RECEPTOR AGONISTS

Field of the Invention

The present invention generally relates to peptides that are useful as agonists of opioid receptors. The invention also relates to pharmaceutical compositions comprising these peptides and the use of the peptides for inducing, promoting or otherwise facilitating pain relief and in the prophylaxis or treatment of conditions, such as but not limited to, inflammation and those that require or benefit from the alleviation of pain or inflammation.

Background of the Invention

Pain management is complex and often unsatisfactory. Many analgesic agents have side effects that cause other medical problems, particularly with long term use. Among the analgesic agents available, opioid analgesics are considered the most effective class of drugs available for the management of pain. Morphine is the 'gold standard' strong opioid analgesic to which all new opioid analgesic compounds are compared. Morphine is also recommended by the World Health Organisation as the drug of choice for the relief of moderate to severe cancer pain, the alleviation of moderate to severe pain in the post- surgical setting and for the relief of pain following trauma and cardiac infarction.

However, opioid analgesics, including morphine, are well documented to produce a range of unwanted side effects. Severe side effects include allergic reactions, such as difficulty breathing, swelling of lips, tongue, face and/or throat and hives; constipation; respiratory depression; seizures; cold, clammy skin; severe weakness, severe dizziness; and unconsciousness. Other side effects include sedation, nausea, vomiting, dry mouth, loss of appetite, dizziness, tiredness, lightheadedness, muscle twitching, sweating, pruritis, urinary retention and loss of libido. Furthermore, long term use of opioid analgesics can result in tolerance where increasing amounts of opioid analgesics are required to provide a constant level of pain relief. Some opioid analgesics, such as morphine, may upon moderate or long term use, also result in patient dependency. In some patients, such as the chronically

ill, the opioid side-effects render it impossible to continuously administer sufficiently high doses to adequately control pain.

Opioid receptors are located in the central and peripheral nervous system. Opioid receptor agonists that act by activating opioid receptors in the central nervous system, particularly in the brain, are well documented and represent a first line therapy for the alleviation of moderate to severe pain. Opioid receptor agonists that activate peripheral opioid receptors are useful for alleviation of peripheral pain.

There is a need for the development of alternative opioid receptor agonists that are able to alleviate pain or deliver an acceptable level of pain relief, especially where unwanted side effects can be reduced or eliminated.

Any discussion of prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

Summary of the Invention

The present invention is predicated in part on the discovery of a new class of peptides that are agonists of opioid receptors, particularly kappa (K) opioid receptors. The peptides may provide pain relief through an action in the central nervous system or more preferably through an action in the peripheral nervous system.

In one aspect of the present invention there is provided a peptide having an amino acid sequence comprising SEQ ID NO:1:

RrPaa _ϊ -Z-XaarHaarCrQrR;)

wherein

Paa _! is absent or a positively charged amino acid residue;

Z is a positively charged amino acid residue (Paa ₂ ), a mono- or di-amino, aminoalkyl or aminoalkoxy carbocyclic, heterocyclic, aryl or heteroaryl carboxylic acid residue, or a mono- or di-amino, or a mono- or di-(aminoalkyl)amino alkanoic acid residue; Xaaj is absent or is any natural or non-natural amino acid residue; Haai is a hydrophobic amino acid residue;

C ₁ is selected from cysteine, homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine and norselenocysteine and C ₂ is selected from cysteine, homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine, norselenocysteine and cysteamine; wherein the side chains of C ₁ and C ₂ are oxidatively linked, or C ₁ and C ₂ taken together form a 6 to 12 membered carbocyclic, heterocyclic, aryl or heteroaryl ring which is optionally linked to Haa _t through a linker, L ₁ ; R ₁ is absent, an N-terminal capping group, R. ₃ -Xaa ₂ -(C ₃ ) _n ~Xaa ₃ ; or R ₁ is an amino acid or a peptide sequence, either of which are optionally capped with an N-terminal capping group;

R ₂ is absent, a C-terminal capping group, Xaa ₄ , or a peptide sequence optionally capped with a C-terminal capping group, or R ₂ is a covalent bond to a heteroatom in the amino acid side chain of Xaa _lλ and when C ₂ is cysteamine, R ₂ is absent; R ₃ is absent, an N-terminal capping group; or R ₃ is an amino acid or a peptide sequence, either of which are optionally capped with an N-terminal capping group; Xaa ₂ and Xaa ₃ are independently absent or are any natural or non-natural amino acid residue; Xaa ₄ is any natural or non-natural amino acid residue which is optionally linked through its side chain to the side chain of Xaa _t and is optionally capped with a C-terminal capping group;

C ₃ is selected from cysteine, homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine and norselenocysteine, wherein when two C ₃ are present, the side chains are oxidatively linked, or two C ₃ taken together form a 6 to 12 membered carbocyclic, heterocyclic, aryl or

- A -

heteroaryl ring which is optionally linked to Xaa ₃ or Paa _t through a linker, L ₂ and/or Xaa ₂ through a linker, L ₁ ; and n is 0, 1 or 2, or a pharmaceutically acceptable salt thereof.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In yet another aspect of the present invention, there is provided a method of activating an opioid receptor comprising exposing the opioid receptor to an effective amount of an opioid agonist peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

In one embodiment, the opioid receptor is a peripheral opioid receptor. In another embodiment, the opioid receptor is a kappa opioid receptor. In yet another embodiment, the opioid receptor is a peripheral kappa opioid receptor.

In a further aspect of the present invention, there is provided a method of inducing, promoting or otherwise facilitating pain relief or analgesia in a subject comprising administering an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

In yet a further aspect of the present invention, there is provided a method of treatment or prophylaxis of inflammation or a condition or disorder associated with inflammation comprising administering an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will

be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The Peptides of the Invention

The opioid receptor agonists of the present invention are peptides having an amino acid sequence comprising SEQ ID NO:1 :

Ri-Paai-Z-Xaai-Haai-Ci-C2-Ra

wherein

Paa _t is absent or a positively charged amino acid residue;

Z is a positively charged amino acid residue (Paa ₂ ), a mono- or di-amino, aminoalkyl or aminoalkoxy carbocyclic, heterocyclic, aryl or heteroaryl carboxylic acid residue, or a mono- or di-amino, or a mono- or di-(aminoalkyl)amino alkanoic acid residue; Xaa} is absent or is any natural or non-natural amino acid residue; Haa _t is a hydrophobic amino acid residue;

C ₁ is selected from cysteine, homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine and norselenocysteine and C ₂ is selected from cysteine, homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine, norselenocysteine and cysteamine; wherein the side chains of C ₁ and C ₂ are oxidatively linked, or C ₁ and C ₂ taken together form a 6 to 12 membered carbocyclic, heterocyclic, aryl or heteroaryl ring which is optionally linked to Haai through a linker, L ₁ ;

R ₁ is absent, an N-terminal capping group, R ₃ -Xaa ₂ -(C ₃ ) _n -Xaa ₃ ; or R ₁ is an amino acid or a peptide sequence, either of which are optionally capped with an N-terminal capping group;

R ₂ is absent, a C-terminal capping group, Xaa ₄ or a peptide sequence optionally capped with a C-terminal capping group, or R ₂ is a covalent bond to a heteroatom in the amino acid side chain of Xaa _t and when C ₂ is cysteamine, R ₂ is absent;

R ₃ is absent, an N-terminal capping group or R ₃ is an amino acid or a peptide sequence, either of which are optionally capped with an N-terminal capping group;

Xaa ₂ and Xaa ₃ are independently absent or are any natural or non-natural amino acid residue; Xaa4 is any natural or non-natural amino acid residue which is optionally linked through its side chain to the side chain of Xaat and is optionally capped with a C-terminal capping group;

C ₃ is selected from cysteine, homocysteine, norcysteine, 4-mercaptoproline,

N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine and norselenocysteine, wherein when two C ₃ are present, the side chains are oxidatively linked, or two C ₃ taken together form a 6 to 12 membered carbocyclic, heterocyclic, aryl or heteroaryl ring which is optionally linked to Xaa ₃ or Paa _t through a linker, L ₂ and/or Xaa ₂ through a linker, L ₁ ; and n is 0, 1 or 2, or a pharmaceutically acceptable salt thereof.

The peptide of the invention may be presented as a derivative or prodrug or together with a cell-targeting moiety such as an antibody.

The term "derivative" as used herein in connection with the opioid receptor agonist peptides, such as a peptide of the SEQ ID NO. I ₅ refers to a peptide which differs from SEQ ID NO. 1 by one or more amino acid additions or side chain modifications or where an amide bond is replaced with an amide bond analogue such as a sulphonamide ester, a reduced amide, an inverted amide or a 1,2,3-triazole, or where the peptide backbone has been otherwise modified as known in the art. Such derivatives that do not have the ability to act as agonists of opioid receptors do not fall within the scope of the present invention.

Additions encompass the addition of one or more naturally-occurring or non-conventional amino acid residues.

The opioid receptor agonist peptides may also be peptides in which one or more of the amino acids has undergone side chain modifications. Examples of side chain modifications contemplated include modifications of amino groups such as by reductive alkylation, by reaction with an aldehyde followed by reduction with NaBH ₄ ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 -phosphate followed by reduction with NaBH ₄ . The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via 0-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide compounds; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. Any modification of cysteine residues must not affect the ability of the peptide to form the necessary disulphide bonds. It is also possible to replace the sulphydryl groups of cysteine with selenium equivalents such that the peptide forms a diselenium bond or a sulfide-selenium bond in place of one or more of the disulphide bonds.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Proline residues may be modified by, for example, hydroxylation in the 4-position, or by aliphatic or aromatic substitution on the proline ring system.

Backbone modifications include the reduction of an amide bond or replacement of an amide bond with a carba analogue, an inverted amide, a phosphate, a phosphone, a sulphonamide, a 1,2,3-triazole group or other analogues known in the art.

Suitable naturally occurring proteogenic amino acids are shown in Table 1 together with their one letter and three letter codes. Table 1

A list of some amino acids having modified side chains and other unnatural amino acids is shown in Table 2.

TABLE 2: List of non-naturally occurring amino acids and derivatives

Non-conventional Code 1 letter Non-conventional Code 1 letter amino acid code amino acid code

α-aminobutyric acid Abu L-N-methylalanine α-amino-α-methylbutyrate Mgabu L-N-methylarginine NMR aminocyclopropane- Cpro L-N-methylasparagine NMN carboxylate L-N-methylaspartic acid NMD aminoisobutyric acid Aib L-N-methylcysteine NMC aminonorbornyl- Norb L-N-methylglutamine NMQ carboxylate L-N-methylglutamic acid NME

L-cyclohexylalanine CHA L-N-methylhistidine NMH cyclopentylalanine Cpen L-N-methylisoleucine NMI

D-alanine ala a L-N-methylleucine NML

D-arginine arg r L-N-methyllysine NMK

D-asparagine asn n L-N-methylmethionine NMM

D-aspartic acid asp d L-N-methylnorleucine NMNLE

D-cysteine cys C L-N-methylnorvaline NMNVA

D-glutamine gin q L-N-methylornithine NMORN

D-glutamic acid glu e L-N-methylphenylalanine NMF

D-histidine his h L-N-methylproline NMP

D-isoleucine ile i L-N-methylserine NMS

D-leucine leu 1 L-N-methylthreonine NMT

D-lysine lys k L-N-methyltryptophan NMW

D-methionine met m L-N-methyltyrosine NMY

D-ornithine om L-N-methylvaline NMV

D-phenylalanine phe f L-N-methylethylglycine NMETG

D-proline pro P L-N-methyl-t-butylglycine NMTBUG

D-serine ser S L-norleucine NLE

D-threonine thr t L-norvaline NVA

D-tryptophan trp W α-methyl-aminoisobutyrate Maib

D-tyrosine tyr y α-methyl-γ-aminobutyrate Mgabu

D-valine val V α-methylcyclohexylalanine Mchexa

D-α-methylalanine mala α-methylcylcopentylalanine Mcpen

D-α-methylarginine marg α-methyl-α-napthylalanine Manap

D-α-methylasparagine masn α-methylpenicillamine Mpen

D-α-methylaspartate masp N-(4-aminobutyl)glycine NgIu

D-α-methylcysteine mcys N-(2-aminoethyl)glycine Naeg

D-α-methylglutamine mgln N-(3-aminopropyl)glycine Norn

D-α-methylhistidine mhis N-amino-α-methylbutyrate Nmaabu

D-α-methylisoleucine mile α-napthylalanine Anap

D-α-methylleucine mleu N-benzylglycine Nphe

D-α-methyllysine mlys N-(2-carbamylethyl)glycine NgIn

D-α-methylmethionine mmet N-(carbamylmethyl)glycine Nasn

D-α-methylornithine morn N-(2-carboxyethyl)glycine NgIu

D-α-methylρhenylalanine mphe N-(carboxymethyl)glycine Nasp

D-α-methylproline mpro N-cyclobutylglycine Ncbut

D-α-methylserine mser N-cyclodecylglycine Ncdec

D-N-methylserine nmser N-cycloheptylglycine Nchep

D-α-methylthreonine mthr N-cyclohexylglycine Nchex

D-α-methyltryptophan mtrp N-cyclodecylglycine Ncdec

D-α-methyltyrosine mtyr N-cylcododecylglycine Ncdod

D-α-methylvaline mval N-cyclooctylglycine Ncoct

D-N-methylalanine nmala N-cyclopropylglycine Ncpro

D-N-methylarginine nmarg N-cycloundecylglycine Ncund

D-N-methylasparagine nmasn N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylaspartate nmasp N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylcysteine nmcys N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamine nmgln N-(I -hydroxyethyl)glycine Nthr

D-N-methylglutamate nmglu N-(hydroxyethyl))glycine Nser

D-N-methylhistidine nmhis N-(imidazolyIethyI))glycine Nhis

D-N-methylisoleucine nmile N-(3-indolylyethyl)glycine Nhtrp

D-N-methylleucine nmleu N-methyl-γ-aminobutyrate Nmgabu

D-N-methyllysine nmlys D-N-methylmethionine nmmet

N-methylcyclohexylalanine Nmchexa N-methylcyclopentylalanine Nmcpen

D-N-methylornithine nniorn D-N-methylphenylalanine nmphe

N-methylglycine NaIa D-N-methylproline nmpro

N-methylaminoisobutyrate Nmaib D-N-methylserine nmser

N-(I -methylpropyl)glycine Nile D-N-methylthreonine nmthr

N-(2-methylpropyl)glycine Nleu N-(I -methylethyl)glycine Nval

D-N-methyltryptophan nmtrp N-methyl-napthylalanine Nmanap

D-N-methyltyrosine nmtyr N-methylpenicillamine Nmpen

D-N-methylvaline nmval N-(p-hydroxyphenyl)glycine Nhtyr γ-aminobutyric acid Gaba N-(thiomethyl)glycine Ncys

L-^-butylglycine TBUG penicillamine Pen

L-ethylglycine ETG L-α-methylalanine MALA

L-homophenylalanine HPHE L-α-methylasparagine MASN

L-α-methylarginine MARG L-α.-methyl-7-butylglycine MTBUG

L-α-methylaspartate MASP L-methylethylglycine METG

L-α-methylcysteine MCYS L-α-methylglutamate MGLU

L-α-methylglutamine MGLN L-α-methylhomophenylalanine MHPHE

L-α-methylhistidine MHIS N-(2-methylthioethyl)glycine Nmet

L-α-methylisoleucine MILE L-α-methyllysine MLYS

L-α-methylleucine MLEU L-α-methylnorleucine MNLE

L-α-methylmethionine MMET L-α-methylornithine MORN

L-α-methylnorvaline MNVA L-α-methylproline MPRO

L-α-methylphenylalanine MPHE L-α-methylthreonine MTHR

L-α-methylserine MSER L-α-methyltyrosine MTYR

L-α-methyltryptophan MTRP L-N-methyl-homophenylalanine Nmhphe

L-α-methylvaline MVAL N-(N-(3 ,3 -diphenylpropyl) Nnbhe

N-(N-(2,2-diphenylethyl) Nnbhm carbamylmethylglycine carbamylniethylglycine L-pyroglutamic acid PYR U

1 -carboxy- 1 -(2,2-diphenyl- Nmbc D-pyroglutamic acid pyr u ethylamino)cyclopropane 0-methyl-L-serine Omser

4-hydroxyproline HYP O-methyl-L-homoserine Omhser ornithine Orn 5-hydroxylysine Hlys

2-aminobenzoyl (anthraniloyl) ABZ α-carboxyglutamate GIa

D-cyclohexylalanine cha phenylglycine Phg

4-phenyl-phenylalanine Bib L-pipecolic acid (homoproline) PBP

L-citrulline CIT L-homoleucine HLE α-cyclohexylglycine CHG L-lysine (dimethyl) DMK

L-l,2,3,4-tetrahydroisoquinoline- L-2-naphthyIalanine NAL

3-carboxylic acid TIC L-dimethyldopa or L-dimethoxy- DMD

L-thiazolidine-4-carboxylic acid THZ phenylalanine

L-homotyrosine HTyr L-3 -pyridylalanine PYA

L-2-furylalanine FLA L-histidine (benzoyloxymethyl) HBO

L-histidine (3 -methyl) HME N-cycloheptylglycine Nchep

N-(3 -guanidinopropyl)gly cine Narg L-diphenylalanine DPA

O-methyl-L-tyrosine MeY O-methyl-L-homotyrosine Omhtyr

O-glycan-serine g-Ser L-β-homolysine BHK

Meta-tyrosine m-Tyr O-glycan-threoine g-Thr

Nor-tyrosine nor-Tyr Ortho-tyrosine o-Tyr

L-N ₅ N' ,N"-trimethyllysine TMK L-N,N' -dimethyllysine DMK homolysine Homolys L-homoarginine HomoARG norlysine Nor-Lys neotryptophan neo-tryp

N-glycan Asparagine g-Asn

3-benzothienylalanine BTA 7-hydroxy- 1,2,3 ,4-tetrahydro- isoquinoline-3 -carboxylic acid HTI

4-fluorophenylalanine MFF diaminopropionic acid DPR

4-methylphenylalanine MEF homocysteine HCY bis-(2-picolyl)amine 3,4-dimethoxyphenylalanine DMF pentafluorophenylalanine PFF 4-chlorophenylalanine CLF indoline-2-carboxylic acid INC L-1 ,2,3,4-tetrahydronorharman-

2-aminobenzoic acid ABZ 3 -carboxylic acid TPI

3-amino-2-naphthoic acid ANZ (ANC) Adamantylalanine ADA

Asymmetric dimethylarginine ADMA Symmetrical dimethylarginine SDMA

L-tetrahydroisoquinoline -1- 3 -carboxythiomorpholine CTM carboxylic acid TIQ D-1 ,2,3,4-tetrahydronorharman-

D-tetrahydroisoquinoline- 1 - 3-carboxylic acid tpi carboxylic acid tiq 3-Aminobenzoic acid

1-Amino-cyclohexane acetic acid 3-Amino-l -carboxymethyl-

D/L-Allylglycine pyridin-2-one

4-Aminobenzoic acid 1 -amino- 1 -cyclohexane

1 -amino-cyclobutane carboxylic acid carboxylic acid 2-aminocyclopentane carboxylic 2 or 3 or 4-aminocyclohexane acid carboxylic acid 1 -amino- 1 -cyclopropane

1 -amino- 1 -cyclopentane carboxylic acid carboxylic acid 2-aminoindane-2-carboxylic acid l-aminoindane-l -carboxylic acid 4-amino-tetrahydrothiopyran-4- 4-amino-pyrrolidine-2-carboxylic carboxylic acid TTC acid azetidine-2-carboxylic acid

2-aminotetraline-2-carboxylic acid b-(benzothiazol-2-yl)-alanine azetidine-3 -carboxylic acid neopentylglycine

4-benzyl-pyrolidine-2-carboxylic 2-carboxymethyl piperidine acid b-cyclobutyl alanine tert-butylglycine allylglycine b-(benzothiazolyI-2-yl)-alanine diaminopropionic acid b-cyclopropyl alanine homo-cyclohexyl alanine HCH diaminobutyric acid (2S,4R)- 4-hydroxypiperidine-2 5,5-dimethyl-l,3-thiazolidine-4- carboxylic acid carboxylic acid octahydroindole-2-carboxylic

(2R,4S)4-hydroxypiperidine-2 acid carboxylic acid (2S,4R) and (2S,4R)-4-(2-naphthyl)

(2S,4S) and (2S,4R)-4-(2- pyrrolidine-2-carboxylic acid naphthylmethoxy)-pyrolidine-2- Nipecotic acid carboxylic acid (2S,4R)and (2S,4S)-4-(4-phenylbenzyl)

(2S, 4S) and (2S,4R)4-phenoxy- pyrrolidine-2-carboxylic acid pyrrolidine-2-carboxylic acid (3 S)- 1 -pyrrolidine-3 -carboxylic acid (2R,5S)and(2S,5R)-5-phenyl- (2S,4S)-4-tritylmercapto- pyrrolidine-2-carboxylic acid pyrrolidine-2-carboxylic acid

(2S,4S)-4-amino- 1 -benzoyl- (2S,4S)-4-mercaptoproline MPC pyrrolidine-2-carboxylic acid ABP t-butylglycine TBG t-butylalanine TBA N,N-bis(3 -aminopropyl)glycine

(2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid 1 -amino-cyclohexane- 1 -carboxylic acid 1-aminomethyl-cyclohexane-acetic acid N-mercaptoethylglycine

3,5-bis-(2-amino)ethoxy-benzoic acid diaminobutyric acid DAB

3,5-diamino-benzoic acid selenocysteine SEC

4,4'-biphenylalanine BPA 2-methylamino-benzoic acid

(or N-methylanthranylic acid) NMA

These types of modifications may be important to stabilise the peptide if administered to an individual, or may provide added affinity for a receptor providing increased activity or specificity.

The amino acid sequences of the present invention, may be represented as the L-configuration by three letter or one letter codes in capital letters or having initial capital letters (refer to Table 1). For example, L-alanine may be represented by Ala, ALA or A. The D-confϊguration is represented by codes that are all lower case letter. For example, D-alanine may be represented by ala or a (refer to Table 2).

As used herein, the term "natural amino acid" refers to amino acids that occur in nature and commonly form the building blocks of proteins. Examples of natural amino acids are given in Table 1.

As used herein, the term "non-natural amino acid" refers to amino acids that do not occur in nature. Non-natural amino acids may be derivatives of natural amino acids or may be synthetic compounds containing an amino group and a carboxylic acid group suitably disposed to be incorporated into a peptide, for example, α, β and γ-amino acids. Non-natural amino acids may be in the L- or D-configuration. Examples of suitable non-natural amino acids are given in Table 2.

The term "positively charged amino acid" as used herein refers to a natural or non-natural amino acid residue having a side chain functional group capable of bearing a positive charge. In some embodiments when the positively charged amino acid has an amino group in its side chain, the amino group may be further substituted with a guanyl group, to form an H ₂ NC(^NH)-NH- group in the side chain. Examples of suitable positively charged

amino acids include, but are not limited to, L-lysine, L-arginine, L-histidine, D-lysine, D-arginine, D-histidine, L-α-methyllysine, D-α-methyllysine, L-α-methylarginine, D-α-methylarginine, L-α-methylhistidine, D-α-methylhistidine, L-3-methylhistidine, D-3-methylhistidine, L-homolysine, D-homolysine, norlysine, L-homoarginine, D-homoarginine, L-β-homolysine, D-β-homolysine, L-dimethyllysine, D-dimethyllysine, L-N-methyllysine, D-N-methyllysine, L-N-methylarginine, D-N-methylarginine, 3- pyridyl-alanine, N-l-(2'-pyrazolinyl)arginine, 2-(4-piperinyl)-glycine, 2-(4-piperinyl)- arginine, 2-[3-(2S)-pyrrolininyl]glycine, 2-[3-(2S)-pyrrolininyl]-arginine, 5-hydroxylysine, dianiinopropionic acid, symmetrical dimethyl arginine, asymmetrical dimethyl arginine, (N ^ε -guanyl)-L-lysine, (N ^ε -guanyi)-D-lysine, (N ^ε -guanyl)-L-α-methyllysine, (N ^ε -guanyl)-D- α-methyllysine, (N ^ζ -guanyl)-L-homolysine, (N ^ς -guanyl)-D-homorysine, (N ^η -guanyl)-L-β- homolysine, (N ^η -guanyl)-D-β-homolysine, (N ^δ -guanyl)-L-norlysine and (N ^δ -guanyl)-D- norlysine. The guanyl groups may be optionally further and independently substituted at a nitrogen with C _1-6 alkyl.

As used herein, the term "hydrophobic amino acid" refers to a natural or non-natural amino acid bearing a hydrophobic, non-polar side chain. Suitable hydrophobic side chains include aliphatic straight chain, branched, caged or aromatic side chains. Examples of suitable hydrophobic amino acid residues include, but are not limited to, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, cyclohexylalanine, cyclopentylalanine, α-methylalanine, α-methylvaline, α-methylleucine, α-methylisoleucine, α-methylmethionine, α-methylphenylalanine, α-methyltryptophan, α-methylcyclohexylalanine, α-methylcyclopentylalanine, α-methyl-α-naphthylalanine, α-naphthylalanine, ethylglycine, t-butylglycine, z'-butylalanine, homophenylalanine, methylethylglycine, α-methylhomophenylalanine, 2-furylalanine, neotryptophan, diphenylalanine, phenylglycine and adamantylalanine. Each of these may be in the D- or L-configuration.

The term "aliphatic amino acid" as used herein refers to a natural or non-natural amino acid bearing a linear or branched saturated hydrocarbon side chain. For example, such side chains have the formula C _n H _2n + ₂ where n is 1 to 20. This term also includes saturated

cyclic or caged hydrocarbon side chains such as cyclopentyl, cyclohexyl, adamantyl or norbornyl groups. In some cases, one or more carbon atoms in the side chain are replaced by a heteroatom such as O, N or S. Examples of suitable aliphatic amino acids include, but are not limited to, alanine, leucine, isoleucine and valine in either the L- or D-configuration.

The term "aromatic amino acid" as used herein refers to natural and non-natural amino acid residues having a side chain bearing an aromatic ring. The aromatic group may be carbocyclic or heterocyclic. Examples of suitable aromatic amino acids include, but are not limited to, L-phenylalanine, L-tryptophan, L-tyrosine, L-histidine, D-phenylalanine, D-tryptophan, D-tyrosine, D-histidine, neotryptophan, phenylglycine, naphthylalanine, 3-pyridylalanine, diphenylalanine, and substituted derivatives of these aromatic amino acids. Substituted derivatives are substituted at one or more aromatic carbon atoms. Suitable substituents include, but are not limited to, methyl, ethyl, propyl, isopropyl, halo, phenyl, carboxyl, nitro, cyano, SO ₃ H, sulfomethyl, sulphonamide, NH ₂ and NHAc, where halo is selected from fluoro, chloro, bromo and iodo. Non-natural aromatic amino acids also include tyrosine residues, including those where the hydroxy group is in the 2, 3 or 4 position on the aromatic ring, in which the hydroxy group is further substituted. The hydroxy group may be substituted, for example, sulfo group to form O-sulphotyrosine or a phospho group to form O-phosphotyrosine. Examples of non-natural aromatic amino acids include, but are not limited to, neotryptophan, phenylglycine, naphthylalanine, 3-pyridylalanine, diphenylalanine, o-tyrosine, m-tyrosine, O-sulfotyrosine, O-phosphotyrosine, monohalo-tyrosine, dihalo-tyrosine, monohalotryptophan, dihalo- tryptophan, nitrophenylalanine, 4-phenylphenylalanine, 2,6-dimethyltyrosine, 5-aminotyrosine, 4-hydroxyphenylglycine and 4-hydroxy-methyltyrosine. Each of these non-natural aromatic amino acids may be in the D- or reconfiguration.

The term "peptide sequence" as used herein refers to a sequence of two or more amino acid residues. The choice of amino acid residues in the peptide sequence is not particularly limited. The amino acid residues in the peptide sequence may be selected to assist with binding to the opioid receptor or may assist in the transport of peptides across membranes

so they may come in contact with opioid receptors at specific sites. The amino acids may also infer stability to the peptide, for example, by cyclisation.

The term "oxidatively linked" as used herein refers to the formation of a bond between any two residues having a mercaptol or selenol group in their side chain. For example, two of cysteine, homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine, penicillamine, selenocysteine, homoselenocysteine or norselenocysteine residues, and in some cases cysteamine, under oxidation conditions. For example, a disulfide bond may be formed between two cysteine residues, two homocysteine residues, two penicillamine residues or a cysteine and homocysteine residue or cysteine and penicillamine residue or the oxidative bond may be formed between two selenocysteine residues, two selenohomocysteine residues or a selenocysteine residue and a homoselenocysteine residue, to form a diselenium bond, or the oxidative bond may be formed between a cysteine or homocysteine residue and a selenocysteine or homoselenocysteine residue to form a sulfur-selenium bond. Suitable oxidative conditions include, but are not limited to, buffered DMSO or isopropanol/ammonium carbonate solutions.

As used herein, the term "N-terminal capping group" refers to a group covalently bonded to the N-terminal nitrogen atom. The N-terminal capping group may assist in stabilising the peptide in vivo or in vitro. For example, the N-terminal capping group may reduce hydrolysis by in vivo proteolytic enzymes or may reduce degradation of the peptide under storage conditions. The N-terminal capping group may assist in receptor binding providing substituents for further attractive binding in the receptor active site. The N-terminal capping group may also be chosen to allow penetration of the peptide to the site of pain or inflammation, for example, through membranes, through the extracellular matrix or through cell walls.

In one embodiment, the N-terminal capping group is selected from a group having the formula (I):

A (CH ₂ ) _m Z

wherein

A is a straight chain or branched C ₁ -Ci ₀ alkyl group or an optionally substituted aryl or optionally substituted heteroaryl group, Z is absent, -C(=O)-, -S(=O>, -S(O) ₂ -, -OP(O)-, -OP(=O)(OH)- or -OP(OH)-, and m is 0 or an integer from 1 to 6. In some embodiments A is a Ci-Ci ₂ alkyl group and m is 0 or A is a phenyl, naphthyl, tetrahydronaphthyl, pyridyl, indolyl, quinolinyl, coumarinyl, adamantyl or benzodioxanyl group, Z is -C(=0)- or -S(O) ₂ - and m is O or an integer from 1 to 3. Preferred optional substituents include for the aryl or heteroaryl group include, but are not limited to, one to three substituents selected from hydroxy, C _1-6 alkyl, C ₁ -C ₆ alkoxy, halo, aryl, aryloxy, and nitro, especially hydroxy, methyl, methoxy, fluoro, chloro, bromo, iodo, phenyl, phenoxy and nitro. Examples of suitable N-terminal capping groups include, but are not limited to, 4- hydroxyphenylCO-, 4-hydroxyphenylCH ₂ CO-, 4-hydroxyphenyl(CH ₂ ) ₂ CO-, 3- hydroxyphenylCO-, 3-hydroxyphenylCH ₂ CO-, 3-hydroxyphenyl(CH ₂ ) ₂ CO-, 2- hydroxyphenylCO-, 2-hydroxyρhenylCH ₂ CO-, 2-hydroxyphenyl(CH ₂ ) ₂ CO-, 4- methoxyphenylCO-, 4-methoxyphenylCH ₂ CO-, 4-methoxyphenyl(CH ₂ ) ₂ CO-, 3- methoxyphenylCO-, 3-methoxyphenylCH ₂ CO-, 3-methoxyphenyl(CH ₂ ) ₂ CO-, 2- methoxyphenylCO-, 2-methoxyphenylCH ₂ CO-, 2-methoxyphenyl(CH ₂ ) ₂ CO-, 3,4- dimethoxyphenylCO-, 3,4-dimethoxyphenylCH ₂ CO-, 3,4-dimethoxyphenyl(CH ₂ ) ₂ CO- ₅ phenylCO-, phenylCH ₂ CO-, phenyl(CH ₂ ) ₂ CO-, phenyl(CH ₂ ) ₃ CO-, naphthyl-2-CO-, naphthyl-2-CH ₂ CO-, naphthyl-2-(CH ₂ ) ₂ CO-, naphthyl-2-(CH ₂ ) ₃ CO-, 1,2,3,4- tetrahydronaphthyl-2-CO-, 1 ,2,3,4-tetrahydronaphthyl-2-CH ₂ CO-, 1 ,2,3,4- tetrahydronaphthyl-2-(CH ₂ ) ₂ CO-, 1 ,2,3,4-tetrahydronaphthyl-2-(CH ₂ ) ₃ CO-, 4-ρhenyl- phenylCO-, 4-phenyl-phenylCH ₂ CO-, 4-phenyl-phenyl(CH ₂ ) ₂ CO-, 3-phenyl-phenylCO-, 3-phenyl-phenylCH ₂ CO-, 3-phenyl-ρhenyl(CH ₂ ) ₂ CO-, 4-ρhenoxyphenylCO- ₅ 4- phenoxyphenylCH ₂ CO-, 4-phenoxyphenyl(CH ₂ ) ₂ CO-, 3-phenoxyphenylCO-, 3- phenoxyphenylCH ₂ CO-, 3-ρhenoxyphenyl(CH ₂ ) ₂ CO-, 4-halophenylCO-, 4- halophenylCH ₂ CO-, 4-halophenyl(CH ₂ ) ₂ CO-, 3-halophenylCO-, 3-halophenylCH ₂ CO-, 3- halophenyl(CH ₂ ) ₂ CO-, 2-halophenylCO-, 2-halophenylCH ₂ CO-, 2-halophenyl(CH ₂ ) ₂ CO-, 3,4-dihalophenylCO-, 3,4-dihalophenylCH ₂ CO-, 3,4-dihalophenyl(CH ₂ ) ₂ CO- ₅ 4- nitrophenylCO-, 4-nitrophenylCH ₂ CO-, 4-nitroρhenyl(CH ₂ ) ₂ CO-, 3-nitrophenylCO-, 3-

nitrophenylCH ₂ CO-, 3-nitrophenyl(CH ₂ ) ₂ CO- ₅ 2-nitroρhenylCO-, 2-nitrophenylCH ₂ CO- ₅ 2-nitrophenyl(CH ₂ ) ₂ CO-, 3-indolylCO-, 3-indolylCH ₂ CO- ₅ 3-indolyl(CH ₂ ) ₂ CO- _} 3-indolyl(CH ₂ ) ₃ CO-, N-methyl-indolylCO-, N-methyl-3-indolylCH ₂ CO-, N-methyl-3- indolyl(CH ₂ ) ₂ CO-, N-methyl-3-indolyl(CH ₂ ) ₃ CO-, 4-indolylCO-, 4-indolylCH ₂ CO-, 4-indolyl(CH ₂ ) ₂ CO-, 4-indolyl(CH ₂ ) ₃ CO-, 2-pyridylCO-, 2-pyridylCH ₂ CO-, 2-pyridyl(CH ₂ ) ₂ CO-, 2-pyridyl(CH ₂ ) ₃ CO-, 3-quinolinyl-CO-, 3-quinolinylCH ₂ CO- ₅ 3-quinolinyl(CH ₂ ) ₂ CO- ₅ 3-quinolinyl(CH ₂ ) ₃ CO-, 2-quinolinylCO-, 2-quinolinylCH ₂ CO- ₅ 2-quinolinyl(CH ₂ ) ₂ CO- ₅ 2-quinolinyl(CH ₂ ) ₃ CO-, coumarinCO-, coumarinCH ₂ CO-, coumarin(CH ₂ ) ₂ CO-, coumarin(CH ₂ ) ₃ CO-, adamantylCO-, benzodioxanylCO-, (R or S)- l,4-benzodioxane-2-CO-, CH ₃ CO-, CH ₃ CH ₂ CO-, CH ₃ CH ₂ CH ₂ CO-, CH ₃ (CH ₂ ) ₃ CO-, CH ₃ (CH ₂ ) ₄ CO- ₅ CH ₃ (CH ₂ ) ₅ CO-, CH ₃ (CH ₂ ) ₆ CO-, CH ₃ (CH ₂ ) ₇ CO-, CH ₃ (CH ₂ ) ₈ CO-, CH ₃ (CH ₂ ) ₉ CO-, CH ₃ (CH ₂ ) ₁₀ CO- and CH ₃ (CH ₂ )πCO-.

In another embodiment the N-terminal capping group is a guanyl group [H ₂ NC(=NH)], or a substituted guanyl group in which one or both of the nitrogen atoms are further independently substituted with C _1-6 alkyl. For example, suitable substituted guanyl groups include, but are not limited to, CH ₃ NHC(=NH)-, H ₂ NC(=NCH ₃ )- and CH ₃ NHC(=NCH ₃ )-.

As used herein, the term "C-terminal group" refers to a group covalently bonded to the C-terminal carbon atom or carboxy group. Suitable C-terminal capping groups include

C-terminal amides, esters, aldehydes and ketones. For example, suitable C-terminal capping groups include, but are not limited to, CONH ₂ , CONH(alkyl), CON(alkyl) ₂ ,

CONHphenyl, CON(phenyl) ₂ , CONH(alkylphenyl), CON(alkyl)(phenyl); C0 ₂ alkyl,

CO ₂ phenyl, CO ₂ alkylphenyl, COH, COalkyl, COphenyl and COalkylphenyl where the "CO" group is derived from the C-terminal carboxylic acid.

In preferred embodiments, the C-terminal capping group is -CON(R) ₂ wherein each R is independently selected from hydrogen or C ₁ -C ₆ alkyl. Examples include, but are not limited to, -CONH ₂ , -CONHCH ₃ or -CON(CH ₃ ) ₂ , especially -CONH ₂ .

As used herein, the term "carbocyclic ring" or "carbocycle" refers to a cyclic saturated, unsaturated or caged non-aromatic hydrocarbon 6 to 10 carbon atoms in the ring. Suitable carbocyclic groups include cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptanyl, cycloheptenyl, cycloheptadienyl, cyclooctanyl, cyclooctenyl, cyclooctadienyl, cyclononyl, cyclononenyl, cyclononadienyl, cyclodecyl, cyclodecenyl, cyclodecadienyl and caged carbocyclic groups such as adamantyl and norbornyl groups.

As used herein, the term "aryl" is intended to mean any stable, monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl and binaphthyl.

The term "heterocyclic" or "heterocyclyl" as used herein, refers to a cyclic hydrocarbon in which one to four carbon atoms have been replaced by heteroatoms independently selected from N, S, O and Se. A heterocyclic ring may be saturated or unsaturated. Examples of suitable heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl, pyranyl, piperidinyl, piperazinyl, pyrazolinyl, dithiolyl, oxathiolyl, dioxanyl, dioxinyl, morpholino, thiomorpholino, oxazinyl, azepinyl, diazepinyl, thiazepinyl, oxepinyl and thiapinyl.

The term "heteroaryl" as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include, but are not limited to, acridinyl, carbazolyl, cirmolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, thiophenyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, imidazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline, thiazolyl, isothiazolyl, 1,2,4-triazolyl, 1 ,2,4-oxadiazolyl, 1,2,4-thiadiazolyl, benzodioxanyl, benzazepinyl, benzoxepinyl, benzodiazepinyl, benzothiazepinyl and benzothiepinyl. Preferred heteroaryl groups have 5- or 6-membered rings, such as pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,

pyrrolyl, thiazolyl, isothiazolyl, 1,2,4-triazolyl and 1,2,4-oxadiazolyl and 1,2,4- thiadiazolyl.

As used herein, the term "halogen" or "halo" refers to fluorine (fluoro), chlorine (chloro), bromine (bromo) and iodine (iodo).

As used herein, the term "alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1 to 20 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, C _1-6 alkyl which includes alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, ^-propyl, /-propyl, n-butyl, /-butyl, t-butyl, «-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 5-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl, octyl, nonyl, decyl and dodecyl.

The term "mono- or di- amino, aminoalkyl or aminoalkoxy carbocyclic, heterocyclic, aryl or heteroaryl carboxylic acid" refers to a carbocyclic, heterocyclic, aryl or heteroaryl ring as defined above having a carboxylic acid substituent attached to a carbon atom of the ring and one (mono) or two (di) substituents selected from -NH ₂ , -Cj- ₆ alkylNH ₂ and -OCi- ₆ alkylNH ₂ . Suitable -Q-βalkylNIfe groups include -CH ₂ NH ₂ , -CH ₂ CH ₂ NH ₂ , -(CH ₂ ) ₃ NH ₂ , -(CH ₂ ) ₄ NH ₂ , -(CH ₂ ) ₅ NH ₂ and -(CH ₂ ) ₆ NH ₂ , especially -C _1-3 alkylNH ₂ . Suitable -OC _1-6 alkylNH ₂ groups include -OCH ₂ NH ₂ , -OCH ₂ CH ₂ NH ₂ , -O(CH ₂ ) ₃ NH ₂ , -O(CH ₂ ) ₄ NH ₂ , -O(CH ₂ ) ₅ NH ₂ and -O(CH ₂ ) ₆ NH ₂ , especially -OC ₂ . ₃ alkylNH ₂ , such as -OCH ₂ CH ₂ NH ₂ . Examples of suitable mono- or di- amino, aminoalkyl or aminoalkoxy carbocyclic, heterocyclic, aryl and heteroaryl carboxylic acids include 2-amino-naphthoic acid, 3,5- diaminobenzoic acid, 4-ammo-l-benzoyl-pyrrolidine-carboxylic acid and 3,5- diaminoethoxybenzoic acid.

The term "mono- or di-amino or mono- or di-(aminoalkyl)amino alkanoic acid" refers to an alkyl group substituted with a carboxylic acid and further substituted with one or two amino groups (NH ₂ ) or an amino group substituted with one or two -C _1-6 alkylNH ₂ groups.

Suitable alkanoic acids include diaminoacetic acid, 2-aminoacetic acid, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, 2,3-diaminopropanoic acid, 3,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 3,4-diaminobutanoic acid, 4,4-diaminobutanoic acid, 2,5-diaminopentanoic acid, 3,5-diaminopentanoic acid, 4,5-diaminopentanoic acid, 5,5-diaminopentanoic acid, 2,6-diaminohexanoic acid, 3,6-diaminohexanoic acid, 4,6-diaminohexanoic acid, 5,6-diaminohexanoic acid, 6,6-diaminohexanoic acid, N-aminomethylglycine, N,N-bis(aminomethyl)glycine, N-aminoethylglycine, N,N-bis(aminoethyl)glycine, N-aminopropylglycine, N,N-bis(aminopropyl)glycine, N-aminomethyl-N- aminoethylglycine and the like.

In some embodiments, the free amino group of the di- amino, aminoalkyl or aminoalkoxy carbocyclic, heterocyclic, aryl or heteroaryl carboxylic acid or the di-amino or di- (arninoalkyl)amino alkanoic acid is substituted with a guanyl group. The guanyl groups may be optionally further and independently substituted at a nitrogen with C _1-6 alkyl.

As used herein, the term "linker" refers to a group of 1 to 3 atoms in length that covalently links the carbocyclic, heterocyclic, aryl or heteroaryl group to the peptide. Linkers may be adapted to link a group to a C-terminal or N-terminal end of a peptide. For example, a linker L ₁ , suitable for linking the carbocyclic, heterocyclic, aryl or heteroaryl group representing C ₁ and C ₂ to the C-terminal carboxyl group of the Haa _t residue or is suitable for linking the carbocyclic, heterocyclic, aryl or heteroaryl group representing (C ₃ ) ₂ to the C-terminal carboxyl group of Xaa ₂ . Suitable linkers include heteroatoms selected from -O-, -S- and -NR ₄ - where R ₄ is H or C _1-4 alkyl, or an alkylene group in which one or more carbon atoms may be optionally substituted or where a carbon atom of an alkylene group is replaced by a heteroatom. Suitable linkers L ₁ include, but are not limited to, -O-, -S-, -NH-, -N(CH ₃ )-, -N(CH ₂ CH ₃ )-, -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -OCH ₂ -, -OCH ₂ CH ₂ -, -CH ₂ O-CH ₂ -, -CH ₂ O-, -CH ₂ CH ₂ O-, -SCH ₂ -, -SCH ₂ CH ₂ -, -CH ₂ SCH ₂ -, -CH ₂ S-, -CH ₂ CH ₂ S-, -NHCH ₂ -, -NHCH ₂ CH ₂ -, -CH ₂ NHCH ₂ -, -CH ₂ NH- and -CH ₂ CH ₂ NH-; especially -NH-, -N(CH ₃ )-, -CH ₂ - and -O-.

Linkers that are suitable for linking a group to the N-terminus of a peptide, are designated L ₂ . These linkers, for example, may link a carbocyclic, heterocyclic, aryl or heteroaryl group representing (C ₃ ) ₂ to the N-terminus of Xaa ₃ or Paa^ Suitable groups include alkylene in which one or more carbon atoms may be optionally substituted or acyl groups. Suitable linkers L ₂ include -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH ₂ OCH ₂ -, -CH ₂ NHCH ₂ -, -CH ₂ SCH ₂ -, -C(O)-, -CH ₂ C(O)- and -CH ₂ CH ₂ C(O)-, especially -C(O)-, -CH ₂ C(O)- and -CH ₂ CH ₂ C(O)-.

In preferred embodiments, the peptides of SEQ ID NO:1 include one or more of the following features:

Pa& _\ is selected from arginine, lysine, histidine, symmetrical dimethylarginine, asymmetrical dimethylarginine, α-methylarginine, α-methyllysine, α-methylhistidine, N-methylarginine, N-methyllysine, dimethyllysine, homolysine, homoarginine, norlysine, 3-pyridylalanine, N-l-(2-pyrazolinyl)arginine, 2-(4-piperinyl)glycine,

2-(4-piperinyl)arginine, 2-[3-(2S)-pyrrolinyl]glycine, 2-[3-(2S)-pyrrolinyl]arginine, 5-hydroxylysine, (N ^ε -guanyl)-lysine, (N ^ε -guanyl)-α-methyllysme, (N ^η -guanyl)-homolysine and (N ^δ -guanyl)-norlysine, or Paai is absent; especially arginine, lysine, N-methylarginine, N-methyllysine and dimethyllysine; more especially arginine. In each case, Paa] may be in the D- or L-configuration.

Z is Paa ₂ , 3-aminonaphthoic acid, 3,5-diaminobenzoic acid, 3-amino-5- guanylaminobenzoic acid, 5-amino-3-guanylaminobenzoic acid, 4-ammo-l-benzoyl- pyrrolidine-2-carboxylic acid, N,N-bis(3-aminopropyl)glycine, N-(3-aminopropyl)-N- (guanyl-3-aminopropyl)-glycine, diaminopropanoic acid (diaminopropionic acid), guanylamino-aminopropionic acid, 3,5-bis(2-aminoethyl)benzoic acid, 3,5-bis(2- aminoethoxy)benzoic acid and 4-aminobutyric acid.

Paa ₂ is selected from arginine, lysine, histidine, symmetrical dimethylarginine, asymmetrical dimethylarginine, α-methylarginine, α-methyllysine, α-methylhistidine,

N-methylarginine, N-methyllysine, dimethyllysine, homolysine, homoarginine, norlysine,

3 -pyridylalanine, N- 1 -(2-pyrazolinyl)arginine, 2-(4-piperinyl)glycine,

2-(4-piperinyl)arginine, 2-[3-(2S)-pyrrolinyl]glycine, 2-[3-(2S)-pyrrolinyl]arginine, 5-hydroxylysine, (N ^ε -guanyl)-lysine, (N ^ε -guanyl)-α-methyllysine, (N ^η -guanyl)-homolysine and (N -guanyl)-norlysine; especially arginine, lysine and N-methylarginine; more especially arginine. In each case, Paa ₂ may be in the L- or D-configuration.

Xaa _t is absent, glycine or an aliphatic amino acid, an amide containing amino acid or a diamino acid. Preferred Xaai include glutamine, glycine asparagine, homoglutamine, γ-N-methylglutamine, γ-N-dimethylglutamine, lysine, homolysine, ornithine, aminobutyric acid and diaminobutyric acid, hi some embodiments, the lysine, homolysine or ornithine side chain amino group forms an amide bond with the C-terminal carboxy group of C ₂ or a carboxy group in the side chain of Xaa ₄ , thereby forming a cyclic peptide. In preferred embodiments, Xaa] is glutamine.

Haa _t is an aliphatic or aromatic amino acid. For example Haat is selected from alanine, leucine, isoleucine, valine, cyclohexylalanine, biscyclohexylalanine, adamantylalanine, phenyalanine, tryptophan, tyrosine, neotryptophan, phenylglycine, 1- or 2-naphthylalanine, 3 -pyridylalanine, 3-benzothienylalanine, 4-halophenylalanine, 3-halophenylalanine, 2-halophenylalanine, diphenylalanine, 4-methylphenylalanine, bis(2-picolyl)amine, 3,4-dimethoxyphenylalanine, 4,4'-biphenylalanine, 4-aminobutyric acid, homocyclohexylalanine, α-cyclohexylglycine, t-butylglycine, t-butylalanine, 1- aminomethylcyclohexane-acetic acid, 1-aminocyclohexane-acetic acid,

1-aminocyclohexane-carboxylic acid and pentafiuorophenylalanine; especially isoleucine, cyclohexylalanine, naphthylalanine, 3-benzothienylalanine, 4-halophenylalanine, diphenylalanine, 4-methylphenylalanine, bis(2-picolyl)amine, 3,4- dimethoxyphenylalanine, pentafiuorophenylalanine, 4-fluorophenylalanine and 4- chlorophenylalanine. In each case IUa ₁ may be in the L- or D-configuration.

C ₁ and C ₂ are both cysteine, homocysteine, norcysteine, 4-mercaptoproline, N- mercaptoethylglycine, penicillamine or selenocysteine, or one of C ₁ or C ₂ is cysteine and the other is homocysteine, norcysteine, 4-mercaptoproline, N-mercaptoethylglycine or

penicillamine, one of C ₁ and C ₂ is homocysteine and the other is norcysteine, 4- mercaptoproline, N-mercaptoethylglycine or penicillamine, or one of C ₁ and C ₂ is 4- mercaptoproline, N-mercaptoethylglycine or norcysteine and the other is penicillamine or where C ₁ is cysteine, homocysteine, norcysteine, 4-mercaptoproline or N- mercaptoethylglycine and C ₂ is cysteamine, and in each case C ₁ and C ₂ are oxidatively linked by the formation of a disulfide bond or in the case of selenocysteine, a diseleno bond; especially where C ₁ and C ₂ are both cysteine, penicillamine or homocysteine, or where one of C ₁ and C ₂ is cysteine and the other is penicillamine or homocysteine, and C ₁ and C ₂ are oxidatively linked by the formation of a disulfide bond. In any case C ₁ and C ₂ may be in either D- or L-configuration. When C ₂ is cysteamine, R ₂ is absent. Alternatively C ₁ and C ₂ together form a 6 to 12 membered carbocyclic or heterocyclic ring optionally fused to a second ring, such as a phenyl ring; especially a 7 to 10 membered carbocyclic or heterocyclic ring optionally fused to an aromatic ring. In preferred embodiments, the carbocyclic or heterocyclic ring is selected from cycloheptanyl, cyclooctanyl, cyclononyl, azepinyl, diazepinyl, thiazepinyl, oxepinyl, thiepinyl, benzodiazepinyl, benzazepinyl, benzothiazepinyl, benzoxepinyl or benzothiepinyl. In some embodiments, the heterocyclic ring is formed by an alkyl bridge, such as -CH ₂ -, -(CH ₂ ) ₂ - or -(CH ₂ ) ₃ - between the sulphur or selenium atoms of C ₁ and C ₂ . For example, a heterocyclic ring may be formed by bridging two cysteines with a -CH ₂ - group (-S-CH ₂ -S-) to form a 9 membered ring, or bridging a cysteine and a homocysteine with a -CH ₂ - group to form a 10 membered ring.

R ₁ is absent, an N-terminal capping group selected from 4-hydroxyphenylCO, 4-hydroxyphenylCH ₂ CO-, 4-hydroxyphenyl(CH ₂ ) ₂ CO-, 3 -hydroxyphenylCO,

3-hydroxyphenylCH ₂ CO-, 3-hydroxyphenyl(CH ₂ ) ₂ CO-, 2-hydroxyphenylCO-, 2-hydroxyphenylCH ₂ CO-, 2-hydroxyphenyl(CH ₂ ) ₂ CO, 4-methoxyphenylCO-,

4-methoxyphenylCH ₂ CO-, 4-methoxyphenyl(CH ₂ ) ₂ CO-, 3 -methoxyphenylCO, 3 -methoxyphenylCHaCO-, 3 -methoxyphenyl(CH ₂ ) ₂ CO-, 2-methoxyphenylCO-, 2-methoxyphenylCH ₂ CO-, 2-methoxyphenyl(CH ₂ ) ₂ CO-, 3 ,4-dimethoxyphenylCO, 3 ,4-dimethoxyphenylCH ₂ CO-, 3 ,4-dimethoxyphenyl(CH ₂ ) ₂ CO-, phenylCO-, phenylCH ₂ CO-, phenyl(CH ₂ ) ₂ CO-, phenyl(CH ₂ ) ₃ CO-, naphthyl-2-CO-, naphthyl-2- CH ₂ CO-, naphthyl-2-(CH ₂ ) ₂ CO-, naphthyl-2-(CH ₂ ) ₃ CO-, l,2,3,4-tetrahydronaphthyl-2-

CO-, 1 ,2,3 ,4-tetrahydronaρhthyl-2-CH ₂ CO-, 1 ,2,3 ,4-tetrahydronaphthyl-2-(CH ₂ ) ₂ CO-, 1 ,2,3,4-tetrahydronaρhthyl-2-(CH ₂ ) ₃ CO-, 4-ρhenyl-phenylCO-, 4-phenyl-phenylCH ₂ CO-, 4-phenyl-phenyl(CH ₂ ) ₂ CO-, 3-phenyl-ρhenylCO-, 3-phenyl-phenylCH ₂ CO-, 3-phenyl- phenyl(CH ₂ ) ₂ CO-, 4-phenoxyphenylCO-, 4-phenoxyphenylCH ₂ CO-, 4-phenoxyphenyl(CH ₂ ) ₂ CO-, 3-phenoxyphenylCO-, 3-phenoxyphenylCH ₂ CO-,

3-ρhenoxyphenyl(CH ₂ ) ₂ CO-, 4-halophenylCO-, 4-halophenylCH ₂ CO-,

4-halophenyl(CH ₂ ) ₂ CO-, 3-halophenylCO-, 3-halophenylCH ₂ CO-,

3-halophenyl(CH ₂ ) ₂ CO-, 2-halophenylCO-, 2-halophenylCH ₂ CO-, 2- halophenyl(CH ₂ ) ₂ CO-, 3,4-dihalophenylCO-, 3,4-dihalophenylCH ₂ CO-, 3,4-dihalophenyl(CH ₂ ) ₂ CO-, 4-nitrophenylCO, 4-nitrophenylCH ₂ CO-,

4-nitrophenyl(CH ₂ ) ₂ CO-, 3-nitrophenylCO-, 3-nitrophenylCH ₂ CO-,

3-nitrophenyl(CH ₂ ) ₂ CO-, 2-nitrophenylCO-, 2-nitrophenylCH ₂ CO-,

2-nitrophenyl(CH ₂ ) ₂ CO- ₅ 3-indolylCO-, 3-indolylCH ₂ CO-, 3-indolyl(CH ₂ ) ₂ CO-, 3-indolyl(CH ₂ ) ₃ CO-, N-methyl-indolylCO-, N-methyW-indolylCHzCO-, N-methyl-3- indolyl(CH ₂ ) ₂ CO-, N-methyl-3-indolyl(CH ₂ ) ₃ CO-, 4-indolylCO-, 4-indolylCH ₂ CO-, 4-indolyl(CH ₂ ) ₂ CO-, 4-indolyl(CH ₂ ) ₃ CO-, 2-pyridylCO-, 2-pyridylCH ₂ CO-, 2-pyridyl(CH ₂ ) ₂ CO-, 2-pyridyl(CH ₂ ) ₃ CO-, 3-pyridylCO-, 3-pyridyl(CH ₂ )CO-, 3- pyridyl(CH ₂ ) ₂ CO-, 3-pyridyl(CH ₂ ) ₃ CO-, 2-quinolinylCO-, 2-quinolinyl(CH ₂ )CO-, 2- quinolinyl(CH ₂ ) ₂ CO-, 2-quinolinyl(CH ₂ ) ₃ CO-, 3-quinolinylCO-, 3-quinolinyl(CH ₂ )CO-, 3- quinolinyl(CH ₂ ) ₂ CO-, 3-quinolinyl(CH ₂ ) ₃ CO-, cyclohexylCO-, cyclohexyl(CH ₂ )CO-, cyclohexyl(CH ₂ ) ₂ CO-, cyclohexyl(CH ₂ ) ₃ CO-, coumarinCO-, coumarin(CH ₂ )CO-, coumarin(CH ₂ ) ₂ CO-, coumarin(CH ₂ ) ₃ CO-, l,4-benzodioxane-2-CO-, l,4-benzodioxane-2- CH ₂ CO-, l,4-benzodioxane-2-(CH ₂ ) ₂ CO-, l,4-benzodioxane-2-(CH ₂ ) ₃ CO-, adamantylCO-, adamantylCH ₂ CO-, adamantyl(CH ₂ ) ₂ CO-, adamantyl(CH ₂ ) ₃ CO-, CH ₃ CO-, CH ₃ CH ₂ CO-, CH ₃ CH ₂ CH ₂ CO-, CH ₃ (CH ₂ ) ₃ CO-, CH ₃ (CH ₂ ) ₄ CO-, CH ₃ (CH ₂ ) ₅ CO-, CH ₃ (CH ₂ ) ₆ CO-, CH ₃ (CH ₂ ) ₇ CO-, CH ₃ (CH ₂ ) ₈ CO-, CH ₃ (CH ₂ ) ₉ CO-, CH ₃ (CH ₂ ) ₁₀ CO-, CH ₃ (CH ₂ ) _! iCO-, guanyl [H ₂ NC(=NH)] or R ₁ is R ₃ -Xaa ₂ (C ₃ ) _n -Xaa ₃ -;

R ₂ is absent, -H, -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -OC _1-10 alkyl, -C _MO alkyl, a covalent bond between the C-terminal carboxy group and an amino group in the side chain of Xaa^ especially absent or -NH ₂ , or R ₂ is Xaa ₄ ;

Xaa ₂ is a polar positively charged or uncharged amino acid residue such as lysine or arginine or a hydrophobic amino acid residue, especially an aliphatic cyclic or aromatic amino acid residue. In preferred embodiments Xaa ₂ is lysine, arginine, phenylalanine, tryptophan, tyrosine, neotryptophan, phenylglycine, naphthylalanine, cyclohexylalanine, N-methylalanine, 2-methylaminobenzoic acid (N-methylanthranylic acid), adamantylalanine, (2S,4S)-4-amino-l -benzoyl-pyrrolidine carboxylic acid, pyroglutamate, proline, hydroxyproline, L-pipecolic acid, induline-2-carboxylic acid, 2-amino-2-naphthoic acid, tetrahydroquinoline-1 -carboxylic acid, 1, 2,3, 4-tetrahydroisoquinoline-3 -carboxylic acid, 7-hydroxy-l ₅ 2,3,4-tetrahydroisoquinoline-3-carboxylic acid and 1,2,3,4- tetrahydroharman-3 -carboxylic acid; especially phenylalanine, tyrosine, pyroglutamate, proline, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, L-pipecolic acid, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 7-hydroxy-l,2,3,4-tetrahydroisoquinoline-3- carboxylic acid; more especially pyroglutamate;

Xaa ₃ is preferably absent or is a positively charged amino acid residue, or is a hydrophobic amino acid residue; especially where Xaa ₃ is absent or is L-pipecolic acid or is proline or arginine, more especially Xaa ₃ is absent or is L-pipecolic acid or proline.

Xaa ₄ is an amino acid residue having a carboxylic acid in its side chain, such as aspartic acid or glutamic acid that forms a covalent amide bond with an amino group in the side chain of Xaai, for example, when Xaa} is a lysine or Xaa ₄ is an amino acid residue having an amino group in its side chain, such as lysine. XBa ₄ is optionally capped with a C-terminal capping group.

R ₃ is absent or an N-terminal capping group selected from 4-hydroxyphenylCO, 4-hydroxyphenylCH ₂ CO-, 4-hydroxyphenyl(CH ₂ ) ₂ CO-, 3-hydroxyphenylCO-,

3 -hydroxyphenylCHbCO-, 3 -hydroxyphenyl(CH ₂ ) ₂ CO-, 2-hydroxyphenylCO-,

2-hydroxyphenylCH ₂ CO-, 2-hydroxyphenyl(CH ₂ ) ₂ CO-, 4-methoxyphenylCO-, 4-methoxyphenylCH ₂ CO-, 4-methoxyphenyl(CH ₂ ) ₂ CO-, 3-methoxyphenylCO-,

3-methoxyphenylCH ₂ CO-, 3-methoxyphenyl(CH ₂ ) ₂ CO-, 2-methoxyρhenylCO-,

2-methoxyphenylCH ₂ CO-, 2-methoxyphenyl(CH ₂ ) ₂ CO-, 3 ,4-dimethoxyphenylCO-, 3,4-dimethoxyphenylCH ₂ CO-, 3,4-dimethoxyphenyl(CH ₂ ) ₂ CO- _; phenylCO-, phenylCH ₂ CO-, phenyl(CH ₂ ) ₂ CO- ₃ phenyl(CH ₂ ) ₃ CO-, naphthyl-2-CO-, naphthyl-2- CH ₂ CO-, naphthyl-2-(CH ₂ ) ₂ CO- ₅ naphthyl-2-(CH ₂ ) ₃ CO-, l,2,3,4-tetrahydronaphthyl-2- CO-, l,2,3,4-tetrahydronaphthyl-2-CH ₂ CO- ₃ l,2,3 _s 4-tetrahydronaphthyl-2-(CH ₂ ) ₂ CO-, 1 ,2,3,4-tetrahydronaphthyl-2-(CH ₂ ) ₃ CO-, 4-phenyl-phenylCO-, 4-phenyl-phenylCH ₂ CO-, 4-phenyl-phenyl(CH ₂ ) ₂ CO-, 3-phenyl-phenylCO-, 3-phenyl-phenylCH ₂ CO- ₅ 3-ρhenyl- phenyl(CH ₂ ) ₂ CO-, 4-phenoxyphenylCO- ₃ 4-phenoxyphenylCH ₂ CO-,

4-phenoxyphenyl(CH ₂ ) ₂ CO-, 3-phenoxyphenylCO-, 3-phenoxyphenylCH ₂ CO-, 3-phenoxyphenyl(CH ₂ ) ₂ CO-, 4-halophenylCO-, 4-halophenylCH ₂ CO-,

4-halophenyl(CH ₂ ) ₂ CO-, 3-halophenylCO-, 3-halophenylCH ₂ CO-, 3-halophenyl(CH _2- ) ₂ CO-, 2-halophenylCO-, 2-halophenylCH ₂ CO-, 2-halophenyl(CH ₂ ) ₂ CO-,

3,4-dihalophenylCO-, 3,4-dihalophenylCH ₂ CO- ₅ 3,4-dihalophenyl(CH ₂ ) ₂ CO-,

4-nitroρhenylCO-, 4-nitrophenylCH ₂ CO-, 4-nitrophenyl(CH ₂ ) ₂ CO-, 3-nitrophenylCO-, 3-nitrophenylCH ₂ CO-, 3-nitrophenyl(CH ₂ ) ₂ CO- ₅ 2-nitroρhenylCO-, 2-nitrophenylCH ₂ CO-, 2-nitrophenyl(CH ₂ ) ₂ CO-, 3-indolylCO-, 3-indolylCH ₂ CO-, 3-indolyl(CH ₂ ) ₂ CO-, 3-indolyl(CH ₂ ) ₃ CO-, N-methyl-indolylCO-, N-methyl-3-indolylCH ₂ CO-, N-methyl-3- indolyl(CH ₂ ) ₂ CO-, N-methyl-3-indolyl(CH ₂ ) ₃ CO-, 4-indolylCO-, 4-indolylCH ₂ CO-, 4-indolyl(CH ₂ ) ₂ CO-, 4-indolyl(CH ₂ ) ₃ CO-, 2-pyridylCO-, 2-pyridylCH ₂ CO-, 2-pyridyl(CH ₂ ) ₂ CO-, 2-pyridyl(CH ₂ ) ₃ CO-, 3-pyridylCO-, 3-pyridyl(CH ₂ )CO-, 3- pyridyl(CH ₂ ) ₂ CO-, 3-pyridyl(CH ₂ ) ₃ CO-, 2-quinolinylCO-, 2-quinolinyl(CH ₂ )CO-, 2- quinolinyl(CH ₂ ) ₂ CO-, 2-quinolinyl(CH ₂ ) ₃ CO-, 3-quinolinylCO- ₅ 3-quinolinyl(CH ₂ )CO-, 3- quinolinyl(CH ₂ ) ₂ CO-, 3-quinolinyl(CH ₂ ) ₃ CO-, cyclohexylCO-, cyclohexyl(CH ₂ )CO-, cyclohexyl(CH ₂ ) ₂ CO-, cyclohexyl(CH ₂ ) ₃ CO- ₅ coumarinCO-, coumarin(CH ₂ )CO-, coumarin(CH ₂ ) ₂ CO-, coumarin(CH ₂ ) ₃ CO-, 1 ,4-benzodioxane-2-CO-, l,4-benzodioxane-2- CH ₂ CO-, l,4-benzodioxane-2-(CH ₂ ) ₂ CO-, l,4-benzodioxane-2-(CH ₂ ) ₃ CO-, adamantylCO-, adamantylCH ₂ CO- ₅ adamantyl(CH ₂ ) ₂ CO- _? adamantyl(CH ₂ ) ₃ CO-, CH ₃ CO-, CH ₃ CH ₂ CO-, CH ₃ CH ₂ CH ₂ CO-, CH ₃ (CH ₂ ) ₃ CO- ₅ CH ₃ (CHz) ₄ CO-, CH ₃ (CH ₂ ) _S CO-, CH ₃ (CH ₂ ) ₆ CO- ₅ CH ₃ (CH ₂ ) ₇ CO-, CH ₃ (CH ₂ ) ₈ CO- ₅ CH ₃ (CH ₂ ) ₉ CO- ₅ CH ₃ (CH ₂ ) ₁₀ CO-, CH ₃ (CH ₂ )nCO- and guanyl;

C ₃ is cysteine; and

n is 0 or 2; especially 0.

Examples of suitable opioid receptor agonist peptides include those listed in Table 3.

UJ

U)

U)

OJ

UJ

I

I I

-J

I

1 The NH ₂ of Xaa _t side chain forms an amide bond with the terminal carboxyl group of C ₂ .

2 In each case C ₁ and C ₂ are cyclised by formation of a disulfide bond or in the case of * C ₁ and C ₂ are bridged by S-CH ₂ -S-. 3 The amino group of the lysine side chain and the carboxylic acid of the glutamic acid or aspartic acid residue have cyclised by forming an amide bond.

The peptides useful in the methods of the present invention may be in the form of a salt, ester, amide, prodrug or, where appropriate, a cyclised derivative. The peptides useful in the present invention may have a free carboxyl at the C-terminal, however compounds with an amidated carboxyl terminus or other modifications, such as esterification at the C- terminal may also be useful. In some embodiments the peptides have an amidated carboxy group. The peptides useful in the present invention may have a free N-terminus, or the N-terminus may be capped using a suitable capping group.

The peptides may be in the form of a pharmaceutically acceptable salt. Examples of suitable salts include, but are not limited to, chloride, acetate, lactate and glutamate salts. Conventional procedures for the preparation of suitable salts are well known in the art.

The peptides useful in the present invention may also be in the form of prodrugs. Prodrugs are understood to include all derivatives of peptides according to the invention which are readily convertible in vivo into the required active peptide. Conventional procedures for the preparation of suitable prodrugs according to the invention are described in text books, such as "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985.

The peptides useful in the present invention may be prepared using standard peptide synthetic methods followed by oxidative disulfide bond formation. For example, the linear peptides may be synthesised by solid phase methodology using Fmoc chemistry as described in Fmoc Solid Phase Peptide Synthesis, A Practical Approach, edited by W.C. Chan, P.D. White, Oxford Press, 2000, or BOC chemistry, as described by Schnoltzer et al, Int. J. Peptide Protein Res., 40, 180 (1992). Following deprotection and cleavage from the

solid support the reduced peptides are purified using preparative chromatography. The purified reduced peptides may be oxidised in buffered systems, for example, 30% DMSO/5% AcOH/65% water, 30% DMSO/0.1 M NH ₄ HCO ₃ at pH 6, 30% isoproρanol/0.1 M NH ₄ HCO ₃ at pH 8 or isoproρanol/DMSO/0.1 M NH ₄ HCO ₃ at pH 8. The oxidised peptides may be purified using preparative chromatography such as reverse phase HPLC.

Other procedures known in the art for selective oxidation of the cysteine residues may also be used such as those described in Tarn et al, 2000, Biochem. Biophys. Res. Commun., 267(3), 783-790; Yu et al, 2000, J Biol. Chem., 275(6), 3943-3949 and Tarn et al, 1999, Proc. Natl. Acad. ScI USA., 96(16), 8913-8918.

Some of the peptides useful in the present invention may also be prepared using recombinant DNA technology. A nucleotide sequence encoding the desired peptide sequence, or its precursor, may be inserted into a suitable vector and protein expressed in an appropriate expression system. In some instances, further chemical modification of the expressed peptide may be appropriate, for example C-terminal amidation or post translational modification of particular residues. Under some circumstances it may be desirable to undertake oxidative bond formation of the expressed peptide as a chemical step following peptide expression. This may be preceded by a reductive step to provide the unfolded peptide. Those skilled in the art may readily determine appropriate conditions for the reduction and oxidation of the peptide.

Pharmaceutical Compositions

In one aspect of the invention, the opioid receptor agonist peptides may be incorporated into a pharmaceutical composition together with a pharmaceutically acceptable carrier.

As will be readily appreciated by those skilled in the art, the formulation of, route of administration of and the nature of the pharmaceutically acceptable carrier will depend on the specific disease or condition to be treated. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences", Mack Publishing

Co., Easton, Pa, latest edition. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art.

In the preparation of any formulation containing the peptide actives, care should be taken to ensure that the activity of the peptide is not destroyed in the process and that the peptide is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the peptide by means known in the art such as microencapsulation. Similarly, the route of administration should be chosen such that the peptide reaches its site of activity.

Suitable routes of administration may, for example, include oral, topical, rectal, transmucosal, intestinal administration or parenteral administration including intramuscular, subcutaneous, intramedullary, intra-articular, as well as intrathecal, epidural, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection.

The compositions of this invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non-ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, hydrophilic beeswax derivatives and hyaluronic acid.

Alternatively, the peptides of the present invention can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral

administration. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving,

granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilising processes.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses.

Pharmaceuticals which can be used orally include push-fit capsules made of gelatine, as well as soft, sealed capsules made of gelatine and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in a mixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilisers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilisers may be added.

Dosage forms of the active compounds of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an active compound of the invention may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release may be achieved by using other polymer matrices, liposomes and/or microspheres. Controlled release may also be achieved using a transdermal patch, particularly a transdermal patch in which the rate of release of one or both of the active agents is controlled by a co-polymer release membrane or in which the active agent(s) is embedded in a biodegradable matrix that dissolves at a known rate. Transdermal patches which allow slow and sustained delivery of a drug at a known rate are known in the art.

The peptides of the present invention may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebuliser, or as a microfme powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In such a case, the particles of the formulation may advantageously have diameters of less than 50 micrometers, suitably less than 10 micrometers.

Methods of treatment and Prophylaxis

The peptides of the present invention are useful as agonists of opioid receptors. Although the peptides of the invention may agonise any type of opioid receptor located in the central or peripheral nervous system, such as kappa (K) opioid receptors (KORs), mu (μ) opioid receptors (MORs) and delta (δ) opioid receptors (DORs), in some embodiments, the peptides are selective for one type of opioid receptor over the other types. In particular embodiments, the peptides are selective at activating KORs. Selectivity for KORs potentially avoids side effects or at least reduces side effects associated with agonism of other types of opioid receptors.

In another aspect of the invention, the peptides are useful as agonists of opioid receptors located in the peripheral nervous system. In preferred embodiments, the peptides of the invention are peripherally restricted selective agonists of peripheral opioid receptors. In some embodiments the peptides are selective agonists of peripheral opioid receptors and are also selective for agonising one type of opioid receptor over other types. In particular embodiments, the peripherally delivered peptides are selective agonists of peripheral kappa opioid receptors. Selective activation of KORs located in the peripheral nervous system potentially avoids side effects or at least reduces side effects associated with agonism of KORs in the central nervous system and any side effects that may be associated with other types of opioid receptors.

In one aspect of the invention, there is provided a method of agonising an opioid receptor comprising exposing the opioid receptor to an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptide selectively activates a kappa opioid receptor. In other embodiments, the peptide activates an opioid receptor located in the peripheral nervous system, especially where the peptide can act as a selective agonist of opioid receptors located in the peripheral nervous system without activating opioid receptors in the central nervous system. In some embodiments, the peripherally delivered peptide selectively agonises a kappa opioid receptor located in the peripheral nervous system, especially where the peptide selectively agonises a peripheral kappa opioid receptor over kappa opioid receptors located in the central nervous system.

The terms "selective" and "selectively" as used herein, mean that the activity of the peptide as an agonist of a receptor at a particular location or a particular type of receptor is considerably greater than any activity at receptors at other locations or of other types. For example, a selective agonist of a peripheral opioid receptor will have at least 50% greater activity for an opioid receptor located in the peripheral nervous system over opioid receptors located in the central nervous system. In some embodiments, the selective agonist will have greater than 60%, 70%, 80%, 90% or 99% activity with peripheral KORs compared to opioid receptors located in the central nervous system. In some embodiments, the peptides are greater than 60%, 70%, 80%, 90% or 99%, or greater than 99% selective for KORs and have no detectable activity at MORs and DORs. It is likely that peptides that are ~ 100% selective for the peripheral opioid receptors are unable to cross the blood-brain barrier and thus their action will be restricted to the activation of only peripheral KORs.

In some embodiments, the peptide is selective for both kappa opioid receptors over other types of receptors and is selective for opioid receptors located in the peripheral nervous system. These peptides are selective agonists of peripheral kappa opioid receptors.

The terms "agonise", "agonising" and "agonist" refer to activation of an opioid receptor or a peptide that activates an opioid receptor, thereby triggering analgesia. Agonists of opioid receptors mimic the activity of the endogenous compounds that bind to the opioid receptors to produce analgesia, such as enkephalin, dynorphin A, endomorphin and β-endorphin.

As used herein, the term "central nervous system" refers to the brain and the spinal cord.

As used herein, the term "peripheral nervous system" refers to the parts of the nervous system, nerves and neurons that reside or extend outside of the central nervous system. The peripheral nervous system provides nerves and neurons to organs and muscles, for example. The nerves and neurons of the peripheral nervous system are not protected by the blood-brain barrier or by bone and are therefore more exposed to damage by toxins or mechanical means. The terms "peripheral opioid receptor" and "opioid receptor located in the peripheral nervous system" are used interchangeably herein to refer to opioid receptors that are located in nerves and neurons in the peripheral nervous system.

In some embodiments, the opioid receptors are located in vivo and exposure to the peptide of SEQ ID NO:1 is by administration of the peptide to the subject. In other embodiments, the opioid receptors are present on isolated cells in vitro and exposure is by contacting the cells with a peptide of SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

In another aspect of the invention, there is provided a method of inducing, promoting or otherwise facilitating pain relief or analgesia in a subject comprising administering an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

The methods of the invention may be used to produce analgesia or provide pain relief or control pain. The pain may be acute or chronic pain and may be nociceptive pain, neuropathic pain or mixed category pain. Nociceptive pain is caused by nerves

(nociceptors) which sense and respond to parts of the body that suffer damage or are about to suffer damage. Nociceptive pain may be localised, constant and include aching or throbbing or may be visceral pain associated with internal organs which may be poorly localised and episodic. Nociceptive pain usually decreases as and if the damage heals. Examples of nociceptive pain include acute trauma, osteoarthritis, rheumatoid arthritis, musculo-skeletal pain and inflammatory pain particularly after trauma, spinal pain, dental pain, myofascial pain syndromes, headache, episiotomy pain, and burns; deep and visceral pain, such as heart pain, muscle pain, eye pain, orofacial pain, for example, odontalgia, abdominal pain, gynaecological pain, for example, dysmenorrhea, and labor pain, post operative pain, irritable bowel syndrome, inflammatory bowel syndrome, inflammatory bowel disease, diabetic neuropathy, gut pain, shingles and gout. In some embodiments, the pain is post-operative pain.

Neuropathic pain is caused by injury or malfunction in the nervous system. The pain is often triggered by an injury but the injury may not involve actual damage to the nervous system. Neuropathic pain frequently includes cold, burning, lancinating, numbing or electric shock type pain and may also include allodynia, pain resulting from a non-painful stimulus such as a light touch. Neuropathic pain may persist for months or years and is often chronic. Examples of neuropathic pain include pain associated with nerve and root damage, such as pain associated with nerve disorders, for example, nerve entrapment and brachial plexus avulsions, amputation, neuropathies, neuralgia, tic douloureux, atypical facial pain, nerve root damage, pain and/or chronic nerve compression, and arachnoiditis; pain associated with carcinoma, often referred to as cancer pain; pain associated with

AIDS, low back pain; sciatica; headache, including migraine, acute or chronic tension headache, cluster headache, temporomandibular pain and maxillary sinus pain; ankylosing spondylitis; post-herpetic pain; phantom pains; diabetic neuropathy; and scar pain.

Mixed category pain includes a complex mixture of nociceptive and neuropathic pain. Examples of mixed category pain include migraine headaches and myofascial pain.

In another embodiment of the invention, there is provided a method of treating one or more of post-operative pain, neuropathic pain, nociceptive pain or mixed category pain comprising administering an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

In preferred embodiments, the pain relief or analgesia is used to control acute pain, such as, but not limited to, post-operative pain. One example is the use of the peptides of the invention to control localised pain resulting from surgery. Pain relief or analgesia induced by agents that do not affect or have low activity in the central nervous system and have few or reduced side effects may allow patients to spend a reduced amount of time in hospital and in some cases allow discharge of a patient on the same day as the surgery was performed. The present invention may be particularly useful in the control of pain after biopsies, such as colonoscopies.

The methods of the invention may also be particularly useful in providing pain relief for gastrointestinal disorders such as colonic hyperanalgesia, visceral pain and Irritable Bowel Syndrome.

In yet a further aspect of the present invention, there is provided a method for the treatment or prophylaxis of inflammation and inflammatory pain or a condition or disorder associated with inflammation comprising administering to a subject an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

As used herein, "inflammation" refers to the response of body tissues to injury or infection and is characterised by pain, redness, swelling and heat. Inflammation may be chronic or acute.

Conditions or disorders associated with inflammation include, but are not limited to, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, Crohn's disease, asthma, multiple sclerosis, psoriasis, psoriatic arthritis, Alzheimer's disease, atherosclerosis,

diabetes, cirrhosis (viral or alcoholic), pulmonary inflammation including pulmonary fibrosis and COPD, ACS including myocardial infarction, obesity, sepsis, AIDS, ulcerative colitis, rhinitis and degenerative cartilage loss.

Any subject who could benefit from the present methods or compositions is encompassed. The term "subject" includes, without limitation, humans and non-human primates, livestock animals, companion animals, laboratory test animals, captive wild animals, reptiles and amphibians, fish, birds and any other organism. The most preferred subject of the present invention is a human subject. A subject, regardless of whether it is a human or non-human organism may be referred to as a patient, individual, subject, animal, host or recipient.

An effective amount of a peptide of the invention is one that is effective in agonising opioid receptors, KORs or peripheral KORs and providing analgesia or an anti-inflammatory effect. The effective amount is an amount necessary to at least partially attain the desired response, or the desired level and duration of pain relief, or delay the onset or inhibit the progression or halt altogether, the onset or progression of the disease or condition associated with inflammation being treated. The amount varies depending on the health and physical condition of the individual being treated, the taxonomic group of the individual, the degree of pain relief required, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by routine trials. In one embodiment, the amount of inhibitor that is administered is in the range of 0.001 mg to 1 g, preferably 0.005 mg to 500 mg, 0.01 mg to 250 mg, 0.03 mg to 100 mg, 0.05 mg to 50 mg.

Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation.

As used herein, "producing analgesia" or "providing, inducing or otherwise facilitating pain relief is used in its broadest context. These terms do not imply that the subject suffers no pain, although in some cases, this may be achieved. These terms include reducing the severity of pain or delaying the onset of pain.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease or condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or delaying the onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.

In another aspect of the invention, there is provided a use of a peptide having SEQ ID NO.l or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inducing, promoting or otherwise facilitating pain relief or analgesia or for treating or preventing inflammation or a condition or disorder associated with inflammation.

In a preferred aspect of the invention there is provided a method of inducing, promoting or otherwise facilitating pain relief in a subject by selectively agonising a kappa opioid receptor comprising administering an effective amount of a peptide having an amino acid sequence comprising SEQ ID NO.l, especially where the kappa opioid receptor agonist is a peripheral kappa opioid receptor agonist.

The peptides of the present invention can be used in combination with other molecules possessing analgesic and/or anti-inflammatory properties, including hyaluronic acid, other opiates, propanolol, COX inhibitors, local anaesthetics, NMDA antagonists, calcium channel antagonists and trafficking antagonists, GABA agonists, alpha2-agonists, NET inhibitors, SERT inhibitors, mixed NET/SERT inhibitors, ILl and bradykinin antagonists, and TNF alpha antagonists and antibodies etc.

The invention will now be described with reference to the accompanying Examples. However, it is to be understood that the particularity of the following description is not to supersede the generality of the preceding description of the invention.

Brief Description of the Figures

Figure 1 provides a time plot of the mechanical paw withdrawal threshold (PWT) using a Randall-Sellito device. An intraplantar injection of a peptide of SEQ ID NO:3 was made at three days after intraplantar injection of CFA (mean + SEM).

Figure 2 is a bar graph showing mean PWT at 0.3 μg, 3 μg, 30 μg and 300 μg of peptide of SEQ ID NO:3.

EXAMPLES

Example 1: Peptide Synthesis

Materials and methods

Reagents

Protected Fmoc-amino acid derivatives were purchased from Novabiochem or Auspep P/L. The following side chain protected amino acids were used: Cys(Trt), Pen(Trt) His(Trt), Hyp(tBu), Tyr(tBu), Lys(Boc), Trp(Boc) ₅ Arg(Pbf), Asn(trt), Asρ(OtBu), GIu(OtBu) ₅ Gln(Trt), Ser(tBu), Thr(tBu), Tyr(tBu). All other Fmoc amino acids or acids were unprotected. Dimethylformamide (DMF), dichloromethane (DCM) ₃ diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), all peptide synthesis grade, supplied by Auspep P/L (Melbourne, Australia) . 2-( 1 H-benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate (HBTU), Triisopropyl silane (TIPS), Dimethyl sulfide (DMS) HPLC grade acetonitrile and methanol was supplied by Sigma Aldrich. The resin used was a

Fmoc-Rink resin (0.65mmol/gm) supplied by AuspepP/L. Ethylene dithiol (EDT) was supplied by Merck.

Peptide synthesis

The peptides described in Table 3 were synthesized on an Advanced ChemTech (ACT- 396) automated peptide synthesizer or on a Bohdan-Mini block on Rink amide resin (C- terminal amides) or Wang resin (C-terminal acids), using HBTU in-situ activation protocols to couple the Fmoc-protected amino acid to the Rink-resin. Attachment of only the first amino acid to Wang resin was achieved using MSNT/1-methylimidazole activation in dichloromethane. After chain assembly and final Fmoc deprotection, the peptides were cleaved from the resin by stirring at room temperature in TFA:H ₂ O:TIPS:EDT (87.5:5:5:2.5) for 2-3 hours prior to removal of the resin. Cold diethyl ether was then added to the mixture and the peptide precipitated out. The precipitate was collected by centrifugation and subsequently washed with further cold diethyl ether to remove scavengers. The final product was dissolved in 50% aqueous acetonitrile and lyophilized to yield a fluffy white solid. The crude, reduced peptide was examined by reverse phase HPLC for purity, and the correct molecular weight confirmed by Electrospray mass spectrometry. Pure, reduced peptides (lmg/ml) were oxidised by stirring at RT in 30% DMSO/ 5% AcOH/ 65% water, for 20-24 hours. The solutions were then diluted so that the concentration of DMSO was <5 %, prior to RP-HPLC purification and lyophilization.

Selective disulfide bond peptides

Peptides were synthesised as described above using a standard Fmoc synthesis protocol with the following exception. For each of the different isomers one pair of cysteine residues was assembled using Cys(Trt) protection while, if present, the other pair used the orthogonal protection of Cys(Acm).

Chain cleavage, purification and oxidation of the first disulfide bond were carried out as described above to yield the single disulfide containing (-ACM) ₂ peptide.

Selective disulfide bond formation

The pure, oxidised (-Acm) ₂ peptide was dissolved at a concentration of ImM in 80% acetic acid/water. Iodine (10eq) dissolved in a small volume of ethyl acetate was added and the mixture stirred at RT. The progress of the Acm deprotection and subsequent oxidation was monitored by direct injection MS at regular intervals until completion (approximately 30-90 minutes). The reaction was then quenched by addition of ascorbic acid solution (lOmg/mL) resulting in a decolourisation of the solution. After dilution with five times the volume of water and adjusting to pH 3, the fully oxidised peptide was loaded onto a RP-HPLC column and purified as described below.

HPLC analysis and purification

Analytical HPLC runs were performed using a Shimadzu HPLC system LClOA with a dual wavelength UV detector set at 214 nm and 254 nm. A reversed-phase C-18 column (Zorbax 300-SB Cl 8; 4.6 x 50mm) with a flow rate of 2mL/minute was used. Gradient elution was performed with the following buffer systems: A, 0.05% TFA in water and B, 0.043% TFA in 90% acetonitrile in water, from 0%B to 80%B in 20 minute. The crude peptides were purified by semi-preparative HPLC on a Shimadzu HPLC system LC8A associated with a reversed-phase C- 18 column (Vydac C- 18, 25 cm x 10 mm) running at a flow rate of 5 ml/min with a 1% gradient of 0-40%B. The purity of the final product was evaluated by analytical HPLC.

Electrospray Mass Spectrometry (ESI-MS)

Electrospray mass spectra were collected inline during analytical HPLC runs on an Applied Biosystems API-150 spectrometer operating in the positive ion mode with an OR

of 20, Rng of 220 and Turbospray of 350 degrees. Masses between 300 and 1800 amu were detected (Step 0.2 amu, Dwell 0.3 ms).

Example 2: Determination of binding affinity of peptides at kappa opioid receptors

Human Kappa opioid receptor radioligand binding assay

The ability of the peptides described to act as inhibitors of the human kappa opioid receptor (h Kappa) can be measured by their competitive inhibition of ³ H-U69,593 from membrane prepared from CHO-Kl mammalian cells stably expressing h Kappa opioid receptor.

A stable CHO-Kl h-Kappa opioid receptor cell line was established. Stable cells were seeded into a 150mm dish and once confluent the cells were scraped, washed, homogenized and centrifuged using 5OmM Tris-HCl (pH 7.4), 5mM MgCl ₂ . For each 150mm dish membrane was resuspended in 500μL Tris-HCl (pH 7.4), 5mM MgCl ₂ with 10% glycerol. Assay buffer is 5OmM Tris-HCl (pH 7.4), 5mM MgCl ₂ and 0.1% BSA. Total assay volume was 80μL (made up of 20μL of each component) and each data point was performed in triplicate. Peptides at various concentrations (10 ^"4 to 10 ^"1 M) or control ligand (naltriben) were added to the assay plate (96 well white with clear bottom) followed by lOOμg/well FlashBlue WGA-coated scintillation beads (Perkin Elmer cat # FBBOOl) which was followed by the addition of h kappa opioid receptor membrane (4μL of prepared membrane +16μL assay buffer). Finally InM ³ H-U69,593 (Perkin Elmer cat # NET952) was added. The assay plate was sealed with a sealing film and incubated at room temperature for 1 hour with shaking. After incubation the plate was counted using a Microbeta Trilux instrument (Perkin Elmer) with the results analysed using GraphPad Prism.

The results are shown in Table 4, where each peptide sequence is identified by a SEQ ID NO: as given in Table 3.

Table 4

Example 3: Determination of selectivity of peptides for kappa opioid receptors

Membrane purchased from Perkin Elmer Life Sciences for the human ORLl opioid like receptor (RBHORLM), human delta2 opioid receptor (RBHODM) and human mu opioid receptor (cat # RBHOMM). Assay buffer for the ORLl binding assay is 50 mM HEPES (pH 7.4), 1 mM EDTA ₅ 10 mM MgCl ₂ and 0.5% BSA. Assay buffer for delta2 opioid receptor binding assay is 50 mM Tris-HCl (pH 7.4), 5 mM MgCl ₂ and 0.1% BSA. Assay buffer for the mu opioid receptor binding assay is 50 mM Tris-HCL (pH7.4), 5 mM MgCl ₂ and 0.5% BSA. Total assay volume was 80 μL (made up of 20 μL of each component) and each data point was performed in triplicate. Peptides at various concentrations (10 ^'4 to 10 ^'11 M) or control ligand (for ORLl opioid like receptor - orphanin FQ; for delta2 opioid receptor - naltriben; for mu opioid receptor - DAMGO) were added to the assay plate (96

well white with clear bottom) followed by 100 μg/well FlashBlue WGA-coated scintillation beads (Perkin Elmer cat # FBBOOl) which was followed by the addition of the required opioid membrane (for ORLl - 0.2 μL/well + 19.8 μL ORLl assay buffer; for delta2 - 0.4 μL/well + 19.6 μL delta2 assay buffer, for mu - 0.5 μL/well +19.5 μL mu assay buffer). Finally appropriate amount of radioligand was added (for ORLl - 1.04 nM ³ H-nociceptin; for delta2 1.6 nM ³ H-naltrindole and for mu 0.8 nM ³ H-diprenorphine) was added. The assay plate was sealed with a sealing film and incubated at room temperature for 1 hour with shaking. After incubation the plate was counted using a Microbeta Trilux instrument (Perkin Elmer) with the results analysed using GraphPad Prism.

The results are shown in Table 5.

Table 5

Avg. Kappa Mu DeHa ₂ ORLl

SEQ ID binding binding binding binding NO: Sequence (nM) (nM) (nM) (nM)

43 URRQICC 338 >100000 >100000 > 100000 δ Un-Q[CHA]CC 7 > 100000 >100000 > 100000

90 UCCRRQ[CHA]CC 4 >100000 >100000 >100000

3 [TIC]rrQ[CHA]CC 1 >30,000 > 100,000 > 16,000

5 [HTI]rrQ[BTA]CC 2 >3,500 >100,000 >20,000

2 Bzo-PrrQ[CHA]CC 0.33 >70,000 >100,000 >18,000

13 URRQ[CHA]CC 12 >100000 >100000 >100000

91 UCCRRQICC 151 > 100000 >100000 >100000

95 NCCRRQ[CHA]CC 4 > 100000 > 100000 > 100000

98 NCCRRRQ[CHA]CC 5 >100000 >100000 >100000

97 NCCRRQ[NAL]CC 8 > 100000 >100000 >100000

4 Un-Q[CHA][PEN]C 2 >100000 >2,500 >70,000

100 Nap-(2)-C(O)-PrrQ[CHA]CC 0.16 >l,500 >100000 >8,000

124 BzoPrrQ[CHG]CC 0.76 >100000 >100000 >100000

126 BzoPrrQ[TBA]CC-NH ₂ 1 >100000 >100000 >100000

122 BzoPrrQ[HCH]CC-NH ₂ 0.42 >4,000 >100000 >8,000

2,5-di-[H ₂ NC(=NH)NH]-

123 PhC(O)-G[CHA]CC-NH ₂ 0.53 >100000 >100000 >14,000

H ₂ NCC=NH)-R[ANC]-

121 NH(CH ₂ ) ₃ C(O)-[CHA]CC-NH ₂ 0.4 >25,000 >100000 >3,500 cyclohexylC(O)-PrrQ[CHA]CC-

131 NH ₂ 2 >100000 >100000 >100000

Bzo-Pr-NH(CH ₂ ) ₃ CO-

132 [CHA]CC-NH ₂ 0.32 >13,000 >100000 >100000

Example 4: ERK and cAMP assays

SureFire™ ERK Assay

Phosphorylation of cellular ERK in cell Iy sates was measured using the SureFire™ ERK assay. The assay screens for modulators of receptor activation as well as agents acting intracellularly, such as inhibitors of upstream events by measuring the phosphorylation of ERK1/2 at Thr202/Tyr204 by MEK.

The assay is performed in accordance with the protocol provided in the SureFire™ ERK assay kit using CHO-Kl cells stably transfected with kappa opioid receptor. Control compounds that were used include an endogenous KOR ligand, dinorphin A (SEQ ID NO: 173) and small molecules U69,593 and U50,488.

LANCE™ cAMP Assay

The LANCE™ cAMP assay kit (Perkin Elmer) is indended for the quantitavive determination of adenosine-3,5-cyclic monophosphate (cAMP) in cell culture and cell membrane preparations.

cAMP is one of the most important second messengers, mediating diverse physiological responses of neurotransmitters, hormones and drugs. Intracellular concentration of cAMP is tightly regulated by two membrane-bound enzymes, adenylyl cyclase (AC) and phosphodiesterase (PDE). AC activity promotes the synthesis of cAMP from ATP while

PDE degrades cAMP to AMP. The activity of AC is controlled through various G-protein coupled receptors (GPCRs) ₅ via their interaction with one of two distinct GTP binding proteins, G _αs or G _α ;. These proteins are heterotrimeric molecules composed of the subunits G _α (s or i), G _p and G _γ . Agonist activation of the GPCRs leads to the binding of GTP to the G _α subunit, causing a conformational change that leads to the dissociation of the trimer into G _α and G _βγ . Upon dissociation, G _as is primarily involved in AC stimulation whereas G _« i and G _pγ are inhibitory. The measurement of intracellular cAMP is thus an ideal method for measuring the effect of test compounds in GPCR-ediated AC activation or inhibition.

The assay is performed in accordance with the protocol provided in the LANCE™ cAMP assay kit using CHO-Kl cells stably transfected with kappa opioid receptor. Control compounds that were used include an endogenous KOR ligand, dinorphin A (SEQ ID NO: 173) and small molecules U69,593 and U50,488.

Results

The results of the SureFire™ ERK and LANCE™ cAMP assays are shown in Table 6.

Table 6

Av Surefire Av LANCE

Seq # in Av Binding logEC50 (nM) logECso (nM) patent Sequence 1OgIC ₅₀ (nM) (ERK) (cAMP)

2 {Bzo}PrrQ[CHA]CC 0.33 0.35 0.05

102 {Bzo} [HYP]rrQ[CHA]CC 0.98 3.89 0.10

122 {Bzo}PrrQ[HCH]CC-NH2 0.42 1.94 0.14

126 {Bzo}PrrQ[TBA]CC-NH2 1.14 1.51 0.30

124 {Bzo}PrrQ[CHG]CC 0.76 0.58 0.36

101 <4-Cl-Ph-COPrrQ[CHA]CC 0.24 2.95 0.43

100 <Nap-(2)-CO>PrrQ[CHA]CC 0.16 1.09 0.71

131 <cyclohex-CO>PrrQ[CHA]CC-NH ₂ 2.29 14 0.85

43 URRQICC 338 9332 0.91

97 NCCRRQ[NAL]CC 7.41 1288 1.34

121 <H ₂ N-C=NH>R[ANC]<NH(CH ₂ ) ₃ CO>[CHA]CC-NH ₂ 0.41 9.33 1.58

123 2,5-bis-[H ₂ N-C(=NH)-NH]-Ph-CO>G[CHA]CC-NH ₂ 0.52 5.24 1.62

106 <3,4-Cl ₂ -Ph-CO>PrrQ[CHA]CC 2.15 6.16 1.73

120 {Bzo}Pr<NH(CH ₂ ) ₃ CO>[CHA]CC-NH ₂ 0.32 44 5.12

129 {Bzo}PrrQ<l -NHCHrcyclohex-l -CH ₂ CO>CC-NH ₂ 1.81 218 5.12

104 [ANC]rrQ[CHA]CC 0.81 1584 7.24

12 <4-MeO-Ph-CH ₂ CO>rrQ[CHA]CC 12 7.41

ON

4 UrrQ[CHA] [PEN]C 2.69 426 8.31

100 <2-Nap-CH ₂ -CO>PrrQ[CHA]CC 3.01 158 8.70

157 {Bzo}P<NH(CH ₂ ) ₃ CO>[CHA]CC-NH ₂ 6.60 169 10.00

91 UCCRRQICC 151 3388 10.20

25 UrrQ[BTA]CC 25 12.00

8 UrrQ[CHA]CC 8.70 831 12.30

3 [TIC]rrQ[CHA]CC 0.96 154 16.20

90 UCCRRQ[CHA]CC 7.24 630 18.10

40 RRQICC 162 2041 22.90

130 <H ₂ N-C=NH>R[ABP]<NH(CH ₂ ) ₃ CO>[CHA]CC-NH ₂ 1.90 245 23.90

10 K[PIP]rrQ[CHA]CC 9.54 41.60

127 <H ₂ N-C=NH>R[ABP]G[CHA]CC-NH ₂ 1.25 131 58.80

133 <H ₂ N-C=NH>rQ[CHA]CC-NH ₂ 6.45 251 74.10

13 URRQ[CHA]CC 10 831 208

5 [HTI]rrQ[BTA]CC 2.29 87 245

159 {Bzo}PrrG[CHA]CC 0.35 1.00 0.19

167 [ABP]rrQ[CHA]CC-NH ₂ 0.46 1.86 0.29

168 {Bzo}Prrq[CHA]CC 0.74 1.47 0.55

169 {Bzo}PrrR[CHA]CC 0.51 3.38 1.12

170 {Bzo}PrrA[CHA]CC 7.24 1.86 14.40

171 {Bzo}PrrQ[CHA] [PEN]C 10 213 14.70 173 [TIC]iτQ[CHA][SEC][SEC] 5.75 114 66.00

173 YGGFLRRIRPKLKWDWQ (dynorphin A) 0.42 0.98 0.13

U69,593 small molecule 2.39 4.67 1.07

^{7 6} -

U50,488 small molecule 3.16 5.88 1.90

oo

I

Example 5: In vivo study in inflammatory pain model

METHODS Animals: Male Sprague-Dawley rats, initially weighing between 160 and 25Og, were used for all experiments. Animals were obtained from the Gore Hill Research Laboratory and housed individually in the Pain Management Research Institute animal holding facility, under a 12:12 h light/dark cycle at 22 ± 1 ⁰ C, with environmental enrichment and free access to food and water. AU experiments were carried out in the light cycle. Experiments were approved by the Royal North Shore Hospital/University of Technology Sydney Animal Care and Ethics Committee (protocol number 0508-027A).

Inflammatory pain model: 150 μL of Complete Freund's Adjuvant (CFA, Sigma, Sydney, Australia) was injected subcutaneously into the plantar surface of the left hind paw under brief isoflurane (1 - 3 % in O ₂ ) anaesthesia.

Assessment of inflammation and hyperalgesia: Paw volume was measured using a plethysmometer (Ugo Basile, Italy). Values shown are the difference in volumes of the two hind paws (left minus right paw volume).

To measure mechanical hyperalgesia, animals were gently restrained in a sock and the hind paw was placed in a paw pressure analgesymeter (Randall-Sellito device, Ugo Basile, Italy). This device applies a steadily increasing force (rate = 17 g.s ^"1 ) which is removed once an animal attempts to withdraw its paw, or upon reaching the cut-off threshold of 250 g. This gave a measure of the mechanical paw withdrawal threshold (PWT). Mechanical PWT was measured twice for the left hind paw and the readings averaged.

Intra-plantar test compound injection: Animals were briefly anaesthetised in isoflurane. Drugs were injected into the plantar surface of the left hind paw in a volume of 100 μL, using a 1 mL syringe with a 30 gauge needle.

Protocol: The following protocol was used (annotated as days relative to intraplantar CFA):

Days -5 to -2: Acclimatise to holding facility and testing environment Day -1 : Measure mechanical hyperalgesia, paw volume. Day 0: Measure mechanical hyperalgesia, paw volume.

Inject CFA.

Day 3 : Inject test compound at time 0 min relative to testing times below.

Measure mechanical hyperalgesia at -30, -5, 5, 10, 30, 60, 120, 240 min. Measure paw volume at -50, 60, 120, 240 min.

Day 4: Measure mechanical hyperalgesia, paw volume.

Day 5 : Measure mechanical hyperalgesia, paw volume.

Data analysis: Only animals which had paw withdrawal thresholds of between 150 - 25O g prior to CFA injection and displayed a decrease in paw withdrawal threshold of greater than 40 g following CFA injection were included. Mean changes in behavioural scores produced by drug injection for each animal were calculated as the integral of post-injection values (from 0 - 4 h) relative to pre-injection mean baseline (area-under-the-curve, AUC).

Plots of mechanical PWT and paw volume are presented as mean ± S.E.M for each group. Plots of mechanical PWT and paw volume are for individual animals are also shown. It should be noted that the mean ± S.E.M. data are presented for clarity, even with agents in which only 2 - 3 animals have been tested. Statistical analysis has not been performed because of the low animal numbers for all agents (even with n = 4 for the U69593 and vehicle experiments it is calculated that there is greater than a 30 % chance of obtained false negatives). Given the variability in the data, which is similar to that reported for comparable studies, the conclusions concerning observed drug effects should be taken with caution. If statistical comparisons are required then at least 6 - 8 animals per agent would be required.

The peptide used in the study was SEQ ID NO:3.

RESULTS

In this study the following compounds were examined: • Vehicle for SEQ ID NO:3 (phosphate buffered saline, n = 2).

• SEQ ID NO:3: 300 μg (n = 2).

At three days after intraplantar injection of CFA mechanical PWT of the left hind paw decreased by 86 ± 7 g (range = 42 - 137 g) from a pre-CFA value of 200 ± 7 g (range = 156 - 250 g), and left-right paw volume difference increased by 1.6 ± 0.1 ml (range = 1.0 - 3.2 mL, n = 16).

The data for the peptide of SEQ ID NO: 3 is shown in Figure 1. Intraplantar administration of the peptide of SEQ ID NO: 3 300 μg (n = 2) produced an increase in mechanical PWT which peaked at 1 - 4 h post-injection and was maintained at 24 and 48 h post-injection. In one animal the peptide of SEQ ID NO: 3 produced a transient decrease in mechanical PWT at 5 - 30 min. At 24 and 48 h post-injection the mechanical PWT was at/above the cut-off of 250 g for both animals.

The peptide of SEQ ID NO: 3 produced immediate reddening and swelling which was evident as an increase in paw volume a 1 - 4 h post-injection and resolved at 24 to 48 h.

Varying dosages of peptide of SEQ ID NO: 3 showed that the peptide provided effective pain relief at dosages of 3 μg, 30 μg and 300 μg, this is depicted as mechanical PWT AUC for all doses (Figure 2).

Example 6: Assessment of peptides in in vivo models

To ascertain the kappa agonists effectiveness to facilitate the alleviation of pain various in vivo models will be assessed. These will include, but not be limited to, such in vivo models that assess acute pain, chronic pain, neuropathic pain, inflammatory pain, pain associated with irritable bowel disease and also gastrointestinal motility.

Some specific pain models are outlined below:

A. For assessment of acute pain and inflammation

METHODS

Animals: Male Sprague-Dawley rats, initially weighing between 160 and 25Og, were used for all experiments. Animals were obtained and housed individually in an animal holding facility, under a 12:12 h light/dark cycle at 22 ± 1 ⁰ C, with environmental enrichment and free access to food and water. All experiments were carried out in the light cycle under the appropriate animal care and ethics guidelines.

Inflammatory pain model. 150 μl of Complete Freund's Adjuvant (CFA, Sigma, Sydney, Australia) was injected subcutaneously into the plantar surface of the left hind paw under brief isoflurane (1 - 3 % in O ₂ ) anaesthesia.

Assessment of inflammation and hyperalgesia: Paw volume was measured using a plethysmometer (Ugo Basile, Italy). To measure mechanical hyperalgesia, animals were gently restrained in a sock and the hind paw was placed in a using a paw pressure analgesymeter (Ugo Basile, Italy). This device applies a steadily increasing force which is removed once an animal attempts to withdraw its paw, or upon reaching the cut-off threshold of 300 g. This gave a measure of the mechanical paw withdrawal threshold (PWT). Mechanical PWT was measured twice for both left and right hind paws and the readings averaged.

Intra-plantar test compound injection: Animals were briefly anaesthetised in isoflurane. Kappa agonists were injected into the plantar surface of the left hind paw in a volume of 100 μL, using a ImI syringe with a 30 gauge needle. Protocol: The following procedure was carried out on all animals: Days -5 to -2: Acclimatise to holding facility and testing environment Day -1 : Measure mechanical hyperalgesia, paw volume.

Day 0: Measure mechanical hyperalgesia, paw volume.

Inject CFA. Day 3: Inject test compound at time 0 min relative to testing times below.

Measure mechanical hyperalgesia at -30, -5, 5, 10, 30, 60, 120, 240 min.

Measure paw volume at -50, 60, 120, 240 min. Day 4: Measure mechanical hyperalgesia, paw volume.

Day 5 : Measure mechanical hyperalgesia, paw volume. Data analysis: Plots of mechanical PWT, thermal PWL and rotarod latency are presented as mean ± S.E.M. Mean changes in behavioural scores produced by drug injection were calculated as the integral of post-injection values (from 0 to 240 min) relative to pre-injection mean baseline (area-under-the-curve, AUC).

Several peptides have been tested using the above method. These peptides have shown to be efficacious in this model.

B. For assessment of acute pain

The model described by Brerman et al (Pain 64 (1996) 493-501) was developed for the assessment of compounds in postoperative pain - this being a common form of acute pain. In summary an incision is made into the foot pad (through the skin, fascia and into the muscle) of a rat (under anaesthesia) after which withdrawal responses are measured using von Frey filaments around the wound over a number of days. This model will allow for the assessment of compounds ability to alleviate pain associated with surgery. It is not suggestive that this is the only model suitable for the assessment of postoperative pain.

C. For the assessment of neuropathic pain

The Bennett model (Bennett GJ & Xie YK, Pain 33 (1988) 87-107) is a common in vivo model used to assess neuropathic pain disorders. In summary loosely constrictive ligatures are placed round the common sciatic nerve of the rat. Hyperalgesia and allodynia were formed as a result of the surgery.

D. Assessment of chronic pain

The Bennett model (Bennett GJ & Xie YK, Pain 33 (1988) 87-107) has also been demonstrated to be effective in the assessment of chronic pain.

E. For the assessment of gastrointestinal disorders the following models may be used:

Gastrointestinal motility (Macht DI & Barba-Gose J, J Am Pharm Assoc (1931) 20 558 or Omusu M et al, Arzneim Forsch/Drug Res (1988) 38 1309), Inflammatory Bowel Disease (Hogaboam CM et al, Eur J Pharmacol (1996) 309, 261), Acetic Acid Writhing model (Inoue K et al, Arzneim Forsch/Drug Res (1991) 41(1) 235).

F. For the assessment of pain and inflammation associated with arthritis the following model could be used. CFA-induced chronic arthritis model (Winter CA & Nuss GW, Arthritis Rheum (1966) 9 394-404).

It is by no means suggestive that these are the only models that will be used however, they provide examples of some of the models that could be used to assess kappa agonists ability to alleviate the various types of pain. The models may also be altered by using alternative modes of administration, such as intravenous, oral, intrathecal or intra-articular and in vivo models are not limited to the rat, other animals, such as mice, guinea pigs, dogs and monkeys could also be used.