AU2603101A - Method of treating gastrointestinal tract disease with purinergic receptor agonists - Google Patents

Description

WO 01/45691 PCTIUSOO/35439 METHOD OF TREATING GASTROINTESTINAL TRACT DISEASE WITH PURINERGIC RECEPTOR AGONISTS 5 INTRODUCTION This application claims priority to U.S. Provisional Application Serial No. 60/171,710, filed December 22, 1999, which is hereby incorporated herein in its entirety 10 by reference. Technical Field This invention relates to a method of regulating mucus secretions and fluid transport in the gastrointestinal system of a patient by administering purinergic receptor agonists such as certain uridine, adenine, or cytidine 5'-di- and triphosphates, 15 dinucleoside polyphosphates and their analogs thereof. Background of the Invention There are many situations where it is therapeutically desirable to increase the amount of mucin secretion, bicarbonate secretions, and/or degree of hydration in gastrointestinal systems. The gastrointestinal system operates principally to extract 20 energy and metabolic building blocks from the nutrient materials presented to it. The digestive tract includes the buccal cavity (primary salivary glands), esophagus, stomach, small intestine, large intestine, rectum, and ancillary organs (pancreas, liver and gall bladder). When the mucosal barrier is impaired in the digestive tract, it results in diseases such as dry mouth, gastro-esophageal reflux disease, peptic ulcer, inflammatory bowel 25 disease, etc. Abnormal fluid and electrolytic transport in the lower gastrointestinal tract results in disorders such as constipation and diarrhea. Mucus is a viscous material that coats many epithelial surfaces and is secreted into fluids such as saliva. It is composed chiefly of mucins and inorganic salts suspended in water. Mucus adheres to many epithelial surfaces, where it serves as a diffusion barrier 30 against contact with noxious substances (e.g. gastric acid, digestive enzymes and bacteria) and as a lubricant to minimize shear stresses. Such mucous coatings are particularly WO 01/45691 PCTIUSOO/35439 prominent on the epithelia of the gastrointestinal, respiratory and genital tracts. Mucous is also an abundant and important component of saliva, giving it virtually unparalleled lubricating properties. Mucus-secreting cells such as goblet cells are abundant in the epithelium of the gastrointestinal tracts. Numerous submucosal mucous glands are 5 scattered along the esophagus and especially accumulated below the upper and above the lower esophageal sphincters. Many of the acinar epithelial cells in salivary glands secrete mucus. The major structural molecules of the mucus layer are mucins, which are a family of large, heavily glycosylated proteins. The dense "sugar coating" of mucins gives them considerable water-holding capacity and makes them resistant to proteolysis, which may 10 be important in maintaining mucosal barriers. Bicarbonate secretion plays an important role in the maintenance of mucosal health in the gastrointestinal tract. The production of bicarbonate and mucus by the esophagus in response to local acidification provides an inherent mechanism for resisting acid-induced damage. The secretion of salivary protective factors, including bicarbonate, 15 as well as bicarbonate secreted from esophageal submucosal glands, are important in preventing esophageal mucosal injury associated with gastrointestinal reflux disease. Mucosal bicarbonate also provides an important mechanism for protection against acid damage in the proximal duodenum, in which adherent mucus provides a stable protective layer supporting surface neutralization of acid by mucosal bicarbonate. [Nucleotides 20 stimulate bicarbonate secretion in guinea pig pancreatic duct (Ishiguro et al. 1999, J. Physiol. 519 Pt 2:551-558) and CFTR knockout mouse gall bladder epithelium (Clarke et al. 2000, Am. J. Physiol. Gastrointest. Liver Physiol. 279:G]32-138)]. Proper regulation of fluid and electrolytic absorption and secretion at appropriate regions along the gastrointestinal system is required for normal digestive function. 25 Impairment of fluid transport leads to a variety of disorders, including constipation and diarrhea. Constipation is associated with a delay in the transit of fecal matter through the large intestine. The increased resident time of feces in the large intestine leads to increased fluid absorption by -the colonic epithelium, and results in dehydration of feces and the subsequent production of dry, hard feces in the descending colon. Conversely, 2 WO 01/45691 PCT/USOO/35439 diarrhea results from rapid movement of fecal matter through the large intestine, resulting from either increased fluid secretion in the small intestine or by reduced fluid absorption in the colon. Xerostomia, commonly known as dry mouth, results from the underproduction of 5 saliva. Dry mouth is caused by radiation treatment or diseases that damage salivary glands and decrease salivary flow. Gastroesophageal reflux disease is the condition where the degree of exposure of esophageal mucosa to gastric contents is greater than normal. The most common manifestation is heartburn. Pharmacological treatment involves the use of H2 antagonists (e.g., Tagamet*, Zantac*, Pepcid*, Axid®) and proton 10 pump inhibitors such as Prilosec* or Prevacid*, for treatment of acute disease. Peptic ulcer diseases include gastric ulcer, pyloric channel ulcer and duodenal ulcer. Ulceration results from a complex interplay of acid and chronic inflammation induced by Helicobacterpylori infection. Patients with duodenal ulcers have high acid secretion. Increased acid secretion causes changes in the wall of the duodenum, setting the stage for 15 invasion by H pylori. Drugs for treating peptic ulcer diseases include Histamine-2 (H2) blockers (Tagamet*, Zantac*, Axid*, Pepcid*, etc.), sucralfate, proton pump inhibitors, and antacids. Inflammatory bowel disease is classified into two types: ulcerative colitis and Crohn's disease. Ulcerative colitis affects the colon/rectum and involves the mucosa or the innermost lining of the colon wall. Crohn's disease is a transmural disease 20 involving all layers of the bowel and may involve any part of the gut, from mouth to anus. Medical treatment of inflammatory bowel disease includes aminosalicylates and corticosteroids. Corticosteroids have substantial long-term toxicity. As an alternative to conventional therapies, medical researchers have sought to develop new treatments for gastrointestinal diseases. 25 The following references disclose the role of mucus integrity and mucin secretion in some diseases of the gastrointestinal tract. Rhodes et al. (Gut, 26:1312-1318 (1985)) suggested that colonic mucus undergoes continual desulphation and desialation in vivo as a result of faecal enzyme activity; altered susceptibility of colonic mucus may be important in the pathogenesis of colonic disease. Somasundaram et al. (Clin. Exp. 3 WO 01/45691 PCTIUSOO/35439 Pharmacol. Physiol., 14:309-318 (1987)) report that the integrity of the gastric mucosa and its ability to secrete mucus are essential for protection of gastric mucosa against ulceration induced by aggressive factors active in any stress situation. Desai et al. (J. Pharm. Pharmacol. 47:734-738 (1995)) showed that SKF 38393, a specific dopamine 5 D1 -receptor agonist, was effective in preventing gastric and duodenal ulceration in rats. Sarosiek et al. (Digestion, 56 Suppl. 1:15-23 (1995)) reported that the rate of secretion of esophageal mucin, EGF and PGE2, under the impact of HC1/pepsin in patients with reflux esophagitis, was significantly impaired. Saitoh et al. (Dig. Dis. Sci. 41:1768-1774 (1996)) showed that compared with healthy subjects, the total yields of mucin from 10 ulcerative colitis patients were low due to a deficiency of neutral mucin, whereas those from Crohn's disease patients were high due to high-molecular weight mucin. Sarosiek et al. (Gastroenterology, 110:675-681 (1996)) suggest that an increase in the secretion rate of inorganic and organic protective components in saliva may be useful to the treatment of gastroesophageal reflux disease. Zeeh (Gastroenterology, 110:1077-1083 (1996)) 15 reported that administration of keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats. Abbas et al. (Indian J. Exp. Biol. 36:182-186 (1998)) report that the antiulcerogenic effect of GABA and baclofen may be due to their predominant effects on mucosal defensive factors like enhanced mucin secretion and decreased cell shedding or mucosal damage. Nath et al. (Clin. Exp. Pharmacol. Physiol. 20 25:564-567 (1998)) report that polyriboinosinic-polyribocytidylic acid had a potent anti gastric ulcer effect on rats; polyriboinosinic-polyribocytidylic acid was shown to cause a decrease in free and total acid and pepsin and an increase in mucin content in Shay rat. Newton et al. (Gut, 43:470-475 (1998)) report that H. pylori in vivo causes structural changes in the adherent gastric mucus layer but the mucus barrier thickness is not 25 compromised. The following references disclose the compositions of purinergic receptor agonists and/or treatment of diseases. Uridine 5'-triphosphate has been shown to increase both the rate and total amount of mucin secretion by Goblet cells in vitro (Lethem et al., Am. J. Respir. Cell Mol. Biol., 9:315-322 (1993)). USPN 5,900,407 (Yerxa et al.) discloses a 4 WO 01/45691 PCT/USOO/35439 method for the stimulation of tear secretion in a subject in need of treatment. The method comprises administering to the ocular surfaces of the subject a purinergic receptor agonist such as uridine 5'-triphosphate, cytidine 5'-triphosphate, adenosine 5'-triphosphate, or their analogs and derivatives, in an amount effective to stimulate tear fluid secretion. 5 USPN 5,837,861 (Pendergast et al.) discloses P2Y 2 purinergic receptors of dinucleotide polyphosphates having structure of Formula I, wherein X is oxygen, methylene, or difluoromethylene; n=0 or 1; m=0 or 1; n+m=O, 1 or 2; and B and B' are each independently a purine residue or a pyrimidine residue linked through the 9- or 1-position. The compounds are useful in the treatment of chronic obstructive pulmonary diseases, 10 bronchitis, certain pneumonias, cystic fibrosis, sinusitis, and otitis media. USPN 5,763,447 (Jacobus et al.) discloses a method of promoting drainage of mucous secretions in the congested airway of an immobilized patient. The method comprises administering to the airway of the patient a uridine phosphate such as uridine 5' triphosphate (UTP), or P,'P 4 -di(uridine-5')tetraphosphate, in an amount effective to 15 promote drainage of fluid in the congested airway, including sinuses, by hydrating mucous secretions or by stimulating ciliary beat frequency in the airway. USPN 5,789,391, 5,981,506, 5,972,904 and 5,958,897 are directed to a method of promoting drainage of congested mucous secretions in the sinuses of a subject in need. The method comprises administering to the sinuses of the subject a uridine phosphate such as uridine 20 5'-triphosphate (UTP) or P', P 4 -di(uridine-5') tetraphosphate, an analog of UTP, or any other analog, in an amount effective to promote drainage of congested fluid in the sinuses by hydrating mucous secretions or by stimulating ciliary beat frequency in the sinuses. USPN 5,968,913 is directed to a pharmaceutical compositions of UTP for use in promoting increased mucociliary clearance of retained mucous secretions of the human 25 airways, middle/inner ears or sinuses. USPN 5,763,447 is directed to a method of preventing or treating pneumonia, including ventilator-associated pneumonia, in a bedridden or immobilized subject in need of such treatment. The method comprises administering to the airways of the patient a uridine phosphate such as uridine 5' triphosphate (UTP), Pl, P 4 -di(uridine-5')tetraphosphate, or their analogs, in an amount 5 WO 01/45691 PCT/USOO/35439 effective to promote drainage of fluid in the congested airways. WO 99/09998 discloses a method of using uridine 5'-diphosphate and analogs thereof to treat lung disease. The compounds described in the above references ('391, '506, '904, '897, '913 and '447 Patent and WO 99/09998), which have purinergic receptor activity, are incorporated 5 herein by reference. USPN 5,733,916 (Neely) discloses a method of preventing or treating ischemia-reperfusion injury or endotoxin-related lung injury by administration of a composition containing a selective A, adenosine receptor antagonist and/or a P 2 X purinoceptor antagonist. Somers et al. (Laboratory Investigation, 78:1375-1383 (1998)) report that P2Y 6 receptor was highly expressed in the T cells infiltrating active 10 inflammatory bowel disease, whereas P2Y 6 expression was absent from the T cells of unaffected bowel. Boyer et al., (Br. J. Pharmacol. 118:1959 (1996)) synthesized and tested a series of chain-extended 2-thioether derivatives of adenosine monophosphate (AMP) as agonsists for activation of the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat 15 glioma cells, and the cloned human P2U-receptor stably expressed in 132 1N1 human astrocytoma cells. Specific dinucleotide phosphate compounds known in other prior art are listed in Table I, along with their corresponding references. These compounds have not been used in the prior art to increase the mucus secretion or to correct for fluid and electrolytic 20 imbalance in the gastrointestinal tract, and Applicants intend to include them in this invention. 6 WO 01/45691 PCT/USOO/35439 TABLE I DINUCLEOTIDE PHOSPHATE COMPOUNDS IN THE LITERATURE 5

NP

2 N NP 2 N' Np 3 N Np 3 N' Np 4 N Np 4 N' Ap 2 A Ap 2 NAD Up 3 U Ap 3 T Up 4 U Ap 4 U Gp 2 G Ap 2 TAD Ap 3 A M 7 Gp 3 G Ap 4 A Ap 4 C m 7 Gp 2 m 7 G Ap 2 C-NAD Xp 3 X m' 7 GpG Cp 4 C Ap 4 G Up 2 U Ap 2 C-PAD m 7 Gp 3 m 7 G m 2 'Gp 3 G Gp 4 G Gp 4 U (5-BrU)p 2 (5-BrU) Ap2BAD Gp 3 G Ap 3 U Xp 4 X Gp 4 C (AZT)p 2 (AZT) m'Gp 2 G (5-BrU)p,(5-BrU) Ap 3 (5-BrU) Dp 4 D Up 4 C (5-FU)p 2 (5-FU) Ap 2 G Cp 3 C Up 3 (5-BrU) eAp 4 eA Ap 4 T 'P2I Ap 2 U Ip3I Gp 3 A m 7 Gp 4 m 7 G m 7 Gp 4 G Ap 2 (5-BrU) Ap-CH 2 -ppA Gp 3 C (5-BrU)p 4 (5-BrU) m 2

,

7 Gp 4 G Up 2 (5-BrU) Ap-CF 2 .ppA Gp 3 Gm dAp 4 dA m 2 2

P

4 G (AZT)p 2 (5-FU) Gp 3 Am 3'-dAp 4 3'-dA (5-BrU)p 4 A Ap 2 T m 7 Gp 3 m'Am dGp 4 dG (5-BrU)p 4 U Gp 2 A m7Gp 3 Gm ApCH 2 p 3 A Ap 4 (8-BrA) Ip 2 A Ap 3 C Ip4I Ap 4 X 2 Ap 3 G Ap 2

CH

2 p 2 A Ap 4 I m 7 Gp 3 A Ap 2

CF

2 p 2 A Ap 4 dA Ip 3 A Dp 2

CH

2 p 2 D Ap 4 d(5-FU) Ip 3 G Dp 2

CF

2 p 2 D Ap 4 araA 2'dGp 3 A Ap 2

CH

2 p 2 U 2'dGp 3 -2'dG Ap 2

CH

2 p 2 G rn 7 Gp 3 Am Ap 3 CHpT Gp 3 U ahaAp 4 A m GP 3 Cm ahaAp 4 G m 7 Gp 3 Um m 7 Gp 3 G App-CH 2 -pT Ap-CF 2 -ppA Np 5 N Np 5 N' Np 6 N Np 6 N' Ap 5 A Ap 5 l Ap 6 A ApJl Up 5 U Ap 5 U Up 6 U Ap 6 U (5-BrU)p 5 (5-BrU) Ap,(5-BrU) (5-BrU)p 6 (5-BrU) Up 6 (5-BrU) Gp 5 G Up 5 (5-BrU) Gp 6 G Ap 6 (5-BrU) 2'dGp,2'dG IPI 0 A = Adenosine eA = Ethenoadenosine U = Uridine M 7 G = 7-Methylguanosine G = Guanosine m 2

,

7 G = 2,7-Dimethylguanosine T = Thymidine = 2,2,7-Trimethylguanosine 5 X = Xanthosine NAD nicotinamide riboside TAD = Tiazofurin C-NAD C-nicotinamide riboside BAD = Benzamide riboside C-PAD C-picolinanide riboside D = 2,6-Diaminopurine N = Nucleoside Gm = 2'-O-methylguanosine Am 2'-O-methyladenosine :0 Urn = 2'-O-methyeidine m 6 Am = N6-methyl-2'-O-methyladenosine 7 WO 01/45691 PCTIUSOO/35439 Cm = 2'-O-methylcytidine aha = 8-(6-aminohexyl) X= Xanthosine AZT =Thymine-3'-azido2',3'-dideoxy-D-riboside 5-BrU = 5-bromouridine 5-FU = 5-fluorouridine 5 (1) M.A.G. Sillero et al., Eur. J. Biochem., 76, 331 (1977) (2) C.G. Vallejo et al., Biochim. Biophys. Acta, 483, 304 (1976) (3) H. Coste et al., J. Biol. Chem., 262, 12096 (1987) (4) K.E. Ng et al., Nucleic Acid Res., 15, 3573 (1987) 10 (5) J. Stepinski et al., Nucleosides & Nucleotides, 14, 717 (1995) (6) A. Zatorski et al., J. Med. Chem., 39, 2422 (1996) (7) P. Rotilan et al., FEBS, 280, 371 (1991) (8) P.C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370 (1992) (9) J. Walker et al., Biochemistry, 32, 14009 (1993) 15 (10) R.H. Hiderman et al., J. Biol. Chem., 266, 6915 (1991) (11) J. Luthje et al., Eur. J. Biochem., 173, 241 (1988) (12) R.H. Silverman et al., Microbiological Rev., 43, 27 (1979) (13) C.D. Lobaton et al., Eur. J. Biochem., 50, 495 (1975) (14) G. Lowe et al., Nucleosides & Nucleotides, 10, 181 (1991) 20 (15) G.M. Blackburn et al., Nucleosides & Nucleotides, 10, 549 (1991) (16) J.C. Baker et al., Mutation Res., 208, 87 (1988) (17) G. Klein et al., Biochemistry, 27, 1897 (1988) (18) E. Castro et al., Br. J. Pharmacol., 100, 360 (1990) (19) D.R. Elmaleh et al., Proc. Natl. Acad. Sci., 81, 918 (1984) 25 (20) R. Bone et al., J. Biol. Chem., 261, 16410 (1986) (21) Fed. Amer. Soc. Exper. Bio., Abstr. Part I, no. 1878 (1991) (22) M.T. Miras-Portugal et al., Ann. NY Acad. Sci., 603, 523 (1990) (23) A. Guranowski et al., Biochemistry, 27, 2959 (1988) (24) F. Grummt et al., Plant Mol. Bio., 2, 41 (1983) 30 (25) A.G. McLennan et al., Nucleic Acid Res., 12, 1609 (1984) (26) P. Zamecnik et al., Analytical Biochem., 134, 1 (1983) (27) E. Rapaport et al., Proc. Natl. Acad. Sci., 78, 838 (1981) (28) T. Kimura et al., Biol. Pharm. Bull., 18, 1556 (1995) (29) E. Schulze-Lohoff et al., Hypertension, 26, 899 (1995) 35 (30) B.K. Kim et al., Proc. Natl. Acad. Sci., 89, 11056 (1992) (31) P.C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370 (1992) (32) H. Morii et al., Eur. J. Biochem., 205, 979 (1992) (33) E. Castro et al., Pflugers Arch., 426, 524 (1994) (34) H. Schluter et al., Nature, 367, 186 (1994) 40 (35) E. Castro et al., Br. J. Pharmacol., 206, 833 (1992) (36) T. Casillas et al., Biochemistry, 32, 14203 (1993) (37) J. Pintor et al., J. Neurochem., 64, 670 (1995) (38) E. Castro et al., J. Biol. Chem., 270, 5098 (1995) (39) V.A. Panchenko et al., Neuroscience, 70, 353 (1996) 45 (40) E. Castro et al., Br. J. Pharmacol., 100, 360 (1990) (41) J. Pintor et al., Gen. Pharmac., 26, 229 (1995) (42) J. Pintor et al., Br. J. Phamacol., 115, 895 (1995) (43) A. Kanavarioti et al., Tett. Lett., 32, 6065 (1991) (44) Stutts, M. J., III, et al. WO 96/40059 50 (45) Theoclitou, et al. J. Chem. Soc. Perkin Trans I, 2009-2019 (1996) (46) Guranowski, A., et al. Nucleosides and Nucleotides, 14, 731-734 (1995) (47) De Flora, A., et al. WO 96/02554A1 (48) Visscher, J. et al. Nucleic Acids Research, 20, 5749-5752 (1992) (49) Holler, E.; Holmquist, B, et al. Biochemistry, 22, 4924-4933 (1983) 55 (50) Orr, R. M.; et al. Biochem. Pharmacol. 673-677 (1988) (51) Plateau, P., Fromant, et al. Biochemistry, 24, 914-922 (1985) (52) Hagmeier, E., et al. Journal of Chromatography, 237, 174-177 (1982) (53) Scheffzek, K, et al. Biochemistry, 35, 9716-9727 (1996) 8 WO 01/45691 PCTIUSOO/35439 (54) Stridh, S., et al. Antiviral Research, 97-105 (1981) (55) Tarusova, N. B., et al. Zh. Org. Khim., 24(7), 1474-1480 (Russian); through Chem. Abs. 110:154770 (1988) (56) Hata, T., et al. Chem. Lett., 987-990 (1976) 5 (57) Huhn, G. F., et al. Separation Science and Technology, 28, 1959-1970 (1993) (58) Tumanov, Yu. V., et al. Bioorg. Khim., 13, 921-927 (Russian); through Chem Abs., 109:6867d (1987) (59) Devash, Y. US Patent No. 4,855,304 (60) Pintor, J., et al. Molecular Pharmacology 51, 277-284 (1997) 10 (61) Stutts et al. US Patent No. 5,635,160. (62) Thiermermann, C., et al. WO 98/55494 (63) Zamechik, P.C., et al. US Patent No. 5,049,550 (64) Pankiewicz, K.W., et al. W098/15563 (65) Kim, B.K., et al. U.S. Patent No. 5,681,823 15 (66) Stutts, et al., U.S. Patent No. 5,935,555 P2Y purinergic receptors are purine and pyrimidine nucleotide receptors that couple to G proteins; they are 308 to 377 amino acid proteins with molecular weights of 41 to 53 kDa after glycosylation. P2Y receptors such as P2Y,, P2Y 2 and P2Y 6 receptors 20 are present in the gastrointestinal tract (Ralevic et al., Pharm. Rev. 50:415-492 (1998)). Because of the demonstrated ability of purinergic receptor agonists to stimulate mucus/mucin secretion in and around the eye (USPN 5,900,407), and in lung and sinuses (USPN 5,837,861), Applicants were motivated to investigate whether P2Y purinergic receptor ligands could affect mucus and/or mucin secretion, and to correct abnormal fluid 25 transport in the gastrointestinal tract, and thus be effective in treating diseases and disorders of the upper and lower gastrointestinal tract. Applicants have discovered that all P2Y receptors, including P2Y 4 and P2Y are present in gastrointestinal tissues. Applicants also discover that mucus and mucin secretion, bicarbonate secretion and fluid transport in these tissues can be regulated via 30 P2Y purinergic receptor-mediated mechanisms. P2Y purinergic receptor ligands, administered orally or systemically, provide a novel method of treating gastrointestinal disorders. 9 WO 01/45691 PCT/USOO/35439 SUMMARY OF THE INVENTION The invention provides a method of regulating mucus/mucin secretions, and fluid transport in the gastrointestinal tract. The invention provides a method for treating gastrointestinal disease in which the mucosal barrier of the gastrointestinal system is 5 impaired. The invention additionally provides a method for correcting disorders of fluid secretion or absorption in the gastrointestinal tract resulting in either diarrhea or constipation. The method comprises administering to a patient a pharmaceutical composition comprising a purinergic P2Y receptor ligand, in an amount effective to regulate mucus/mucin and bicarbonate secretions and fluid transport in the 10 gastrointestinal tract. Methods of administering include oral and systemic administration. The diseases treated include diseases and disorders of the buccal cavity, esophagus, stomach, small intestine, large intestine, rectum, and ancillary organs such as pancreas, liver and gall bladder. The pharmaceutical composition used in this invention comprises a P2Y 15 purinergic receptor agonist. P2Y agonists increase secretion of water, bicarbonate and mucin in the mucosal epithelia of the gastrointestinal tract. P2Y agonists include uridine 5'- di-and triphosphate (UDP, UTP) and their analogs (Formulae Ia and Ib), adenosine 5' monophosphate (AMP) and its analogs, adenosine 5'- di-and triphosphate (ADP, ATP) and their analogs (Formulae Ila and Ilb), cytidine 5'- di-and triphosphate (CDP, CTP) and 20 their analogs. (Formulae Ila and IlIb), and dinucleotide polyphosphate compounds (general Formula IV). BRIEF DESCRIPTION OF THE FIGURES The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office 25 upon request and payment of the necessary fee. Figure 1 shows the P2Y, receptor in situ hybridization results of stomach tissues (gastric epithelia) with (a) control sense probe and (b) antisense probe. 10 WO 01/45691 PCTIUSOO/35439 Figure 2 shows the P2Y 2 receptor in situ hybridization results of esophagus epithelia with (a) control sense probe and (b) antisense probe. Figure 3 shows the P2Y 2 receptor in situ hybridization results of the large intestine (colon) epithelia with (a) sense probe and (b) antisense probe. 5 Figure 4 shows the P2Y 2 receptor in situ hybridization results of the small intestine jejunumm) epithelia with (a) sense probe and (b) antisense probe. Figure 5 shows calcium mobilization induced by P2Y receptor agonists in human colonic epithial cells. Figure 6 shows calcium mobilization induced by P2Y receptor agonists in HT-29 10 human colonic epithelial cells. DETAILED DESCRIPTION OF THE INVENTION The invention provides a method of regulating mucous secretions, bicarbonate secretion, and fluid transport in the gastrointestinal tract. The invention also provides a method of treating gastrointestinal disease in which the mucosal barrier of the organ is 15 impaired, or in which an imbalance of fluid absorption rf secretion occurs in the small and large intestine. The method comprises administering to a mammal a pharmaceutical composition comprising a purinergic P2Y receptor ligand, in an amount effective to regulate mucus or mucin secretions, bicarbonate secretion, or fluid transport in the gastrointestinal tract. The method enhances the mucin release, pH and hydration, or 20 regulates the fluid transport in the gastrointestinal tract. Gastrointestinal diseases suitable for treatment by this invention include diseases or disorders affecting the buccal cavity (primary salivary), esophagus, stomach, small intestine, large intestine, rectum and ancillary organs such as pancreas, liver and gall bladder. For example, dry mouth, mouth ulcer, gum disease, esophageal reflux disease, 25 peptic ulcer, inflammatory bowel disease (ulcerative colitis and Crohn's disease), mycositis, diarrhea and constipation can be treated by the present method. In addition, gastrointestinal problems associated with cystic fibrosis diseases such as dry mucin and decreased absorption of nutrient by epithelial cells in the gastrointestinal tract can also be 11 WO 01/45691 PCTIUSOO/35439 treated by the present method. In addition, gastrointestinal problems caused by cancer and chemotherapy can also be treated by this method. Mucin has been shown to be important in protecting mucosal surfaces from environmental exposure; it acts as an acid barrier and has been found to bind to 5 pathogens. Mucin is thus a part of the natural mucosal defense system in the body, and stimulation of its secretion may lead to protection of the mucosal surface epithelium. The method of the present invention is to increase the mucous secretions in gastrointestinal tracts, such as stomach and esophagus, thus strengthening the natural defense system. The epithelial lining of the human esophagus consists of squamous epithelium and 10 submucosal glands that serve as a natural barrier between the lumen and blood and act as protective lining against the physical perturbations of the invested food and against the acidic gastric juices from the stomach. The esophageal submucosal glands contain mucous, serous, and myoepithelial cell types. Submucosal glands in the airways and conjunctiva contain P2Y 2 receptors in mucosal epithelia of the esophagus. Activation of 15 P2Y 2 receptors by natural and synthetic agonists increases mucus secretion into and hydration of the mucosal layer of the esophagus. A variety of pathophysiological conditions lead to the erosion of the protective mucosal barrier of the esophagus, resulting in gastroesophageal reflux disease (GERD). The symptoms of GERD include mild to severe heartburn, esophageal inflammation 20 (esophagitis), regurgitation, dysfunctional swallowing, and chest pain. GERD is caused by a variety of factors, including abnormal function of the lower esophageal sphincter (which allows reflux of gastric juices into the lower esophagus), delayed stomach emptying, reduced rates of esophageal clearance, and diminished salivation. When the esophagus is exposed to high acid content during gastric reflux, it results in a breakdown 25 of the protective mucus layer. The present invention discloses that in an animal model of esophagitis, P2Y 2 receptor agonists can restore the integrity of the disrupted esophageal mucosal layer by naturally stimulating mucin, bicarbonate, and fluid production by squamous epithelia and/or submucosal glands. 12 WO 01/45691 PCT/USOO/35439 Gastric ulcers and gastric reflux are conditions characterized in part by a breakdown in the mucosal defense barrier of the upper gastrointestinal epithelium. Excessive acid secretion in the stomach can lead to a breakdown in the natural mucus layer that protects the epithelial cells from acid damage. Gastric ulcer is associated with, 5 but not limited to stress, diet, H. pylori infection, chemotherapy or radiotherapy, other autoimmune diseases such as Sjogren's syndrome, etc., surgery, psychosomatic disorders, stress, anxiety, and pharmacological drug-related side effects. The crypts of Lieberktihn along the small intestine play a principal role in mediating fluid secretion into the lumen of the small intestine. Chloride efflux across 10 apical membrane chloride channels at the apical membrane of epithelial cells along these crypts provide the driving force for osmotically obliged fluid secretion in the small intestine. Constipation and diarrhea result from abnormal fluid transport (absorption verses secretion) across the small and large intestines. The present invention discloses a method to correct for imbalance of fluid transport leading to either constipation or 15 diarrhea by targeting activation of P2Y receptors along the small and large intestines. In pathological conditions, such as exposure to cholera toxin, the apical chloride channels are constitutively active, thus leading to uncontrolled fluid secretion into the small intestine, extreme diarrhea, and mortal bodily dehydration. This observation has provided a scientific rationale for treating constipation and diarrhea. 20 The activation of chloride and fluid secretion across the small intestines is known to provide additional fluid to chyme before it becomes fecal matter in the large intestine. This addition of fluid to the chyme will thus offset the hyper-absorption of fluid in the large intestine leading to constipation. The present invention discloses that activation of P2Y receptors by agonists provide a mechanism for providing such increase in chloride 25 and fluid secretion into the small intestine and can be used therapeutically to treat constipation. The colonic epithelia of the large intestine normally absorb fluid and function to remove excessive fluid from the entering chyme from the small intestine and convert it into feces. Diarrhea results from excessive fluid in the entering chyme, or malabsorption 13 WO 01/45691 PCTIUSOO/35439 of fluid across the colon. Fluid absorption along the colonic epithelia is mediated by sodium absorption via apical membrane sodium channels and basolateral membrane sodium potassium transporters. The fluid absorption properties of this epithelium can be modulated electrogenically by calcium-activated potassium channels at the basolateral 5 membrane. Increase in basolateral membrane potassium conductance hyperpolarizes the apical and basolateral membranes and thus increases the electrogenic driving force for sodium influx across the apical membrane. This results in a concomitant increase in sodium and potassium absorption by the epithelium, and increases ion-coupled fluid absorption. The present invention discloses that activation of P2Y purinoceptors by 10 agonists increases basolateral membrane potassium conductance and facilitates fluid removal from feces. Therefore direct administration of P2Y receptor agonists can be used therapeutically to treat diarrhea. The present method comprises administering to a patient a pharmaceutical composition that regulates mucus/mucin secretion, hydration and fluid transport in the 15 gastrointestinal tract. The present method has advantages over other commonly used treatments. The method regulates a patient's own production and secretion of mucus as well as the levels of mucosal hydration. Thus the method maintains the natural protective and lubricant characteristics of mucosa of gastro-intestinal system and directly addresses the problem resulting from mucus impairment. The present invention is concerned 20 primarily with the treatment of human subjects, but may also be employed for the treatment of other mammalian subjects, such as dogs and cats, for veterinary purposes. Applicants have discovered that (a) many purinergic P2Y receptors (P2Y,, P2Y 2 , P2Y 4 , P2Y 6 , and P2Y, ) are present in gastrointestinal tissues such as salivary glands, esophagus, stomach, small intestine, colon, duodenum, and rectum; (b) a potent 25 purinergic receptor agonist increases the secretion of mucin, and regulates fluid transport in the mucosal epithelia of the gastrointestinal tract. P2Y agonists include nucleotide mono-, di-, and triphosphates and dinucleotide polyphosphates. Nucleotide monophosphates useful in this invention include adenosine 5'-monophosphate (AMP) and its derivatives such as 2-thioether-substituted AMP, e.g., 14 WO 01/45691 PCT/USOO/35439 2-hexylthio AMP (Br. J Pharmacol. 118:1959 (1996)). Nucleotide di-and triphosphates useful in this application include uridine 5'-di- and triphosphate (UDP and UTP) and their analogs of general formulae Ia and Ib; adenosine 5'-di- and triphosphate (ADP and ATP) and their analogs of general formulae Ila and Ilb; and cytosine 5'-di- and triphosphate 5 (CDP and CTP) and their analogs of general formulae Ila and IlIb. UDP and its analogs are depicted by general formula Ia: Formula Ia 10 R2 R3N-' 0 N 0 0 15

HO---R

1 --- O-CH 2 O H wherein: 20 X,, and X, are each independently either 0- or S-; Y is H or OH; R, is selected from the group consisting of 0, imido, methylene, and dihalomethylene (e.g., dichloromethylene, difluoromethylene);

R

2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, 25 alkoxyl, nitro and azido;

R

3 is selected from the group consisting of nothing, H, alkyl, acyl (including arylacyl), and arylalkyl; and

R

4 is selected from the group consisting of -OR', -SR', NR', and NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted 30 alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R 4 is double bonded from an oxygen or sulfur atom to the carbon at the 4 position of the pyrimidine ring. 15 WO 01/45691 PCT/USOO/35439 As used herein, the term "alkyl" refers to Cr- 0 inclusive, linear, branched, or cyclic, saturated or unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, 5 butynyl, pentynyl, hexynyl, heptynyl, allenyl and optionally substituted arylalkenyl and arylalkyny groups. As used herein, the term "acyl" refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO-, wherein R is an alkyl or an aryl group). As such, the term "acyl" specifically includes arylacyl groups. Specific examples of acyl groups include acetyl and 10 benzoyl. As used herein, the term "aryl" refers to 5 and 6-membered hydrocarbon and heterocyclic aromatic rings. Specific examples of aryl groups include but are not limited to cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, isothiazole, isoxazole, pyrazole, pyrazine, pyrimidine, and the like. The term "alkoxyl" as used herein refers to C I, inclusive, linear, branched, or cyclic, saturated or unsaturated 15 oxo-hydrocarbon chains, including for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, and pentoxy. The term "aryloxyl" as used herein refers to aryloxy such as phenyloxyl, and alkyl, halo, or alkoxyl substituted aryloxyl. As used herein, the terms "substituted alkyl" and "substituted aryl" include alkyl and aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl or alkyl group are replaced 20 with another atom or functional group, including for example, halogen, aryl, alkyl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. The terms "halo," "halide," or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups. Compounds illustrative of the compounds of Formula (la) include those disclosed 25 in WO 99/09998; the reference is incorporated herein by reference. Formula Ia compounds, for example, include: uridine 5'-diphosphate (UDP); uridine 5'-0-(2 thiodiphosphate)(UDPBS); 5-bromouridine 5'-diphosphate (5-BrUDP); 5-(1 phenylethynyl)-uridine 5'-diphosphate (5-(1-phenylethynyl)UDP); 5-methyluridine 5' diphosphate (5-methylUDP); 4-hexylthiouridine 5'-diphosphate (4-hexylthioUDP); 4 16 WO 01/45691 PCT/USOO/35439 mercaptouridine 5'-diphosphate (4-mercaptoUDP); 4-methoxyuridine 5'-diphosphate ( 4 methoxyUDP); 4-(N-morpholino)uridine 5'-diphosphate (4-(N-morpholino)UDP; 4 hexyloxyuridine 5'-diphosphate ( 4-hexyloxyUDP); N,N-dimethylcytidine 5' diphosphate (N,N-dimethylCDP); N-hexylcytidine 5'-diphosphate (N-hexylCDP); and 5 N-cyclopentylcytidine 5'-diphosphate (N-cyclopentylCDP). Preferred compounds of Formula Ia include UDP and UDPpS and 4-thio UDP. Certain compounds of Formula Ia (e.g., UDP, dUDP, UDPpS, and 4-mercaptoUDP) are known and may be made in accordance with known procedures or variations thereof, which will be apparent to those skilled in the art. For example, the identification and 10 preparation of certain thiophosphate analogues of nucleoside diphosphates (such as UTP p-S) are set forth in U.S. Patent No. 3,846,402 (Eckstein et al.), and in R.S. Goody and F. Eckstein, J. Am. Chem. Soc. 93: 6252-6257 (1971). Alternatively, UDP, and other analogs thereof are also commercially available from vendors such as Sigma (St. Louis, MO) and Pharmacia (Uppsala, Sweden). 15 UTP and its analogs are depicted by general formula Ib; Formula lb R4

R

3 N R2 20 0 0 0 0 N HO-P-R1-P-O-P-0 0 k1 k 2 k 3 ' H Y wherein: 25 X 1 , X2 and X 3 are each independently either 0- or S-, Y is H or OH;

R

1 , R 2 , R 3 and R 4 are defined as in Formula Ia. Preferably, X2 and X 3 are 0-, R, is oxygen or imido, and R2 is H. Particularly preferred compounds of Formula lb include uridine 5'-triphosphate (UTP) 30 and uridine 5'-O-(3-thiotriphosphate) (UTPyS). 17 WO 01/45691 PCT/US00/35439 ADP and its analogs are depicted by general Formula Ila: Formula Ila

NR

3

R

4 5 N 'NR2 o O N N NZ HO-P-R1-P -O k1 k 2 H 10 H Y wherein:

R

1 , X 1 , X 2 and Y are defined as in Formula Ia; Z is H, Cl, or SR, wherein R is alkyl (CI-C 2 0 , saturated or unsaturated);

R

3 and R 4 are H while R2 is nothing and there is a double bond between N-1 15 and C-6 (adenine), or

R

3 and R 4 are H while R 2 is nothing and Z is SR, or

R

3 and R 4 are H while R, is 0 and there is a double bond between N-1 and C-6 (adenine 1-oxide), or

R

3 , R 4 , and R 2 taken together are -CH=CH-, forming a ring from N-6 to N-1 20 with a double bond between N-6 and C-6 (1,N6-ethenoadenine). Particularly preferred compounds of Formula Ila include 5'-adenosine diphosphate (ADP) and 2-methyl-SADP. 18 WO 01/45691 PCT/USOO/35439 ATP and its analogs are depicted by general Formula Ilb: Formula IIb

NR

3

R

4 5I N NR2 0 0 0 HO- -R1- -O- -O O k 1 k2 3 H H 10 H Y wherein:

R

1 , X 1 , X 2 , X 3 and Y are defined as in Formula Ib, and

R

2 , R 3 , R 4 and Z are defined as in Formula Ila. Preferred compounds of Formula Ilb include 5'-adenosine triphosphate (ATP). 15 CDP and its analogs are depicted by general Formula Ila: Formula IIla R5N/ R 6 RyN 20 O.: N 0 0 HO-P-R1--0 0 HH wherein: H Y 25 R 1 , X 1 , X 2 and Y are defined as in Formula Ia;

R

5 and R 6 are H while R 7 is nothing and there is a double bond between N-3 and C-4 (cytosine), or

R

5 , R 6 and R 7 taken together are -CH=CH-, forming a ring from N-3 to N-4 with a double bond between N-4 and C-4 (3,N 4 -ethenocytosine), optionally the hydrogen 19 WO 01/45691 PCTIUSOO/35439 of the 4- or 5-position of the etheno ring is substituted with alkyl, substituted alkyl, aryl, substituted aryl (heteroaryl, etc.), alkoxyl, nitro, halo, or azido. CTP and its analogs are depicted by general Formula IIb: 5 Formula IlIb

R

5 " NR 6 RyN 10

HO-P-R

1 -P-0--0 0

X

1 2 k 3 H Y wherein: Rj,X,, X 2 , X 3 and Y are defined as in Formula Ib, and 15 R 5 , R 6 and R 7 are defined as in Formula Ila. Preferred compounds of Formula IlIb include cytidine 5'-triphosphate (CTP) and 4-nitrophenyl ethenocytidine 5'-triphosphate. For simplicity, Formulas I, II, and III, herein illustrate the active compounds in the naturally occurring D-configuration, but the present invention also encompasses 20 compounds in the L-configuration, and mixtures of compounds in the D- and L-configurations, unless otherwise specified. The naturally occurring D-configuration is preferred. 20 WO 01/45691 PCT/USOO/35439 P2Y agonists also include dinucleotide phosphates of general Formula (IV): Formula IV 5 O 0 0 0 1 1 1 l 1 1 | 1 B O0 P-O -P-X-P O-P- B' OH O 0 OH Y Z n m Z' Y' wherein: 10 X is oxygen, methylene, difluoromethylene, or imido; n=0, 1 or 2; m=0, 1 or 2; n + m=0,1, 2, 3, or 4; Z =OH or H; 15 Z'= OH or H; Y=H or OH; Y' = H or OH. The sugar moieties are as depicted in the D-configuration, but may be L-, or D and L-. The D-configuration is preferred. The nucleoside residue can be in the alpha- or 20 beta- and D- or L-configurations, but most preferably the beta-D-configuration. B and B' are each independently a purine residue or a pyrimidine residue, as defined in Formula V and VI, respectively, linked through the 9- or I-position, respectively. 21 WO 01/45691 PCT/USOO/35439 Formula V R2 7 ''Rx N 6''' N' 51 9 4 2 N H N R 3 wherein: R, is hydrogen, chlorine, amino, monosubstituted amino, disubstituted amino, 5 alkylthio, arylthio, or aralkylthio, wherein the substituent on sulfur contains up to a maximum of 20 carbon atoms, with or without unsaturation;

R

2 is hydroxy, amino, mercapto, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, acyloxy, monosubstituted alkylamino, heterocyclic, monosubstituted cycloalkylamino, monosubstituted aralkylamino, monosubstituted 10 arylamino, diaralkylamino, diarylamino, dialkylamino (wherein alkyl groups are optionally linked to N 7 to form a substitute ring), acylamino, diacylamino, or NHRy; Rx is 0 (adenine 1-oxide derivatives), or is absent (adenine derivatives); provided that when R 2 is NHRy, Ry and Rx may be taken together form a 5 membered fused imidazole ring (1, N-ethenoadenine derivatives), optionally substituted 15 on the 4- or 5-positions of the etheno moiety with alkyl, aryl or aralkyl moieties as defined below;

R

3 is hydrogen, azido, alkoxy, aryloxy, aralkyloxy, alkylthio, arylthio, or aralkylthio as defined below; or co-A(C 16 alkyl)OCONH (C,_ 6 alkyl)B- wherein A and B are independently amino, mercapto, hydroxy or carboxyl; or pharmaceutically acceptable 20 esters, amides or salts thereof; or absent. Thus, the substituted derivatives of adenine include adenine 1-oxide; 1,N 6 -(4- or 5-substituted etheno) adenine; 6-substituted adenine; or 8-substituted aminoadenine, where R' of the 6- or 8-HNR' groups are chosen from among: arylalkyl (C 1

.

6 ) groups 22 WO 01/45691 PCTiUSOO/35439 with the aryl moiety optionally functionalized as described below; alkyl; and alkyl groups with functional groups therein, such as: ([6-aminohexyl]carbamoylmethyl)-, and o acylated- amino(hydroxy, thiol and carboxy)alkyl(C- 1 )- and their w-acylated-amino (hydroxy, thiol and carboxy) derivatives where the acyl group is chosen from among, but 5 not limited to, acetyl, trifluoroacetyl, benzoyl, substituted-benzoyl, etc., or the carboxylic moiety is present as its ester or amide derivative, for example, the ethyl or methyl ester or its methyl, ethyl or benzamido derivative. The o-amino(hydroxy, thiol) moiety may be alkylated with a C 14 alkyl group. J is carbon or nitrogen, with the provision that when J is nitrogen, R3 is not 10 present; wherein the alkyls are straight-chain, branched or cyclic; wherein the aryl groups are optionally substituted with lower alkyl, amino, alkylamino, NO 2

N

3 , carboxylic, amido, sulfonamido, or halo groups; and B and B', can also be a pyrimidine with the general formula of Formula VI, linked 15 through the 1- position to ribosyl residue: Formula VI R6 R7 N R5 ,r 4 N R 6 2'

R

8 N R4 1 wherein: R4 is hydrogen, hydroxy, oxo, mercapto, amino, cyano, C 7 12 arylalkoxy, C, 6 20 alkylthio, C,_ 6 alkoxy, C 1

.

6 alkylamino, or diC, 4 alkylamino, wherein the alkyl groups are optionally linked to form a heterocycle; R5 is hydrogen, oxo, acetyl, benzoyl, C,- 6 alkyl, CM, alkanoyl, or aroyl; R6 is hydroxy, oxo, mercapto, C 14 alkoxy, C 7

.

12 arylalkoxy, C,.

6 alkylthio, amino, S phenyl, C,, disubstituted amino, triazolyl, C,_ 6 alkylamino, or di-C 14 alkylamino, wherein 23 WO 01/45691 PCTUSOO/35439 said dialkyl groups are optionally linked to form a heterocycle or linked to N' to form a substituted ring; or R, and R 6 taken together form a 5-membered fused imidazole ring between positions 3 and 4 of the pyrimidine ring and form a 3,N 4 -ethenocytosine derivative, 5 wherein said etheno moiety is optionally substituted on the 4- or 5-positions with C,, alkyl, phenyl or phenyloxy; wherein at least one hydrogen of said C 14 alkyl, phenyl or phenyloxy is optionally substituted with a moiety selected from the group consisting of: halogen, hydroxy, C- 4 alkoxy, C 14 alkyl, C,,, aryl, C,- 1 2 arylalkyl, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic acid, amino, C, alkylamino, and di- C 1 10 alkylamino, wherein said dialkyl groups are optionally linked to form a heterocycle;

R

7 is selected from the group consisting of: hydrogen, hydroxy, cyano, nitro, and

C

2 -s alkenyl; wherein said alkenyl moiety is optionally linked through an oxygen to form a ring, wherein at least one hydrogen of said alkenyl moiety on the carbon adjacent to said oxygen is optionally substituted with a substituent selected from the group consisting of: 15 C 1

_

6 alkyl or phenyl; substituted C 2 -, alkynyl, halogen, substituted C- 4 alkyl, CF 3 , C1- 3 alkenyl, C 2

-

3 alkynyl, allylamino, bromovinyl, ethyl propenoate, or propenoic acid; or

R

6 and R 7 together form a 5 or 6-membered saturated or unsaturated ring bonded through N or 0 or S at R, such ring optionally contains substituents that themselves contain functionalities; provided that when R 8 is amino or substituted amino, R 7 is 20 hydrogen; and

R

8 is selected from the group consisting of: hydrogen, amino or di-C. 4 alkylamino, C,.

4 alkoxy, C 7

-,

2 arylalkoxy, C 1 4 alkylthio, C 7

_

12 arylalkylthio, carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, and phenylthio. In the general structure of Formulae I-III above, the dotted lines in the 2- to 6 25 positions are intended to indicate the presence of single or double bonds in these positions; the relative positions of the double or single bonds being determined by whether the R 4 , R 5 and R 6 substituents are capable of keto-enol tautomerism. In the general structures of Formula 1-III above, the acyl groups comprise alkanoyl or aroyl groups. The alkyl groups contain 1 to 8 carbon atoms, particularly I to 4 carbon 24 WO 01/45691 PCTIUSOO/35439 atoms optionally substituted by one or more appropriate substituents, as described below. The aryl groups including the aryl moieties of such groups as aryloxy are preferably phenyl groups optionally substituted by one or more appropriate substituents, as described below. The above-mentioned alkenyl and alkynyl groups contain 2 to 8 carbon atoms, 5 particularly 2 to 6 carbon atoms, e.g., ethenyl or ethynyl, optionally substituted by one or more appropriate substituents as described below. Appropriate substituents on the above-mentioned alkyl, alkenyl, alkynyl, and aryl groups are selected from halogen, hydroxy, C 14 alkoxy, C- 4 alkyl, C 6

-

12 aryl, C 6

.

1 2 arylalkoxy, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic, amino and 10 substituted amino wherein the amino is singly or doubly substituted by a C, alkyl, and when doubly substituted, the alkyl groups optionally being linked to form a heterocycle. The invention further provides novel pharmaceutical compositions comprising compounds of general Formula IV, which newly feature: (1) a novel dinucleotide with a sugar moiety selected from the group consisting of: arabinofuranosyl, 3' 15 deoxyribofuranosyl, xylofuranosyl, and lyxofuranosyl; (2) a novel dinucleotide with an azapurine base; and (3) a novel dinucleotide with a 6-substituted purine. In the first type of novel composition, when the sugar moiety is 3'-deoxyribofuranosyl, Z and Z' are H. In the second type of novel composition with an azapurine base, J is nitrogen and R 3 is absent. In the third type of novel composition with the 6-substituted purine, the 6 20 monosubstituted amino purine base is excluded. Preferred dinucleotide polyphosphate compounds useful in this invention are P1,

P

4 -di (urdine-5')-tetraphosphate, dUP 4 U, U 2

P

3 , U 2

P

5 , dCP 4 U, CP 4 U, IP 5 I, AP 4 A, CPU,

UP

3 A and A 2

P

3 . Some compounds of Formula I, II and III can be made by methods known those 25 skilled in the art; some compounds are commercially available, for example, from Sigma Chemical Co. (St. Louis, MO 63178). Compounds of Formulae Ia (UDP and its analogs) can be prepared according to WO 99/09998. Compounds of Formulae lb, Ilb and IlIb (UTP, ATP, CTP and their analogs) can be prepared according to USPN 5,763,447. Compounds of Formula IV can be made in accordance with known procedures described 25 WO 01/45691 PCT/USOO/35439 by Zamecnik, et al., Proc. Natl. Acad Sci. USA 89, 838-42 (1981); and Ng and Orgel, Nucleic Acids Res. 15:3572-80 (1987), Pendergast et al., USPN 5,837,861, or variations thereof. The compounds of the present invention also encompass their non-toxic 5 pharmaceutically acceptable salts, such as, but not limited to, an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as manganese, magnesium or calcium; or an ammonium or tetraalkyl ammonium salt, i.e., NX' (wherein X is C 1 4). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. The present 10 invention also encompasses the acylated prodrugs of the compounds disclosed herein. Those skilled in the art will recognize various synthetic methodologies which may be employed to prepare non-toxic pharmaceutically acceptable salts and acylated prodrugs of the compounds. The pharmaceutical utility of P2Y agonist compounds of this invention are 15 indicated by the inositol phosphate assay for P2Y activity. This widely used assay, as described in E. Lazarowski, et al., Brit. J. Pharm. 116, 1619-27 (1995), relies on the measurement of inositol phosphate formation as a measurement of activity of compounds activating receptors linked via G-proteins to phospholipase C. In addition, the pharmaceutical utility of P2Y agonist compounds of this invention 20 are indicated by the intracellular calcium mobilization assay for P2Y activity. In this assay, cultured cells are stimulated with the increasing concentrations of P2Y receptor agonists. Intracellular calcium levels are monitored by measuring the changes in fluorescence intensity of a calcium-sensitive dye using the FLIPR (Molecular Devices Corp., Sunnyvale, CA) or equivalent instrumentation. 25 P2Y agonist compounds increase mucus production in in vitro preparations of esophageal, gastric mucosal, jujenum, proximal and distal colon epithelia. Mucus secretion can be assayed by a variety of techniques, including impression cytology, enzyme-linked immunosorbent assay (ELISA), and dot blots using mucin-specific antibodies. (See Danjo et al., Invest. Ophthalmol. Vis. Sci., 39: 2602-2609 (1988); 26 WO 01/45691 PCT/USOO/35439 Jumblatt et. al., Invest. Ophthalmol. Vis. Sci. 40:43-49 (1999); and Jumblatt et. al., Invest. Ophthalmol. Vis. Sci. 39: 5803 (1988)). Our results show robust, prolonged, and significant increases in mucus production when P2Y receptor agonists following administration to the luminal surface of epithelial preparations. Mucin production can be 5 repeatedly increased by repetitive stimulation with agonists. P2Y agonists significantly alter short circuit currents (Isc) in epithelial preparations from the gastrointestinal system, including esophagous, jujenum and proximal and distal colon. The changes in Isc are consistent with increases in transluminal chloride flux or transerosal potassium flux, and are thus expected to mobilize 10 fluid absorption or secretion across the epithelia accordingly. The effectiveness of P2Y agonists for amelioration of symptoms associated with gastrointestinal disease can be shown in an animal model. For example, Helicobacter pylori infection by acetic acid administration to the antral mucosa of cynomolgus monkeys is a model for chronic gastritis; the model shows histological and clinical 15 phenotype similar to those of human gastric ulcers. Oral administration of P2Y receptor agonists to monkeys with gastritis shows significant recovery of staining of periodic acid Schiff-positive substances and increases anti-mucin immunoreactivity, both of which reflect an increase in mucin secretion. Reduced histological incidents of gastric ulcerations are also an indication of the effectiveness of the P2Y receptor agonists. 20 The desired compounds of the present invention may be administered orally, systemtically, intra-operatively, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term systemic as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. 25 The pharmaceutical formulation in this invention comprises a ligand compound and a pharmaceutically acceptable carrier. One or more ligand compounds may be present in association with one or more non-toxic pharmaceutically acceptable carriers or diluents or adjuvants and, if desired, other active ingredients. One such carrier would be sugars, where the compounds may be intimately incorporated in the matrix through 27 WO 01/45691 PCT/USOO/35439 glassification or simply admixed with the carrier (e.g., lactose, sucrose, trehalose, mannitol) or other acceptable excipients for oral or systemic delivery. For oral use, the pharmaceutical composition is in a suitable form such as tablets, lozenges, aqueous or oily suspensions, viscous gels, chewable gums, dispersible powders 5 or granules, emulsion, hard or soft capsules, syrups or elixirs. Compositions intended for oral use are prepared according to any method known to the art for the manufacture of pharmaceutical compositions. Such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable 10 preparations. Tablets usually contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for 15 example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed. For oral use, hard gelatin capsules are prepared by mixing the active ingredient 20 with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. Soft gelatin capsules are prepared by mixing the active ingredient with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil. For oral use, chewable gums are prepared by embedding the active ingredient in gums; the active ingredient is slowly released upon chewing. This form is suitable for 25 treating mouth ulcers. For oral use, an aqueous suspension is prepared by addition of water to dispersible powders and granules with a dispersing or wetting agent, suspending agent and one or more preservatives. Suspending agents include, for example, sodium carboxymethylcellulose, methylcellulose and sodium alginate. Dispersing or wetting 28 WO 01/45691 PCT/USOO/35439 agents include naturally-occurring phosphatides, condensation products of an allylene oxide with fatty acids, condensation products of ethylene oxide with long chain aliphatic alcohols, condensation products of ethylene oxide with partial esters from fatty acids and a hexitol, and condensation products of ethylene oxide with partial esters derived from 5 fatty acids and hexitol anydrides. Preservatives include, for example, ethyl, and n-propyl p-hydroxybenzoate. An aqueous suspension may also contain one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. Those skilled in the art will recognize the many specific excipients and wetting agents encompassed by the general description above. 10 For systemic administration, the pharmaceutical formulation is prepared in a sterile medium. The active ingredient, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Adjuvants such as local anaesthetics, preservatives and buffering agents can also be dissolved in the vehicle. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic 15 acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are sterile water, saline solution, or Ringer's solution. The pharmaceutical application may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary 20 temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the compound. Such excipients include cocoa butter and polyethylene glycols. Dosage levels of the order of from about 10-2000 mg of active ingredients are useful in the treatment of the above-indicated conditions. Preferred doses are about 50 1000 mg, and more preferred doses are about 75-850 mg of active ingredients. These 25 doses can be given several times a day as needed. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the 29 WO 01/45691 PCTUSOO/35439 age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The invention is illustrated further by the following examples which are not to be 5 construed as limiting the invention in scope or spirit to the specific procedures described in them. EXAMPLES 10 Example 1. Identification of P2Y Receptor in Human Tissues The presence of P2YI, P2Y 2 , P2Y 4 , P2Y 6 and P2YI 1 purinergic receptors in gastrointestinal tissues was determined in vitro using RT-PCR techniques of human RNA purchased from a commercial sources. Human normal stomach poly A* mRNA was purchased from Clontech (Palo Alto, CA). First strand cDNA was synthesized 15 (Advantage RT-for PCR kit; Clontech, Palo Alto, CA) from 1 OOng of stomach polyA' mRNA using an oligo (dT)18 primer and MMLV reverse transcriptase (60 min, 42*C). Control reactions in the absence of reverse transcriptase were also carried out. Human normal esophagus, rectum, duodenum and salivary gland cDNA were purchased from Invitrogen (Carlsbad, CA). First strand cDNAs for normal human colon, liver, and small 20 intestine were from Clontech's multiple tissue cDNA panels. RT-PCR was performed with stomach tissues and PCR was performed with other tissues. Sequence specific primers for P2Y,, P2Y 2 , P2Y 4 , P2Y 6 and P2Y, 1 genes are listed as follows: P2Y (accession number U42029) 25 forward 5' CGATCTGTATCAGCGTGCTGGTGTG 3', reverse 5' TCTAGAAGCTTTCCTTGTGGCTCGG 3'; P2Y 2 (accession number S74902) forward 5' AGGAGATGTGTTGGGCAGCAGTGAGGAC 3'; reverse 5' ACCAGGGTTTTCTGGCCAACCTGTGACT 3'; 30 WO 01/45691 PCT/USOO/35439 P2Y 4 (accession number X91852) forward 5' ATGCAACGGCCACCTACATGTTCC 3'; reverse 5' GTACTCGGCAGTCAGCTTCCAACA 3'; P2Y 6 (accession number U52464) 5 forward 5' ATGGCATGGCTCTCACTGTCATCG 3'; reverse 5' TTGGTGAGCTTCTGGGTCCTGTGAG 3'; P2YII (accession number AF03 0335) forward 5' ATACTGGTGGTTGAGTTCCTGG 3'; reverse 5' ACCAGGCTATACGCTCTGTAGG 3'. 10 PCR was performed on 3pl of the cDNA of all tissues listed above using the forward and reverse primer sets (1 t 1 of each primer) designed to amplify each P2Y (P2Y,, P2Y 2 , P2Y4, P2Y 6 , P2Y,1) partial cDNA. The reaction also contained 400mM of each deoxy nucleotide triphosphate, 3.5mM MgCl 2 and 1 pl of the Advantage cDNA polymerase mix (Clontech, Palo Alto, CA). The reaction conditions were: initial 2.5 min 15 at 94*C and then 30 sec at 94'C, 30 sec at either 60*C (P2Y 4 , P2Y, 1 ) or 65'C (P2Y,, P2Y 2 , P2Y 6 ), 1 min at 72'C for 35 cycles and finally, 10 min at 72*C. Some of the PCR products were cloned into the pCR 2.1-TOPO vector (TOPO TA Cloning kit; Invitrogen, Carlsbad, CA) and sequenced completely using an automated DNA sequencer. All tissues tested were positive for P2Y receptors. The results are summarized in 20 Table 2. 31 WO 01/45691 PCT/USOO/35439 Table 2. Identification of P2Y receptor in human tissues. Human P2Y, Human P2Y 2 Human P2Y 4 Human P2Y 6 Human P2Y, Stomach + + + + + Salivary Gland + + + + + Esophagus + + + + + Duodenum + + + + + Small Intestine + + + + + Colon + + + + + Rectum + + + + + Liver + + + + + Pancreas + + + + + 5 Example 2. Cellular Localization of P2Y Nucleotide Receptor Gene Expression in Monkey Gastrointestical Epithelial Tissues by Nonisotopic In Situ Hybridization Tissues. Study tissues were obtained from Pathology Associates International, 10 Frederick, MD. Tissues included in this study were stomach, esophagus, small intestine jejunumm), and large intestine (colon). Tissues were removed from a 3.25 year old Indian Rhesus Macaque immediately following euthanasia and snap frozen in O.C.T. embedding medium. Frozen tissues were stored at -80'C prior to cryosectioning. Tissues were cut in 5 ptm sections and mounted on microscope slides for hematoxylin & eosin (H&E) 15 staining, and in situ hybridization (ISH). Assessment of Tissue Sections. H&E-stained tissue sections were prepared to evaluate the quality and orientation of study tissues. Examination of H&E slides indicated that all tissues were suitable for ISH. Riboprobe Synthesis. A PCR product containing nucleotides 253-651 from a 20 human P2Y 2 -R cDNA was obtained from a sponsor. P2Y,-R nucleotides 272-627 were 32 WO 01/45691 PCT/USOO/35439 reamplified with P2Y 2 primers (forward primer sequence: 5'AGGAGATGTGTTGGGCAGCAGTGAGGAC 3' reverse primer sequence: reverse 5'ACCAGGGTTTTCTGGCCAACCTGTGACT 3') designed to incorporate either an upstream T3 promotor or a downstream T7 promotor. The resulting PCR products were 5 used to synthesize digoxigenin-labeled riboprobes by in vitro transcription (IVT). Antisense and sense riboprobes were synthesized using T7 and T3 RNA polymerases, respectively, in the presence of digoxigenin- 11 -UTP (Roche Molecular) using a MEGAscript IVT kit (Ambion) according to the manufacturer. Following IVT, template DNA was degraded with DNase-1, and unincorporated digoxigenin was removed by 10 ultrafiltration. Riboprobe integrity was assessed by electrophoresis through a denaturing polyacrylamide gel. Apparent molecular size was estimated by comparison with the electrophoretic mobility of a 100-1000 base pair RNA ladder (Ambion). Probe yield and labeling was evaluated by blot immunochemistry. Riboprobes were dispensed in 5 PL aliquots and stored at -80*C until used for ISH. 15 In Situ hybridization. Frozen tissues were cut into 5 ptm sections, mounted on SuperFrost Plus slides (Fisher Scientific), and post-fixed for 15 minutes in 4% paraformaldehyde in PBS at pH 7.4. Tissue sections were then treated for 30 minutes with 0.1% active diethylpyrocarbonate in PBS at pH 7.4. Sections were prehybridized in the absence of probe, then incubated overnight in hybridization buffer containing 400 20 ng/mL of either antisense or sense probe. Following hybridization, slides were subjected to a series of post-hybridization stringency washes to reduce nonspecific staining. Hybridization was visualized by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin Fab and nitroblue tetrazolium chloride-bromochloroindolyl phosphate (Roche Molecular) according to the manufacturer. Tissue sections were 25 counter stained with nuclear fast red. Negative controls included stomach and esophagus tissues stained with the sense P2Y 2 -R probe. Results. The results from in situ hybridization experiments are shown for the sense probe (negative control) and antisense probe for the stomach (Figure 1), esophagus (Figure 2), colon (Figure 3), and jejunum (Figure 4). All tissues show positive staining in 33 WO 01/45691 PCT/USOO/35439 the mucosal epithelium, indicative of P2Y 2 receptor gene expression, with the antisense probe (Figures Ib, 2b, 3b, and 4b), whereas no staining was observed with the control sense probe (Figures la, 2a, 3a, and 4a). More specifically, P2Y 2 gene expression was noted in the epithelium of the gastric pit and in the neck and base of the gastric gland in 5 the stomach; in the stratified squamous epithelium of the esophagus; in absorptive enterocytes and mucus-secreting goblet cells of villus epithelium and secretory crypt epithelium of the jejunum; and in columnar absorptive cells, mucus-secreting goblet cells, and secretory enteroendocrine crypt cells of the colon. The demonstration of P2Y 2 receptor gene expression in gastrointestinal epithelium, including both secretory and 10 absorptive cell types, supports a role for P2Y 2 receptors in gastrointestinal mucosal physiology, and as a target for the treatment of gatrointestinal diseases in which enhanced mucus secretion and/or fluid secretion are therapeutic. Example 3: Measurement of intracellular calcium mobilization in cultured epithelial cells 15 from the gastrointestinal tract. A conventional technique is used to detect intracellular calcium mobilization induced by P2Y receptor agonists. The technique is familiar to those well versed in the art. Cells are seeded in 96-well plates and used for calcium mobilization assays. On the day of the assay, the growth medium is aspirated and replaced with a solution of Fluo-3 20 AM (2.5 tM final concentration) in an assay buffer consisting of (mM): KCl (10.0), NaCl (118), CaCl 2 (2.5), MgC1 2 (1.0), HEPES (20), glucose (10), pH 7.4. Probenecid (Sigma Chemical Co.) is added to the dye load and dye wash medium at a working concentration of 2.5mM to increase dye retention in the cells. After a 60 minute incubation with Fluo-3 AM at 25"C, cells are washed free of dye (Columbus Plate Washer, TECAN U.S., Inc., 25 Research Triangle Park, N.C.) and are stimulated with increasing concentrations of P2Y receptor agonists. Intracellular calcium levels are simultaneously monitored in each well by measuring the changes in fluorescence intensity using the FLIPR (Molecular Devices Corp., Sunnyvale, CA). T84 cells, a human colonic epithelial cell line, were subjected to the calcium 30 mobilization assay described above. The results show that the P2Y receptor agonists ATP 34 WO 01/45691 PCT/USOO/35439 and UTP are stimulate calcium mobilization in these cells, consistent with activation of the P2Y 2 receptors (Figure 5). 2-methylthioADP, a P2Y, receptor-selective agonist, did not simulate calcium mobilization, indicating the lack of P2Y, receptors in these cells. Similarly, human colonic HT-29 cells exhibit a robust P2Y response to ATP and UTP, 5 consistent with P2Y 2 receptor activation (Figure 6). The activation of intracellular calcium mobilization by ATP and UTP in T84 and HT-29 cells indicate the pharmaceutical utility of P2Y receptor agonists in the gastrointestinal tract. [UTP stimulated calcium mobilization has been reported in HT-29 cells: Otero et al. Mol Cell Biochem 2000 Feb;205(1-2):115-23]. 10 Example 4: Measurement of gastrointestinal epithelial mucus production, bicarbonate secretion, and short circuit current in epithelial cultures and explants. Conventional techniques are used for investigating epithelial electrical responses mediated by agonists of P2Y 2 and P2Y 4 purinoceptors. The techniques are familiar to 15 those well versed in the art. Native explants and cultured mucosal epithelial cells from the esophagus, stomach, jujenum and colon epithelia are isolated or grown as monolayers, where the integrity of junctional complexes separating apical (mucosal) and basolateral (serosal) membranes remains intact. Epithelial tissues or monolayers are mounted in a modified Ussing chamber that allows for maintenance of epithelial polarity and affords 20 the ability to separately perfuse Ringer's solution to apical and basolateral surfaces. Short-circuit currents and total transepithelial resistances are continuously measured using conventional electrophysiological techniques. Bicarbonate secretion is measured by monitoring pH using a pH-stat system. Changes in these parameters that are consistent with chloride ion secretion, bicarbonate secretion, or alteration of the transmembrane flux 25 of other ions indicate that P2Y receptor agonists modify secretion, absorption, and/or mucosal hydration in the gastrointestinal tract. Mucus production by goblet cells residing in epithelial glands is assayed by a variety of techniques familiar to those well versed in the art. Native explant and cultured monolayers of esophageal and gastric mucosal epithelia are assayed for mucin production 30 by impression cytology, which consists of exposing fixed surface area of epithelium with 35 WO 01/45691 PCT/USOO/35439 polyvinylidene difluoreide (PVDF) membrane and staining PVDF membrane with periodic-acid and Schiff's (PAS) reagent. The amount of PAS-positive staining is inversely proportional to mucin secretion. In esophageal and gastric mucosal epithelia, agonists of P2Y 2 and P2Y 4 perinoceptors are shown to decrease PAS staining, which is 5 consistent with an increase mucus secretion. P2Y purinoceptor-induced increases in mucus secretion are verified by enzyme-linked immunosorbent assay (ELISA), and immunoblots using mucin-specific antibodies. Positive results indicate robust, prolonged, and significant increases in mucus production when purinoceptor agonists following administered to luminal surface of epithelial preparation. Mucin production can be 10 repeatedly increased by repetitive stimulation with purinoceptor agonists. Example 5: Purinoceptor agonists for amelioration of symptoms associated with ulcerative colitis. Helicobacter pylori infection by acetic acid administration to the antral mucosa of 15 cynomolgus monkeys is a model for chronic gastritis, and shows histological and clinical phenotype similar to those of human gastric ulcers. Oral administration of P2Y receptor agonists to monkeys with gastritis shows significant recovery of staining of periodic acid Schiff-positive substances and increases in anti-mucin immunoreactivity, both of which reflect an increase in mucin secretion. Reduced histological incidents of gastric 20 ulcerations are also an indication of the effectiveness of the P2Y receptor agonists. A human subject, suffering from ulcerative colitis or chronic gastritis, is treated by a method in the present invention. The patient is given an endoscopy, followed with a biopsy of the gastric mucosa. Ulcerative colitis is diagnosed following confirmation of mucosal inflammation and erosion of the gastric mucosal layer. The present invention 25 treats the patient by an oral administration of a suitable formulation of the P2Y-receptor agonist, which coats the esophageal and gastric mucosal layer and stimulates mucous production under the gastric mucosa. The composition is administered as a slow-release oral form, preferable in the form of chewing gum or lozenges, and is administered multiple times during the day as needed. Disease activity is monitored on the basis of the 30 Clinical Activity Index, Endoscopic Index, Histological Index, and Global Efficacy 36 WO 01/45691 PCT/USOO/35439 Assessments by the clinical investigator. Improvements in one or more of these parameters indicate that P2Y agonists ameliorate the symptoms of ulcerative colitis. Example 6: Purinoceptor agonists for altering fluid absorption by the small intestine and 5 distal colon and for amelioration of symptoms associated with diarrhea or constipation. A human subject, suffering from either constipation of diarrhea, is treated by methods in the present invention as follows. The patient presenting either diarrhea or constipatory symptoms is given an oral formulation of compound, said compound formulated as tablet that can discharge the active compound in an amount therapeutically 10 and specifically into the small intestine (for constipation) or large intestine (for diarrhea). The activate compound specifically agonizes P2Y receptors in respective tissue and promotes fluid secretion in small intestine and fluid absorption in large intestine. 48 hour stool output, measured in grams, and the duration of diarrhea or constipation, as assessed by patient questionnaire and/or clinical observation, are determined following treatment. 15 Positive results indicate that P2Y agonists are effective in the treatment of diarrhea and/or constipation. The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the 20 foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the spirit or scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification. 37

Claims (13)

1. A method of regulating mucus or mucin secretions, bicarbonate secretion or fluid transport in the gastrointestinal tract of a mammal, said method comprising: 5 administering to said mammal a pharmaceutical composition comprising a purinergic P2Y receptor ligand, in an amount effective to regulate mucus or mucin secretions, bicarbonate secretion or fluid transport in the gastrointestinal tract.

2. A method of treating gastrointestinal diseases or disorders in which the mucosal 10 barrier or bicarbonate secretion of the gastrointestinal system is abnormal, or in which the fluid transport across the lumenal tract is abnormal, said method comprising: administering to a patient a pharmaceutical composition comprising a purinergic P2Y receptor agonist compound, in an amount effective to regulate mucus or mucin secretions or correct abnormal fluid transport in the gastrointestinal system. 15

3. The method according to Claim 1 or 2, wherein said P2Y receptor is selected from the group consisting of: P2Y,, P2Y 2 , P2Y 4 , P2Y 6 and P2Y 1 .

4. The method according to Claim 3, wherein said purinergic receptor agonist is a 20 nucleotide diphosphate selected from the group consisting of compounds of Formula Ia, Ila and Ila: Formula Ia 25 R 4 R2 R3N ' 0 N 0 0 30 HO-0-LR -- O-CH 2 0 p a H H H 38 WO 01/45691 PCTIUSOO/35439 wherein: X,, and X 2 are each independently either 0- or S~ Y is H or OH; 5 R, is selected from the group consisting of 0, imido, methylene, and dihalomethylene (e.g., dichloromethylene, difluoromethylene); R 2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, alkoxyl, nitro and azido; R 3 is selected from the group consisting of H, alkyl, acyl (including arylacyl), and 10 arylalkyl; and R 4 is selected from the group consisting of -OR', -SR', NR', and NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R 4 is double bonded from an oxygen or sulfur atom to the carbon at the 4 15 position of the pyrimidine ring; Formula Ila NR 3 R 4 N 'NR 2 o O N N z 20 HO-P-RS-P-O O H H H Y wherein: 25 R 1 , X,, X 2 , and Y are defined as in Formula Ia; Z is H, Cl, or SR, wherein R is C, - C 2 0 saturated or unsaturated alkyl; R 3 and R 4 are H while R 2 is nothing and there is a double bond between N-I and C-6, or 39 WO 01/45691 PCT/USOO/35439 R 3 and R 4 are H while R 2 is 0 and there is a double bond between N-I and C-6, or R 3 , R 4 , and R 2 taken together are -CH=CH-, forming a ring from N-6 to N-I with a double bond between N-6 and C-6; 5 Formula I1a R5 N R 6 Ry N 0 0N 10 ~HO-P-R,-P-00 10 1 I2 H wherein: H Y R 1 , X , X 2 , and Y are defined as in Formula Ia; R, and R 6 are H while R, is nothing and there is a double bond between N-3 15 and C-4, or R 5 , R 6 and R, taken together are -CH=CH-, forming a ring from N-3 to N-4 with a double bond between N-4 and C-4 (3,N 4 -ethenocytosine), optionally the hydrogen of the 4- or 5-position of the etheno ring is substituted with alkyl, substituted alkyl, alkoxyl, nitro, halo and azido. 20

5. The method according to Claim 4, wherein said nucleotide diphosphate is selected from the group consisting of: 5'- uridine diphosphate, 5'- adenosine diphosphate and 5' cytidine diphosphate. 25

6. The method according to Claim 3, wherein said purinergic receptor agonist is a nucleotide triphosphate selected from the group consisting of: compounds of Formula Ib, Ib and IlIb: 40 WO 01/45691 PCT/USOO/35439 Formula Ib R4 R 3 )N R2 5 O O O0 N HO-P-R 1 -P-0--0 1 2 3 wherein: 10 X, X 2 and X 3 are each independently either O- or S-, Y is H or OH; R, is 0, imido, methylene or dihalomethylene; R2 is H or Br; R 3 is selected from the group consisting of nothing, H, alkyl, acyl and 15 arylalkyl; and R 4 is selected from the group consisting of -OR', -SR', NR', and NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R 4 is double bonded from an oxygen or sulfur atom to the 20 carbon at the 4-position of the pyrimidine ring; Formula lIb NR 3 R 4 N 'NR 2 25 O O O N N Z H O - P-R - P-0 - - 0 H H H Y 41 WO 01/45691 PCTIUSOO/35439 wherein: R 1 , XI, X 2 , X 3 and Y are defined as in Formula Ib, Z is H, Cl, or SR, wherein R is C, - C 20 saturated or unsaturated alkyl; R 3 and R 4 are H while R 2 is nothing and there is a double bond between 5 N-1 and C-6, or R 3 and R 4 are H while R 2 is 0 and there is a double bond between N-i and C-6, or R 3 , R 4 and R 2 taken together are -CH=CH-, forming a ring from N-6 to N-i with a double bond between N-6 and C-6; 10 Formula IlIb R 5 'N R 6 R7' N 15O 150 0 00 N HO-P-R1-P-0-P-00 1 2 3 H Y 20 wherein: R,X, X 2 , X 3 , and Y are defined as in Formula Ib, and R 5 and R 6 are H while R, is nothing and there is a double bond between N-3 and C-4, or R, R 6 and R 7 taken together are -CH=CH-, forming a ring from N-3 to N-4 25 with a double bond between N-4 and C-4, optionally the hydrogen of the 4- or 5-position of the etheno ring is substituted with alkyl, substituted alkyl, alkoxyl, nitro, halo and azide. 42 WO 01/45691 PCT/USOO/35439

7. The method according to Claim 6, wherein said nucleotide triphosphate is selected from the group consisting of: 5'-uridine triphosphate, 5'-adenine triphosphate and 5' cytidine triphosphate.

8. The method according to Claim 2, wherein said purinergic receptor agonist is a 5 dinucleotide polyphosphate selected from the group consisting of: compounds of Formula IV: Formula IV O 0 0 0 B O 0 P- P-X-P- O-P- B' OH O 0 OH Y Z n . m Z' Y' 10 wherein: X is oxygen, methylene, difluoromethylene, or imido; n = 0, 1 or 2; 15 m=0, 1 or 2; n + m=0,1, 2, 3, or 4; Z =OHorH; Z' = OH or H; Y=H or OH; 20 Y'=HorOH; the sugar moieties are as depicted in the D-configuration, but may be L-, or D- and L-, and the D-configuration is preferred; the nucleoside residue is in either the alpha- or beta- and D- or L-configurations, and most preferably the beta-D-configuration; 25 43 WO 01/45691 PCT/USOO/35439 B and B' are each independently a purine residue or a pyrimidine residue, as defined in Formula V and VI, respectively, linked through the 9- or 1-position, respectively: Formula V 5 R2 7 -''Rx N 6 N 9 4 2 N H N R, 3 wherein: R, is hydrogen, chlorine, amino, monosubstituted amino, disubstituted amino, alkylthio, arylthio, or aralkylthio, wherein the substituent on sulfur contains up to a 10 maximum of 20 carbon atoms, with or without unsaturation; R 2 is hydroxy, amino, mercapto, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, acyloxy, monosubstituted alkylamino, heterocyclic, monosubstituted cycloalkylamino, monosubstituted aralkylamino, monosubstituted arylamino, diaralkylamino, diarylamino, dialkylamino, wherein alkyl groups are 15 optionally linked to N, to form a substitute ring, acylamino, diacylamino, or NHRy; Rx is 0 as in adenine 1-oxide derivatives, or is absent as in adenine derivatives; provided that when R 2 is NHRy, Ry and Rx may be taken together form a 5 membered fused imidazole ring as in 1, N-ethenoadenine derivatives, optionally substituted on the 4- or 5-positions of the etheno moiety with alkyl, aryl or aralkyl 20 moieties as defined below; R 3 is hydrogen, azido, alkoxy, aryloxy, aralkyloxy, alkylthio, arylthio, or aralkylthio as defined below; or co-A(C,_ 6 alkyl)OCONH (C,_ 6 alkyl)B- wherein A and B are independently amino, mercapto, hydroxy or carboxyl; or pharmaceutically acceptable 44 WO 01/45691 PCT/USOO/35439 esters, amides or salts thereof; or absent; wherein the substituted derivatives of adenine include adenine 1-oxide; 1,N 6 -(4- or 5-substituted etheno) adenine; 6-substituted adenine; or 8-substituted aminoadenine, where R' of the 6- or 8-HNR' groups are chosen from the group consisting of: arylalkyl 5 (C,. 6 ) groups with the aryl moiety optionally functionalized as described below; alkyl; and alkyl groups with functional groups therein, as in: ([6-aminohexyl]carbamoylmethyl)-, and o-acylated- amino(hydroxy, thiol and carboxy)alkyl(C 2 10 )- and their o-acylated amino (hydroxy, thiol and carboxy) derivatives where the acyl group is chosen from among, but not limited to, acetyl, trifluoroacetyl, benzoyl, substituted-benzoyl, or the 10 carboxylic moiety is present as its ester or amide derivative, such as the ethyl or methyl ester or its methyl, ethyl or benzamido derivative, and the o-amino(hydroxy, thiol) moiety may be alkylated with a CM alkyl group; J is carbon or nitrogen, with the provision that when J is nitrogen, R 3 is not present; 15 wherein the alkyls are straight-chain, branched or cyclic; wherein the aryl groups are optionally substituted with lower alkyl, amino, alkylamino, NO 2 , N 3 , carboxylic, amido, sulfonamido, or halo groups; and B and B', can also be a pyrimidine with the general formula of Formula VI, linked through the 1- position to ribosyl residue: 20 Formula VI IR6 R7 N R5 r 5 31 6 2' R8 N R4 1 45 WO 01/45691 PCT/USOO/35439 wherein: R 4 is hydrogen, hydroxy, oxo, mercapto, amino, cyano, C_ 12 arylalkoxy, C 16 alkylthio, C - alkoxy, CI_- alkylamino, or diC 1 4 alkylamino, wherein the alkyl groups are optionally linked to form a heterocycle; 5 R 5 is hydrogen, oxo, acetyl, benzoyl, C 1 _ alkyl, C 1 , alkanoyl, or aroyl; R, is hydroxy, oxo, mercapto, C 1 4 alkoxy, C,_ 1 arylalkoxy, C, 6 alkylthio, amino, S phenyl, C, disubstituted amino, triazolyl, C 1 6 alkylamino, or di-C, 4 alkylamino, wherein said dialkyl groups are optionally linked to form a heterocycle or linked to N 3 to form a substituted ring; or 10 R, and R 6 taken together form a 5-membered fused imidazole ring between positions 3 and 4 of the pyrimidine ring and form a 3,N 4 -ethenocytosine derivative, wherein said etheno moiety is optionally substituted on the 4- or 5-positions with C,, alkyl, phenyl or phenyloxy; wherein at least one hydrogen of said C 1 alkyl, phenyl or phenyloxy is optionally substituted with a moiety selected from the group consisting of: 15 halogen, hydroxy, C 14 alkoxy, C 14 alkyl, C 6 1 0 aryl, C 712 arylalkyl, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic acid, amino, C 1 alkylamino, and di- C 4 alkylamino, wherein said dialkyl groups are optionally linked to form a heterocycle; R 7 is selected from the group consisting of: hydrogen, hydroxy, cyano, nitro, and C 2 _s alkenyl; wherein said alkenyl moiety is optionally linked through an oxygen to form a 20 ring, wherein at least one hydrogen of said alkenyl moiety on the carbon adjacent to said oxygen is optionally substituted with a substituent selected from the group consisting of: C 1 . alkyl or phenyl; substituted C 2 -s alkynyl, halogen, substituted C 1 alkyl, CF 3 , C 2 -3 alkenyl, C 2 3 alkynyl, allylamino, bromovinyl, ethyl propenoate, or propenoic acid; or R and R, together form a 5 or 6-membered saturated or unsaturated ring bonded 25 through N or 0 or S at R,, such ring optionally contains substituents that themselves contain functionalities; provided that when R 8 is amino or substituted amino, R 7 is hydrogen; and 46 WO 01/45691 PCTIUSO0/35439 R 8 is selected from the group consisting of: hydrogen, amino or di-C 4 alkylamino, C 1 alkoxy, C 7 . 12 arylalkoxy, C, 4 alkylthio, C 7 .arylalkylthio, carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, and phenylthio. 5

9. The method according to Claim 8, wherein said dinucleotide polyphosphate is selected from the group consisting of: U 2 P 4 , dUP 4 U, U 2 P 3 , U 2 Ps, dCP 4 U, CP 4 U, 1PsI, AP 4 A, CP 3 U, UP 3 A and A 2 P 3

10. The method according to Claim 1 or 2, wherein said administering is administering 10 an oral form of said pharmaceutical composition, such that a therapeutically effective amount of said compound contacts the tissues of said gastroinstestinal system of said mammal.

11. The method according to Claim 1 or 2, wherein said administering is injecting said 15 pharmaceutical composition in an injectable form, such that a therapeutically effective amount of said compound contacts the tissues of said gastrointestinal system via systemic absorption and circulation.

12. The method according to Claim 1 or 2, wherein said administering is accomplished 20 by administering a suppository form of said pharmaceutical composition, such that a therapeutically effective amount of said compound contacts the tissues of said gastrointestinal system via systemic absorption and circulation.

13. The method according to claim 2, wherein said gastrointestinal disorder is gastroesophageal reflux disease (GERD). 47