(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`1111111111111111 IIIIII 1111111111111111111111111111111111111111 IIII IIIIIII IIII 11111111
`
`(43) International Publication Date
`10 October 2002 (10.10.2002)
`
`PCT
`
`(10) International Publication Number
`WO 02/078683 Al
`
`(51) International Patent Classification 7:
`A61K 31/00,
`38/00, A0lN 61/00, C12Q 1/00, C07K 2/00, 4/00, 5/00,
`7/00, 14/00, 16/00, 17/00, G0lN 33/53, 33/48, 33/567,
`33/574
`
`(21) International Application Number: PCT/US02/09551
`
`(22) International Filing Date: 28 March 2002 (28.03.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/279,438
`60/279,437
`60/300,850
`60/303,806
`60/307,358
`60/348,646
`
`29 March 2001 (29.03.2001)
`29 March 2001 (29.03.2001)
`27 June 2001 (27.06.2001)
`10 July 2001 (10.07.2001)
`25 July 2001 (25.07.2001)
`17 January 2002 (17.01.2002)
`
`us
`us
`us
`us
`us
`us
`
`(71) Applicant: SYNERGY PHARMACEUTICALS, INC.
`[US/US]; Suite 450, Two Executive Drive, Somerset, NJ
`08873 (US).
`
`(72) Inventors: SHAILUBHAI, Kunwar; 600 Wick Lane,
`Blue Bell, PA 19422 (US). NIKIFOROVICH, Gregory;
`751 Aramis Drive, St. Louis, mo 63141 (US). JACOB,
`Gary, S.; 12541 Mason Forest Drive, Creve Coeur, MO
`63141 (US).
`
`(74) Agents: SANZO, Michael, A. et al.; Pillsbury Winthrop
`LLP, 1600 Tysons Boulevard, McLean, VA 22102 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, 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, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, UZ, VN,
`YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, 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.
`
`iiiiiiii
`
`---iiiiiiii
`-iiiiiiii -----
`
`iiiiiiii
`
`iiiiiiii ----
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`,-...I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`
`< (54) Title: GUANYLATE CYCLASE RECEPTOR AGONISTS FOR THE TREATMENT OF TISSUE INFLAMMATION AND
`
`~ CARCINOGENESIS
`Q0
`\0 (57) Abstract: A method of treatment of inflamed, pre-cancerous or cancerous tissue or polyps in a mammalian subject is disclosed.
`~ The treatment involves administration of a composition of at least one peptide agonist of a guanylate cyclase receptor and/or other
`Q
`small molecules that enhance intracellular production of cGMP. The at least one peptide agonist of a guanylate cyclase receptor may
`--.. be administered either alone or in combination with an inhibitor of cGMP-dependent phosphodiesterase. The inhibitor may be a small
`~ molecule, peptide, protein or other compound that inhibits the degradation of cGMP. Without requiring a particular mechanism of
`action, this treatment may restore a healthy balance between proliferation and apoptosis in the subject's population of epithelial cells,
`0 and also suppress carcinogenesis. Thus, the method may be used to treat, <i>inter alia<i/>, inflammation, including gastrointestinal
`> inflammatory disorders, general organ inflammation and asthma, and carcinogenesis of the lung, gastrointestinal tract, bladder, testis,
`
`;;, prostate and pancreas, or polyps.
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`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
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`Cross Reference to Related Applications
`the benefit of U.S. provisional application
`The present application claims
`nos. 60/279,438, filed on March 29, 2001; 60/279,437, filed on March 29, 2001; 60/300,850,
`filed on June 27, 2001; 60/303,806, filed on July 10, 2001; 60/307,358, filed on July 25, 2001;
`and 60/348,646, filed on January 17, 2002.
`
`Field of the Invention
`The present invention relates to the therapeutic use of guanylate cyclase receptor
`agonists as a means for enhancing the intracellular production of cGMP. The agonists may be
`used either alone or in combination with inhibitors of cGMP-specific phosphodiesterase to
`prevent or treat cancerous, pre-cancerous and metastatic growths, particularly in the
`gastrointestinal tract and lungs. In addition, the agonists may be used in the treatment of
`inflammatory disorders such as ulcerative colitis and asthma.
`
`Background of the Invention
`Uroguanylin, guanylin and bacterial ST peptides are structurally related peptides that
`bind to a guanylate cyclase receptor and stimulate intracellular production of cyclic guanosine
`in the activation of the cystic fibrosis
`monophosphate ( cGMP) (1-6). This results
`transmembrane conductance regulator (CFTR), an apical membrane channel for efflux of
`chloride from enterocytes lining the intestinal tract (1-6). Activation of CFTR and the
`subsequent enhancement of transepithelial secretion of chloride leads to stimulation of sodium
`and water secretion into the intestinal lumen. Therefore, by serving as paracrine regulators of
`CFTR activity, cGMP receptor agonists regulate fluid and electrolyte transport in the GI tract
`(1-6; US patent 5,489,670).
`
`The process of epithelial renewal involves the proliferation, migration, differentiation,
`senescence, and eventual loss of GI cells in the lumen (7,8). The GI mucosa can be divided
`into three distinct zones based on the proliferation index of epithelial cells. One of these zones,
`the proliferative zone, consists of undifferentiated stem cells responsible for providing a
`constant source of new cells. The stem cells migrate upward toward the lumen to which they
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`are extruded. As they migrate, the cells lose their capacity to divide and become differentiated
`
`for carrying out specialized functions of the GI mucosa (9). Renewal of GI mucosa is very
`
`rapid with complete turnover occurring within a 24-48 hour period (9). During this process
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`mutated and unwanted cells are replenished with new cells. Hence, homeostasis of the GI
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`5 mucosa is regulated by continual maintenance of the balance between proliferation and
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`apoptotic rates (8).
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`The rates of cell proliferation and apoptosis in the gut epithelium can be increased or
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`decreased in a wide variety of different circumstances, e.g., in response to physiological
`
`stimuli such as aging, inflammatory signals, hormones, peptides, growth factors, chemicals and
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`IO
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`dietary habits. In addition, an enhanced proliferation rate is frequently associated with a
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`reduction in turnover time and an expansion of the proliferative zone (I 0). The proliferation
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`index has been observed to be much higher in pathological cases of ulcerative colitis and other
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`GI disorders (11). Thus, intestinal hyperplasia is the major promoter of gastrointestinal
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`inflammation and carcinogenesis.
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`In addition to a role for uroguanylin and guanylin as modulators of intestinal fluid and
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`ion secretion, these peptides may also be involved in the continual renewal of GI mucosa.
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`Previously published data in WO 01/25266 suggests a peptide with the active domain of
`
`uroguanylin may function as an inhibitor of polyp development in the colon and may constitute
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`a treatment of colon cancer. However, the mechanism by which this is claimed to occur is
`
`questionable in that WO 01/25266 teaches uroguanylin agonist peptides that bind specifically
`
`to a guanylate cyclase receptor, termed GC-C, that was first described as the receptor for E.
`
`coli heat-stable enterotoxin (ST) (4). Knockout mice lacking this guanylate cyclase receptor
`
`show resistance to ST in intestine, but effects of uroguanylin and ST are not disturbed in the
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`kidney in vivo (3). These results were further supported by the fact that membrane
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`depolarization induced by guanylin was blocked by genistein, a tyrosine kinase inhibitor,
`
`whereas hyperpolarization induced by uroguanylin was not effected (12, 13). Taken together
`
`these data suggest that uroguanylin also binds to a currently unknown receptor, which is
`
`distinct from GC-C.
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`Other papers have reported that production of uroguanylin and guanylin is dramatically
`
`decreased in pre-cancerous colon polyps and tumor tissues (14-17). In addition, genes for both
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`uroguanylin and guanylin have been shown to be localized to regions of the genome frequently
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`associated with loss of heterozygosity in human colon carcinoma (18-20). Taken together,
`these findings indicate that uroguanylin, guanylin and other peptides with similar activity may
`be used in the prevention or treatment of abnormal colon growths. This proposal is bolstered
`by a recent study demonstrating oral administration of uroguanylin inhibits polyp formation in
`5 mice (15,16).
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`Uroguanylin and guanylin peptides also appear to promote apoptosis by controlling
`cellular ion flux. Alterations in apoptosis have been associated with tumor progression to the
`metastatic phenotype. While a primary gastrointestinal (GI) cancer is limited to the small
`intestine, colon, and rectum, it may metastasize and spread to such localities as bone, lymph
`nodes, liver, lung, peritoneum, ovaries, brain. By enhancing the efflux of K+ and influx of
`Ca++, uroguanylin and related peptides may promote the death of transformed cells and thereby
`inhibit metastasis.
`
`One of the clinical manifestations of reduced CFTR activity is the inflammation of
`airway passages (21). This effect may be due to CTFR regulating the expression of NF-kB,
`chemokines and cytokines (22-25). Recent reports have also suggested that the CFTR channel
`is involved in the transport and maintenance of reduced glutathione, an antioxidant that plays
`an important role in protecting against inflammation caused by oxidative stress (39).
`Enhancement of intracellular levels of cGMP by way of guanylate cyclase activation or by way
`of inhibition of cGMP-specific phosphodiesterase would be expected to down-regulate these
`inflammatory stimuli. Thus, uroguanylin-type agonists should be useful in the prevention and
`treatment of inflammatory diseases of the lung (e.g., asthma), bowel (e.g., ulcerative colitis and
`Crohn's disease), pancreas and other organs.
`
`Overall, it may be concluded that agonists of guanylate cyclase receptor such as
`uroguanylin have potential therapeutic value in the treatment of a wide variety of inflammatory
`conditions, cancer (particularly colon cancer) and as anti-metastatic agents. The development
`of new agonists is therefore of substantial clinical importance.
`
`Summary of the Invention
`The present invention is based upon the development of new agonists of guanylate
`cyclase receptor, and new uses of naturally occurring agonists. The agonists are analogs of
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`uroguanylin, many of which have superior properties either in terms of improved receptor
`
`activation, stability, activity at low pH or reduced adverse effects. The peptides may be used to
`
`treat any condition that responds to enhanced intracellular levels of cGMP. Intracellular levels
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`of cGMP can be increased by enhancing intracellular production of cGMP and/or by inhibition
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`of its degradation by cGMP-specific phosphodiesterases. Among the specific conditions that
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`can be treated or prevented are inflammatory conditions, cancer, polyps, and metastasis.
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`In its first aspect, the present invention is directed to a peptide consisting essentially of
`
`the amino acid sequence of any one of SEQ ID NOs:2-21 and to therapeutic compositions
`
`10 which contain these peptides. The term "consisting essentially of' includes peptides that are
`
`identical to a recited sequence identification number and other sequences that do not differ
`
`substantially in terms of either structure or function. For the purpose of the present application,
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`a peptide differs substantially if its structure varies by more than three amino acids from a
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`peptide of SEQ ID NOs:2-21 or if its activation of cellular cGMP production is reduced or
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`enhanced by more than 50%. Preferably, substantially similar peptides should differ by no
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`more than two amino acids and not differ by more than about 25% with respect to activating
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`cGMP production. The most preferred peptide is a bicycle having the sequence of SEQ ID
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`NO:20.
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`The peptides may be in a pharmaceutical composition in unit dose form, together with
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`one or more pharmaceutically acceptable excipients. The term "unit dose form" refers to a
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`single drug delivery entity, e.g., a tablet, capsule, solution or inhalation formulation. The
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`amount of peptide present should be sufficient to have a positive therapeutic effect when
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`administered to a patient (typically, between 100 µg and 3 g). What constitutes a "positive
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`therapeutic effect" will depend upon the particular condition being treated and will include any
`
`significant improvement in a condition readily recognized by one of skill in the art. For
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`example, it may constitute a reduction in inflammation, a shrinkage of polyps or tumors, a
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`reduction in metastatic lesions, etc.
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`The invention also encompasses combination therapy utilizing a guanylate cyclase
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`receptor agonist administered either alone or together with an inhibitor of cGMP-dependent
`
`phosphodiesterase, an anti-inflammatory agent or an anticancer agent. These agents should be
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`present in amounts known in the art to be therapeutically effective when administered to a
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`include alkylating agents, epipodophyllotoxins,
`patient. Anti-neoplastic agents may
`nitrosoureas, antimetabolites, vinca alkaloids, anthracycline antibiotics, nitrogen mustard
`agents, and the like. Particular anti-neoplastic agents may include tamoxifen, taxol, etoposide
`and 5-fluorouracil. Antiviral and monoclonal antibody therapies may be combined with
`chemotherapeutic compositions comprising at least one guanylate cyclase receptor agonist in
`devising a treatment regimen tailored to a patient's specific needs.
`
`In another aspect, the invention is directed to a method for preventing, treating or
`retarding the onset of cancer, particularly cancer of epithelial cells, or polyps in a subject by
`administering a composition comprising an effective amount of a guanylate cyclase receptor
`agonist, preferably a synthetic guanylate cyclase receptor agonist. The term "effective amount"
`refers to sufficient agonist to measurably increase intracellular levels of cGMP. The term
`"synthetic" refers to a peptide created to bind a guanylate cyclase receptor, but containing
`certain amino acid sequence substitutions not present in known endogenous guanylate cyclase
`agonists, such as uroguanylin. The agonist should be a peptide selected from those defined by
`SEQ ID NOs:2-21 and which are listed in Tables 2 and 3. Also included in the invention are
`methods of treating primary cancers, other than primary colon cancer, by administering an
`effective dosage of a peptide selected from the group consisting of: uroguanylin; guanylin; and
`E. coli ST peptide. Any known form of uroguanylin or guanylin can be used for this purpose,
`although the human peptides are preferred.
`
`The invention also includes methods of preventing or treating tumor metastasis from a
`primary tumor mass. Metastatic tumor cells having guanylate cyclase receptors may be
`targeted by peptides generated according to the invention. In a preferred embodiment, the
`targeted receptor is found on cells of gastrointestinal (GI) cancers and on metastasized cells
`derived from those cancers. Such receptors are typically transmembrane proteins with an
`extracellular ligand-binding domain, a membrane-spanning domain, and an intracellular
`domain with guanylate cyclase activity. Although the invention is not bound by any particular
`mechanism of action, it is believed that the peptides will act by binding to these cellular
`receptors and inducing apoptosis. Metastatic tumors may also be treated by administering any
`known form of uroguanylin or guanylin (preferably human) or by administering E. coli ST
`peptide.
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`Peptides may be administered either alone or together with one or more inhibitors of
`cGMP dependent phosphodiesterase. Examples of cGMP dependent phosphodiesterase
`inhibitors include suldinac sulfone, zaprinast, and motapizone. Treatable forms of cancer
`include breast cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer,
`prostate cancer, renal cancer, and testicular cancer. Colon carcinogenesis may be prevented by
`inhibiting pre-cancerous colorectal polyp development via administration of a composition
`according to the invention. It is believed that the peptides should be especially effective with
`respect to the treatment of colon cancer and in preventing the metastasis of colon tumors.
`
`In another aspect, the invention is directed to a method for treating, preventing, or
`retarding the onset of organ inflammation (e.g., inflammation associated with the GI tract,
`asthma, nephritis, hepatitis, pancreatitis, bronchitis, or cystic fibrosis) of a subject by
`
`administering a composition comprising an agonist of a guanylate cyclase receptor that
`
`enhances intracellular production of cGMP. Preferred peptide agonists are selected from the
`group defined by SEQ ID NOs:2-21 shown in Tables 2 and 3, or uroguanylin, or guanylin, or
`E.coli ST peptide. These peptides may optionally be administered with one or more inhibitors
`of cGMP dependent phosphodiesterase, e.g., suldinac sulfone, zaprinast, or motapizone. In a
`preferred embodiment, the invention is directed to a method of treating an inflammatory
`disorder in a mammalian gastrointestinal tract. The inflammatory disorder may be classified as
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`an inflammatory bowel disease, and more particularly may be Crohn's disease or ulcerative
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`colitis. Administration may be enteric, and employ formulations tailored to target enterocytes.
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`In a broader sense, the invention includes methods of inducing apoptosis in a patient by
`administering an effective amount of a peptide having the sequence of any one of SEQ ID
`25 NO:2 - SEQ ID NO:21, or uroguanylin, or guanylin or E. coli ST peptide. An "effective
`amount" of peptide, in this sense, refers to an amount sufficient to increase apoptosis in a target
`tissue. For example, sufficient peptide may be given to induce an increased rate of cell death in
`
`a neoplastic growth.
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`The most preferred peptide for use in the methods described above is the peptide
`defined by SEQ ID NO:20. The sequence is as follows (see also Table 3):
`
`Asn1 Asp2 Glu3 Cys4 Glu5 Leu6 Cys7 Val8 Asn9 Val10 Ala11 Cys12 Thr13 Gly14 Cys15 Leu16
`*
`**
`*
`**
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`and wherein there is one disulfide linkage between the cysteine at position 4 and the
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`cysteine at position 12; and a second disulfide linkage between the cysteine at position 7 and
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`the cysteine at position 15 (SEQ ID NO:20). This peptide has been found to have enhanced
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`biological activity as an agonist of cGMP production due to its enhanced binding constant for
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`the guanylate cyclase receptor, and is superior to uroguanylin with regard to temperature and
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`protease stability and with regard to its biological activity at the physiologically favorable pH
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`range (pH 6 to 7) in the large intestine.
`
`The guanylate cyclase receptor agonists used in the methods described above may be
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`administered either orally, systemically or locally. Dosage forms include preparations for
`
`inhalation or injection, solutions, suspensions, emulsions, tablets, capsules, topical salves and
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`lotions, transdermal compositions, · other known peptide formulations and pegylated peptide
`
`analogs. An effective dosage of the composition will typically be between about 1 µg and
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`about 10 mg per kilogram body weight, preferably between about 10 µg to 5 mg of the
`compound per kilogram body weight. Adjustments in dosage will be made using methods that
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`are routine in the art and will be based upon the particular composition being used and clinical
`
`considerations. Agonists may be administered as either the sole active agent or in combination
`
`with other drugs, e.g., an inhibitor of cGMP-dependent phosphodiesterase. In all cases,
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`additional drugs should be administered at a dosage that is therapeutically effective using the
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`existing art as a guide. Drugs may be administered in a single composition or sequentially.
`
`Detailed Description of the Invention
`
`The present invention is based upon several concepts. The first is that there is a cGMP- .
`
`dependent mechanism which regulates the balance between cellular proliferation and apoptosis
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`and that a reduction in cGMP levels, due to a deficiency ofuroguanylin/guanylin and/or due to
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`the activation of cGMP-specific phosphodiesterases, is an early and critical step in neoplastic
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`transformation. A second concept is that the release of arachidonic acid from membrane
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`phospholipids, which leads to the activation of cPLA2, COX-2 and possibly 5-lipoxygenase
`during the process of inflammation, is down-regulated by a cGMP-dependent mechanism,
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`leading to reduced levels of prostaglandins and leukotrienes, and that increasing intracellular
`levels of cGMP may therefore produce an anti-inflammatory response. In addition, a cGMP(cid:173)
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`dependent mechanism, is thought to be involved in the control of proinflammatory processes.
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`Therefore, elevating intracellular levels of cGMP may be used as a means of treating and
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`controlling inflammatory bowel diseases such as ulcerative colitis and Crohn's disease and
`other organ inflammation (e.g., associated with asthma, nephritis, hepatitis, pancreatitis,
`bronchitis, cystic fibrosis).
`
`Without intending to be bound by any theory, it is envisioned that ion transport across
`the plasma membrane may prove to be an important regulator of the balance between cell
`that will be affected by compositions altering cGMP
`proliferation and apoptosis
`concentrations. Uroguanylin has been shown to stimulate K+ efflux, Ca++- influx and water
`transport in the gastrointestinal tract (3). Moreover, atrial natriuretic peptide (ANP), a peptide
`that also binds to a specific guanylate cyclase receptor, has also been shown to induce
`apoptosis in rat mesangial cells, and to induce apoptosis in cardiac myocytes by a cGMP
`mechanism (26-29). It is believed that binding of the present agonists to a guanylate cyclase
`receptor stimulates production of cGMP. This ligand-receptor interaction, via activation of a
`cascade of cGMP-dependent protein kinases and CFTR, is then expected to induce apoptosis in
`target cells. Therefore, administration of the novel peptides defined by SEQ ID NOs:2-21, as
`shown in Tables 2 and 3, or uroguanylin, or guanylin or E. coli ST peptide is expected to
`eliminate or, at least retard, the onset of inflanunatory diseases of the GI tract and general
`organ inflammation (e.g., asthma, nephritis, hepatitis, pancreatitis, bronchitis, cystic fibrosis).
`
`In another aspect, the , invention is directed to a method for preventing, treating or
`retarding the onset of cancer, particularly cancer of epithelial cells, in a subject by
`administering a composition comprising an effective amount of a guanylate cyclase receptor
`agonist, preferably a synthetic a guanylate cyclase receptor agonist. The term "effective
`amount" refers to sufficient agonist to measurably increase intracellular levels of cGMP. The
`term "synthetic" refers to a peptide created to bind a guanylate cyclase receptor, but containing
`certain amino acid sequence substitutions not present in known endogenous guanylate cyclase
`agonists, such as uroguanylin. The agonist should be a peptide selected from those defined by
`SEQ ID NOs:2-21 and which are listed in Tables 2 and 3. Also included in the invention are
`methods of treating primary and metastatic cancers, other than primary colon cancer, by
`administering an effective dosage of a peptide selected from the group consisting of:
`uroguanylin; guanylin; and E. coli ST peptide. Any known form of uroguanylin or guanylin
`can be used for this purpose, although the human peptides are preferred.
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`the balance between cellular
`The cGMP-dependent mechanism that regulates
`proliferation and apoptosis in metastatic tumor cells may serve as a mechanism for targeting
`and treating metastatic tumors. The liver is the most common site of metastasis from a primary
`colorectal cancer. Toward later stages of disease, colorectal metastatic cells may also invade
`other parts of the body. It is important to note that metastatic cells originating from the primary
`site in the gastrointestinal tract typically continue to express guanylate cyclase receptors and
`therefore, these cells should be sensitive to apoptosis therapy mediated by intestinal guanylate
`cyclase receptors. Peptides having uroguanylin activity, when used either alone or in
`combination with specific inhibitors of cGMP-phosphodiesterase, also retard the onset of
`carcinogenesis in gut epithelium by restoring a healthy balance between cell proliferation and
`apoptosis via a cGMP-mediated mechanism.
`
`As used herein, the term "guanylate cyclase receptor" refers to the class of guanylate
`cyclase receptors on any cell type to which the inventive agonist peptides or natural agonists
`described herein bind.
`
`As used herein, the term "guanylate cyclase receptor-agonist" refers to peptides and/or
`other compounds that bind to a guanylate cyclase receptor and stimulate cGMP production.
`The term also includes all peptides that have amino acid sequences substantially equivalent to
`at least a portion of the binding domain comprising amino acid residues 3-15 of SEQ ID NO:l.
`This term also covers fragments and pro-peptides that bind to guanylate cyclase receptor and
`stimulate cGMP production. The term "substantially equivalent" refers to a peptide that has an
`amino acid sequence equivalent to that of the binding domain where certain residues may be
`deleted or replaced with other amino acids without impairing the peptide's ability to bind to a
`guanylate cyclase receptor and stimulate cGMP production.
`
`Strategy and design of novel guanylate cyclase receptor agonists
`Uroguanylin is a peptide secreted by the goblet and other epithelial cells lining the
`gastrointestinal mucosa as pro-uroguanylin, a functionally inactive form. The human pro-
`peptide is subsequently converted to the functionally active 16 amino acid peptide set forth in
`SEQ ID NO: 1 (human uroguanylin sequence, see Table 2) in the lumen of the intestine by
`endogenous proteases. Since uroguanylin is a heat-resistant, acid-resistant, and proteolysis(cid:173)
`resistant peptide, oral or systemic administration of this peptide and/or other peptides similar to
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`the :functionally active 16 amino acid peptide sequence of SEQ ID NO: 1 may be effectively
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`employed in treatment methods.
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`Peptides similar to, but distinct from, uroguanylin are described below, including some
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`5 which produce superior cGMP enhancing properties and/or other beneficial characteristics
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`(e.g., improved temperature stability, enhanced protease stability, or superior activity at
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`preferred pH's) compared to previously known uroguanylin peptides. The peptides may be
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`used to inhibit GI inflammation and for treating or preventing the onset of polyp formation
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`associated with gut inflammation. Epithelial tissues susceptible to cancer cell formation may
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`also be treated. The guanylate cyclase receptor agonists described have the amino acid
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`sequences shown in Tables 2 and 3. The "binding domain" for agonist-receptor interaction
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`includes the amino acid residues from 3-15 of SEQ ID NO:l.
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`Molecular modeling was applied to the design of novel guanylate cyclase receptor
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`agonists using methods detailed in (30). It consisted of energy calculations for three
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`interact with guanylate cyclase receptors, namely for human
`compounds known to
`uroguanylin, bicycle [ 4,12; 7,15]Asn1-Asp2-Asp3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-Val10-Ala11
`Cys12-Thr13-Gly14-Cys15-Leu16 (UG, SEQ ID NO:1); human guanylin, bicyclo [4,12;
`7,15]Pro1-Gly2-Thr3-Cys4-Glu5-Ile6-Cys7-Ala8-Tyr9-Ala10-Ala11-Cys12-Thr13-Gly14-Cys15 (GU,
`SEQ ID NO:22); and E. coli small heat-stable enterotoxin, tricycle [6,10; 7,15; 11-18] Asn1-
`Ser2-Ser3-Asn4-Tyr5-Cys6-Cys7-Glu8-Leu9-Cys10-Cys11-Asn12-Pro13-Ala14-Cys15-Thr16-Gly17-
`Cys18-Tyr19 (ST, SEQ ID NO:23). Geometrical comparisons of all possible low-energy
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`-
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`conformations for these three compounds were used to reveal the common 3D structures that
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`served as the "templates" for the bioactive conformation, i.e., for the conformation presumably
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`adopted by GU, UG and ST during interaction with receptor. It allowed designing novel
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`analogs with significantly increased conformational population of the bioactive conformation
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`at the expense of other low-energy conformations by selecting individual substitutions for
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`various amino acid residues.
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`Energy calculations were performed by use of build-up procedures (30). The ECEPP/2
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`potential field (31,32) was used assuming rigid valence geometry with planar trans-peptide
`bonds, including that for Pro13 in ST. Thero angle in Pro13 was allowed to vary. Aliphatic and
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`aromatic hydrogens were generally included in united atomic centers of CHn type; Ha-atoms
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`and amide hydrogens were described explicitly.
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`The main calculation scheme involved several successive steps. First, the sequences of
`the two monocyclic model fragments (three fragments for ST), Ac-cyclo (Cysi ~ ... -Cysi) -NMe,
`were considered, where all residues except Cys, Gly and Pro were replaced by alanines; the i
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`and j values corresponded to the sequences of GU, UG and ST. At this step, all possible
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`combinations of local minima for the peptide backbone for each amino acid residue were
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`considered, i.e., the minima in the Ramachandran map of E, F, C, D, A and A* types
`(according to the notation in (33)) for the Ala residue; of E*, F*, c*, D*, A , E, F, CD and A*
`types for the Gly residue; and of F, C and A types for Pro. For each backbone conformation,
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`one optimal possibility to close a cycle employing the parabolic potential functions, intrinsic to
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`the ECEPP force field, was found by checking an energy profile of rotation around the dihedral
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`angle Xl for the D-Cys residue.
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`Totally, as many as ca. 180,000 conformations for each of the cyclic moieties w