522, IJS5066586, US5089626, US5049565, US5087702. US5L24335, US5102880, US5128327, US5151435, US5202322, US51S7159, US5198438, US51S2288, US5036048, US5140036, US5087634. IJS5196537, US5I 53347, US5191086, US5190942, US5ϊ77097. ϊJS5212177, US5208234, US5208235, US52I 2J95. US5 I3O439, US5045540, US5041 152, and US5210204, and pharmaceutically acceptable salts and esters thereof; α/β adrenergic blockers such as nlpradiiol, arotiuoIoL aniosυiaiol, hretyϋum tosyiafe (CAS RN: 61-75-6), dihydroergtamine mesylate (such as ergotamaπ-3\ 6', i 8-trκ>ne,9,- 10-dihydro 12'-hydroxy-2'-methyi -5 ^! -{pheny lmeihyl )- ,{5'(α})-, αionometlianesitlfonate, e.g., DHE 45© Injection, Novartis), csrvedilol (such us {t)~ i-{CϊM'ha2θl-4-yloxy)-3-[[2-(.o-πiethoxyphenoxy}ethyl]amm o|-2-piOpaiiol, e.g., foreg®, SmithKHπe Beecham), labetalol (such as 5-[I -hydroxy-2-[(l-methyV3- phenyipropyi) amino! ethyljsalieylamide røonohydrochioride, e.g., Normodync®, Sohering}, brdylium tosylate (Btmenemethanammium, 2-bromo-M-etliy!-N,N ^T - diπiethyi-, salt with 4-methylbenzenesυlfonϊc acid f l:i) CAS RN 61-75-6),

- ϊ S5 -

phentolamine mesylate (Phenol, 3-[[(4,5~dJhydro~iH-imidazoi-2-yl)methyl ](4- methylphenyl)aminoj-, monomethanesυlfonate (salt) CAS RN 65-28-1), sαlypeiime tartrate (5f-ϊ- ! , 3-Dioxolo[4,5-f3indole, 7-[2-[4-(2~methoxyphenyl)-l ^■ • piperazinyljeihyl]-, (2il»3R)-2,3-dihydroxybutancdTOale (l :l) CAS RN 5591 -43-5), /olertme hydrochloride {Pipeline, l-plieny14-[2-(l H-tetrazo[-5-yl)ethyl|-, snonohydroehlαride (SCK 9Cl) CAS RN 7241 -94-3} and Ae like; u adrenergic receptor blockers, such as aU ^' uzαsin (CAS RN; 81403-68-1 ), terazosin, urapidiL prazosin (Minϊprøsφ;), tamsulosin, bioiazosin, Iriπw.osin. doxazosin, naftopidiL jndoramin, WI-J P 164, XENOIO, fenspiride hydrochiorkie (which may be prepared as disclosed in US3399192), proroxan (CAS RN 33743-96-3), and kbetaio! hydrochloride and combinations thereof; α 2 agonists such as røethyldopa, rnelhyldopa MCL, lofexidine, tiameπidine, nioxonidine, πimenidine, gua.n€>be.nz, and the like: aldosterone inhibitoπs, and the like; renin inhibitors including λiiskireu (SPPIOO; Nαvartis/Speedei): angiopoietin-2~binding agents such as thctse disclosed in WO03/030833; anti-angina agents such as ranolazine (hydrochloride! -Piperazineacetamidy, N-(2,ό- dimetiiyiρbcnyl)-4~[2-hydroxy-3-(2-raethoxyphenoxy)propyi)- , dihydrochloride CAS RN 95035-56-6). bctaxoiol hydrochloride (2-Propanol, l-j ^" 4-[2 (cycloρroρybϊϊeftoxy}ethyi]pheno?;y]"3-[ (1 -methybthy] }aminoV, hydrochloride CAS RN 63659- 19-8 ), butoprozinc hydrochloride (Methanone, [4- [3{dibulylainim))pmpoxyjphenyl](2-eUiyi-3-i«do1iziny{)-j monohydrochJondc CAS ! ^? ,N 62134-34-3), cinepazet maleaiei-Piperazineacetic acid, 4-[l-oxo-3-{3,4.5- iriaicUκ>χ\phcnyl)-2-proρeπyl)-, ethyl cater. (2Z)-2-butenedioate (1:1) CAS RN 50679-07-7). iosiien (Benzenesulfonamide, 4-mcthyl-N-[[[{ J S)-I -π5cthy1-2- CλS UN 32295-184), verapamilhydrocbloride (Benzeneuceiomtrile. a-[3-[[2-(3,4-dimcthoxyphenyl)ethyljraethy[ami«o]propy!]-3, 4- dhncthoxy-tt-{ l-methylethyl)-, monohydrochloride CAS RN \ 52-114). molsidomine ( 1 ;2,3-Oxadia2θIium, 5-[(ethoxycarbonyl)aminoj-3-(4-moφhoIinyl)- _> inner salt CAS R N 25? 17-80-U), and ranolaziπe hydπiehbride ( I -Piperazineacetaraide, ^" N-(2,6- diπ}cthylρhcny1) ₄ ~| ^" 2~hydroxy-3-(2~meth- oxyphencxy)propyl]-, dihydrochloride (^AS

- 186 -

RN 95635-56-6); tαsifen (Benzenesuifonamide, 4-methyl-N-[[[(lS)-1 -irιethyI-2- ρhenykthyl]amino]earbony£]- CAS RN 32295-184); adrenergic stimulants such as guantaciπe hydrochloride (such as N-amidiπo-2-(2,6-dichlorophenyi) aeetaraide hydrochloride, e.g., Tenex® Tablets available from Robins); methyidopa- hydrochlorothiazide (such as levo-3-(3 ,4-diliydroxyphenyl )-2-rnethylaianine} combined with Hydrochlorothiazide (such as 6~chloro-3,4-dihydiX>2H -1 ,2,4- bcnzotliiadiazinc-?- sulfanamide 1,1-dioxide, e.g., the combination as, e.g., Aldoril® Tablets available from Merck). rneihyUlαpa-chloroihiazkle (such as δ-chk>ro-2H~l , 2,4-bcnzoihiadϊazine-7-suHOnaπvkie 1 ,1-dioxide and niethykfopa as described above, u.g., Aldoclor®, Merck), eionidsne hydrochloride (such as 2-(2.6- dich!orophenyiamino}-24niJdazϋline hydrochloride and chlorthalidone (such as 2- eh!oro-5-{i -hydroxy-3-oxo-i -isoindolinyl) benzenesulfonainide). e.g.. Cαmbipres®, Bochrmger (rigelheim), clonidine hydrochloride (such as 2-(2,6- dichIorophen\ia»iino}-2 -imidazoline hydrochloride, e.g., C'ataprcs€λ Boehrύiger ϊngdheirn), clonidiπe (1 Ii-lrnidaxo!~2-araine, N-(2,6-dichlorophenyl)4,5-dihγdro- CAS RN 4205-90-7K Hyzaar (Merck; a combination oflosanan &s\ά hydrochlorothiazide), Co-Diovan (Novartis; a combination of valsartan and iry'droehloTOlhiazide, Lotrel {Novartis; a combination of benazepril and amkxiipine) and Caduet (Pfizer; a combination of amlodipine and atorvastalin _. κ and those agents disclosed in U S 2003006922! .

The peptides and agonists described herein can be used in combination therapy with one or more of the following agents useful in the treatment of respiratory and other disorders including bin not limited to: ( i ) β-?ig(mis£s including but not limited to: albuterol (PROVENTIL®,

SALBIJTAMOl®, VENTOLIN®}, bamhuteral, bitoterol, denbuiero!, fenoierol, rormotetυl, isøethanne (BRONKOSOL®, SRONKOMEϊER®), metaprotertajol (λLUFENTφ, MKTAPREL®), pirbuterol (MAXAIR®), reprolerol riirriterol, saimcteroL tcrbulaiine (βRElηAIREφ, BRETH !NEt ^; , BRiCANYUH- adrenalin. isoproterenol (ϊSUPRELS-), epinephrine bitartrate (PRLMATENE®), ephedriπe. orc.φτenhr _: c, fenote.ro! and .isoetharine;

- 187 -

i 2 } steroids, i nci uding but not limited to betamethasone, beclomelhasonc di propionate, betamethasone, hudesonide, bunedosidc, butixoeort, dexamethasorse, lliuiisolide, Ouoeortm, fluticasone, hydrocortisone, methyl prednisone, nioroeiasone, predonisoione, predomsorse, tipredane, tixoeortaiL triamcinolone, and triamcinolone aeeionide;

(3) β2-agθMSt-Cϋrtieosteroid combinations [e.g., saime-teroMlutieasorie (ADVASR®), formoterol-budesonid (SYMBICORT®)];

(4) ieukαtriene D4 receptor antagonists/leukotriene aiitagonists/LTD4 antagonists (i.e., any compound that is capable of blocking, inhibiting, reducing or otherwise interrupting the interaction between Ieukotriertes mid the Cys LTi receptor) including but not limited to: zafπiukast, monteJukast, montelukast sodium (SING ULA IR®), pranlukcist, irakskasi pobilukast, SKB-106,203 and compounds described as basing LID4 antagonizing activity described in U.S. .Patent No. 5,565,473; (5 S 5 -lipoxygenase inhibitors and/or kukotriene biosynthesis inhibitors [e.g., ziicuton and BAYKX)S (CA registry 128253-31-6)];

(6) histamine HI receptor aπtagonists/antihistamines (i.e., any compound that is capable of blocking, inhibiting, reducing or otherwise interrupting the interaction between histamine and its receptor) including but not limited to: astemizole, acrivastine, antazoline, azatadine, azclastine, astami/.oie, bron>opheniramine _; brømophenirarmne mabate. carbinoxamine, carebastine, cetirixinε, chlorphenirirmirse, chh)ropheniraπτhie malcatc, cimetidine .clemastine, cyclizine, cyproheptadine, descarboethoxyloratadine, dexchloφheniramine, dimetbindene, diphenhydramine, ϋi]Acπylpyτalir)e, doxylamine succinate, doxylamine, ebastine, dletirixiαe, epiπastine, famotidine, fexofenadine, hydroxyzine, hydi"oxyxiτie. ketotifen, levoeabastinc, levocetirizine, kvocetirizine, loratadine, jnecliziiie. niepsτaminc, ϊiiequitazinc. methdiϊazme, mianserin, πiizoiastine, noherastine, norasternizole, noraztemizose, phenindamine, phcniraniiiic, picumast, promethazine, pynlamine, pyrilamiae, ranitidine, terαdastine, tεrfenadine, trimeprazine, ϋ-ipekπaiϊύiie, and triproiidine; (7) an anticholinergic including but not limited to: atropine, beπztropine, biperiden, Outropium, hyoscyamine l ^' e.g. Lcvsiti®; Levbid®; l.evsin/SL®,

^■ 188 -

Amsspaz®. Levsinex timeeaps®, NuLev®), ilutropiuirc. Ipratropium, ipratropium bromide, raeth scopolamine, oxybuttnin, risperøepine, scopolamine, and tiαtropiiun;

(S) an anti-tussive including but not limited kx dextromethorphan, codeine, and hydromorphoπe: (9) a decongestant including but not limited to: pseudoephedrine and phenylpropanolamine;

( ! 0} an expectorant including but not limited to; guafenesin, gaaieolsuf fate, terpsn, ajTHϊioniurn chloride, glyce.ro! guaieolaie, and iodinated glycerol;

CI l ) a bro.nchodilator including but not limited to: theophylline and amirtoph yljiiie; 112) an ami -inflammatory including but not limited to: fluribiprofen, didøphenac, iαdometliacin, kctoprofen, S-keιroprophen, tenoxicain;

( 13} a PDE (phosphodiesterase) inhibitor including but not limited to those disclosed herein;

( 14} a recombinant humanized monoclonal antibody (e.g. xokπ ^' r (also called ornaliziuϊuibh rhuMab, and taiizumab];

^■ 15) Ά humanized Iimg smfactaat including recombinant forms of surfactant piυtcins SP-B, SP-C or SP-D [e.g. SURFAXIN&, formerly known as dsc-104

(Di sco very Labora tori es ) 1.

(16) agents that inhibit epithelial sodium channels (ENaC) such as amilorkle and related compounds;

; 17) antimicrobial agents used to treat pulmonary infections such as acyclovir, amikacin, amoxicillin, doxycyciine, trimethoprin sulfamethoxazole, amphotericin B, azithromycin, clarithromycin, roxithromycin, clarithromycin., cephalosporins* ^' cefoxitin, cef-metazoie etc), ciprofloxacin, ethanibαfol. gyntimycin, ganciclovir, imipenem, isoniazid, itraconazole, penicillin, ribavirin, rifampin, rlmaπtkline. streptomycin, tobramycin, and vancomycin;

C 18} ag&nts that activate chloride secretion through Ca+ ⁴ - dependent chloride channels (such as purmergk receptor f P2Y(2) agonisis);

(19) agents that decrease sputum viscosity, such as human recombinant DNase I. (Palmozyme®);

- 1 S9 -

(20) nonsteroidal anti-inflammatory agents (acemetacin, acetaminophen, acetyl salicylic acid, alclofenac, alrairjoprαien, apazone, aspirin. benoxaprofen, bezpiperylon, bucloxic acid, carprofen, elklanac. diclofenac, diclofenac, diilunisal, tlifiusiiial, etodolac, SVinhuferu fbibufen, feπelσfenae, fenclozic acid, tcnopmfcn, tentia^ac tepraxone, tlufenamic acid, ilufenisal, flufenisal, fluprofcπ, fiurbiprofen, Hiirbiprofoi. fυrofenac, ibtifenac, ibuprofen, irtdomethacin, indomethacin, indoprofbn, i.soxcpac. isoxicam, ketoprofen, kctoprofen, ketorolac, meclofenamic acid, medofenamsc acid, mefenamic acid, mefenamic acid, miroprofen, πiofebutazoπ.e, πabuπietorx ¹ nxaproziπ. naproxen, naproxen, nillimiic acid , oxaprozm, oxpinat', oxyphenbutazone, pheπacetin. phenylbutazone, phenylbutazone, plroxlcam, piroxicam, pirprofcn, pranoprofcn. sudoxicam,teπoxica« , sulfasalazine, sulindac, sulindac, tiaprofenic acid, tiopinac, tioxaprofen, tolfeπajuic acid, tolmetin, tohne.iπ, z.idometaciπ. zomepiiac, and zomepirac); and (2 ! ) aerosolized antioxidant therapeutics such as S-Nitrosogiutathione.

Tiic peptide!* and agonists described herein can be used in combination therapy with an anti-obesity agent. Suitable such agents include, but are not limited to:

I If) H SI)-I (1 1 -beta hydroxy steroid dehydrogenase type 1 ) inhibitors, such as BVT 3498, BVT 2733, 3 -{ i -adamantyl H-ethyl-S-Cethylthio)- 4H- I ,2,4-triazole, 3-(l- adanianiy!)-5-f3,- ^: i,5-trimet.hoxypheny] )-4-methyl-4H-l ,2,4-{riazofe, 3- adamantanyl- 4,5,6,7,8,9,10J L! 2,3a-decahydτo~],2,4-triazo)o(43-a][ ) ijannulene, and those α>πipυuτids disclosed m WOOI/90091, WO01/90090, WO01/90092 and WO02/0720S4;

5UT antagonists such as those in WCXJ3/037871. WO03/037887, and the ϋke;

5HTIa modulators such as carbidopa, bcnstrra/.ide and those disclosed in US6207699, WO03/03 ϊ439, and the like;

- 190 -

SHT2c (serotonin receptor 2c) agonists, such as BVT933, DPCA372! 5. ϊK.2M, FNU 22394, WAYI6I503, R-I065, SB 243213 (Glaxo Smith Kline) and YM 348 and those disclosed in US3914250, WOOO/77010, WO02/36596, WOθ2/48124, WO02/10J 69, WOOI /6654S, WO02/44152, WO02/51844, WO02/4Q456, and WO02/40457;

5ϊ-IT6 receptor modulators, such as those in WO03/030901, WO03/035061 , WO03/039547, and the like;

acyi-estrogeπs, such as oieoyi -estrone, disclosed in del Mar-Grasa, M. ct al,, Obesity Research, 9:202-9 (2001 } and Japanese Patent Application Nυ. JP 2000256190;

anorectic hi cyclic compounds such as 1426 (Aventis) and 1954 (Aventis), and {he compounds disclosed in WO00/ϊ8749, WOOi /32638, WO01/62746, WOO 1/62747, and WO03/015769:

CB 1 (cannabinoid-i receptor) antagonist/inverse agonists such as rimønabant (Acomplia; Sarrøfi), SR-I4777S (Sanofi), SR-1417I6 (Sanofi), BAY 65-2520 (Bayer), and SLV 3 19 (Solvay), and those disclosed in patent publications

US4973587, US5O13S37, US508 H22. US5112820, US5292736, US5532237, US562494K US60280S4, US6509367, US6509367, WO9633159, WO97/29079, WO9S/3 I 227, WO98/33765, WO98/3706L WO98/41519, WO98/43635, WO98/43636, WO99/02499, WOOO/10967, WOOO/ 10968, WCXJi /09120, WO01/58S69, WOOi /64632, WOO 1/64633, WOO 1/64634, WOO I /70700,

WOOI/96330. WO02/076949, WO03/006007, WO03/007887, WO03/020217, WO03/f)2^647, WO03/026648, WO03/027069, WO03/027076, WO03/027 ! 14, WO03/037332, WO03/040107, WO03/086940, WO03/084943 and EP658546;

CCK-A (cholecystokinin-A) agomsis, such as AR-R 15849, GI 181771 (GSK), JMV- I SO, A-7137S, λ-71623 and SR 146131 (Sanofi), and those described in US5739ϊ06;

CNTF i ^' Oiiiary neurotrophic factors), such as GI-1 S1771 (Glaxo-SmiihKline), SR 14613 I (Sanofi Synthelabo). butabindide, PDI 70,292, and PD 149164 (Pfizer):

- 193 -

CNTF derivatives, such as Axokineφ (Regeoeron), aαd those disclosed in WO94/09ϊ34, WO98/22128. and WO99/43813;

dipeptidyt peptidase IV (DP-IV) inhibitors, such as isoleucine thiazoiidide, valine pyrrolidide, NVP-DPP728. LAF237, PWOl , P 3298, TSL 225 (Iryptnphyϊ- 1 ,2,3,4- tetraliydroisuquiiioline-3-carboxyiic acid; disclosed by Yamada et at, Bioorg. & Med, Chem. Lett. S (199S) 1537- 1540), TMC-2A/2B/2C, CD26 inhibtors, FE 9990! 1 , P931U/K364, ViP Oi 77, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrαlidides as disclosed by Ashworth et aK Bioorg. & Med. Chem. Lett., Vol. 6, No. 22, pp 1 163- ! 166 and 2745-2748 (1996) and the compounds disclosed patent publications. WO99/3850! , WO99/46272, WO99/67279 (Probiodrug), WO99/67278 (Prøbiodrug). WO99/61431 (Probwdrug), WO02/0S3128/WO02/062764, WO03/0U0180. ^' WO03/000181, WO03/000250, WO03/002530, WO03/00253 L WO03/002553, WO03/002593, WO03/004498, WO03/004496,WO03/017936, WO03/024942, WO03/024965, WO03/033524, WO03/037327 and EP 1258476:

growth hormone secretagogue receptor agonisis/antagonists, such as NN703, hcxardiπ, MK-0677 (Merck), SM- 130686, CP-42439I (Pfizer), LY 444,71 i (Eli Lilly), L-692,429 and L- 163,255, and such as those disclosed in USSN 09: ^; 662448, I)S _T «ϊ)visiona! application 60/203335, US635895 L US2002049196, US2002/022637,

WO01 /56592 and WO02/328SS;

! !3 (histaiϊiinε 113) antagonist/inverse agonists, such as tiiiαperanvidc, 3-( \ .H-inikiazoi- 4-yl)propy[ N-(4-pentenyl)earb«nate), clobenpropit, iodopheπpropit, imoproxifan, (Kiec-Koππnowicz, K, el ai., Fkirmazie, 55:349-55 (2000)), piperidinc-c.ontaini.ng hisiamlτκ: 113-rcccpior aπtagυnists (Lazewska, ϊ>. et a!,, Pharniazie, 56:927-32 (2001 ), bcπzoplicr« ^' .ϊne derivatives and related compounds (Sasse, A. ct a!,, Arch. ?ham).(Wcinheim} 334:45-52 (2001 )}, substituted N-phenyl carbamates (Rcidemeister, S. et al, Phaπnazie, 55:83-6 (2000)}, and proxifan derivatives (Sasse, A, et a!., J. Med. Chem.. 43:3335-43 (2000)) and histamine H3 receptor modulators such as those disclosed in WO02/ 15905, WO03/G24928 and WO03/024929;

. 192 -

ϊeptm derivatives, such as those disclosed in US5552S24 US5552523, USS552522, U S 5521283, WO96/23513, WO96/235R WO96/235I S, WO96/23516, WC)%/2351 ?, WO96/23518, WO96/235R and WO96/2352Q;

leptin, including recombinant human ieptin (FE-G-OB, Hoffman La Roche) and rccooibinar.it melhionyl human leptin (Amgen);

lipase inhibitors, such as telrahydrolipstatin ioriistat/Xenica!®), Triton WRI 339, RHC80267, Npstaim, tcasaponin, diethylumbelϋferyl phosphate, FL-386, WAY- 12! 898, Bay-N-3176, vaϋlactonc, cstαraciπ, cbelacione A, ebelactone B, and RFiC XO26 ^" , aBd those disclosed in patent publications WOOl /77094, US45080S9, IJS44528I3, USUS5512565, US539I571 , USS602151 , US4405644, US4189438, and US4242453;

lipid metabolism modulators such as masϊimc acid, erythrodiol ursolic acid uvaol, beiυiimc acid, belulin, and the like and compounds disclosed in WO03/011267;

Mc4r (mclanocortin 4 receptor) agonists, such as CH1RS6036 (Chiron), ME-! 01.42, ME- 10145, and HS-13 ^" i (Melaeure), and those disclosed m PCT publication Nos, WO99/64G02, WO00/74679, WO0J/99I 752, WOOI/25192, WOOl/52880, WO01/74B44, WO01/70708, WOOl/70337, WO01/91752, W()02/05y()95, WO02/059107, WO02/059J08, WO02/0591 ϊ7, WO02/06276, WO02/12I66, WO02/1 1715, WO02/I2178, WO02/ 15909, WO02/3S544, WCXJ2/06S3S7, WO02/068388, WO02/067869, WO02/081430, WO03/06604, WO03/007949, WOt)3/ϋυy847, WO03/009850, WO03/013509, and WO03/031410;

McSr fπielanocortm 5 rsceptor) modulators, such as those disclosed in WO97/19952, WO00/ϊ 5826, WOOO/ 15790, US2003009204I ;

nielaniπ-i'oαcentratmg boπirøπe 1 receptor (MCiIR) ajitagouisis, such as T-226296 (Takeda), SB 56B849, SIN P-7941 (Synaptic), and those disclosed in paten { publseations WOO I /21 169, WOOl /82925, WOOϊ/87834, WO02/051809, WO02/06245, WO02/076929, WO02/076947, WO02/04433, WO02/5 I 809,

- 193 -

WO02/083134, WO02/G94799, WO03/004027, WO03/ 13574, WO03/ϊ 5769. WO03/02864ϊ , WOO.3/035624, WOG3/033476, WO03/033480, JPI 3226269, and

JP 1437059;

mGluRS modulators such as those disclosed in WO03/029210, WO03/047581 . WO03/G4S137, WO03/051315, WO03/051833, WO03/053922, WO03/Q59904, and the like;

serotonergic agents, such as fenfluramine (such as Pondirαin® {Bcnzenecϋianamύie, N-ethyl-alpha-methyl-S-Ctrifluorometbyl)-, hydrochloride), Robbins), dexfeπfluramine (such as Redux® (Benzeneethanamine, N-ethyl-alpha- nieihyl"3"( ^s -rifluoromcthy!)-, hydrochloride), Interncuron) and sibυtraniiπe

((Meridian, K.nol3/Reduc{Jl ^{1 M} ) including racemic mixtures, as optically pure isomers ( ^• ; ^■ } and <-}, arsd phaπnaceutically acceptable sails, solvents, hydra iess, clalhrat.es and prodrugs tiiereol ^" including sibutramine hydrochloride mofiohydrate salts thereof; and those compounds disclosed in US4746680, US4806570, and IJS5436272, US2GG20G06964, WOO 1/27068, and WOOl /62341 ;

NK (norepinephrine) transport inhibitors, such as GW 320659, despiranime, "αϋsiipram, and nomifensine:

NPY 1 antagonists, such as BIBP3226. J-1 15814 BIBO 3304, LY-35789 ^" , CP- 671906, GI-264879A, and those disclosed in US600I836, WO96/ 14307, WO01/23387, WO99/51600, WOO 1/85690, WOOL'85098, WOOi /85173, and WO0I AS952S;

NPY5 fricuropcplide Y Y5) antagonists, such as 152,804, GW-569180A, GW- 594884A ₁ GW-5S7081X, GW- 5481 18X. FR235208, FR226928, FR240662, FR252384, 12291)91 , GI-264879A, CGP71683A, LY-377897, tλ ^' -366377. PO- 16017(5. SR- 12O562A. SR- 120819 A, JCl ^< - 104, and H409/22 and tiiose eonψounds disclosed in patent publications US6I40354, US619! 160, US62184ϊ)8, US6258837, US6313298, US6326375, US6329395, US6335345, US6337332, US6329395, US6340683, EPOI 010691, EP-Ol 044970, WO97/ 19682, WO97/20820, WO97/2082I,

- !94 -

WO97/2G822, WO97Q0823, WO98/27063, WOOO/! 07409, WOOG/185714, WOOO/185730, WO00/64880, WO00/68197, WO00/69849, WO/01 13917, WO01/09120. WOOI /14376, WOOi/85714, WOOl/85730, WOϋl/07409, WOO 1/02379. WO01/233S8, WOO 1/23389, WOOl /442Of , WOOl /62737, WOO !/6273S, WOOI /09120, WO02/20488, WO02/22592, WO02/4S152,

WO02/4964S, WOG2O51 &06. WO02/094789, WO03/009S45, WO03/014083, WO03/G22849, WO03/02S726 and Norman et ai., J. Med. Chera. 43:4288-4312 (2000) ^;

opioid antagonists, such as nalmeime (REVEX φ), 3-mcthoxyϊialtrcxonc, TrscthylTiailrexcme, naloxone, and naltrexone (e.g. PT9CH; Pain Therapeutics, Inc.) aod those disclosed in US2005O0O4155 aod WOO0/21 509;

orcxin antagonists, such as SB-334S67-A and those disclosed in patent publications WOOI/%302, WO01/6Jϊ609, W(X)2/44172, WO02/S 1232, WO02.''5183S. WO02/0S9S00, WO02/090355, WO03/02356I , WO03/032991 , and WO03/037847;

PDE inhibitors (e.g. compoimtls which slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP CcGMF) by inhibition of the phosphodiesterases, which can lead to a relative increase in the intracellular concentration of c AM P and cGMP; possible PDE inhibitors are primarily those substances which are to be numbered among the class consisting of the FDE3 inhibitors, the class consisting of the PDE4 inhibitors and/or the class consisting of the PDE5 inhibitors, in particular those substances which can be designated m mixed types of PDE3/4 inhibitors or as mixed types of PDO/4/5 inhibitors) such as those disclosed in patent publications DEl 470341, DE210H43H, DE212332S, DE2305339, DE2305575, DE2315S01 , DE240290S, DE24 B935. DE245I417, DE2459G90, DE264646 ^Q , DE272748 J , DE282504S, DB283716L DE2S45220. DE2847621 , DE2934747, DE3021792, DE303S166, DE3044568. EP00071 S, EP000840S. EP0010759, EP0059948, EP(K)75436, bP00%5] 7, EPOI 12987, t-POl 16948. EP0150937, EPOJ 5838O, EPOϊ6 I 632,

- S 95 -

HPl ⁾ iδl9l8, EP0I67121. ^" EF0199127, EP0220044, IϊP0247725, EP0258191 , EP0272910, EP0272914, EP0294647, EP030O726, EP0335386, EP035778S, E-P0389282, ϊ.P ^Q 406958, EP0426 I 80, EP0428302, EP043581 1, EP047080S, EP0482208, EP0490823, EP0506W, EP051 I865, EP05271 17. EP0626939, EP0664289, EF06713S9, EP0685474, EP068S475, EP0685479, JP92234389, JP94329652, JP950I 0S75, US496356I. US5141931 , WO9J 17991, WO9200968, WO9212961 , WO9307146 _; WO9315044, WO93 ! 5045, WO9318024, WO93 i 9068, WO9319720, WO931974 ⁷ . WO9319749, WO93 1975 ! , WO9325S 17, WQ9402465, WO940A423, WO9412461, WO9420455, WO9422852, WO9425437 _; WO9427947, WO9500516, WO9501980, WO9503794, WO9504fM5. WO9504046. WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO95 J 4667. WO95146S0, WO951468U WO9517392, WO95ϊ7399, WO9519362, WO9522520, WO95243B L WO9527692, WO9528926, WO9535281 , WO9535282, WO96002I8, WO9601825, WO%02541 , WO961 1917, DE31429S2, DE 1 116676, DE2162096, EP0293063. EP04637S6- EP0482208, EP0579496, EP0667345 US6331543,

IJ S 20050004222 (including those disclosed in formulas I-XHI and paragraphs 37-39, 85-0545 and 557-577), WO9307124, EPO! 63965, EP0393500, EP05I0562, RFO553174, WO9501338 and WO9603399, as well as PDES inhibitors (such as RX- Rλ-69, SCH-51866, KT-734, vcsnarinone, zaprlnast, SKF-96231 , ER-21355, BF/GP- 3H5. NM-702 and sildenafil {V?agra ^iM )), FDE4 inhibitors (such as efazoiate, 1C163197. RP73401 , imazoϋdmone (RO-20-1724), MEM 1414 (R i 533/R 1500; Pharnuicia Roche), denbutylϋne, rolipram, oxagrelale, nitraqmizoπe, Y-590, DH- 6471 , SKF-94120. motapizone, lixaziπone, mdolidafj, olprinone, atizoram. KS-506-G. dtpamfylline, BMY-43351, atizoram, arofylline, filaiTiinast, PDB-093, UCB-29646, CDP-840, SKF-107S06, piclamiiast. RS-! 7597, RS-25344-000, SB-207499,

TIBENELAST, SB-210667, SB-21 1572. SB-21 1600, SB-212066, SB-2I2179, GW- 3fi00, CDP-S40, mopidaj-nol, anagrdide, ibudilast, anirinone, pimohcndan, cϊlostazol, quavdiϊone and N-(3.5-dichloropyrid-4-yl)-3-cyclopropylrnethoxy4- diliuorom€itoxybenzamide, PDE3 inhibitors (such as ICl 153, 100, beraorandanc (R WJ 22867). MCM 54, UD-CG 212, sulmazole, amptøone. cilostamide, carbazeran, piroximone, imaxodaa, Cl-930, siguazodaπ, adibεndan, saterinone, SKF-95654, SDZ-

MKS-492, 349-U-85. emoradan, EMD-5399S, EMD-57033, NSP-306, NSP-307, revizinone, NM-702, WIN-62582 and WfN-63291, enoximone and milrinone, PDE3/4 inhibitors (such as beiiafentrinc, trequinsin, ORG-30029, zardaverine, L- 686398, SDZ-ISQ-844, ORG-20241, EMD-54622. a«d tolafcntriπc) and other PDE 5 inhibitors (such as vinpocetin, papaverine, eπprofyliine, cilomilass, fenoxJrnøne. pentoxifylline, roflumilast, tadalafil(Ci.aiis®), thα>plαyl1ine, and vardcnafil{Lcvitraφ);

Neuropeptide Y2 (NPY2) agonists include hut are not limited to: peptide YY and fragments arsd variants thereof (e.g. YY3- -36 (PYY3-36 )(N ^S . ηngl J. Med. 349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLN L VTRQRY (SEQ ID NO:XXX}} τθ and PYY agonists such as those disclosed in WQ02/47712, WO03/02659L WO03/057235, rød WO03/027637;

serotonin reuptake ii^ibitors, such as, paroxetine, .fluoxetine (Prozac ^!?v! ). iluvoxamine, sertraline, citalopram, and imiprarnine, and those disclosed in. US6162805, US6365633, WO03/00663, WO01/27Q60, and WO01/ϊ6234! ;

15 thyruid hormone β agonists, such as lCB-261 1 (KaroBioBMS), miά ihosε disclosed in WO02/15S45 _r WO97/21993, WO99/00353, GB98/284425, U.S. Provisional λppHeatkm ^" No. 60/183,223, and Japanese Patent Application No. JF 2000256190:

UCP-I (uncoupling protein-!), 2. or 3 activators, such as phyianic acid, 4-|(E)-2~ {SjδJ^-tetrahydro-Sp^jS-ietraniethyi^-napthalenylJ-l -propenylJbenϋoic acid 0 (TTNPB), retinoic acid, and those disclosed in WO09/00123;

|53 (beta adrenergic receptor 3 ) agonists, such as λJ9677rfλK67? (Dainippon/Takeda). L75O355 (Merck), CP331648 (Pilzer), CU316,243, SB 418790, 3RL-37344, L-79656S, BMS-1%085, BR.L-35135A, CGP12I 77A, BTλ-243, GW

427353, Trceadrme, Zeneca D71 14, N-5984 (Nisshin K>υrin). LY-377604 (Lilly), SR b 591 19A, asid those disclosed in US554i2()4. US577O615, USS491 134, US5776983, US48S064, US5705515, US5451677, WO94/1816L WO95/29159, WO97/46556, WO98/04526 and WO9S/32753, WO0I/747S2, WO02/32897, WO03/0ϊ4ϊ 13,

WO03/0I6276, WO03/016307^003/024948, WO03/024953 and WO03/03788 ϊ ;

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noradrenergic agents including, hut not limited to, dicthylpropioπ (such as T'enuateC ¹

! -propanone, 2 -(diethylaniino)-l -phenyl-, hydrochloride}, Merrell), dextroamphetamine (also known as dextroamphetamine sulfate, dex amphetamine, dexedrmej Dexampex, Ferndex _* ϋxydess ii, Robese, Spancap #1), nwindol ((or 5-(p- chioropheny!}--*,S-dihydro-3H-imidazo[2,l -a]isoindo3-5-ol) such as Saπorex®,

Novartis or Mazanor®, Wyeth λyerst), phenylpropanolamine (or BcnzeneraethanoK aipha-^I-aniinoethvi)-, hydrochloride), phentei'mmc ((or Phenol, 3-[[4,5-duhydro~i H- i πiidazol-2~yl)eth> ¹ ! j(4-methy !pheny-l)amino], moπohydrctcWoπde) such as Adipex- Pφ, Lernmon, FAS11N®, Sniith-KIiπe Beecham and loαamin®, Medeva) _? phemlimetrszinc ((or (2S,3S)~3,4-Dιτnt'thyl~2phenylmorpholine L-(^ )-tartra!e ( 1 :1 )} such as Metraφ (Forest) , Plegine® (Wyeth-Ayerst}, Pre!u~2® (Boekinger ingelheisn), and Staiobexφ (Lcmmon), pheπdarnine tartrate (such as lliephann® (2,3,4,9" ^' rctrahydro-2-methyl-9-phenyi- JH-iiidciiol| ^' 2J-c]pyridinc L-(í)-tartrate (i : 1 }), Hoffnianri-LaRoche), meihamphetamine (such as Desoxyπ€ ^:; , Abbot ((S)-N, (u!pha)"dimeihy}bcπzencethanamine hydrochloride)), and phendlmeira^ine tartrate (such as Bontrilφ Slow-Release Capsules, Airmrin (-3,4-Dimcthyl-2- pheny 1 mo φh oline Tartrate) ;

fatty acid oxidation upreguiatαr/inducers such as Famoxin® (Genset):

.monaminc oxidase inhibitors including but no. limited to betloxaione, nκ>clobemκi&, brofaromine, phcnαxathme. esυprone, betbl, toloxatone, pirlindol, afniila-ϊiiπe, sereioreniine, bazinaprirse, lazabemide, milacemide, caroxazoπe and other certain compounds as disclosed by WOO 1/12176; and

other anti-obesity agents such as 5HT-2 agonists, ACC (acetyl-CoA carboxylase) inhibitors such as those described in WOO3/O721O7, alpha-lipoie aeid (aJpha-LA}, A 0096(54. appetite suppressants such as those in WO03/40107, ATL-%2 (Aikyrøe PLC), henzoeaine, benzphetaminc hydrochloride (Didrex), bladderwraek (focus Ycsiculosuis), BRS3 (bombesin receptor subtype 3) agonists, bupropion, caffeine, CCK agonists, chilosan, ehromium, conjugated linoleic acid, corticotropin-releasing hormcsne agonists, dehydroepiandrosterone, DGATl (diaeylgiyccrol aeyltransl erase

- !98 ~

I } inhibitors, DGAT2 (diacyiglyeerol aeyitomsferase 2} inhibitors, dicarbβxyiate transporter inhibitors, ephedra, exendin-4 (an inhibitor of glp-1) FAS (fatty acid synthase;} inhibitors (such as Cerυlenin and C75}, fat resorption inhibitors (such as those in WO03/053451, and the like), fatty acid transporter inhibitors, natural water soluble fibers (such as psyllium, piantago, guar, oat, pectin), gaiartin antagonists, galega (Croat's Rue, French Lilac), garcinia cambogia, germander (teueriuro cliaroaedπ's), gbreiiπ antibodies and ghreiin antagonists (such as those disclosed in WOC ⁾ 1/87335, and W 002/0825Q), peptide hormones miά variants thereof which affect the islet cell secretion, such as the hormones of the secretm/gastric inhibitory peptide (G!P)/γ;i8oacirve intestinal peptide (VϊP)/ρituitary adenylate cyclase activating peptide {PACAPJ/ghicagon-Hkx peptide Il {CiLP-liygiicentiivglucagon gene family and/or those of the adrenomeduHiwamyiiii/ calcitonin gene related peptide ((XJRP) gyrse family mcludiπgCjtP-1 (glueagon-iike peptide 1) agonists (e.g. (I ) exeτidi3>4, (2) those GLP-i molecules described in US20050130891 inemdrag GLP-i{7-34), OLP-K7-35), GLP-I (7-36) or GLP- 1(7-3?) in its C-teπmnally carboxylatεd or amidatcd ioππ or as modi tied GLP-! peptides and modifications thereof including those described in paragraphs 17-44 of US 2(K)SOl 30891.and derivatives derived from IJ LP- I -(7-3-OCOOi ! and the corresponding acid amide are employed which have the following general formula:

R -NH -J-IAEGTITSDVSYLKGQAAKEFIAWLVK-CON H ₂

wherein R ^~ !i or an organic compound having from 1. to K) carbon atoms. Preferably. R is the residue of a carboxylic acid. Particularly preferred are the following Cetrboxylic acid residues; fbrrnyf, acetyl, prαpionyl, isopropionyl, methyl ethyl, propyl, isoprøpyi n-butyl. sec -butyl tert-butyl.) and gip-1 (gjucagon-like peptide-! ), giueocorticoki aπlagonists, glucose transporter inhibitors, growth hormone secretogogues {such as those disclosed and specifically described in US5536716K interleukiπ-ό (fL-6) and modulators thereof (as in WO03/057237, and the like), L- carnitine, Mc3r (πielanoeortin 3 receptor) agonists, VlCH 2 R (melanin concentrating hormone 2R) agonist/antagonists, melanin concentraling hormone antagonists, melanoeortin agonists (such as .Melanotan Il or those described in WQ 99/64002 and

WO Qf>'746?9}. nora&me herba, phosphate transporter inhibitors, phytopharm compound 57 (CP 644,673), pyruvate, SCD-I (stεaroyl-CoA desaturase-i.) inhibitors, T71 (Tularik, Inc., Boulder CO). Topiramate (Topimax®, indicated as an anticonvulsant which has been shown to increase weight loss), transcription factor modulators {such as those disclosed in WO03/026576}, β-hydroxy steroid dehydrogenase-! inhibitors (β -HSD-I ), β-hydrøxy-(3-methy1butyrate, p57 {Pfizer}, ZonisaiXiide indicated as an anti- epileptic which has been shown to lead to weight iosvs), and the agents disclosed in US200301 19428 paragraphs 20-26,

The peptides and agonists described herein can be used in therapeutic combination with one or more anti-diabetic agents, including but not limited to: FFARγ agonists such as glitazones (e.g., WAY-120,744, AD 5075, babghtazone, cigiitazone, dargϋiazone (CP-86325, Pfizer), englitazone (CP-68722, Pfizer). isagliiazone (MϊT/J&J), MCC-SS5 (Mitsibishi diseiosed in US5594016), pioglitazone (.such as such as Actos ^" pioglitazone; Takcda), rossglitazorte (Avaαdia ^v ;Smith K.ϋne Beecham), røsigϋtaxoπe roaicate, trogiitaxone (Rezolin®, disclosed in US45729J2), • ^■ 5vog!ita2one (CS-OI L Sankyo}, GL-262570 (Glaxo Welcome}, BRL49653 {disclosed in WO98/0533I ), CLX-0921, 5-BTZD ₁ GW-0207, LG- 10064!, JJT-50I (JFNT7P&U), L-S95645 (Merck), R-! 19702 (Sankyo/Pfizer), NN-2344 (Dr. Rcddy/NN), YM-440 (Yamanouchi), LY-300512, LY-St ^Q SI S, R483 (Roche), T131 (Tularik). and rbe like and compounds disclosed hi US4687777, US5002953, US574J8O3, US59655S4, US6! 50383, US6150384, US6166042, US6166043, US6172090, US621 1205, QS6271243, US628809S, US6303640, US6329404, US5994554, W097/10813, WO97/27857,WO97/281 l5,WO97/ ^" 28137,WO97/27847, WOQ0/764S8, WO03/000685,WO03/0271 12.WO03/035602,

WO03/048I30,WO03/055S67, and pharmaceutically acceptable sails iheruof: biguanidcs such as rnetfoπniB hydrochloride {N,N-dimethyliroidodicarb(mimidic diarnide hydrochloride, such as G!ucophage ^rM , Bristol-Myers Squibb); metibmiiπ hydrochloride with gSyburide, such as Glucovaiicc™, Bπstol-Mycrs Squibb); htiibmπn (fmidodicarhonimidic diaraide, IN -butyl-); eioibmiine (1 -Butyl-2- ctbylbigiiaπidc, Schcring A. G.): other metfoττ«in salt forms (including where the salt

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