(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`1111111111111111 IIIIII 111111111111111 IIIII IIIII IIIII IIIII IIII IIIIIII IIII 11111111
`
`(43) International Publication Date
`12 April 2001 (12.04.2001)
`
`PCT
`
`(10) International Publication N um her
`WO 01/25266 Al
`
`(51) International Patent Classification 7:
`A61K 38/10, A61P 35/00
`
`C07K 7/08,
`
`(21) International Application Number: PCT/US00/21998
`
`(22) International Filing Date: 4 October 2000 (04.10.2000)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/157,950
`
`6 October 1999 (06.10.1999) US
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO,NZ,PL,PT,RO,RU,SD,SE,SG,SI,SK,SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, VW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, 7»/), Eurasian
`patent(AM,AZ,BY,KG, KZ,MD, RU, TJ, TM),European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG,
`CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments.
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`::::::
`::::::
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`--(cid:173)
`iiiiiiiiiiiiiii ---
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`\0
`\0
`N
`ll)
`.._
`N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`~ (54) Title: UROGUANYLIN AS AN INTESTINAL CANCER INHIBITING AGENT
`Q
`0 (57) Abstract: Disclosed is a method of retarding the development of polyps and prevention, inhibition and treatment of cancer in
`> the intestine of a subject by administration of a composition comprising a peptide with the active domain of uroguanylin, or any
`~ agonist peptide or compound binding to the guanylate cyclase receptor GC-C in the intestine.
`
`(71) Applicant (for all designated States except US): PHAR(cid:173)
`MACIA CORPORATION [US/US]; Corporate Patent
`Department, P.O. Box 5110, Chicago, IL 60680-5110
`(US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only) : SHAILUBHAI,
`Kunwar [US/US]; 15438 Harrisberg Court, Chesterfield,
`MO 63017 (US). CURRIE, Mark, G. [US/US]; 404
`Mason Ridge Drive, St. Charles, MO 63304 (US).
`
`(74) Agents: BENNETT, Dennis, A. et al.; Pharmacia Cor(cid:173)
`poration, Corporate Patent Department, P.O. Box 5110,
`Chicago, IL 60680-5110 (US).
`
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`UROGUANYLIN AS AN INTESTINAL CANCER INHIBITING AGENT
`
`This application claims priority from U.S. provisional
`application# 60/157,950, filed October 6, 1999, which is
`incorporated herein by reference.
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`BACKGROUND OF THE INVENTION
`
`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.
`Certain individuals develop multiple colorectal adenomas and
`subsequent carcinomas early in life because of a genetic
`defect in the APC gene responsible for causing a condition
`called familial adenomatous polyposis (FAP). Dihlmann et
`al, Dominant negative effect of the AFC 1309 mutation: a
`possible explanation for genotype-phenotype correlations in
`familial adenomatous polyposis, Cancer Res. 1999 Apr. 15,
`59(8): 1857-60. 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
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`(GC) known as GC-C. Krause, W.J. et al, The guanylin and
`uroguanylin peptide hormones and their receptors, Acta.
`Anat. (Basel) 160:213-231 (1997). GC-C receptors are
`localized on the luminal surface of enterocytes throughout
`the GI tract. Swenson, E.S. et al, The guanylin/STa
`receptor is expressed in crypts and apical epithelium
`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
`transmembrane conductance regulator (CFTR), the apical
`membrane channel for efflux of chloride from enterocytes
`lining the intestinal tract. Forte, L.R. et al, Salt and
`15 water homeostasis: uroguanylin is a circulating peptide
`hormone with naturiuretic activity, 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. Forte, L.R.
`et al, Guanylin regulatory peptides: structures, biological
`activities 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.
`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. Forte, et el, Salt and Water Homeostasis:
`Uroguanylin Is a Circulating Peptide Hormone With
`Natriuretic Activity, Am. J. Kid. Dis. 1996, 28, No.2, 296-
`304. Uroguanylin is an acid-stable and proteolysis(cid:173)
`resistant peptide, which will remain in tact to act on the
`intestinal lumen directly rather than being absorbed
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`systemically. Uroguanylin and guanylin are produced
`throughout the intestinal mucosa and in the myocardium.
`Forte et al, Salt and water homeostasis:uroguanylin is a
`circulating peptide hormone with natriuretic activity Am. J.
`5 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.
`Additionally, human guanylin 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.
`Binding of uroguanylin or guanylin to the guanylin
`cyclase receptor stimulates the intracellular production of
`the cGMP ultimately resulting in the stimulation of salt and
`water secretion into the intestinal lumen. Uroguanylin and
`guanylin receptors are found on the lurninal surface of
`epithelial cells lining the intestinal tract and renal
`proximal tubules as well as in other organs. Forte et al,
`Salt and Water Homeostasis: Uroguanylin Is a Circulating
`Peptide Hormone with Natriuretic Activity, Arn. 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. Forte et
`al, Lymphoguanylin: Cloning and Characterization of a Unique
`30 Member of the Guanylin Peptide Family, Endocrinology Vol.
`140, No. 4, p.1800-1806. These 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. Unlike uroguanylin the STs from enteric
`35 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
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`conditions. Forte et al, Guanylin: a peptide regulator of
`epithelial 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.
`
`SUMMARY OF THE INVENTION
`Among the objects and features of the present invention
`may be noted the provision of a method of modulating polyps
`in the intestine of a subject, in need thereof; said
`"modulating" or "modulation" includes retarding the
`development of polyps, preventing, treating, and inhibiting
`polyps. Also, 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.
`Briefly, therefore , 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:
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`25 wherein each of X11 X2 , X3 , X4 , X5 , X 61 and X7 is an amino acid
`residue, X8 and X9 are independently hydrogen or at least one
`amino acid residue, and the polypeptide is cross-linked by a
`disulfide bond between the cystine residue immediately
`adjacent the amine group of X1 and the cystine residue
`immediately adjacent the amine group of X6 and by a disulfide
`bond between the cystine residue immediately adjacent the
`amine group of X3 and the cystine residue immediately
`adjacent the carboxy group of X7 together with a
`pharmaceutically acceptable carrier.
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`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, pro(cid:173)
`uroguanylin, and human pro-uroguanylin, guanylin,
`lymphoguanylin, prolymphoguanylin and heat stable
`enterotoxin, together with a pharmaceutically acceptable
`carrier.
`Additionally, the invention is directed to a process
`f o r 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.
`Other objects of this invention will be in part
`apparent and, in part, pointed out hereinafter.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`Figure l(a) depicts the effect of human uroguanylin on
`the stimulation of I sc where fresh mouse duodenum consisting
`of mucosa and submucosa (~lcm2
`) 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 l(b) depicts the effect of human uroguanylin on
`the stimulation of I sc where human intestinal mucosa ( ~lcm2
`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.
`
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`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. After an incubation of 72 hours, indicated
`concentrations of human uroguanylin were added in the media
`and cells were allowed to grow until they formed
`semiconfluent monolayers. Subsequently, 5-bromo-2-
`deoxyuridine (BrdU) was added (final concentration l00µM)
`and cells were re-incubated for an additional 24 hours. The
`incorporation of BrdU was measured at 450 nm as per
`manufacturer's instructions.
`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.
`Approximately 2X 10 5 cells were inoculated in 35 mm dishes
`and cultured for 7 days. Semiconfluent monolayers were
`washed with serum-free DMEM, and further incubated with the
`same media containing indicated concentrations of human
`uroguanylin. Subsequently, the cells were quickly collected
`by trypsinization and washed twice with PBS. Harvested
`cells were immediately used for DNA isolation as per the
`instructions of the DNA fragmentation analysis kit
`(Boehringer Mannheim Corp., Indianapolis, IN). The
`fragmentation of DNA was analyzed by electrophoresis using
`1.8% agarose gel followed by staining with ethidium bromide.
`Apoptotic DNA provided with the test kit was used as
`positive control, M (lane 1) and a functionally inactive
`variant of human uroguanylin (V) was used as negative
`control (lane 6). Different concentrations of uroguanylin,
`as indicated were examined (lanes 2 to 5).
`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(cid:173)
`confluent monolayers. Subsequently the cells on slide B
`were treated with human uroguanylin (1 µM) for 48 hours.
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`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
`5 depicts vehicle-treated cells. Slide B depicts uroguanylin(cid:173)
`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 (h Gua) [identified as SEQ. ID. 3], and 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 Ile) at their N-terminii. These N(cid:173)
`terminal residues make bacterial ST insensitive towards
`intestinal pH. Two underlined (Asp-Asp) residues are
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`believed to be important for regulating the functional
`activity of uroguanylin only at the acidic environment of
`the intestinal mucosa.
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`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
`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,
`10 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. Without intending to be bound by any
`theory, applicants believe that 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
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`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
`5 of gastrointestinal epithelial renewal and its relevance to
`the development of adenocarcinomas of the gastrointestinal
`J. Clin. Gastroenterol. 21: 81-11 (1995). The
`tract,
`process of apoptosis is known to be suppressed in colon
`cancer tissues. Baretton, et al, Apoptosis and
`immunohistochemical bcl - 2 expression in colorectal adenomoas
`and carcinomas. Aspectes of Carcinogenesis and prognostic
`significance, Cancer 77:255-264 (1996).
`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.
`Several sources of evidence have implicated a potential role
`of K+ efflux in the induction of apoptosis . Hughes, F.M. et
`al, Intracellular K+ suppresses the activation of apoptosis
`in lymphocytes, J.Biol.Chem. 272:30567-30576 (1997); Hughes,
`F.M. et al, Potassium is a critical regulator of apoptotic
`enzymes in vitro and in vivo, Adv. Enzyme Regul. 39:157-171
`(1999). First, a bacterial pore-forming cytolysin,
`staphylococcal a-toxin, which selectively permeabilizes
`plasma membranes for monovalent cations, was
`found to
`induce apoptosis. Bhakdi, S. et al, Release of interleukin-
`1 beta associated with potent cytocidal action of
`staphylococcal alpha-toxin on human monocytes, Infect.
`Immun. 57:3512-3519 (1989). Second, apoptotic and shrunken
`cells have been shown to contain much lower levels of
`intracellular K+ as compared to that in normal cells.
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`Hughes, F.M et al, Intracellular K+ suppresses the
`activation of apoptosis in lymphocytes, J.Biol.Chem.
`272:30567-30576 (1997). Third, an intracellular K+
`concentration more than 150mM has been shown to selectively
`inhibit Caspase-3, a proteolytic enzyme involved in the
`induction of apoptosis. Hughes, F . M. et al, Potassium is a
`critical regulator of apoptotic enzymes in vitro and in
`vivo , Adv.Enzyme Regul. 39:157-171 (1999). Finally,
`suppressing K+ efflux in whole cells prevents the activation
`of pro-apoptosis nucleases, whereas enhancing the efflux of
`this ion facilitates enzymatic activities of these
`nucleases. Hughes, F.M. 39: 157-171 (1999). Thus,
`intracellular levels of potassium balance appear to be the
`critical regulator of apoptosis.
`Without intending to be bound by any theory, applicants
`believe that there is a relationship between K+ channel
`activity and uroguanylin-induced apoptosis in colon
`carcinoma cells. Uroguanylin and guanylin have been shown
`to stimulate c1 - and K+ efflux to regulate electrolyte and
`20 water transport in the GI tract. Recently, heat-stable
`enterotoxin (STa) of Escherichia coli, a GC-C agonist
`peptide that also increases intracellular accumulation of
`cGMP and stimulates fluid secretion in the lumen of the
`intestine, has been shown to increase K+ efflux and ca+
`influx. Bhattacharya, J. et al, Rise of intracellular free
`calcium levels with activation of inositol triphosphate in a
`human colonic carcinoma cell line (COLO 205) by heat-stable
`enterotoxin of Escherichia coli, Biochem. Biophys. Acta.
`1403:1-4 (1998). Atrial naturiuretic peptide (ANP), 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 . et al, Natriuretic peptides increase a~
`conductance in rat mesangial cells, Pflugers Arch. 43:571-
`577 (1996). Furthermore, pretreatment of rat endothelial
`(10 -7M) or 8-bromo-cGMP (10- 3M) caused a
`cells with either ANP
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`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
`nitric oxide induce endothelial apoptosis via a cGMP-
`dependent mechanism, Arterioscler. Thromb. Vase. Biol.
`19:140-146 (1999). Also, CFTR expression is associated with
`K+ and c1- efflux and shrinkage of cells, characteristic
`biochemical changes found in apoptotic cells. Rotoli, B.M.
`et al, CFTR expression in C127 cells is associated with
`enhanced cell shrinkage and ATP extrusion in Cl ( -) -
`free
`medium, Biochem. Biophys. Res. Commun. 227:755-61 (1996).
`Applicants believe that 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. Applicants believe that 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. Thus, 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.
`Additionally, 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. et
`al, Guanylin cyclase C is a selective marker for metastatic
`colorectal tumors in human extraintestinal tissues, Proc.
`Natl. Acad. Sci. USA. 93:14827-14832. By contrast, the
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`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 adenocarcinoma, 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. Additionally, the expression of
`uroguanylin in human colon cancer and the adjacent normal
`tissues has been examined. Thus, the expression of both
`uroguanylin and guanylin is completely diminished in all
`human colon cancer specimens examined. This study suggests
`that either the reduced expression of uroguanylin and/or
`guanylin leads to or is a result of adenocarcinoma
`formation.
`The applicants also demonstrate that treatment
`with uroguanylin results in the induction of apoptosis in T-
`84, human colon carcinoma cells, and that the oral
`administration of human uroguanylin leads to inhibition in
`polyp formation in the intestinal tract of Min-mouse, an
`animal model for human Familial Adenomatous Polyposis (FAP).
`Both guanylin and uroguanylin genes have recently been
`mapped on the mouse chromosome 4 and to a synthetic position
`on human chromosome lp34-35. Sciaky, D. et al, Mapping of
`guanylin to murine chromonsome 4 and human chromosome lp34-
`35, Genomics 26:427-429 (1995); Whitaker, T.L. et al, The
`uroguanylin gene (Guca lb) is linked to guanylin (Guca 2) on
`mouse chromosome 4, Genomics 45:348-354 (1997). This region
`is frequently associated with the loss of heterozygosity in
`human colon carcinoma. Leister, I. et al, Human colorectal
`cancer: high frequency of deletions at chromosome lp35,
`Cancer Res. 50:7232-7235 (1990).
`In the min-mouse tumor
`model, ademona multiplicity and growth rate are regulated by
`APC, the tumor suppressor gene, which is also localized to
`mouse chromosome 4 in a region syntenic with human
`chromosome lp34-36. Dietrich, W.F. et al, Genetic
`identification of Mom-1, a major modifier locus affecting
`Min-induced intestinal neoplasia in the mouse, Cell 75:631-
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`639 (1992). The APC gene is mutated in the vast majority of
`humans with colorectal cancer. Miyoshi, Y. et al, Somatic
`mutations of the AFC gene in colorectal tumors: mutation
`cluster region in the AFC gene, Hum. Mol. Genet. 1:229-233
`(1992). The principal function of this gene is to regulate
`cell cycle via the wnt signal transduction cascade.
`Cadigan, K.M. et al, Wnt signaling: a common theme in animal
`development, Genes Dev. 11:3286-3305 (1997). Thus, the
`uroguanylin and guanylin peptides may be involved early in
`the process of colon carcinogenesis.
`In accordance with the process of the present
`invention, therefore, 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:
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`25 wherein each of X11 X2 , X3 , X4 , X5 , X6 , and X7 is an amino acid
`residue, X8 and X9 are independently hydrogen or at least one
`amino acid residue, and the polypeptide is cross-linked by a
`disulfide bond between the cystine residue immediately
`adjacent the amine group of X1 and the cystine residue
`immediately adjacent the amine group of X6 and by a disulfide
`bond between the cystine residue immediately adjacent the
`amine group of X3 and the cystine residue immediately
`adjacent the carboxy group of X7 • Preferably, the
`polypeptide is guanylan, uroguanylin, pro-uroguanylin, or
`another polypeptide which contains the active domain of
`uroguanylin.
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`As is known in the art, 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.
`In making such changes, it is preferable
`that amino acids having hydropathic indices within ±2 are
`substituted for one another. More preferred substitutions
`are those wherein the amino acids have hydropathic indices
`10 within ±1. Most preferred substitutions are those wherein
`the amino acids have hydropathic indices within ±0.5.
`Like amino acids can also be substituted on the basis
`of hydrophilicity. U.S. Patent No. 4,554,101 discloses that
`the greatest local average hydrophilicity of a protein, as
`governed by the hydrophilicity of its adjacent amino acids,
`correlates with a biological property of the protein. The
`following 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.