JP2003516316A - Uroganiline as an intestinal cancer inhibitor - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method of preventing, suppressing and treating intestinal polyps expression and cancer in a subject, comprising a peptide having an active domain of uroguanylin, or any agonist that binds to the guanylate cyclase receptor GC-C in the intestine. It involves administering a composition containing the peptide or compound.

Description

Detailed Description of the Invention

This application is US Provisional Application # 60/157 filed October 6, 1999.
, 950 is a patent application claiming priority.

BACKGROUND OF THE INVENTION The present invention relates to the use of certain peptides in the expression of polyps in the intestine and the prevention, suppression or treatment of cancer, more particularly uroguanylin, prouroguanylin, guanylin, And other similar peptides.

The etiology of colorectal cancer is characterized by a multistep process that begins with increased proliferation and / or decreased apoptosis of polyp-expressing colorectal epithelial cells, followed by adenoma formation and terminal adenocarcinoma formation. . One individual subsequently develops multiple colorectal adenomas due to hereditary defects in the APC gene in response to the development of a condition called familial adenomatous polyposis (FAP) and develops cancer early in life. . Dihlmann et al., "The most potent negative effect of APC1309 mutation: a possible explanation for genotype-phenotype interactions in familial adenoma polyposis".
Cancer Res. 1999, Apr. 15, 59 (8): 1857-60.
. In the last few decades, chemoprevention methods have emerged as a useful strategy for cancer prevention in order to control the onset of cancer by pharmacological effects and / or dietary interventions prior to the development of clinically detectable tumors. Was included. Reddy, B.A. S. (199
7): "Chemical prevention of colon cancer by dietary administration of naturally occurring and related synthetic agents"; Adv. Dev. Med. Biol. 400B: 931-936.

Uroguanylin and guanine are structurally related intestinal peptides that are endoluminally mediated by a variety of cell types including enterochromaffins, goblets and others present within the intestinal mucosal lining. Secreted.
The receptors for these peptides that have been identified at the molecular level are the transmembrane forms of guanylate cyclase (GC) known as GC-C. Kraus
e, W.E. J. "Guaniline and uroguanylin peptide hormones and their receptors": Acta. Anat. (Basel) 160: 213-2
31 (1997). The GC-C receptor is located on the luminal surface of intestinal cysts throughout the GI tract. Swenson, E .; S. "Guaniline / STa receptors are expressed in the anterior and apical epithelium of the entire mouse intestine.": Biochem. Bi
ophys. Res. Commun. 225: 1009-1014 (1996).
.

The binding of uroguanylin or guanylin to the extracellular domain of the GC-C receptor stimulates intracellular production of the second messenger cGMP and induces the cystic fibrosis transmembrane conductance regulator.
tance regulator (CFTR), which results in activation of the apical passage for chloride outflow from the intestinal cysts that line the gastrointestinal tract. Forte, L .; R. "Salt and water homeostasis: uroguanylin is a circulating peptide hormone with natriuretic activity.": An. J. Kidney Dis. 28:29
6-304 (1996). Activation of the CFTR chloride pathway protein and subsequent increase in epithelial transsecretion of chloride results in sodium (Na +) into the intestinal lumen.
And leads to stimulation of water secretion. Forte, L .; R. Et al., "Guaniline-regulating peptide: structure, biological activity and etiology mediated by cyclic GMP": Regu.
l. pept. 81: 25-39 (1999). Therefore, the physiological function of these hormones is to regulate fluid and electrolyte transport in the gastrointestinal tract (GI) by acting as a paracrine regulator of CFTR activity.

The precursor of uroguanylin is prouroguanylin, which is degraded by endogenous proteases in the intestinal tract to produce active uroguanylin. Chymotrypsin activates prouroguanylin and degrades prouruguaniline to the active form of uroguanylin. Forte, L .; R. Et al., "Salt and Water Homeostasis: Uroguanylin is a Circulating Peptide Hormone with Natriuretic Activity.": Am. J. Kid. Dis. 1996, 28, No. 2,296-30
4. Uroguanylin is acid stable and is a proteolytic-resistant peptide that stays in the intestinal tract and acts directly in the intestinal cavity rather than being systemically absorbed.

Uroguanylin and guanylin are produced throughout the intestinal mucosa and in the myocardium. Forte, L .; R. "Salt and water homeostasis: uroguanylin is a circulating peptide hormone with natriuretic activity.": An. J
． Kidney Dis. 28: 296-304 (1996). Human Uroguanylin was isolated from human urine and
), And is chemically synthesized by solid phase peptide synthesis as described in US Pat. No. 5,489,670. In addition, human guanylin has been isolated from human enterocytes and chemically synthesized by solid phase peptide synthesis as described in US Pat. No. 5,969,097 entitled Human guanylin. There is.

Binding of uroguanylin or guanylin to the guanylate cyclase receptor stimulates intracellular production of cGMP, ultimately leading to stimulation of salt and water secretion into the intestinal lumen. The uroguanylin and guanylin receptors are found on the luminal surface of epithelial cells lining the intestinal and rectal proximal tract and other organs. Forte, L .; R. "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 a product of E. Pathogens of E. coli and other intestines that activate intestinal guanylyl cyclase and cause secretory diarrhea, which is a major cause of traveler's diarrhea and considerable death in developing countries. Cyclic GM in a manner similar to another family of thermostable enterotoxins (STs) secreted by endophytes
Stimulates increased P levels. Forte et al., "Lymphatic Guaniline: Cloning and Characterization of Specific Members of the Guaniline Peptide Family": Endocrino.
logic, Vol. 140, No. 4, p. 1800-1806. These ST
The peptides act as molecular mimetics of the endogenous mammalian peptides of uroguanylin and prouroguanylin. Forte et al., Endocrinology, V
ol. 140, No. 4, p. 1800. Unlike uroguanylin, ST from enterobacteria does not diminish its potency when pH changes in the colon.

ST is more potent than either uroguanylin or guanylin under both acidic and alkaline conditions. Forte et al., "Guaniline: a peptide regulator of epithelial transport": The FASEB Journal, Vol. 9,64
3-650 (1996). Uroguanylin is believed to regulate liquid and electrolyte transport in a manner similar to ST and guanylin in the GI tract. Thus, as disclosed in prior publications, human uroguanylin can act as a laxative and can be useful in patients with constipation.

SUMMARY OF THE INVENTION Among the objects and features of the present invention, it can be noted that there is provided a method of controlling intestinal polyps in a subject in need thereof. Here, the term "regulate" or "control" includes delaying the onset of polyps and preventing, treating and suppressing polyps. The invention also relates to methods of preventing, controlling and treating cancer in the intestines (small intestine and colon) of a subject in need thereof.

Thus, in brief, the present invention relates to a method of inhibiting intestinal polyps in a subject in need thereof, which method comprises the sequence:

[Chemical 2] (In the formula, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each an amino acid residue, and X ₈ and X ₉ are independently hydrogen or at least one amino acid. And a cystine residue directly adjacent to the amine group of X ₁ and a cystine residue immediately adjacent to the amine group of X ₆ are crosslinked by a disulfide bond. A polypeptide in which a cystine residue directly adjacent to the amine group of X ₃ and a cystine residue immediately adjacent to the carboxy group of X ₇ are cross-linked by a disulfide bond together with a pharmaceutically acceptable carrier Administering a pharmaceutical composition containing the same.

The present invention also relates to a method for controlling polyps in a subject and a method for preventing, suppressing and treating cancer in the intestinal tract, which method comprises the following peptides: uroguanylin, human uroguanylin, guaniline, lymphoguaniline, prolymphguaniline and Administering a pharmaceutical composition containing any one or a combination of heat stable enterotoxins together with a pharmaceutically acceptable carrier.

Further, the present invention provides a method for controlling intestinal polyps in a subject in need thereof and a method for preventing, suppressing and treating intestinal cancer in the subject, which comprises an agonist peptide for guanylyl cyclase receptor GC-C and And / or administering any of the other agonist compounds or combinations thereof in combination with a pharmaceutically acceptable carrier. Other objects of the invention are considered to be in part apparent or in part pointed out in the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 (a) shows the effect of human uroguanylin on the stimulation of I _SC .
In this case, a mouse duodenum (-1 cm ² ) consisting of mucosa and submucosa was placed between the bisected Ussing Chambers and bathed on both sides as described. At the time indicated by the arrow, the indicated concentration of TTX and uroguanylin (ur
oG) and carbachol (apical reservoir)
Was added to. Electrical measurements were monitored with an automatic voltage clamp. FIG. 1 (b) shows the effect of human uroguanylin on the stimulation of I _SC ,
In this case, human intestinal mucosa (-1 cm ² ) was placed between the bisected Ussing chambers and bathed on both sides as described. At the time indicated by the arrow, TTX of the indicated concentration,
Uroguanylin (uroG) and carbachol were added to the tip reservoir. Electrical measurements were monitored with an automatic voltage clamp.

FIG. 2 is a graph showing the effect of human uroguanylin on the inhibition of T-84 human cancer cell proliferation. These cells were seeded in 96-well plates. 72
After incubation for a period of time, the indicated concentration of human uroguanylin was added to the medium and these cells were grown until they formed a semi-confluent monolayer. Then 5-
Bromo-2-deoxyuridine (BrdU) was added (final concentration 100 μM),
The cells were then reincubated for an additional 24 hours. BrdU uptake,
Measured at 450 nm as per manufacturer's instructions.

FIG. 3 shows the results of analysis of fragments of T-84 human colon cancer cells after treatment with human uroguanylin by electrophoresis on a 1.8% agarose gel followed by staining with ethidium bromide. . Approximately 2X10 ⁵ cells, 35m
m dishes were inoculated and then cultured for 7 days. These semi-confluent monolayers were washed with DMEM without serum and further incubated with the same medium containing the indicated concentration of human uroguanylin. Subsequently, these cells were rapidly harvested by trypsinization and then washed twice with PBS. The harvested cells were immediately used for DNA isolation according to the instructions of the DNA fragmentation analysis kit. The DNA fragments were analyzed by electrophoresis on a 1.8% agarose gel followed by staining with ethidium bromide. Apoptotic DNA provided by the test kit was used as a positive control, M (lane 1) and functionally inactive human uroguanylin variant (V) were used as negative controls (lane 6). ). Various concentrations of uroguanylin were tested as indicated (lanes 2-5).

FIG. 4 shows a microscope slide of a semi-confluent monolayer of Caco-2 cells, demonstrating the effect of human uroguanylin on the expression of apoptosis. Cells were cultured on microscope slides until they formed a semi-confluent monolayer. Then,
"In situ cell death detection kit" (Boehriger Mannheiem Corp., Indianapolis, Indi)
The cells on slide B were detected by fluorescence microscopy directly after UNEL reaction according to the instructions of (ana). Slide A shows media treated cells. Slide B
Shows uroguanylin treated cells.

FIG. 5 (a) shows Northern blotting analysis demonstrating that the expression of uroguanylin and guanylin is suppressed in human colon cancer cells. FIG. 5 (b) shows RT-PCR demonstrating that the expression of uroguanylin and guanylin is suppressed in human colon cancer cells, followed by Southern blotting analysis.

FIG. 6 (a) is a graph showing the increase in daily food consumption by Min-mouse after oral administration of human uroguanylin. The total food consumption per day (24 hours) by 5 animals (5) / cage was measured and used for the calculation of total food consumption / mouse / day. Results are expressed as mean ± standard error. FIG. 6 (b) is a graph showing the increase in body weight gain by Min-mouse after oral administration of human uroguanylin. All animals were weighed weekly during the study.
Results are expressed as mean ± SD of weight gain / mouse during the study.

FIG. 7 shows human uroguanylin (h UronG) [identified as SEQ ID NO: 2], human guanylin (h Gua) [identified as SEQ ID NO: 3], and bacterial enterotoxin (E. coli [SEQ ID NO: 2]). No .: 4] and V. cholerae [identified with SEQ ID NO: 5]). Bold and italic letters represent similar residues in these peptides. These residues are thought to be necessary for the functional activity of these peptides. At their N-termini, E. coli ST has three additional residues (Asn-Se).
r-Ser), and V. cholerae has two additional residues (Leu
And Ile). These N-terminal residues render the bacterial ST insensitive to pH. The two underlined residues (Asp-Asp) are believed to be important in regulating the functional activity of uroguanylin only in the acid environment of the intestinal mucosa.

Detailed Description of the Preferred Embodiments Uroguanylin is produced naturally by the goblet cells of the intestinal mucosal lining in the form of prouroguanilin in a functionally inactive form, which is then functional in the intestine by endogenous proteases. Is converted to the active uroguanylin. Uroguanylin is an acid stable, proteolytically resistant peptide. Thus, orally released prouroguanylin and uroguanylin act on the surface of the intestinal lumen and are not systemically absorbed. Similar peptides containing amino acid sequences similar to uroguanylin, prouroguanylin and other active domains are expected to induce apoptosis and cell death in the intestinal mucosal cell lining. Intestinal mucosal cell lining-induced apoptosis is expected to delay polyp formation and subsequent development of intestinal cancer.

Without wishing to be bound by any theory, the Applicant has found that the peptides of the present invention increase the rate of apoptosis, cell death in the intestinal mucosal cell lining, cell proliferation and programmed cells. Exerts their effect by promoting a favorable balance between death and thus delays the growth of polyps and also has receptors for cancer and guanylin, uroguanylin, lymphoguanylin and STa family of peptides in the intestine, We believe that other epithelial-derived cancers will be prevented, suppressed, and treated.

The rate of cell proliferation and cell death in the intestinal mucosa is very rapid. The cells of the intestinal mucosa alternate in a certain state, ensuring a favorable balance between cell proliferation and cell death. The constant and rapid renewal of the GI tract epithelium maintains the integrity of normal mucosa and exerts the repair and recruitment functions of differentiated specialized epithelial cells. Prevention of apoptosis in the intestinal mucosa resulting in an imbalance in the renewal process results in an increased incidence of polyp formation and subsequent incidence of intestinal cancer. Eastw
Wood et al., "Introduction to gastrointestinal organ epithelial renewal and its relevance to the development of gastrointestinal organ adenocarcinoma": J. Clin. Gastroenterol. 21: S1-11 (1
995). The process of apoptosis is known to be suppressed in colon cancer tissues. Barettton et al., "Apoptosis and immunohistochemistry bcl-2 expression in colorectal adenomyomas and cancers. Significance of oncogenic and prognostic signs":
Cancer 77: 255-264 (1996).

A key cellular feature of the apoptotic process is a marked loss of cell volume, which is associated with ion migration and is accompanied by homeostasis achieved by an osmotic balance across the plasma membrane. Hoffman, E .; K. Et al., "Membrane Mechanism of Intracellular Signaling in Cell Volume Regulation": Int. Rev. Cytol. 161:
173-262 (1995). Most mammalian cells contain Na ⁺ / K ⁺ ATPas
This osmotic pressure is achieved and maintained by the continuous action of the e-pump. In this case, a gradient of these monovalent cations across the membrane is created. Several lines of evidence have linked a potential role for K ⁺ efflux in the induction of apoptosis. Hugh
es, F.F. M. Intracellular K ⁺ suppresses activation of apoptosis in lymphocytes: J. Biol. Chem. 272: 30567-30576 (199
7); 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, the bacterial pore-forming cytolysin, Staphylococcus alpha-toxin, which selectively permeates plasma membranes for cation transfer, has been found to induce apoptosis. Bhakdi, S .; Et al., "Interleukin-associated with the potent cytocidal effect of Staphylococcus α-toxin on human monocytes.
1β ”: Infect. Immun. 57: 3512-3519 (1989).
Second, dead and contracting cells were shown to contain even lower levels of intracellular K ⁺ compared to those in normal cells. Hughes, F .; M. etc,"
Intracellular K ⁺ suppresses activation of apoptosis in lymphocytes. ": J. Bi
ol. Chem. 272: 30567-30576 (1997).

Third, intracellular K ⁺ concentrations greater than 150 mM have 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). Furthermore,
Inhibition of K ⁺ efflux in whole cells prevents activation of pro-apoptotic nucleases, whereby enhanced efflux of this ion promotes enzymatic activity of these nucleases. Hughes, F .; M. 39: 157-171 (1999). Therefore, intracellular levels of potassium balance are considered to be critical regulators of apoptosis.

Without wishing to be bound by any theory, Applicants believe that there exists a relationship between K ⁺ channel activity and uroguanylin-induced apoptosis in colon cancer cells. Aloganiline and guaniline have been shown to stimulate Cl ⁻ and K ⁺ efflux and regulate electrolyte and water transport in the GI tract. Recently, heat-stable enterotoxins (STs) of Escherichia coli, which also increase intracellular accumulation of cGMP in the intestinal lumen and stimulate fluid secretion, are associated with K ⁺ efflux and Ca. ⁺ Proven to increase influx. Bhattacharya, J. et al. Etc., "Eschericya
Elevation of intracellular free calcium levels by activation of inositol triphosphate in a human colon cancer cell line (COLO205) by a heat-stable enterotoxin of Escherichia coli ": Biochem. Biophys. Acta
． 1403: 1-4 (1998).

Atrial natriuretic peptide (ANP), which is a peptide that stimulates intracellular accumulation of cGMP by binding to specific GC receptors
Also activates K ⁺ conductance in rat glomerular stromal cells, and c
It has been shown to induce apoptosis in myocardium via a GMP-dependent mechanism. Cermak, R .; Et al., "Atrial Peptides Increase K ⁺ Conductance in Rat Glomerular Stromal Cells.": Pflugers Arch. 43:
571-577 (1996). Furthermore, pretreatment of rat epithelial cells with either ANP (10-7M) or 8-bromo-cGMP (10-3M)
It resulted in a distinct accumulation of the nuclear phospholipid, p53, a tumor suppressor protein known to induce apoptosis in many cell types. Sue
nobu, N .; , "Natriuretic peptides induce epithelial apoptosis by a cGMP-dependent mechanism.": Arterioscler. Th
romb. Vasc. Biol. 19: 140-146 (1999).

CFTR expression is also associated with K ⁺ and Cl ⁻ efflux and cell contraction, characteristic biochemical changes found in abototic cells. Rotoli, B.A.
M. Et al., "CFTR expression in C127 cells results in increased cell contraction and Cl
Accompanied by ATP extrusion in (−)-free medium. ": Biochem. Bi
ophys. Res. Commun. 227: 755-61 (1996). Applicants have shown that uroguanylin, prouroguanylin, guanylin and other similar peptides induce apoptosis of epithelial cells lining the GI tract mucosa by maintaining intracellular concentrations of K ⁺ ions as a result of binding to the GC-C receptor. Believe that it can trigger. Applicants have found that the binding of the GC-C receptor is cGM.
It is believed to activate the CFTR chlorine channel, which stimulates the production of P, thereby causing an increase in K ⁺ efflux. Therefore, the induction of apoptosis also leads to GC
Expected from administration of agonist peptides that bind to the -C receptor and other receptors for guanylin, uroguanylin and lymphoguanylin in the intestine.

Furthermore, guanylin has been shown to completely reduce colon cancer cells and be uniformly expressed in normal intestinal mucosal cells. This finding suggests that guanylin is involved in maintaining colon differentiation or function as a tumor suppressor gene. M
itchell et al., "Guanilin mRNA and expression in human intestinal and colorectal adenocarcinoma": Lab. Invest. 1998, Vol. 78, No. 1,10
1-108. Recent data demonstrate that the guanylyl cyclase receptor, known as GC-C, is expressed in all primary and metastatic colorectal cancers and can act as a specific marker for these tumors. . Ca
rlithers, S .; L. "Guanylyl cyclase C is a selective marker of metastatic colorectal cancer in human extraintestinal tissue.": Proc. Natl. Ac
ad. Sci. USA, 93: 14827-14832. In contrast, guanylin expression has been shown to be down-regulated in colorectal cancer tissues and cell lines. Cohen, M .; B. "Guaniline mRNA expression in human intestinal and colorectal adenocarcinoma": Lab. Invest. 78: 101-108.

The tests illustrated in the examples described herein show that uroguanylin is also completely reduced in colon cancer cells and evenly distributed in normal intestinal mucosal cells. Furthermore, uroguanylin expression in human colon cancer and adjacent normal tissues was tested. That is, the expression of both uroguanylin and guanylin is completely reduced in all human colon cancer specimens tested. This test indicates that decreased expression of either uroguanylin and / or guanylin leads to adenocarcinogenesis.
Or suggests that it is the result. Applicants have also shown that treatment with uroguanylin results in the induction of apoptosis in human colon cancer cells, T-84, and oral administration of uroguanylin is Min-mouse [this mouse is a human familial adenomatous polyposis. (FAP) animal model] leads to inhibition of polyp formation in the intestinal tract.

Both the guanylin and uroguanylin genes have recently been mapped to mouse chromosome 4 and to synthetic positions p34-35 on the human chromosome. Sci
aky, D.A. "Mapping guanylin to mouse chromosome 4 and human chromosome 1p34-35": Genomic, 26: 427-429 (1995); W.
hitaker, T .; L. Et al., "Uroguanylin gene (Guca1b) binds to guanylin (Guca2) on mouse chromosome 4": Genomics, 4
5: 348-354 (1997). This region is often associated with loss of heterozygosity in human colon cancer. ": Leister, I. Et al., "Human Colorectal Cancer:
Missing frequency on chromosome 1p35 ": Cancer Res. 50: 7322-
7235 (1990). In the min-mouse tumor model, adenoma diversity and growth rate are regulated by the tumor suppressor gene APC, which is also localized to mouse chromosome 4 in regions of the human chromosome and synteny. Dietr
ichi, W.I. F. Et al., "Min-1 gene identification: major modifier loci affecting Min-induced intestinal neoplasms in mice": Cell 75: 631-639 (1
992).

The APC gene is mutated in the majority of humans with colorectal cancer. Miyo
shi, Y. "Somatic mutation of APC gene in colorectal tumor: mutation cluster region of APC gene": Hum. Mol. Genet. 1: 229-23
3 (1992). The basic function of this gene is to regulate the cell cycle through the wnt signal transduction cascade. Cadigan, K .; M. etc,"
wnt Signaling: A Common Topic in Animal Expression ": Cene Dev. 11
: 3286-3305 (1997). Therefore, uroguanylin and guanylin peptides may be involved early in the process of colon carcinogenesis.

Accordingly, according to the methods of the present invention, a polypeptide containing an active domain of human uroguanylin or binding to the intestinal guanylyl cyclase receptor GC-C of the subject is administered to the subject. The polypeptide can be administered prophylactically, but is typically administered to a subject who has been established to have a genetic predisposition to intestinal cancer, intestinal polyps, or polyp growth in value. be able to.

In a preferred embodiment of the invention, this peptide has the following sequence identified in SEQ ID NO: 1:

[Chemical 3] (Wherein, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each an amino acid residue, and X ₈ and X ₉ are independently hydrogen or at least A single amino acid residue) between the cystine residue immediately adjacent to the amine group at X ₁ and the cystine residue immediately adjacent to the amine group at X _6. Is cross-linked by a disulfide bond, and the cystine residue directly adjacent to the amine group of X ₃ and the cystine residue directly adjacent to the carboxy group of X ₇ are cross-linked by a disulfide bond. Is a guanylin, uroguanylin, prouroguanylin, or another polypeptide containing the active domain of uroguanylin.

As is known in the art, certain amino acids in peptides or proteins have a similar hydropathic index or score,
It can also be replaced with another amino acid that is capable of producing a peptide or protein that has a similar biological activity, ie, still retains biological function. When carrying out such changes, it is preferable to use an amino acid having a hydropathic index within ± 2 instead of another amino acid. Further preferred replacements are those in which the amino acid has a hydropathic index within ± 1. The most preferred replacement is one in which the amino acid has a hydropathic index within ± 0.5.

Amino acids can also be replaced on the basis of hydrophilicity. U.S. Pat.No. 4,555
No. 4,101 discloses that the maximum local average hydrophilicity of a protein correlates with the biological properties of the protein, as governed by the hydrophilicity of its flanking amino acids. The following hydrophilicity values have been estimated for amino acids: arginine / lysine (+3.0); aspartate / glutamate (+ 3.0 ± 1); serine (+3.0); asparagine / glutamine (+0.2). Glycine (0); Threonine (-0.4);
Proline (-0.5 ± 1); Alanine / Histidine (-0.5); Methionine (-1.3); Valine (-1.5); Leucine / Isoleucine (-1)
． 8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).

Accordingly, one amino acid in a peptide, polypeptide, or protein is replaced by
It can be replaced by another amino acid that has a similar hydrophilicity score and that also has a similar biological activity, ie still possesses the correct biological function. When such a conversion is performed, it is preferable to replace amino acids having a hydropathic index within ± 2 with each other, and it is more preferable to replace an amino acid having a hydropathic index within ± 1 with ± 0.5. Most preferred is the replacement with an amino acid having a hydropathic index within.

As outlined above, amino acid substitutions in the peptides of the present invention result in amino acid substitutions in the inactive domain of the peptide in order to form a protein with the same biological activity as the human uroguanylin peptide. It can be based on the relative similarity of chain substituents. Therefore, X ₁ can be selected from the group of all amino acid residues, but selected from the group of amino acid residues consisting of aspartic acid, glutamic acid, glycine, lysine, asparagine, proline, glutamine, arginine, serine and threonine. It is preferable. Further preferred amino acid residues that can be replaced for X ₁ are glutamic acid, aspartic acid, arginine and lysine.

The most preferred amino acid residue that can be used for X ₁ is glutamic acid.
X ₂ can be selected from all amino acid residues, but suitable amino acids for this replacement are leucine, isoleucine, tyrosine, phenylalanine, tryptophan, valine, methionine, cysteine, alanine, histidine, proline, threonine, glycine. , Asparagine, and glutamine. Further preferred amino acid residues that can be replaced for X ₂ are cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, valine, and tyrosine. Among the more preferred amino acid residues described above, the more preferred amino acid residues for replacement of X ₂ are leucine, isoleucine, tyrosine, valine, and methionine. The most preferred amino acid for replacement of X ₂ is leucine.

Furthermore, as mentioned above, X ₃ and X ₄ can be selected from all amino acid residues, preferred amino acid residues being valine, isoleucine, tyrosine,
Phenylalanine, tryptophan, methionine, cysteine, alanine, histidine, proline, threonine, glycine, glutamine, asparagine, and serine. More preferred amino acid residues that can be replaced for X ₃ and X ₄ are valine, isoleucine, leucine, tyrosine, phenylalanine,
Methionine, cysteine, alanine, histidine, and proline. Among the more preferred amino acid residues above, the more preferred amino acid residues for replacement of X ₃ and X ₄ are valine, isoleucine, leucine, methionine, and cysteine. The most preferred amino acid for replacement of X ₃ and X ₄ is valine.

[0043]   Also, X_FiveCan be selected from all amino acid residues, but preferred amino
Acid residues are alanine, histidine, cysteine, methionine, valine, leucine
, Isoleucine, tyrosine, phenylalanine, proline, threonine, gluta
Min, asparagine, and serine. X_FiveCan be replaced about
More preferred amino acid residues are alanine, histidine, cysteine and proline.
, Threonine, glycine, glutamine, asparagine, and serine. X _Five The most preferred amino acid residue for substitution is alanine.

Furthermore, although X ₆ can be selected from all amino acid residues, suitable amino acid residues for this replacement are threonine, proline, alanine, histidine, cysteine, methionine, valine, leucine, isoleucine. , Tyrosine, glycine, glutamine, asparagine, and serine. Further preferred amino acid residues that can be replaced for X ₆ are threonine, proline, alanine, histidine, cysteine, methionine, glycine, glutamine, asparagine, and serine. Further preferred amino acid residues for the replacement of X ₆ are:
Threonine, proline, alanine, histidine, and glycine. The most preferred amino acid residue for replacement of X ₆ is threonine.

X ₇ can be selected from all amino acid residues, and preferred amino acid residues are glycine, threonine, proline, alanine, histidine, cysteine, methionine, valine, leucine, isoleucine, glutamine, Asparagine, serine, glutamic acid, and aspartic acid. Further preferred amino acid residues that can be replaced for X ₇ are glycine, threonine, proline, alanine, histidine, cysteine, glutamine, asparagine, and serine. Further preferred amino acid residues for the replacement of X ₇ are glycine, threonine, proline, alanine, histidine, glutamine, asparagine, and serine. The most preferred amino acid residue for replacement of X ₇ is glycine.

The polypeptides of the invention serve in the art to act as pharmaceutically acceptable carriers allowing pharmaceutical administration in the form of, for example, injection solutions, suspensions, emulsions, tablets, capsules and ointments. It can be combined with various known excipients and / or auxiliaries. These pharmaceutical compositions can be administered by any acceptable means. For warm-blooded animals, especially for humans, administration can be oral, parenteral, subcutaneous, intravenous, intramuscular and / or intraperitoneal. The particular dose administered will depend on the general health and condition symptoms of the subject, as well as the age and weight of the subject, the stage of the disease state of the subject, any current treatment, and frequency of administration; In particular, this dose will range from about 0.5 mg / kg to about 2.0 mg / kg for human subjects. Generally, the composition will comprise at least about 0.0001% by weight, more typically at least about 0.001% by weight, and even more typically, one or more polypeptides of the present invention, based on the weight of the composition. By the way
At a concentration of at least about 0.01%, more typically at least about 0.1%, and in some embodiments at least about 1%.

Human uroguanylin cDNA has been cloned in bacteria and has been chemically synthesized by solid phase peptide synthesis. Uroguanylin peptides can be chemically synthesized by using the methods described in US Pat. No. 5,489,670 (human uroguanylin) and US Pat. No. 5,140,102 (pentadecapeptide guanylin, which stimulates intestinal guanylyl cyclase). it can. Peptides similar to uroguanylin have been identified in the species mouse, rat, pig, and cow. In most of these peptides, the functionally active domain is highly conserved.
Therefore, the physiological functions of these peptides can be similar, and these peptides can be successfully used as prophylactic agents for intestinal cancer. That is, as long as the functionally active domains of these peptides are conserved, replacements in the inactive domain can be obtained without alteration in the activity of the peptides.

All references, US or foreign patents or applications cited herein are hereby incorporated by reference as if set forth herein. To further illustrate the invention, the following specific laboratory practice operations were performed. However, it should be clear that the invention is not limited to these examples or the detailed description herein.

Examples and Materials Cell Culture: T-84 cells are available from the American Type Culture.
Obtained in passage 52 from Collection. The cells are Ham's F-
Dulbecco's supplemented with 12 medium and 100 μg / ml streptomycin
Co's) denatured Eagle's medium (DMEM) was grown in a 1: 1 mixture. Cells were fed with fresh medium every 30 days and detached at approximately 80% confluence. Human tissues: normal colon and tumor, Missouri University
Obtained after colectomy in adenocarcinomas according to the human experimental method approved by the / Truman VA Hospital Committee. Mucosal samples from normal colon tissue adjacent to colon adenocarcinoma were separated from submucosa by scraping the mucosal surface with a microscope slide to separate mucosa from underlying tissue. Tumor areas were harvested and processed as whole tissue. 82 to 82 representing male and female patients
Tissues from 11 subjects up to age 10 were used in this study.

Example 1 Materials and Methods Cell Proliferation Assay: Approximately 10,000 T-84 cells were seeded in each well of a 96-well plate. After a 3 day incubation period, the indicated concentration of human uroguanylin was added to the medium and the cells were grown until they formed a semi-confluent monolayer. Subsequently, BrdJ labeling agent (5-bromo-2′-deoxyuridine in PBS) was added (final concentration: 100 μM), then the cells were reincubated for a further 24 hours. The monolayer is washed and then according to the manufacturer's instructions (Boehringer M
Annheim Corp., Indianapolis, IN), BrdU incorporation was measured.

Results: Uroguanylin treatment causes a dose-dependent inhibition of the growth of these cells, reaching almost 30% growth inhibition at 1 μM, as shown in FIG. In contrast, the biologically inactive species of this peptide did not show cytostatics, suggesting that growth inhibition is a receptor-mediated event.

Example 2 Materials and Methods Apoptosis analysis: T-84 cells were grown in 35 mm dishes for 7 days. The confluent monolayers were washed once with DMEM without serum and then incubated for 16 hours in the same medium containing various concentrations of human uroguanylin. After this incubation, cells were rapidly harvested by trypsinization and then the cell pellet was washed twice with phosphate buffered saline (PBS). Cells were resuspended in PBS at a concentration of approximately 10 ⁸ cells / ml. To demonstrate the nucleosome ladder, apoptotic DNA was isolated from these cells 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 is also described in "In situ cell death detection kit".
detection kit) (Boehringer Mannheiem Corp., Indianapolis Indiana) was further verified by using the TUNEL assay.

Results: As shown in FIG. 3, DNA isolated from the control (lane 2) and DNA isolated from cells treated with the biologically inactive uroguanylin variant (lane 6) were: It showed very low levels of DNA fragmentation. This is consistent with a low basal rate of apoptosis under serum-free conditions. On the other hand, DNA from uroguanylin-treated cells showed large-scale DNA fragmentation in a dose-dependent manner. Apoptosis by uroguanylin treatment (NDCELCVNVACTGC
L) Peptide was added to the Multiple Peptide System (San D
iego, CA). The biological activity of the synthesized peptides was evaluated by a modified cell-based assay. Induction of apoptosis by treatment with uroguanylin was further demonstrated by using terminal dexoneus cleotidyl (using CaCO-2 cells).
dexoynucleotidyl) tranferase mediated d
UTP-Biotin Nick End Labeling (TUNEL) (also confirmed in the assay. Uroguanylin treatment significantly enhanced apoptotic cells compared to the vehicle treated cells seen in Figure 4. These results indicate that uroguanylin is a human colon tumor. It was confirmed to induce apoptosis in cells (T-84 and CaCo-2).

Example 3 Urguaniline Functional Assay Human urguaniline (NDDCELCVNVACTGCL) peptide was synthesized using a conventional multiple peptide system (San Diego, CA). The physiological activity of synthetic peptides is determined by a modified cell-based assay (a modified c
The analysis was carried out by the well-based assay. Briefly, confluent monolayers of T-84 cells were plated in 24-well plates at 50
Washed twice with 250 μl DMEM containing mM HEPES (pH 7.4) and 3 times for 1 min with 250 μl DMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX).
It was preincubated at 7 ° C. and then with various concentrations of human uroguanylin (10 ⁻⁶ to 10 ⁻¹⁰ M). The medium was aspirated and the reaction was then stopped by the addition of 3% perchloric acid. The plate was centrifuged at 1000 xg for 5 minutes and the supernatant was collected. After neutralization with 0.1 N NaOH, the supernatant was used directly for the determination of cGMP by using the ELISA kit from Caymen Chemical, Ann Arbor, MI. Results are expressed as the average of 3 measurements. Results: Biological activity was found with several isoforms of this peptide, as indicated by the cGMP levels found.

Example 4 Ussing Chamber Test: The seromuscular layer of human intestinal mucosa was separated by rough dissection, using 1-4 mucosal sheets from each sample (-1 cm). Animals were sacrificed by 100% CO ₂ inhalation to harvest the intestinal mucosa from mice. A midline laparotomy was used to excise the intestinal mucosal layer. The dissected intestinal tissue was treated with ice-cold oxygenated Krebs-Ringer-bicarbonate (KRB).
) Opened along the mesentery in solution and pinned luminal side down on a flexible silicone surface. The outer muscular layer was peeled off by a shallow incision using a scalpel and a fine pick. Mouse intestinal tissue and human ileal tissue consisting of mucosa and submucosa were placed between two Ussing half-chambers and bathed on both sides. Electrical measurements were monitored by automatic voltage clamp, direct voltage and current differential and I _SC . Tissues were equilibrated under short-circuit conditions until the I _SC was stabilized, then the potential difference across the epithelium was measured intermittently.

Human uroguanylin peptides were chemically synthesized and the relative potency of the various synthetic forms was evaluated by their ability to stimulate cGMP accumulation in intact T-84 cells. The biological activity of several isoforms displaying similar physico-chemical properties of this peptide was observed. The major isoforms showing strong biological activity were further purified to about 99% purity and used in this test. To confirm that the synthetic form of human uroguanylin was equally effective in murine and human GI mucosa, its activity was tested in mouse Duodenum and human colon mucosa in Ussing chambers.

Results: I _SC response patterns were recorded for sequential treatment of the mouse duodenum (FIG. 1a) and human colon (FIG. 1b) with a particular drug. TTX in both organizations
Addition of [0.1 μM in serosal bath] resulted in a decrease to a stable value of baseline I _SC within 20 minutes. Subsequent addition of uroguanylin [luminal
) 0.1 μM in bath] resulted in a rapid increase in I _SC , which lasted for 60 minutes. Carbachol, a known stimulator of ion transport across membranes, also increased I _SC . These results confirm that synthetic human uroguanylin peptides are effective in stimulating electrolyte transport in humans and in mouse intestinal mucosa.

Example 5 Methods and Materials Min-mouse model: Male Min mice (C57BL / 6J-APC ^Min / +), a strain carrying a dominant mutation with sufficient penetrance in the APC gene,
Obtained from Acksson Laboratory, Bar Harbor, ME at 4-5 weeks of age. All mice were fed a high fat AIN-93G diet, tap water as drinking water and housed in a humidity and temperature controlled room under a 12 hour light / dark cycle. The animals consumed about 5 g of food per day. After a one week isolation period, animals were randomly divided into 3 groups of 10 animals per group. Animals in these groups were fed the same diet containing different concentrations of human uroguanylin (0, 10, and 20 μg / 5 g of diet).

Animals were also given an additional amount of human uroguanylin (vehicle, 10 and 20 μg) in 0.2 ml of PBS containing 20% polyethylene glycol by oral gavage at a frequency of twice weekly. Food consumption and body weight of these animals were measured weekly. At the end of 17 weeks, animals were sacrificed by CO ₂ inhalation and the GI tract was isolated. After clarification with PBS to remove dietary material, the GI tract was duodenum (two (2) inches from the stomach), jejunum (middle, approximately 4-5 inches from the stomach),
The ileum (two (2) inches from the cecum) and the colon were divided. These sections were cut longitudinally, washed with tissue fixative (Streck Laboratories, Inc., Omaha, NE) and then placed between two blotting papers in a tray containing tissue fixative.

The number of polyps and tumors was measured independently by 4 different observers.
Results are expressed as the average of the total number of polyps in each individual animal by 4 different observers. Evaluation of the data obtained from all observers showed non-observer differences that were not significant. Portions of these tissues were observed under constant magnification (10X) and the difference between polyp diameters between animals was measured.

Results: Min-mouse, the most widely used animal model for assessing chemopreventive properties of dietary nutrients and therapeutics, has a dominant mutation in one of the alleles of the APC gene. . Therefore, when these mice are fed a high fat diet, they begin to develop polyps throughout the intestine around 55 days of age. The expression of polyps blocks the migration of intestinal contents, which results in reduced food consumption and reduced weight gain as the disease progresses. The results of studies involving oral administration of uroguanylin to min-mice showed a dose-dependent increase in food consumption, as shown in Figure 6a and a dose-dependent increase in body weight gain, as shown in Figure 6b. showed that. The average weight of the control group was 25.1 ± 0.9 g, and uroguanylin treatment (20 μg)
In the group, 17 was 29.4 ± 1.07 g. Furthermore, the animals treated with uroguanylin were visibly more healthy and active.

At the end of this study, all animals were sacrificed, their GI tracts were dissected, and the number and distribution of polyps in the small intestine and colon tissues was determined, as shown in Table I. The GI tract of the untreated control group had 48.3 ± 7.7 polyps / mouse. The majority of polyps were located throughout the small intestine, with only a few found in the colon. The size of the polyps in the control group of mice was in the range of about 3-5 mm. These control animals also developed globular tumors in the duodenum.
Administration of uroguanylin reduced the total number of polyps (23.3 ± 3.1) by approximately 50%. Furthermore, the polyps of animals in the uroguanylin treated group were exceptionally small in size (<2.0 mm).

No polyps were found in the colon of all animals in this county and no globular tumors were found in these animals. The absence of polyps in the colons of uroguanylin-treated mice indicates that this peptide also blocks the progression of colon cancer, since the appearance of polyps in the colon of Min-mouse occurs only during periods of severe disease. Suggests that you can. These results suggest that oral administration of uroguanylin inhibits polyp formation and progression of polyp formation in this animal model of colon cancer.

Table 1: Inhibition of polyp formation in Min-mouse by oral administration of human uroguanylin

[Table 1]

[0065] All results are shown as mean ± SEM, n = 10. * Uroguanylin was administered orally twice weekly; Group A: vehicle control; Group B: 10 μg u.   Loganiline; and Group C: 20 μg. ** p = <0.05 compared to control group.

Example 4 Uloganiline Expression Methods and Materials in Human Colon Cancer Tissue RNA Isolation: Using a Combination of TRI Reagent Method (Molecular Research Center, Inc., Cincinnati, OH) and RNAeasy Kit (Qiagen, Valencia, CA) , RNA was extracted from the tissue.
The tissue was homogenized in TRI reagent according to the manufacturer's instructions. After separating the phases with chloroform, the aqueous supernatant phase containing total RNA was separated and then mixed with an equal amount of 70% ethanol and cell lysis buffer without β-mercaptoethanol. The resulting mixture was loaded onto the RNAeasy column,
It was then processed according to the instructions provided by the manufacturer.

Northern blotting: Total RNA (20 μg) was electrophoresed on a formaldehyde-agarose gel, then nylon membrane (Zeta-Probe, Bio-Rad Laborat).
ories, Inc., Hercules, CA). These membranes are treated with Express Hyb (Expr
essHyb) solution (Clontech, Palo Alto, CA) at 65 ° C. for 2 hours prehybridization followed by human guanylin, uroguanylin and GC-C.
Hybridized with cDNA overnight at 65 ° C. All cDNA probes were labeled with 32P by random priming (Boehringer Mannheim, Indianapolis, IN). RNA blot is then 2X at room temperature for 5 minutes
Wash twice with SSC-0.1% SDS, then 0.2X SS at 60 ° C.
A 15 minute wash with C-0.1% SDS was performed. Exposure to X-ray film was performed at -80 ° C with an intensifying screen.

RT-PCR: Oligo (deoxythymidine) 18-primed cDNA was analyzed by reverse transcription (Superscript II, Life Technologies, Gaithersburg, MD) at 3 mμ.
g Synthesized from total RNA. Two PCR primers, 5'-primer (5'-
GAACCCAGGGAGCGCGAT-3 ') [identified as SEQ ID NO: 6] and 3'-primer (5'-GGGCTCAGGGGTACC-3') [
Was identified as SEQ ID NO: 7], and the open reading frame of human prouroguanylin cDNA was designed from the flanking region. A PCR product of the expected size of 384 bp was subjected to 25 cycles of 93 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1.5 minutes using Tag DNA polymerase (USBiochemical Corp., Cleveland, OH). Colon cycle after cycle
Amplified from NA.

A pair of primers involved in RT-PCR for guanylin was a 5'-primer (5'-AACTCAGGAACTTTTGCAC-3 ') [identified as SEQ ID NO: 8] and a 3'-primer (5'-CGTAGGCACAGATTTT.
CAC-3 ') [identified as SEQ ID NO: 9]. These primers used PCR conditions of 25 cycles of 93 ° C. for 1 minute, 59 ° C. for 1 minute, and 72 ° C. for 1.5 minutes, and the 174 bp for human guanylin.
cDNA was generated. The PCR-generated cDNA product was electrophoresed on a 1% agarose gel in TAE buffer containing ethidium bromide and then transferred to a nylon membrane. Southern hybridization was performed with uroguanylin and guanylin cDNA probes. Prehybridization was performed with Express Hyb solution at 65 ° C. for 1 hour, then hybridization was performed at 65 ° C. for 3 hours. Blots were washed as above and then exposed to X-ray film at -80 ° C using an intensifying screen.

Results: Expression of uroguanylin, guanylin and GC-C receptors in 11 samples of human colon carcinoma and surrounding normal tissues. Northern blotting analysis showed that the expression of uroguanylin and guanylin was completely suppressed in all samples tested. In contrast, as shown in Figure 5a, adjacent non-cancerous tissue from the same patient showed healthy expression of these transcripts. As shown in Figure 5b, similar expression patterns made these tissue samples more sensitive to RT-P.
It was also found when analyzed based on CR, followed by Southern blotting. Apart from the fact that these samples were from different stages of colon cancer and from patients of different age groups, expression of guanylin and uroguanylin was significant in all 11 tissue samples tested. It was severely suppressed. These results are
It is possible that these intestinal hormones lead to or are the consequences of adenoma formation.

In light of the detailed description of the invention and the examples above, it can be appreciated that the several objects of the invention are achieved. The description and specific examples provided herein are for the purpose of bringing those skilled in the art to the invention, its principles, and its practical application. Those of ordinary skill in the art will adapt to its many forms so that it may be best adapted to the requirements of a particular application,
It can also be applied. Therefore, the particular examples described above are not intended to be exclusive or limiting of the invention.

[Sequence list]

[Brief description of drawings]

FIG. 1A   FIG. 1 (a) shows I in a mouse duodenal sample consisting of mucosa and submucosa. _SC The effect of human uroguanylin on the stimulation of
It is a graph which compares and shows.

FIG. 1 (b) is a graph showing the effect of human uroguanylin on the stimulation of I _SC in human intestinal mucosa compared to TTX and carbachol.

FIG. 2 is a graph showing the effect of human uroguanylin on the inhibition of T-84 human cancer cell proliferation.

FIG. 3 shows the results of analysis of fragments of T-84 human colon cancer cells after treatment with human uroguanylin by electrophoresis on a 1.8% agarose gel, followed by staining with ethidium bromide.

FIG. 4A is a photograph showing the results of observing a microscope slide of a semi-confluent monolayer of medium-treated Caco-2 cells with a fluorescence microscope.

FIG. 4B is a photograph showing the results of observing a microscope slide of a semi-confluent monolayer of Caco-2 cells treated with uroguanylin with a fluorescence microscope.

FIG. 5 (a) shows the northern blotting analysis results demonstrating that the expression of uroguanylin and guanylin is suppressed in human colon cancer cells.

FIG. 5 (b) shows RT-PCR demonstrating that the expression of uroguanylin and guanylin is suppressed in human colon cancer cells, followed by Southern blotting analysis results.

FIG. 6 (a) is a graph showing the increase in daily food consumption by Min-mouse after oral administration of human uroguanylin as mean ± standard error.

FIG. 6B is a graph showing the increase in body weight gain by Min-mouse after oral administration of human uroguanylin as mean ± standard error of weight gain / mouse.

FIG. 7 shows human uroguanylin (h UronG) [identified as SEQ ID NO: 2], human guanylin (h Gua) [identified as SEQ ID NO: 3], and bacterial enterotoxin (E. coli [E. SEQ ID NO: 4] and V. cholerae [identified with SEQ ID NO: 5]).

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Claims (75)

[Claims] 1. A method of controlling intestinal polyps in a subject, comprising the sequence: (In the formula, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each an amino acid residue, and X ₈ and X ₉ are independently hydrogen or at least one amino acid. And a cystine residue directly adjacent to the amine group of X ₁ and a cystine residue immediately adjacent to the amine group of X ₆ are crosslinked by a disulfide bond. A polypeptide in which a cystine residue directly adjacent to the amine group of X ₃ and a cystine residue immediately adjacent to the carboxy group of X ₇ are cross-linked by a disulfide bond together with a pharmaceutically acceptable carrier The above method, comprising administering the pharmaceutical composition containing the same to a subject in need thereof. 2. A method for controlling intestinal polyps in a subject, which comprises a compound or agonist peptide that binds to guanylate cyclase receptor GC-C in the intestine of the subject together with a pharmaceutically acceptable carrier. The above method comprising administering the composition to a subject in need thereof. 3. The method of claim 1, wherein the concentration of peptide in the composition is at least 0.0001 percent by weight of the composition. 4. The method of claim 1, wherein the concentration of peptide in the composition is at least 0.001 percent, based on the weight of the composition. 5. The method of claim 1, wherein the concentration of peptide in the composition is at least 0.01 percent, based on the weight of the composition. 6. The method of claim 1, wherein the concentration of peptide in the composition is at least 0.1 percent, based on the weight of the composition. 7. The method of claim 1, wherein the concentration of peptide in the composition is at least 1 percent, based on the weight of the composition. 8. The method of claim 1, wherein the subject has been identified as having a genetic predisposition to the growth of polyps in the intestine. 9. The method of claim 1, wherein the polyp is defined in the intestine of the subject. 10. The method of claim 1, wherein the subject has been identified as having intestinal cancer. 11. The method of claim 2, wherein the concentration of peptide in the composition is at least 0.0001 percent, based on the weight of the composition. 12. The method of claim 2, wherein the concentration of peptide in the composition is at least 0.001 percent, based on the weight of the composition. 13. The method of claim 2, wherein the concentration of peptide in the composition is at least 0.01 percent, based on the weight of the composition. 14. The method of claim 2, wherein the concentration of peptide in the composition is at least 0.1 percent, based on the weight of the composition. 15. The method of claim 2, wherein the concentration of peptide in the composition is at least 1 percent, based on the weight of the composition. 16. The method of claim 2, wherein the subject has been identified as having a genetic predisposition to the growth of polyps in the intestine. 17. The method of claim 2, wherein the polyp is defined in the intestine of the subject. 18. The method of claim 2, wherein the subject has been identified as having intestinal cancer. 19. The method of claim 1, wherein X ₁ is selected from the group of amino acid residues consisting of aspartic acid, glutamic acid, glycine, lysine, asparagine, proline, glutamine, arginine, serine, and threonine. 20. The method of claim 1, wherein X ₁ is selected from the group of amino acid residues consisting of glutamic acid, arginine, lysine, serine, aspartic acid, asparagine, glutamine, and glycine. 21. The method of claim 1, wherein X ₁ is selected from the group of amino acid residues consisting of glutamic acid, aspartic acid, arginine, and lysine. 22. The method of claim 1, wherein X ₁ is glutamic acid. 23. X ₂ is selected from the group of amino acid residues consisting of leucine, isoleucine, tyrosine, phenylalanine, tryptophan, valine, methionine, cysteine, alanine, histidine, proline, threonine, glycine, asparagine, and glutamine. The method according to claim 1. 24. The method of claim 1, wherein X ₂ is selected from the group of amino acid residues consisting of cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, valine and tyrosine. 25. The method of claim 1, wherein X ₂ is selected from the group of amino acid residues consisting of leucine, isoleucine, tyrosine, valine, methionine. 26. The method of claim 1, wherein X ₂ is selected from the group of amino acid residues consisting of leucine and isoleucine. 27. The method of claim 1, wherein X ₂ is leucine. 28. X ₃ is selected from the group of amino acid residues consisting of valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, methionine, cysteine, alanine, histidine, proline, threonine, glycine, glutamine, asparagine, and serine. The method of claim 1, wherein the method comprises: 29. The method of claim 1, wherein X ₃ is selected from the group of amino acid residues consisting of valine, isoleucine, leucine, tyrosine, phenylalanine, methionine, cysteine, alanine, histidine, and proline. 30. The method of claim 1, wherein X ₃ is selected from the group of amino acid residues consisting of valine, isoleucine, leucine, methionine and cysteine. 31. The method of claim 1, wherein X ₃ is valine. 32. The method of claim 1, wherein X ₃ is isoleucine. 33. X ₄ is selected from the group of amino acid residues consisting of valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, methionine, cysteine, alanine, histidine, proline, threonine, glycine, glutamine, asparagine, and serine. The method of claim 1, wherein the method comprises: 34. The method of claim 1, wherein X ₄ is selected from the group of amino acid residues consisting of valine, isoleucine, leucine, tyrosine, phenylalanine, methionine, cysteine, alanine, histidine, and proline. 35. The method of claim 1, wherein X ₄ is selected from the group of amino acid residues consisting of valine, isoleucine, leucine, methionine and cysteine. 36. The method of claim 1, wherein X ₄ is valine. 37. X ₅ is alanine, histidine, cysteine, methionine, valine, leucine, isoleucine, tyrosine, phenylalanine, proline,
The method of claim 1, wherein the method is selected from the group of amino acid residues consisting of threonine, glycine, glutamine, asparagine, and serine. 38. The method of claim 1, wherein X ₅ is selected from the group of amino acid residues consisting of alanine, histidine, cysteine, methionine, valine, proline, threonine, glycine, glutamine, asparagine, and serine. . 39. X ₅ is alanine, histidine, cysteine, proline,
The method of claim 1, wherein the method is selected from the group of amino acid residues consisting of threonine, glycine, glutamine, asparagine, and serine. 40. The method of claim 1, wherein X ₅ is alanine. 41. X ₆ is threonine, proline, alanine, histidine,
The method according to claim 1, which is selected from the group of amino acid residues consisting of cysteine, methionine, valine, leucine, isoleucine, tyrosine, glycine, glutamine, asparagine, and serine. 42. X ₆ is threonine, proline, alanine, histidine,
The method of claim 1, wherein the method is selected from the group of amino acid residues consisting of cysteine, methionine, glycine, glutamine, asparagine, and serine. 43. X ₆ is threonine, proline, alanine, histidine,
The method of claim 1, wherein the method is selected from the group of amino acid residues consisting of and glycine. 44. The method of claim 1, wherein X ₆ is threonine. 45. X ₇ is selected from the group of amino acid residues consisting of glycine, threonine, proline, alanine, histidine, cysteine, methionine, valine, leucine, isoleucine, glutamine, asparagine, serine, glutamic acid, and aspartic acid. The method of claim 1, wherein 46. The method of claim 1, wherein X ₇ is selected from the group of amino acid residues consisting of glycine, threonine, proline, alanine, histidine, cysteine, glutamine, asparagine, and serine. 47. The method of claim 1, wherein X ₇ is selected from the group of amino acid residues consisting of glycine, threonine, proline, alanine, histidine, glutamine, asparagine, and serine. 48. The method of claim 1, wherein X ₇ is glycine. 49. The method of claim 1, wherein the polypeptide is uroguanylin. 50. The method of claim 1, wherein the polypeptide is human uroguanylin. 51. The method of claim 1, wherein the composition comprises pro-uroguanylin. 52. The method of claim 1, wherein the composition comprises human pro-uroguanylin. 53. The method of claim 2, wherein the composition comprises guanylin. 54. The method of claim 2, wherein the composition comprises lymphologaniline. 55. The method of claim 2, wherein the composition comprises prolymph guanylin. 56. The method of claim 2, wherein the composition comprises a heat stable enterotoxin. 57. The method of claim 1, wherein the composition comprises a polypeptide that is degraded to uroguanylin by an endogenous protease of interest. 58. About 0.5 mg to about 2 m of polypeptide per kg of subject's body weight.
The method of claim 1, wherein g is administered. 59. The method of claim 1, wherein the subject is a human. 60. The method of claim 2, wherein the composition comprises a polypeptide that is degraded to uroguanylin by an endogenous protease of interest. 61. X ₁ is glutamic acid, X ₂ is leucine, X ₃ is isoleucine, X ₄ is valine, X ₅ is alanine, X ₆ is threonine, and X ₇ The method of claim 1, wherein is glycine. 62. A method of preventing, suppressing and treating intestinal cancer in a subject, comprising administering to the subject the composition of claim 1. 63. A method of preventing, suppressing and treating intestinal cancer in a subject, comprising administering to the subject the composition of claim 2. 64. The method of claim 62, wherein the composition comprises uroguanylin. 65. The method of claim 63, wherein the composition comprises uroguanylin. 66. The method of claim 62, wherein the composition comprises pro-uroguanylin. 67. The method of claim 63, wherein the composition comprises pro-uroguanylin. 68. The method of claim 62, wherein the composition comprises human uroguanylin. 69. The method of claim 63, wherein the composition comprises human uroguanylin. 70. The composition of claim 6, wherein the composition comprises human pro-uroguanylin.
The method described in 2. 71. The composition of claim 6, wherein the composition comprises human pro-uroguanylin.
The method according to 3. 72. The method of claim 63, wherein the composition comprises guanylin. 73. The method of claim 63, wherein the composition comprises lymph guanylin. 74. The composition of claim 6, wherein the composition comprises a heat stable enterotoxin.
The method according to 3. 75. The composition of claim 63, wherein the composition comprises pro-lymph guanylin.
The method described in.