Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the use of certain peptides, more particularly the use of uroguanylin, prouroguanylin, guanylin, and other like peptides to retard the development of polyps and prevent, inhibit or treat cancer in the intestine.
• the pathogenesis of colorectal cancer is characterized as a multistep process that begins with increased proliferation and/or decreased apoptosis of colorectal epithelial cells resulting in generation of polyps, followed by adenoma formation and ultimately to adenocarcinoma .
• FAP familial adenomatous polyposis
• Chemoprevention has evolved during the last decade as a viable strategy for cancer prevention, with the aim of controlling the development of cancer through pharmacological and/or dietary intervention prior to the appearance of a clinically detectable tumor. Reddy, B.S. (1997) Chemoprevention of colon cancer by dietary- administration of naturally-occurring and related synthetic agents, Adv. Exp. Med. Biol . 400B : 931-936.
• Uroguanylin and guanylin are structurally related enteric peptide hormones that are secreted intraluminally by different types of cells, include enterochromaffin, goblet and others within the intestinal mucosal lining.
• a receptor for theses peptides that has been identified at the molecular level is a transmembrane form of guanylate cyclase (GC) known as GC-C.
• GC-C receptors are localized on the luminal surface of enterocytes throughout the GI tract.
• guanylin/STa receptor is expressed in crypts and apical epi thelium throughout the mouse intestine, Biochem. Biophys . Res. Commun. 225:1009-1014 (1996).
• Binding of uroguanylin or guanylin to the extracellular domain of GC-C receptors stimulates intracellular production of the second messenger cGMP, resulting in activation of cystic fibrosis transme brane conductance regulator (CFTR) , the apical membrane channel for efflux of chloride from enterocytes lining the intestinal tract.
• CFTR cystic fibrosis transme brane conductance regulator
• uroguanylin is a circulating peptide hormone wi th naturiuretic activi ty, Am. J. Kidney Dis. 28:296-304 (1996).
• Activation of CFTR chloride channel proteins and the subsequent enhancement of transepithelial secretion of chloride leads to stimulation of sodium (Na + ) and water secretion into the intestinal lumen.
• Na + sodium
• Guanylin regulatory peptides structures, biological activi ties mediated by cyclic GMP and pathobiology , Regul . Pept. 81:25-39 (1999). Therefore, one of the major physiological functions of these hormones is the regulation of fluid and electrolyte transport in the gastrointestinal (GI) tract by serving as paracrine regulators of CFTR activity.
• GI gastrointestinal
• the precursor of uroguanylin is prouroguanylin, which is broken down by endogenous proteases in the intestinal tract to produce the active uroguanylin.
• Chymotrypsin activates prouroguanylin to cleave it into its active form of uroguanylin.
• Uroguanylin Is a Circulating Peptide Hormone Wi th Natriuretic Activi ty, Am. J. Kid. Dis. 1996, 28, No.2, 296- 304.
• Uroguanylin is an acid-stable and proteolysis- resistant peptide, which will remain in tact to act on the intestinal lumen directly rather than being absorbed systemically. Uroguanylin and guanylin are produced throughout the intestinal mucosa and in the myocardium. Forte et al , Sal t and water homeostasis : uroguanylin is a circulating peptide hormone wi th natriuretic activi ty Am. J. Kidney Dis. 28:296-304 (1996) . Human uroguanylin has been isolated from human urine and has been chemically synthesized by solid phase peptide synthesis as described in U.S.
• Patent Number 5,489,670 for Human Uroguanylin has been isolated from human intestinal cells and has been chemically synthesized by solid phase peptide synthesis as described in U.S. Patent Number 5,969,097 for Human Guanylin .
• Uroguanylin and guanylin receptors are found on the luminal surface of epithelial cells lining the intestinal tract and renal proximal tubules as well as in other organs. Forte et al, Sal t and Water Homeostasis : Uroguanylin Is a Circulating Peptide Hormone wi th Natriuretic Activi ty, Am. J. Kid. Dis.1996, 28, No. 2, 296-304.
• Uroguanylin has been found to stimulate increases in cyclic GMP levels in a manner similar to another family of heat stable enterotoxins (STs) secreted by pathogenic strains of E. coli and other enteric bacteria that activate intestinal guanylate cyclase and cause secretory diarrhea, which is a major cause of traveler's diarrhea and many deaths in developing countries .
• STs heat stable enterotoxins
• Lymphoguanylin Cloning and Characterization of a Unique Member of the Guanylin Peptide Family, Endocrinology Vol.
• ST peptides act as molecular mimics of the endogenous mammalian peptides of uroguanylin and prouroguanylin.
• Forte et al Endocrinology Vol. 140, No. 4, p.1800.
• the STs from enteric bacteria do not have a decrease in potency when the pH changes in the colon.
• STs are more potent than either uroguanylin or guanylin under both acidic and alkaline conditions.
• Guanylin a peptide regulator of epi thelial transport, The FASEB Journal, vol. 9, 643-650 (1995) .
• Uroguanylin is believed to regulate fluid and electrolyte transport in a manner similar to guanylin and the STs in the GI tract. Therefore, as mentioned in previous publications the human uroguanylin may act as a laxative and be useful in patient suffering from constipation.
• the present invention is directed to a method of preventing, inhibiting and treating cancer in the intestine (small intestine and colon) of a subject in need thereof .
• the present invention is directed to a process for modulating polyps in the intestine of a subject, in need thereof, which comprises the administration of a peptide including the amino acid sequence:
• each of X lf X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 is an amino acid residue
• X 8 and X 9 are independently hydrogen or at least one amino acid residue
• the polypeptide is cross-linked by a disulfide bond between the cystine residue immediately adjacent the amine group of X ⁇ and the cystine residue immediately adjacent the amine group of X 6 and by a disulfide bond between the cystine residue immediately adjacent the amine group of X 3 and the cystine residue immediately adjacent the carboxy group of X 7 together with a pharmaceutically acceptable carrier.
• the invention is further directed to a method for modulation of polyps in a subject, and to a process for the prevention, inhibition or treatment of cancer in the intestinal tract by administration of a pharmacuetical composition comprising any one of or combination of the following peptides: uroguanylin, human uroguanylin, prouroguanylin, and human pro-uroguanylin, guanylin, ly phoguanylin, prolymphoguanylin and heat stable enterotoxin, together with a pharmaceutically acceptable carrier.
• the invention is directed to a process for modulating polyps in the intestine of a subject, and a process for the prevention, inhibition or treatment of cancer in the intestine of a subject, in need thereof, by administration of a pharmaceutical composition comprising any one of or a combination of agonist peptides and/or other agonist compounds to the guanylate cyclase receptor GC-C, together with a pharmaceutically acceptable carrier.
• a pharmaceutical composition comprising any one of or a combination of agonist peptides and/or other agonist compounds to the guanylate cyclase receptor GC-C, together with a pharmaceutically acceptable carrier.
• Figure 1 depicts the effect of human uroguanylin on the stimulation of I sc where fresh mouse duodenum consisting of mucosa and submucosa ( ⁇ lcm 2 ) was mounted between two halves of Ussing Chambers and bathed on both sides as described. At the arrows, indicated concentrations of TTX, uroguanylin (uroG) and carbachol were added to the apical reservoir. Electrical measurements were monitored with an automatic voltage clamp.
• Figure 1(b) depicts the effect of human uroguanylin on the stimulation of I sc where human intestinal mucosa ( ⁇ lcm 2 ) was mounted between two halves of Ussing Chambers and bathed on both sides as described. At the arrows, indicated concentrations of TTX, uroguanylin (uroG) and carbachol were added to the apical reservoir. Electrical measurements were monitored with an automatic voltage clamp.
• Figure 2 depicts a graphic demonstration of the effect of human uroguanylin on the inhibition of proliferation of T-84 human carcinoma cells. Cells were inoculated in 96- well plates.
• Figure 3 depicts the fragmentation of DNA in T-84, human colon carcinoma cells, after treatment with human uroguanylin as analyzed by electrophoresis using 1.8% agarose gel followed by staining with ethidium bromide.
• Figure 4 depicts microscopic slides with semi-confluent monolayers of Caco-2 cells demonstrating the effects of human uroguanylin on the induction of apoptosis.
• Cells were cultured on microscopic slides until they formed semi- confluent monolayers. Subsequently the cells on slide B were treated with human uroguanylin (1 ⁇ M) for 48 hours. Induction of apoptosis was detected by fluorescence microscopy directly after the TUNEL reaction as per the instructions of "In situ cell death detection kit" (Boehringer Mannheim Corp., Indianapolis, Indiana) .
• Slide A depicts vehicle-treated cells.
• Slide B depicts uroguanylin- treated cells.
• Figure 5 (a) depicts a Northern blot analysis demonstrating that the expression of uroguanylin and guanylin is suppressed in human colon carcinoma cells.
• Figure 5 (b) depicts an RT-PCR followed by Southern blotting demonstrating that the expression of uroguanylin and guanylin is suppressed in human colon carcinoma cells.
• Figure 6 (a) depicts a graphic demonstration of the enhancement of daily food consumption by Min-mice after oral administration of human uroguanylin. Total food consumption per day (24 hours) by five (5) animals in one cage was determined and used for calculation of total food consumption per mouse per day. Results are expressed as an average + standard deviation.
• Figure 6(b) depicts a graphic demonstration of the enhancement of body weight gain by Min-mice after oral administration of human uroguanylin. Body weights of all animals were measured weekly throughout the study. Results are expressed as average + standard deviation of gain in body weight per mouse during the study.
• Figure 7 depicts the primary structure of human uroguanylin (h UroG) [identified as SEQ. ID. 2] , human guanylin ( Gua) [identified as SEQ. ID. 3], and bacterial enterotoxins (E. coli [identified as SEQ. ID. 4] & V. cholerae [identified as SEQ. ID. 5]).
• h UroG human uroguanylin
• Gua human guanylin
• bacterial enterotoxins E. coli [identified as SEQ. ID. 4] & V. cholerae [identified as SEQ. ID. 5]
• Bold and italic letters represent the similar residues in these peptides. These residues are believed to be required for the functional activity of these peptides.
• E. coli ST has three additional residues (Asn-Ser-Ser) and V. cholerae has two additional residues (Leu and lie) at their N-terminii.
• N- terminal residues make bacterial ST insensitive towards intestinal pH.
• Two underlined (Asp-Asp) residues are believed to be important for regulating the functional activity of uroguanylin only at the acidic environment of the intestinal mucosa.
• Uroguanylin is secreted naturally by the goblet cells of the intestinal mucosal lining as prouroguanylin, a functionally inactive form, which is then converted to the functionally active uroguanylin in the intestine by endogenous proteases.
• Uroguanylin is an acid-stable, proteolysis-resistant peptide. Therefore, orally delivered prouroguanylin and uroguanylin will act on the lumenal intestinal surface and not be absorbed systemically .
• Oral administration of uroguanylin, prouroguanylin and other like peptides, containing the amino acid sequences similar to the active domain, are expected to induce apoptosis, cell death, in the intestinal mucosal cell lining.
• the induced apoptosis in the intestinal mucosal cell lining is expected to retard the incidence of polyp formation and subsequent intestinal cancer.
• the peptides of the invention exert their effects by increasing the rate of apoptosis, cell death, in the intestinal mucosal cell lining promoting the perfect balance between the cell proliferation and the programmed cell death thereby retarding the growth of polyps and preventing, inhibiting, and treating cancer in the intestine and other epithelial-derived cancer possessing receptors for guanylin, uroguanylin, lymphoguanylin and STa family of peptides.
• the rate of cell proliferation and cell death in the intestinal mucosa is very rapid.
• the cells of the intestinal mucosa are in a steady state of turnover to insure a perfect balance between cell proliferation and cell death.
• the constant rapid renewal of the GI tract epithelium fulfills the functions of maintaining the integrity of normal mucosa, repairing and replenishing differentiated epithelial cells that have specialized functions.
• the prevention of apoptosis in the intestinal mucosal cells creating an imbalance in the renewal process results in an increased incidence of polyp formation and subsequent intestinal cancer. See Eastwood et al , A review of gastrointestinal epi thelial renewal and i ts relevance to the development of adenocarcinomas of the gastrointestinal tract, J. Clin. Gastroenterol .
• a major cellular characteristic of the apoptotic process is a marked loss of cell volume, which is directly related to the movement of ions, with homeostatsis being achieved by the balance of osmotic pressure across the plasma membrane.
• Hoffman, E.K. et al Membrane mechanisms in intracellular signalling in cell volume regulation, Int. Rev. Cytol . 161:173-262 (1995).
• Most mammalian cells achieve and maintain this osmotic pressure through the continuous action of Na + /K + ATPase pump, which creates a gradient of these monovalent cations across the membrane.
• K + efflux in the induction of apoptosis. Hughes, F.M.
• Atrial naturiuretic peptide a peptide that stimulates intracellular accumulation of cGMP by binding to a specific GC receptor, has also been shown to activate K + conductance in rat mesangial cells, and to induce apoptosis in cardiac myocytes by a cGMP-dependent mechanism. Cermak, R.
• Natriuretic peptides increase a K + conductance in rat mesangial cells, Pflugers Arch. 43:571- 577 (1996) .
• pretreatment of rat endothelial cells with either ANP (10 ⁇ 7 M) or 8-bromo-cGMP (10 "3 M) caused a marked accumulation of the nuclear phosphoprotein, p53, a tumor suppresser protein known to induce apoptosis in many cell types.
• Suenobu, N. et al, Natriuretic peptides and ni tric oxide induce endothelial apoptosis via a cGMP- dependent mechanism, Arterioscler . Thromb. Vase. Biol .
• CFTR expression is associated with K + and CI " efflux and shrinkage of cells, characteristic biochemical changes found in apoptotic cells.
• Rotoli, B.M. et al CFTR expression in C127 cells is associated wi th enhanced cell shrinkage and ATP extrusion in Cl (-) - free medium, Biochem. Biophys . Res. Commun. 227:755-61 (1996).
• uroguanylin, prouroguanylin, guanylin and other like peptides may induce apoptosis of epithelial cells lining the GI tract mucosa via maintenance of intracellular concentration of K + ions as a result of binding to the GC-C receptors.
• the binding of the GC-C receptors stimulates the production of cGMP thereby activating the CFTR chlorine channel which causes an increase in K + efflux.
• the induction of apoptosis is also expected from the administration of agonist peptides which bind to the GC-C receptors, and to other receptors for guanylin, uroguanylin and lymphoguanylin in the intestine.
• guanylin has been shown to be completely diminished in colon cancer cells and evenly expressed in normal intestinal mucosal cells. This finding suggest that guanylin is involved in the maintenance of colonic differentiation or functions as a tumor modifier gene. Mitchell et al . , Guanylin mRNA Expression in Human Intestine and Colorectal Adenocarcinoma, Lab. Invest. 1998, Vol. 78, No. 1, 101-108. Recent data demonstrates that the guanylin cyclase receptor known as GC-C receptor is expressed in all primary and metastatic colorectal cancers and it may serve as a specific marker for these tumors. Carrithers, S.L.
• Guanylin cyclase C is a selective marker for metastatic colorectal tumors in human extraintestinal tissues, Proc. Natl. Acad. Sci. USA. 93:14827-14832.
• the expression of guanylin has been shown to be down-regulated in colorectal cancer tissues and cell lines.
• Cohen, M.B. et al Guanylin mRNA expression in human intestine and colorectal adeno carcinoma, Lab. Invest. 78:101-108.
• a study described in the examples to this application shows that uroguanylin is similarly completely diminished in colon cancer cells and evenly distributed in normal intestinal mucosal cells.
• a polypeptide which contains the active domain of human uroguanylin or which binds to the guanylate cyclase receptor GC-C in the intestine of the subject is administered to a subject. While the polypeptide may be administered prophylactically, it will typically be administered to a subject who has been determined to have intestinal cancer, intestinal polyps, or a genetic predisposition for the growth of polyps in the intestine. In a preferred embodiment of the present invention, the polypeptide is a polypeptide having the sequence as identified in SEQ. ID. 1:
• each of X x , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 is an amino acid residue
• X 8 and X 9 are independently hydrogen or at least one amino acid residue
• the polypeptide is cross-linked by a disulfide bond between the cystine residue immediately adjacent the amine group of X ⁇ and the cystine residue immediately adjacent the amine group of X 6 and by a disulfide bond between the cystine residue immediately adjacent the amine group of X 3 and the cystine residue immediately adjacent the carboxy group of X 7 .
• the polypeptide is guanylan, uroguanylin, pro-uroguanylin, or another polypeptide which contains the active domain of uroguanylin.
• certain amino acids in a peptide or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide or protein having similar biological activity, i.e., which still retains biological functionality.
• amino acids having hydropathic indices within ⁇ 2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within +1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within ⁇ 0.5.
• hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 +1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4) .
• amino acids having hydropathic indices within +2 are preferably substituted for one another, those within +1 are more preferred, and those within +0.5 are most preferred.
• amino acid substitutions in the peptides of the present invention can be based on the relative similarity of the amino acid side-chain substituents in the non-active domain of the peptide to create a protein with the same biological activity as the human uroguanylin peptide.
• X x may be selected from the group of all amino acid residues, but preferably is selected from the group of amino acid residues consisting of aspartic acid, glutamic acid, glycine, lysine, asparagine, proline, glutamine, arginine, serine and threonine .
• the more preferred amino acid residues that may be substituted for X x are glutamic acid, aspartic acid, arginine, and lysine.
• the most preferred amino acid residue that may be used for X 1 is glutamic acid.
• X 2 may be selected from all amino acid residues, however the preferred amino acid residues for substitution are leucine, isoleucine, tyrosine, phenylalanine, tryptophan, valine, methionine, cysteine, alanine, histidine, proline, threonine, glycine, asparagine, and glutamine.
• the more preferred amino acid residues that may be substituted for X 2 are cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, valine, and tyrosine.
• the even more preferred amino acid residues for substitution for X 2 are leucine, isoleucine, tyrosine, valine, and methionine.
• the most preferred amino acid residue for substitution for X 2 is leucine.
• X 3 and X 4 may be selected from all amino acid residues, but the preferred amino acid residues are valine, isoleucine, tyrosine, phenylalanine, tryptophan, methionine, cysteine, alanine, histidine, proline, threonine, glycine, glutamine, asparagine, and serine.
• the more preferred amino acid residues that may be substituted for X 3 and X 4 are valine, isoleucine, leucine, tyrosine, phenylalanine, methionine, cysteine, alanine, histidine, and proline.
• the even more preferred amino acid residues that may be substituted for X 3 and X 4 are valine, isoleucine, leucine, methionine, and cysteine. Even more preferable for substitution for X 3 and X 4 are isoleucine and valine. The most preferred amino acid residue for substitution for X 3 and X 4 is valine.
• X 5 may be selected from all amino acid residues, but the preferred amino acid residues are alanine, histidine, cysteine, methionine, valine, leucine, isoleucine, tyrosine, phenylalanine, proline, threonine, glycine, glutamine, asparagine, and serine.
• the more preferred amino acid residues that may be substituted for X 5 are alanine, histidine, cysteine, methionine, valine, proline, threonine, glycine, glutamine, asparagine, and serine.
• amino acid residues for substitution for X 5 are alanine, histidine, cysteine, proline, threonine, glycine, glutamine, asparagine, and serine.
• the most preferred amino acid residue for substitution for X 5 is alanine.
• X 6 may be selected from all amino acid residues, but the preferred amino acid residues for substitution are threonine, proline, alanine, histidine, cysteine, methionine, valine, leucine, isoleucine, tyrosine, glycine, glutamine, asparagine, and serine.
• the more preferred amino acid residues for substitution for X 6 are threonine, proline, alanine, histidine, cysteine, methionine, glycine, glutamine, asparagine, and serine.
• Even more preferred amino acid residues for substitution threonine, proline, alanine, histidine, and glycine.
• the most preferred amino acid residue for substitution for X 6 is threonine.
• X 7 may be selected from all amino acid residues, but the preferred amino acid residues are glycine, threonine, proline, alanine, histidine, cysteine, methionine, valine, leucine, isoleucine, glutamine, asparagine, serine, glutamic acid, and aspartic acid.
• the more preferred amino acid residues for substitution for X 7 are glycine, threonine, proline, alanine, histidine, cysteine, glutamine, asparagine, and serine.
• amino acid residues for substitution for X 7 are glycine, threonine, proline, alanine, histidine, glutamine, asparagine, and serine.
• the most preferred amino acid residue for substitution for X 7 is glycine.
• polypeptides of the present invention can be combined with various excipient vehicles and/or adjuvants well known in this art which serve as pharmaceutically acceptable carriers to permit drug administration in the form of, e.g., injections, suspensions, emulsions, tablets, capsules, and ointments.
• These pharmaceutical compositions may be administered by any acceptable means.
• administration can be oral, parenteral, subcutaneous, intravenous, intramuscular and/or intraperitoneal .
• the specific dose administered will be dependent upon such factors as the general health and physical condition of the subject as well as the subject's age and weight, the stage of the subject's disease condition, the existence of any concurrent treatments, and the frequency of administration; typically, the dose will be in the range of about 0.5 to about 2.0 mg/kg for human subjects.
• the composition will contain one or more of the polypeptide (s) of the present invention in a concentration of at least about 0.0001% by weight, more typically at least about 0.001% by weight, still more typically at least about 0.01%, still more typically at least about 0.1% and, in some embodiments, in a concentration of at least about 1% by weight of the composition.
• Pentadecapeptide which stimulates intestinal guanylate cyclase .
• Peptides similar to uroguanylin peptides have been identified in mouse, rat, porcine, and bovine species.
• the functionally active domain in most of these peptides are highly conserved. Therefore, the physiological functions of these peptides may be similar, and these peptides may be used as intestinal cancer preventative agents as well. Thus, as long as the functionally active domains of these peptides are conserved, substitutions in the non-active domains may be achieved with no change in the activity of the peptides.
• T-84 cells were obtained from the American Type Culture Collection at passage 52. Cells were grown in 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100U penicillin/ml, and 100 ⁇ g/ml streptomycin. Cells were fed fresh medium every third day and split at a confluence of approximately 80%.
• DMEM Dulbecco's modified Eagle's medium
• T-84 cells were grown in 35 mm dishes for 7 days .
• the confluent monolayers were washed once with serum-free DMEM, and incubated with the same media containing different concentrations of human uroguanylin for 16 hours. After this incubation, cells were quickly collected by trypsinization, and the cell pellet was washed twice with phosphate buffer saline (PBS) .
• PBS phosphate buffer saline
• Cells were resuspended in PBS at a concentration of approximately 10 8 cell/ml.
• the apoptotic DNA was isolated from these cells by following the instructions of the DNA fragmentation analysis kit (Boehringer Mannheim Corp., Indianapolis, IN).
• apoptotic DNA was separated by agarose gel electrophoresis followed by staining with ethidium bromide. Induction of apoptosis by uroguanylin was further demonstrated by using the TUNEL assay as per the instructions of the 'In situ cell death detection kit' (Boehringer Mannheim Corp., Indianapolis, IN) . Results :
• Uroguanylin functional assay Human uroguanylin (NDDCELCVNVACTGCL) peptide was custom synthesized by Multiple Peptide System, San Diego, CA. The biological activity of the synthetic peptide was assayed by a modified cell-based assay.
• the confluent monolayers of T-84 cells in 24-well plates were washed twice with 250 ⁇ l of DMEM containing 50 mM HEPES (pH 7.4), preincubated at 37° C for 10 min with 250 ⁇ l of DMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX), followed by incubation with different concentration of human uroguanylin (10 " ⁇ to 10 "10 M) for 30 min. The medium was aspirated, and the reaction was terminated by the addition of 3% perchloric acid. The plate was centrifuged at lOOOxg for 5 min and the supernatant was collected. After neutralization with 0.
• IBMX isobutylmethylxanthine
• Mouse intestine and human colon tissue consisting of mucosa and submucosa, were mounted between two ussing half-chambers and bathed on both sides. Electrical measurements were monitored with an automatic voltage clamp, and direct-connecting voltage and current passing difference and I sc . Tissues were equilibrated under short-circuit conditions until I sc had stabilized and the potential difference across the epithelium was measured intermittently.
• Human uroguanylin peptide was chemically synthesized and the relative potencies of various synthetic forms were evaluated by their abilities to stimulate cGMP accumulation in intact T-84 cells. The biological activity of several isoforms of this peptide that exhibited similar physico- chemical properties was observed. The major isoform, exhibiting a potent biological activity, was further purified to about 99% purity and used for this study. To ensure that the synthetic form of human uroguanylin was equally effective in mouse and human GI mucosa, its activity was examined in the Ussing chamber using the mouse duodenum and the human colonic mucosa.
• Min-mouse model Male Min mice (C57BL/6J-APC Min /+) , a strain containing a fully penetrant dominant mutation in the
• APC gene were obtained at 4-5 weeks of age from The Jackson Laboratory, Bar Harbor, ME. All mice were fed a high-fat AIN-93G diet, tap water to drink and housed in a humidity and temperature controlled room with a 12 hour light-dark cycle. Animals consumed approximately 5 grams of the diet per day. After one week of quarantine period, animals were randomly divided in three groups of 10 animals each. These groups of animals were fed the same diet containing different concentrations of (0, 10, and 20 ⁇ g/5grams of the diet) of human uroguanylin. Animals were also given additional amounts of human uroguanylin (vehicle, 10 and 20 ⁇ g) in 0.2 ml of PBS containing 20% polyethylene glycol by oral gavage twice a week.
• Results are expressed as the average of the total number of polyps for each individual animal by four different observers. Analysis of the data obtained from all observers revealed insignificant inter- observer variance. Sections of these tissues were viewed under a constant magnification (10 X) to gauge the differences between polyp diameter between animals. Results :
• the Min-mouse the most widely used animal model to assess the chemopreventive properties of dietary nutrients and therapeutic agents, carries a dominant mutation in one of the alleles of the APC gene.
• these mice when raised on a high fat diet, they begin to develop polyps throughout the intestine at around 55 days of age. Development of polyps causes blockage in the movement of intestinal contents, which leads to decreased food consumption and reduced gain in body weight as the disease progresses.
• the test results for oral administration of uroguranylin to the min-mouse showed a dose-dependent increase in the food consumption as shown in figure 6a, and in the body weight gain as shown in figure 6b.
• the average body weight for the control group was 25.1+ 0.9 g, and that for the uroguanylin-treated (20 ⁇ g) group was 29.4 + 1.07 g at 17.
• animals treated with uroguanylin were visibly more healthy and active.
• the GI tract in the untreated control group contained 48.3 + 7.7 polyps per mouse. A majority of the polyps were located throughout the small intestine and only a few polyps were found in the colon. The sizes of the polyps in the control group of mice were in the range of approximately 3 to 5 mm. Three animals in the control group had also developed globular tumors in the duodenum. Administration of uroguanylin reduced the total number of polyps (23.3 + 3.1) by approximately 50%.
• polyps in uroguanylin-treated group of mice were significantly smaller in size ( ⁇ 2.0 mm) . There were no polyps observed in the colons of any animals in this group, nor were there any globular tumors in these animals. Since the appearance of polyps in colon of Min-mice occurs only during the severe cases of diseases, the absence of polyps in colon of the uroguanylin-treated group of mice suggest that this peptide might also inhibit the progression of colon cancer. These results suggest that the oral administration of uroguanylin suppresses both the formation as well as the progression of polyp formation in this animal model for colon cancer.
• TRI reagent method Molecular Research Center, Inc., Cincinnati, OH
• RNAeasy Kit Qiagen, Valencia, CA
• RNA (20 ⁇ g) was subjected to electrophoresis in formaldehyde-agarose gels and then transferred to nylon membranes (Zeta-Probe, Bio-Rad Laboratories, Inc., Hercules, CA) . The membranes were prehybridized for two hours at 65°C in ExpressHyb solution
• RNA blots were then washed twice with 2X SSC-0.1% SDS for 5 min at room temperature followed by a 15 min wash at 60 °C with 0.2X SSC- 0.1% SDS. Exposure to X-ray film was performed at -80°C with intensifying screens.
• PCR primers 5 ' -primer (5 ' -GAACCCAGGGAGCGCGAT-3 ') [identified as SEQ. ID. 6] and 3 ' -primer (5 ' -CTGGTGGGCTCAGGGTACC- 3') [identified as SEQ. ID 7], were designed from regions flanking the open reading frame of human pre-prouroguanylin cDNA.
• a PCR product of the expected size of 384 bp was amplified from colon cDNAs after 25 cycles at 93 °C for 1 min, 56°C for 1 min, and 72 °C for 1.5 min using Taq DNA polymerase (U.S.
• the pair of primers for RT-PCR of guanylin were 5 ' -primer (5'- AACTCAGGAACTTTGCAC-3 ') [identified as SEQ. ID. 8] and 3'- primer (5 ' -CGTAGGCACAGATTTCAC-3 ') [identified as SEQ. ID. 9]. These primers produced a 174 bp cDNA for human guanylin using the PCR conditions of 25 cycles at 93 °C for 1 min, 59°C for 1 min and 72 °C for 1.5 min.
• PCR-generated cDNA products were subjected to electrophoresis on 1% agarose gels in TAE buffer containing ethidium bromide and then transferred to nylon membranes.
• Southern hybridization was carried out using the urogyanylin and guanylin cDNA probes. Prehybridization was for 1 hour at 65°C with ExpressHyb solution and then hybridization was for 3 hours at 65°C. Blots were washed as described above and exposed to X-ray films at -80°C with intensifying screens.

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