Background of the Invention

Chronic visceral pain (CVP) is a prevalent and debilitating syndrome with limited treatments. Visceral pain is pain that results from the activation of nociceptors of the thoracic, pelvic, or abdominal viscera (organs). Visceral structures are highly sensitive to distension (stretch), ischemia and inflammation, but relatively insensitive to other stimuli that normally evoke pain such as cutting or burning. Visceral pain is diffuse, difficult to localize and often referred to a distant, usually superficial, structure. It may be accompanied by symptoms such as nausea, vomiting, changes in vital signs as well as emotional manifestations. The pain may be described as sickening, deep, squeezing, and dull. Distinct structural lesions or biochemical abnormalities explain this type of pain in only a proportion of patients. These diseases are grouped under gastrointestinal neuromuscular diseases (GINMD). Others can experience occasional visceral pains, often very intense in nature, without any evidence of structural, biochemical or histolopathologic reason for such symptoms. These diseases are grouped under functional gastrointestinal disorders (FGID) and the pathophysiology and treatment can vary greatly from GINMD. The two major single entities among functional disorders of the gut are functional dyspepsia and irritable bowel syndrome.

Guanylyl cyclase C (GUCY2C), also referred to as GCC, is the receptor for uroguanylin (GUCA2B) in small intestine and guanylin (GUCA2A) in colorectum. This hormone-receptor axis produces cyclic (c)GMP accumulation, inducing intestinal fluid secretion.

A rare population of intestinal enteroendocrine cells, called neuropod cells, have been identified as the richest source of GUCY2C in the body (GUCY2C ^H1GH ). While GUCY2C is expressed by all epithelial cells in intestine, a GUCY2C promoter-driven GFP reporter revealed a novel rare population of cells enriched in GUCY2C mRNA, protein, and activity. These cells have the characteristics of (1) intestinal epithelial cells, (2) endocrine cells (they make hormones for release into the circulation), and (3) neuronal cells (they form synapses with underlying nerves in the intestinal wall). Neuropod cells are concentrated in the upper small intestine, they are enriched in cholecystokinin (anorexigenic), and they signal to the underlying nervous system to restrict appetite and eating through synaptic transmission. GUCY2C ^High cells, concentrated in duodenum but rare in rectum, morphologically resemble enteroendocrine cells with a basal neuropod potentially synapsing on visceral afferents in the lamina propria, offering a cellular substrate for GUCY2C visceral nociceptive signaling.

GUCY2C agonists target GUCY2C expressed by bulk intestinal epithelial cells to regulate fluid and electrolyte secretion that relieves constipation. This forms the basis for use of GUCY2C agonists linaclotide and plecanatide to treat constipation-type irritable bowel syndrome (IBS-C) and chronic idiopathic constipation (CIC). The most frequent adverse side effect of this therapy is diarrhea, which causes patients to discontinue the medication. Beyond secretion, previous studies have identified a role for GUCY2C and its agonists in controlling (1) visceral pain and (2) appetite, obesity, and metabolism. However, the precise cells and mechanisms regulating those processes remain undefined.

GUCY2C agonists relieve CVP in these patients, and in mouse models of visceral pain; these patients are deficient in uroguanylin, suggesting that constipation and CVP may reflect hormone insufficiency silencing GUCY2C. Eliminating GUCY2C in mice (GUCY2C-/-) produced CVP, evoked by cyclic rectal distension, quantified by the abdominal withdrawal reflex and by phospho-ERK signaling in the spinal cord dorsal horn. CVP severity in GUCY2C-/- mice recapitulated TNBS-induced inflammatory bowel disease in wild type (GUCY2+/+) mice. Oral linaclotide relieved TNBS-induced CVP in wild type mice, but was without effect on CVP in GUCY2C-/- mice. The mechanistic basis of GUCY2C-dependent visceral analgesia remains obscure.

Phosphodiesterase 3, also referred to herein as PDE3, is a member of the phosphodiesterase family. A phosphodiesterase (PDE) is an enzyme that breaks a phosphodiester bond. The PDEs belong to at least eleven related gene families, which are different in their primary structure, substrate affinity, responses to effectors, and regulation mechanism. PDE3 is clinically and PDE3 inhibitors have been developed as pharmaceuticals.

PDE3 is a cyclic nucleotide phosphodiesterases. The cyclic nucleotide phosphodiesterases comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling within subcellular domains. PDEs are therefore important regulators of signal transduction mediated by these second messenger molecules. PDE3 enzymes are involved, inter alia, in regulation of cardiac and vascular smooth muscle contractility. Molecules that inhibit PDE3 were originally investigated for the treatment of heart failure. The PDE3 inhibitor milrinone is approved for use in heart failure in intravenous form. Cilostazol is PDE3 inhibitor approved for treatment of intermittent claudication. Zardaverine is a dual-selective PDE3/4 phosphodiesterase inhibitor which studies indicate may have useful anti-cancer properties.

The PDE3 family in mammals consists of two members, PDE3A and PDE3B. The PDE3 isoforms are structurally similar, containing an N-terminal domain important for the localization and a C-terminus end. PDE3A is mainly implicated in cardiovascular function and fertility but PDE3B is mainly implicated in lipolysis.

Summary of the Invention

Some embodiments of the instant invention relate to the use of GUCY2C agonists in combination with PDE3 inhibitors in the treatment of an individual who has been identified as experiencing chronic visceral pain.

Some embodiments of the instant invention relate to compositions or kits comprising GUCY2C agonists and PDE3 inhibitors for use in the treatment of an individual who has been identified as experiencing chronic visceral pain.

Some embodiments of the instant invention relate to methods of treating an individual who has been identified as experiencing chronic visceral pain comprising oral administration of GUCY2C agonists in an amount of a GUCY2C agonist sufficient to reduce pain in combination with oral administration of PDE3 inhibitors.

Some embodiments of the instant invention relate to the use of GUCY2C agonists in combination with PDE3 inhibitors in to suppress appetite in an individual who has been identified as desiring appetite suppression.

Some embodiments of the instant invention relate to compositions or kits comprising GUCY2C agonists and PDE3 inhibitors to suppress appetite in an individual who has been identified as desiring appetite suppression.

Some embodiments of the instant invention relate to methods to suppress appetite in an individual who has been identified as desiring appetite suppression comprising oral administration of compositions comprising GUCY2C agonists in an amount of a GUCY2C agonist suppress appetite in combination with oral administration of PDE3 inhibitors.

Description of Embodiments

The gene expression profile of these cells was analyzed and found to distinguish them from bulk epithelial cells by their lack of the machinery for GUCY2C-mediated fluid and electrolyte secretion; their unique complement of cyclic nucleotide phosphodiesterases, including PDE3 (compared to PDE5 in bulk epithelial cells); and their expression of endocrine hormones regulating appetite and insulin secretion. Tn that context, it is noteworthy that PDE3 is a cGMP-inhibited PDE3 that regulates intracellular concentrations of cAMP, the ultimate downstream mediator of signaling. Further, these specific cells have been demonstrated to regulate the excitability of dorsal root ganglion neurons, including their firing of action potentials, and the extension of those signals to nociceptive regions of the spinal cord. Additionally, these specific cells have been revealed to carry the signal that mediates the analgesic effects of GUCY2C agonists, rather than bulk intestinal epithelial cells. Moreover, neuropod cells regulate DRG excitability by releasing compounds other than cyclic GMP (as thought previously).

RNAseq analysis revealed that GUCY2CHigh neuropod cells were deficient in gene products canonically associated with GUCY2C-driven secretion, including GUCA2A, GUCA2B, CFTR and NHE3. Rather, these cells were enriched in gene sets characteristic of neurons. Importantly, dorsal root ganglia (DRG) cells formed functional connections with GUCY2CHigh neuropod cells in co-culture. Thus, DRG cells with GUCY2C ^Hlgh neuropod cells alone (GUCA2B -deficient, GUCY2C silenced) were hyperexcitable following current injection, with a reduced rheobase and repetitive action potentials (APs). In contrast, adding linaclotide (hormone replete, GUCY2C activated) silenced DRG neuron excitability, raising the rheobase and eliminating repetitive APs. DRG neuron excitability was not affected by linaclotide in co-cultures with neuropod cells from GUCY2C-/- mice. Moreover, the effects of linaclotide on DRG neuron excitability was not recapitulated by extracellular cGMP. These observations suggest that GUCY2C ^Hlgh neuropod cells synapse with DRG visceral afferents and modulate their excitability. They support a model in which GUCA2B sufficiency activates GUCY2C to suppress DRG neuron excitability, while GUCA2B insufficiency silences GUCY2C to produce DRG hyperexcitability and CVP. Finally, they suggest that linaclotide relieves CVP by stimulating GUCY2CHigh neuropod cells to inhibit DRG excitability and nociceptive signaling.

The high concentration of GUCY2C in these cells, their specific regulation of neuronal pathways mediating nociception and analgesia in vitro and in vivo, their unique complement of endocrine hormones regulating appetite and insulin secretion, and their unique complement of expressed phosphodiesterases provide a previously unanticipated paradigm for the therapeutic management of (1) visceral pain and (2) appetite, obesity and metabolism. Thus, neuropod-targeted therapies for these conditions should focus on the anatomical concentration of these cells in the upper small intestine, rather more distally in the lower small intestine or colorectum. Further, the high concentration of GUCY2C in these cells suggests that lower doses of GUCY2C agonists can be used for appetite and pain control, compared to doses used for control of fluid and electrolyte secretion, mitigating diarrhea as an adverse side effect. Moreover, the effects of GUCY2C agonists should be potentiated by inhibitors of PDE3, intensifying cAMP downstream signaling mediating the differential effects of neuropod, compared to bulk intestinal epithelial cells. This latter strategy specifically targets neuropod cells, rather than bulk epithelial cells, and permits a differential intensification of the analgesia and appetite control of GUCY2C and its agonists, without inducing secretion and diarrhea, the established adverse side effects of these agents. Chronic Visceral Pain

Visceral pain is pain related to the internal organs in the midline of the body. Unlike somatic pain — pain that occurs in tissues such as the muscles, skin, or joints — visceral pain is often vague, and can be described as a deep ache, pressure, gnawing, twisting, colicky or dull. Visceral pain is generally poorly localized and characterized by hypersensitivity to a stimulus such as organ distension. Visceral pain represents a major clinical problem, yet far less is known about its mechanisms compared to somatic pains, e.g. from cutaneous and muscular structures. Visceral pain is generally characterized by hypersensitivity to a stimulus such as organ distension. Functional gastrointestinal disorders (FGID) underlie the most prevalent forms of visceral pain. Irritable bowel syndrome (IBS) is one FGID characterized by abdominal pain, discomfort and altered bowel habits and creates tremendous pressure on the healthcare system affecting an estimated 10-15% of Europe and U.S. populations with consequent costs estimated to exceed USS 40 billion. Dysmenhorrea, severe pelvic pain during menstrual cycles, underlies one of the most common gynecologic complaints in young women. It also contributes to economic burdens associated with lost workdays and productivity. Although some visceral pain disorders are not life-threatening, they still contribute significantly to a large segment of healthcare resource consumption and have a considerable negative impact on lives with psychological distress, disturbance of work and sleep and sexual dysfunction. Visceral pain originates in the organs of the chest, belly, or pelvis. Visceral pain can be described as a dull ache. Visceral pain originates in the middle of the body, but may also be felt it in other areas. It leads to sensitivity in the affected area or elsewhere and is typically diffuse and difficult to locate. Visceral pain is often accompanied by other symptoms such as vomiting, sweating, or a racing heart and has a strong connection to psychological symptoms, such as depression.

In the visceral organs, pain receptors are not as closely packed and not as evenly spread out as in other organs, which makes the pain’s origin much harder to pinpoint and treat. Common causes of visceral pain in individuals who has been identified as experiencing chronic visceral pain include: inflammation, menstrual cramps, swelling and stretching of the organs, blockage — particularly of the bowels or urethra, decreased blood flow and tumors — particularly when concentrated in the pelvis or abdomen. These causes are, themselves, often the result of an underlying health condition or disorder such as: inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), cancer, pancreatitis, indigestion and interstitial cystitis (IC).

Appetite Suppression

Appetite suppression refers to regulating the desire for food and its consumption. Individuals desiring appetite suppression include overweight, obese and morbidly obese individuals, individuals who have hyperphagia, individuals desiring weight loss and preventing weight gain, and individual with certain eating disorders.

GUCY2C Agonists

GUCY2C agonists are known. Two native GUCY2C agonists, guanylin and uroguanylin, have been identified (see U.S. Patent Nos 5,969,097 and 5,489,670, which are each incorporated herein by reference. In addition, several small peptides, which are produced by enteric pathogens, are toxigenic agents which cause diarrhea (see U.S. Patent No. 5,518,888, which is incorporated herein by reference). The most common pathogen derived GUCY2C agonist is the heat stable enterotoxin produced by strains of pathogenic E. coli. Native heat stable enterotoxin produced by pathogenic E coli is also referred to as ST. A variety of other pathogenic organisms including Yersinia and Enterobacter, also make enterotoxins which can bind to guanylyl cyclase C in an agonistic manner. In nature, the toxins are generally encoded on a plasmid which can "jump" between different species. Several different toxins have been reported to occur in different species. These toxins all possess significant sequence homology, they all bind to ST receptors and they all activate guanylate cyclase, producing diarrhea.

ST has been both cloned and synthesized by chemical techniques. The cloned or synthetic molecules exhibit binding characteristics which are similar to native ST. Native ST isolated from E. coli is 18 or 19 amino acids in length. The smallest "fragment" of ST which retains activity is the 13 amino acid core peptide extending toward the carboxy terminal from cysteine 6 to cysteine 18 (of the 19 amino acid form). Analogues of ST have been generated by cloning and by chemical techniques. Small peptide fragments of the native ST structure which include the structural determinant that confers binding activity may be constructed. Once a structure is identified which binds to ST receptors, non-peptide analogues mimicking that structure in space are designed.

U.S. Patent Nos. 5,140,102 and 7,041,786, and U.S. Published Applications US 2004/0258687 Al and US 2005/0287067 Al also refer to compounds which may bind to and activate guanylyl cyclase C.

SEQ ID NO: 1 discloses a nucleotide sequence which encodes 19 amino acid ST, designated ST la, reported by So and McCarthy (1980) Proc. Natl. Acad. Sci. USA 77:4011 , which is incorporated herein by reference.

The amino acid sequence of ST la is disclosed in SEQ ID NO:2.

SEQ ID NO:3 discloses the amino acid sequence of an 18 amino acid peptide which exhibits ST activity, designated ST I*, reported by Chan and Giannella (1981) J. Biol. Chem. 256:7744, which is incorporated herein by reference.

SEQ ID NO:4 discloses a nucleotide sequence which encodes 19 amino acid ST, designated ST lb, reported by Mosely et al. (1983) Infect. Immun. 39:1167, which is incorporated herein by reference.

The amino acid sequence of ST lb is disclosed in SEQ ID NO:5.

A 15 amino acid peptide called guanylin which has about 50% sequence homology to ST has been identified in mammalian intestine (Currie, M. G. et al. (1992) Proc. Natl. Acad Sci. USA 89:947-951, which is incorporated herein by reference). Guanylin binds to ST receptors and activates guanyl ate cyclase at a level of about 10- to 100-fold less than native ST. Guanylin may not exist as a 15 amino acid peptide in the intestine but rather as part of a larger protein in that organ. The amino acid sequence of guanylin from rodent is disclosed as SEQ ID NO: 6. SEQ ID N0:7 is an 18 amino acid fragment of SEQ ID NO:2. SEQ ID NO:8 is a 17 amino acid fragment of SEQ ID NO:2. SEQ ID NO:9 is a 16 amino acid fragment of SEQ ID NO:2. SEQ ID NO: 10 is a 15 amino acid fragment of SEQ ID NO:2. SEQ ID NO:1 1 is a 14 amino acid fragment of SEQ ID NO:2. SEQ ID NO: 12 is a 13 amino acid fragment of SEQ ID NO:2. SEQ ID NO:13 is an 18 amino acid fragment of SEQ ID NO:2. SEQ ID NO:14 is a 17 amino acid fragment of SEQ ID NO:2. SEQ ID NO: 15 is a 16 amino acid fragment of SEQ ID NO:2. SEQ ID NO: 16 is a 15 amino acid fragment of SEQ ID NO:2. SEQ ID NO: 17 is a 14 amino acid fragment of SEQ ID NO:2.

SEQ ID NO: 18 is a 17 amino acid fragment of SEQ ID NO:3. SEQ ID NO: 19 is a 16 amino acid fragment of SEQ ID NO:3. SEQ ID NO:20 is a 15 amino acid fragment of SEQ ID NO:3. SEQ ID NO:21 is a 14 amino acid fragment of SEQ ID NO:3. SEQ ID NO:22 is a

13 amino acid fragment of SEQ ID NO:3. SEQ ID NO:23 is a 17 amino acid fragment of SEQ ID NO:3. SEQ ID NO:24 is a 16 amino acid fragment of SEQ ID NO:3. SEQ ID NO:25 is a 15 amino acid fragment of SEQ ID NO:3. SEQ ID NO:26 is a 14 amino acid fragment of SEQ ID NO:3.

SEQ ID NO:27 is an 18 amino acid fragment of SEQ ID NO:5. SEQ ID NO:28 is a 17 amino acid fragment of SEQ ID NO:5. SEQ ID NO:29 is a 16 amino acid fragment of SEQ ID NO:5. SEQ ID NO:30 is a 15 amino acid fragment of SEQ ID NO:5. SEQ ID NO:31 is a

14 amino acid fragment of SEQ ID NO:5. SEQ ID NO:32 is a 13 amino acid fragment of SEQ ID NO:5. SEQ ID NO:33 is an 18 amino acid fragment of SEQ ID NO:5. SEQ ID NO:34 is a 17 amino acid fragment of SEQ ID NO:5. SEQ ID NO:35 is a 16 amino acid fragment of SEQ ID NO:5. SEQ ID NO:36 is a 15 amino acid fragment of SEQ ID NO:5. SEQ ID NO:37 is a 14 amino acid fragment of SEQ ID NO:5.

SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:36 AND SEQ ID NO:37 are disclosed in Yoshimura, S., et al. (1985) FEBS Lett. 181:138, which is incorporated herein by reference.

SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, which are derivatives of SEQ ID NO:3, are disclosed in Waldman, S. A. and O'Hanley, P. (1989) Infect. Immun. 57:2420, which is incorporated herein by reference.

SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, which are derivatives of SEQ ID NO:3, are disclosed in Yoshimura, S., et al. (1985) FEBS Lett. 181:138, which is incorporated herein by reference. SEQ ID NO:46 is a 25 amino acid peptide derived from Y. enterocolitica which binds to the ST receptor.

SEQ ID NO:47 is a 16 amino acid peptide derived from V. cholerae which binds to the ST receptor. SEQ ID NO:47 is reported in Shimonishi, Y., et al. FEBS Lett. 215:165, which is incorporated herein by reference.

SEQ ID NO:48 is an 18 amino acid peptide derived from Y. enterocolitica which binds to the ST receptor. SEQ ID NO:48 is reported in Okamoto, K., et al. Infec. Immun. 55:2121, which is incorporated herein by reference.

SEQ ID NO:49, is a derivative of SEQ ID NO:5.

SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53 are derivatives. SEQ ID NO:54 is the amino acid sequence of guanylin from human.

A 15 amino acid peptide called uroguanylin has been identified in mammalian intestine from opossum (Hamra, S. K. et al. (1993) Proc. Natl. Acad Sci. USA 90: 10464- 10468, which is incorporated herein by reference; see also Forte L. and M. Curry 1995 FASEB 9:643-650; which is incorporated herein by reference). SEQ ID NO:55 is the amino acid sequence of uroguanylin from opossum.

A 16 amino acid peptide called uroguanylin has been identified in mammalian intestine from human (Kita, T. et al. (1994) Amer. J. Physiol. 266:F342-348, which is incorporated herein by reference; see also Forte L. and M. Curry 1995 FASEEB 9:643-650; which is incorporated herein by reference). SEQ ID NO: 56 is the amino acid sequence of uroguanylin from human.

SEQ ID NO:57 is the amino acid sequence of proguanylin, a guanylin precursor which is processed into active guanylin.

SEQ ID NO:58 is the amino acid sequence of prouroguanylin, a uroguanylin precursor which is processed into active uroguanylin.

Two recently approved products in the US, linaclotide (SEQ ID NO:59) and plecanatide (SEQ ID NO:60) may be used as GUCY2C agonists in the methods set forth herein.

U.S. Patent Nos. 5,140,102, 7,041 ,786 and 7,304,036, and U.S. Published Applications US 2004/0258687, US 2005/0287067, 20070010450, 20040266989, 20060281682, 20060258593, 20060094658, 20080025966, 20030073628, 20040121961 and 20040152868, which are each incorporated herein by reference, also refer to compounds which may bind to and activate guanylyl cyclase C.

In some embodiments, GUCY2C agonists which are peptides may be administered in an amount ranging from 100 ug to 1 gram every 4-48 hours. In some embodiments, GUCY2C agonists are administered in an amount ranging from 1 mg to 750 mg every 4-48 hours. In some embodiments, GUCY2C agonists are administered in an amount ranging from 10 mg to 500 mg every 4-48 hours. In some embodiments, GUCY2C agonists are administered in an amount ranging from 50 mg to 250 mg every 4-48 hours. In some embodiments, GUCY2C agonists are administered in an amount ranging from 75 mg to 150 mg every 4-48 hours,

In some embodiments, doses are administered every 4 or more hours. In some embodiments, doses are administered every 6 or more hours. In some embodiments, doses are administered every 8 or more hours. In some embodiments, doses are administered every 12 or more hours. In some embodiments, doses are administered every 24 or more hours. In some embodiments, doses are administered every 48 or more hours. In some embodiments, doses are administered every 4 hours or less. In some embodiments, doses are administered every 6 hours or less. In some embodiments, doses are administered every 8 hours or less. In some embodiments, doses are administered every 12 hours or less. In some embodiments, doses are administered every 24 hours or less. In some embodiments, doses are administered every 48 hours or less.

PDE3 inhibitors

In some embodiments, the active agent comprises PDE3 selective inhibitors. PDE inhibitors are generally discussed in the following references which are each incorporated herein by reference: Uzunov, P. and Weiss, B.: Separation of multiple molecular forms of cyclic adenosine 3',5'-monophosphate phosphodiesterase in rat cerebellum by polyacrylamide gel electrophoresis. Biochim. Biophys. Acta 284:220-226, 1972; Weiss, B.: Differential activation and inhibition of the multiple forms of cyclic nucleotide phosphodiesterase. Adv. Cycl. Nucl. Res. 5:195-211, 1975; Fertel, R. and Weiss, B.: Properties and drug responsiveness of cyclic nucleotide phosphodiesterases of rat lung. Mol. Pharmacol. 12:678- 687, 1976; Weiss, B. and Hait, W.N.: Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. Ann. Rev. Pharmacol. Toxicol. 17:441-477, 1977; Essayan DM. (2001). "Cyclic nucleotide phosphodiesterases.". J Allergy Clin Immunol. 108 (5): 671-80; Deree J, Martins JO, Melbostad H, Loomis WH, Coimbra R. (2008). "Insights into the Regulation of TNF-a Production in Human Mononuclear Cells: The Effects of Non- Specific Phosphodiesterase Inhibition". Clinics (Sao Paulo). 63 (3): 321-8; Marques LJ, Zheng L, Poulakis N, Guzman J, Costabel U (February 1999). "Pentoxifylline inhibits TNF- alpha production from human alveolar macrophages". Am. J. Respir. Crit. Care Med. 159 (2): 508-11; Peters-Golden M, Canetti C, Mancuso P, Coffey MJ. (2005). "Leukotrienes: underappreciated mediators of innate immune responses". J Immunol. 174 (2): 589-94; Daly JW, Jacobson KA, Ukena D. (1987). "Adenosine receptors: development of selective agonists and antagonists". Prog Clin Biol Res. 230 (1): 41-63; MacCorquodale DW. THE SYNTHESIS OF SOME ALKYLXANTHINES. Journal of the American Chemical Society. 1929 July; 51(7):2245-2251; WO/ 1985/002540; US Pat No. 4,288,433; Daly JW, Padgett WL, Shamim MT (July 1986). "Analogues of caffeine and theophylline: effect of structural alterations on affinity at adenosine receptors". Journal of Medicinal Chemistry 29 (7): 1305— 8; Daly JW, Jacobson KA, Ukena D (1987). "Adenosine receptors: development of selective agonists and antagonists". Progress in Clinical and Biological Research 230: 41-63; Choi OH, Shamim MT, Padgett WL, Daly JW (1988). "Caffeine and theophylline analogues: correlation of behavioral effects with activity as adenosine receptor antagonists and as phosphodiesterase inhibitors". Life Sciences 43 (5): 387-98; Shamim MT, Ukena D, Padgett WL, Daly JW (June 1989). "Effects of 8-phenyl and 8-cycloalkyl substituents on the activity of mono-, di-, and trisubstituted alkylxanthines with substitution at the 1-, 3-, and 7- positions". Journal of Medicinal Chemistry 32 (6): 1231-7; Daly JW, Hide I, Muller CE, Shamim M (1991). "Caffeine analogs: structure-activity relationships at adenosine receptors". Pharmacology 42 (6): 309-21; Ukena D, Schudt C, Sybrecht GW (February 1993). "Adenosine receptor-blocking xanthines as inhibitors of phosphodiesterase isozymes". Biochemical Pharmacology 45 (4): 847-51. doi:10.1016/0006-2952(93)90168-V; Daly JW (July 2000). "Alkylxanthines as research tools". Journal of the Autonomic Nervous System 81 (1-3): 44-52. doi:10.1016/S0165-1838(00)00110-7; Daly JW (August 2007). "Caffeine analogs: biomedical impact". Cellular and Molecular Life Sciences: CMLS 64 (16): 2153-69; Gonzalez MP, Teran C, Teijeira M (May 2008). "Search for new antagonist ligands for adenosine receptors from QSAR point of view. How close are we?". Medicinal Research Reviews 28 (3): 329-71; Baraldi PG, Tabrizi MA, Gessi S, Borea PA (January 2008). "Adenosine receptor antagonists: translating medicinal chemistry and pharmacology into clinical utility". Chemical Reviews 108 (1): 238-63; de Visser YP, Walther FJ, Laghmani EH, van Wijngaarden S, Nieuwland K, Wagenaar GT. (2008). "Phosphodiesterase-4 inhibition attenuates pulmonary inflammation in neonatal lung injury". Eur Respir J 1 (3): 633-644; Yu MC, Chen JH, Lai CY, Han CY, Ko WC. (2009). "Luteolin, a non-selective competitive inhibitor of phosphodiesterases 1-5, displaced [(3)H]-rolipram from high-affinity rolipram binding sites and reversed xylazine/ketamine-induced anesthesia". Eur J Pharmacol. 627 (1-3): 269-75; Bobon D, Breulet M, Gerard- Vandenhove MA, Guiot-Goffioul F, Plomteux G, Sastre-y-Hernandez M, Schratzer M, Troisfontaines B, von Frenckell R, Wachtel H. (1988). "Is phosphodiesterase inhibition a new mechanism of antidepressant action? A double-blind double-dummy study between rolipram and desipramine in hospitalized major and/or endogenous depressives". Eur Arch Psychiatry Neurol Sci. 238 (1): 2-6; Maxwell CR, Kanes SJ, Abel T, Siegel SJ. (2004). "Phosphodiesterase inhibitors: a novel mechanism for receptor-independent antipsychotic medications". Neuroscience. 129 (1): 101-7; Kanes SJ, Tokarczyk J, Siegel SJ, Bilker W, Abel T, Kelly MP. (2006). "Rolipram: A specific phosphodiesterase 4 inhibitor with potential antipsychotic activity". Neuroscience. 144 (1): 239—46; and Vecsey CG, Baillie GS, Jaganath D, Havekes R, Daniels A, Wimmer M, Huang T, Brown KM, Li XY, Descalzi G, Kim SS, Chen T, Shang YZ, Zhuo M, Houslay MD, Abel T. (2009). "Sleep deprivation impairs cAMP signaling in the hippocampus". Nature. 461 (7267): 1122-1125.

PDE inhibitors which elevate cGMP specifically are disclosed in U.S. Pat. Nos. 6,576,644, 7,384,958, 7,276,504, 7,273,868, 7,220,736, 7,098,209, 7,087,597, 7,060,721, 6,984,641, 6,930,108, 6,911,469, 6,784,179, 6,656,945, 6,642,244, 6,476,021, 6,326,379, 6,316,438, 6,306,870, 6,300,335, 6,218,392, 6,197,768, 6,037,119, 6,025,494, 6,018,046, 5,869,516, 5,869,486, 5,716,993. Other examples include compounds disclosed in WO 96/05176 and 6,087,368, U.S. Pat. Nos. 4,101,548, 4,001,238, 4,001,237, 3,920,636, 4,060,615, 4,209,623, 5,354,571, 3,031,450, 3,322,755, 5,401,774, 5,147,875, 4,885,301, 4,162,316, 4,047,404, 5,614,530, 5,488,055, 4,880,810, 5,439,895, 5,614,627, GB 2 063 249, EP 0 607 439, WO 97/03985, EP 0 395 328, EP 0428 268, PCT WO 93/12095, WO 93/07149, EP 0 349 239, EP 0 352 960, EP 0 526 004, EP 0 463 756, EP 0 607 439, WO 94/05661, EP 0 351 058, EP 0 347 146, WO 97/03985, WO 97/03675, WO 95/19978, WO 98/08848, WO 98/16521, EP 0 722 943, EP 0 722 937, EP 0 722 944, WO 98/17668, WO 97/24334, WO 98/06722, PCT/JP97/03592, WO 98/23597, WO 94/29277, WO 98/14448, WO 97/03070, WO 98/38168, WO 96/32379, and PCT/GB98/03712. PDE inhibitors may include those disclosed in the following patent applications and patents: DE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801 , DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792, DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948, EP0075436, EP0096517, EP0112987, EP0116948, EP0150937, EP0158380, EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725, EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386, EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479, JP92234389, JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931, WO9117991, W09200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437, WO9427947, W09500516, W09501980, WO9503794, W09504045, W09504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282, W09600218, WO9601825, WO9602541, WO9611917, DE3142982, DE1116676, DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345 and WO9307124, EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399.

Examples of nonselective phosphodiesterase inhibitors include: methylated xanthines and derivatives such as for examples: caffeine, a minor stimulant, aminophylline, IB MX (3- isobutyl-1 -methylxanthine), used as investigative tool in pharmacological research, paraxanthine, pentoxifylline, a drug that has the potential to enhance circulation and may have applicability in treatment of diabetes, fibrotic disorders, peripheral nerve damage, and microvascular injuries, theobromine and theophylline, a bronchodilator. Methylated xanthines act as both competitive nonselective phosphodiesterase inhibitors which raise intracellular cAMP, activate PKA, inhibit TNF-alpha and leukotriene synthesis, and reduce inflammation and innate immunity and nonselective adenosine receptor antagonists. Different analogues show varying potency at the numerous subtypes, and a wide range of synthetic xanthine derivatives (some nonmethylated) have been developed in the search for compounds with greater selectivity for phosphodiesterase enzyme or adenosine receptor subtypes.

PDE3 selective inhibitors include for example, cilostazol, enoximone, amrinone, milrinone, sulmazole, ampozone, cilostamide, carbazeran piroximone, imazodan, siguazodan, adibendan, saterinone, emoradan, revizinone. Some are used clinically for short-term treatment of cardiac failure. These drugs mimic sympathetic stimulation and increase cardiac output. PDE3 is sometimes referred to as cGMP-inhibited phosphodiesterase. Examples of PDE3/4 inhibitors include benafentrine, trequinsin, zardaverine and tolafentrine. Additional PDE3 inhibitors include those set forth in U.S. Pat. Nos. 7,375,100, 7,056,936, 6,897,229, 6,716,871, 6,498,173, and 6,110,471, which are each incorporated herein by reference.

Amrinone, also known as inamrinone, and sold as Inocor, is a pyridine phosphodiesterase 3 inhibitor. Milrinone, is approved and sold under the brand name Primacor, as a pulmonary vasodilator used in patients who have heart failure. Enoximone (INN, trade name Perfan) is an imidazole phosphodiesterase inhibitor. Cilostazol, sold under the brand name Pletal among others, is a medication used to help the symptoms of intermittent claudication in peripheral vascular disease.

Administration of GUCY2C agonists and PDE3 inhibitors

Methods of treating visceral pain and methods of suppressing appetite comprise identifying individual in need of and/or desirous of such treatment, and oral administration of GUCY2C agonists in a therapeutically effective amount in combination with oral administration of an effective amount of PDE3 inhibitors.

In methods of treating visceral pain, a therapeutically effective amount of GUCY2C agonists refers to an amount of GUCY2C agonist sufficient to eliminate or reduce severity of visceral pain in an individual, i.e. a therapeutically effective amount. In some embodiments, the therapeutically effective amount of GUCY2C agonists does not induce diarrhea or only minimally induces diarrhea.

In methods of suppressing appetite, a therapeutically effective amount of GUCY2C agonists refers to an amount of GUCY2C agonist sufficient to reduce appetite and food intake in an individual, i.e. a therapeutically effective amount. In some embodiments, the therapeutically effective amount of GUCY2C agonists does not induce diarrhea or only minimally induces diarrhea. In some embodiments, a GUCY2C agonist and a PDE3 inhibitor are administered in a single composition that results in activation of GUCY2C and inhibition of PDE3 in neuropod cells contemporaneously. Tn some embodiments, a GUCY2C agonist and a PDE3 inhibitor are administered as separate composition administered in a regimen that results in activation of GUCY2C and inhibition of PDE3 in neuropod cells contemporaneously. In some embodiments, a GUCY2C agonist and a PDE3 inhibitor are provided in a single composition. In some embodiments, a GUCY2C agonist and a PDE3 inhibitor are provided in two separate compositions. In some embodiments, kits are provided that comprise a GUCY2C agonist and a PDE3 inhibitor in two separate compositions which are packaged together. The contemporaneous activation of GUCY2C and inhibition of PDE3 in neuropod cells results in enhanced effects in elimination or reduction of severity of visceral pain in an individual experiencing chronic visceral pain. The elimination or reduction of severity of visceral pain resulting from the administration of a GUCY2C agonist and a PDE3 inhibitor is enhanced relative to the elimination or reduction of severity of visceral pain resulting from the administration of a GUCY2C agonist without PDE3.

Diarrhea can be induced when GUCY2C is activated in intestinal cells which have GUCY2C receptors, particularly in intestinal cells of the lower intestine. The activation of GUCY2C results in secretion of water by the intestinal cells, thereby causing diarrhea. Neuropod cells are primarily found in the upper GI tract. While neuropod cells have PDE3, the cells of the lower intestine have PDE5. Thus, administration of PDE3 inhibitors in combination with GUCY2C agonists results in enhanced effects by GUCY2C activation in neuronal cells without enhancing the effects by GUCY2C activation in the cells of the lower intestine. Accordingly, in methods of treating chronic visceral pain, enhancement of the effectiveness of GUCY2C agonists by contemporaneous use of PDE3 inhibitors relative to the effectiveness of GUCY2C agonists when used in the absence of PDE3 inhibitors allows for the use of less GUCY2C agonist to achieve effective reduction of pain. The PDE3 inhibitors do not have same enhancement effect on cells of the lower intestine which have PDE5. As a result, less GUCY2C agonist can be used in the treatment of chronic visceral pain. Less GUCY2C agonist results of less induction of diarrhea.

Likewise, the contemporaneous activation of GUCY2C and inhibition of PDE3 in neuropod cells results in enhanced effects in appetite suppression. The appetite suppression resulting from the administration of a GUCY2C agonist and a PDE3 inhibitor is enhanced relative to the appetite suppression resulting from the administration of a GUCY2C agonist without PDE3. In methods of suppressing appetite, enhancement of the effectiveness of GUCY2C agonists by contemporaneous use of PDE3 inhibitors relative to the effectiveness of GUCY2C agonists when used in the absence of PDE3 inhibitors allows for the use of less GUCY2C agonist to achieve appetite suppression. The PDE3 inhibitors do not have same enhancement effect on cells of the lower intestine which have PDE5. As a result, less GUCY2C agonist can be used to suppress appetite. Less GUCY2C agonist results of less induction of diarrhea.

In some embodiments, a therapeutically effective amount of a GUCY2C agonist such as SEQ ID NO:2, 3 or 5-60, particularly linaclotide, plecanatide, ST IA, ST IB, guanylin or uroguanylin in combination with a PDE3 inhibitor such as cilostazol, enoximone, amrinone, milrinone, sulmazole, ampozone, cilostamide, carbazeran piroximone, imazodan, siguazodan, adibendan, saterinone, emoradan, revizinone, benafentrine, trequinsin, zardaverine and tolafentrine, particularly cilostazol, milrinone, enoximone and amrinone, are used in methods of treating visceral pain.

In some embodiments, a therapeutically effective amount of a GUCY2C agonist such as SEQ ID NO:2, 3 or 5-60, particularly linaclotide, plecanatide, ST IA, ST IB, guanylin, or uroguanylin in combination with a PDE3 inhibitor such as cilostazol, enoximone, amrinone, milrinone, sulmazole, ampozone, cilostamide, carbazeran piroximone, imazodan, siguazodan, adibendan, saterinone, emoradan, revizinone, benafentrine, trequinsin, zardaverine and tolafentrine, particularly cilostazol, milrinone, enoximone and amrinone, are used in methods to suppress appetite.

Table of Sequence