`
`
`
`I IIIII IIIIIIII III
`
`
`
`
`
`IIIII 111111 IIII
`
`(12) United States Patent
`Shailubhai et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,041,786 B2
`May 9, 2006
`
`(54) GUANYLATE CYCLASE RECEPTOR
`AGONISTS FOR THE TREATMENT OF
`TISSUE INFLAMMATION AND
`CARCINOGENESIS
`
`(75)
`
`Inventors: Kunwar Shailubhai, Blue Bell, PA
`(US); Gregory Nikiforovich, St. Louis,
`MO (US); Gary S. Jacob, Creve Coeur,
`MO (US)
`
`(73) Assignee: Callisto Pharmaceuticals, New York,
`NY (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 362 days.
`
`(21) Appl. No.: 10/107,814
`
`(22) Filed:
`
`Mar. 28, 2002
`
`(65)
`
`Prior Publication Data
`
`US 2003/0073628 Al
`
`Apr. 17, 2003
`
`OTHER PUBLICATIONS
`
`Shailubhai K, et al. Clinical Cancer Res. (Proc. 1999 AACR
`NCI EORTC International Conference) 1999; 5 (Suppl.);
`Abstract #0734.*
`Pitari GM, et al. Proc. Natl. Acad. Sci. USA. Jul. 3, 2001; 98
`(14): 7846-51.*
`Natham A, et al. Bioconjug Chem. Jan.-Feb.; 1993 4 (1):
`54-62.*
`Caliceti P, et al. Biochimica et Biophysica Acta. 2001; 1528:
`177-86.*
`Hinds K, et al. Bioconjug. Chem. 2000; 11: 195-201.*
`Forte LR. Regul. Pept. May 31, 1999; 81 (1-3): 25-39.*
`Hikada Y, et al. Biochemistry. 1998; 37: 8498-507.*
`Hikada Y, et al. J. Biol. Chem. Aug 18, 2000; (33); 25155-
`62.*
`Klodt J, et al. J. Pept. Res. Sep. 1997; 50 (3): 222-30.*
`Garcia KC, et al. J. Biol. Chem. Oct 25, 1993; 268 (30):
`22397-401.*
`Baxter GF. Basic Res. Cardiol. Mar. 2004; 99 (2): 71-5.*
`Takada I, et' al. Mol. Endocrinol. 2000; 14 (5): 733-40.*
`Bergers G, et al. Current Opinion in Genetics and Develop-
`ment. 2000; 10: 120-7.*
`Gura T. Science. 1997; 278: 1041-2.*
`
`Related U.S. Application Data
`
`(Continued)
`
`(60) Provisional application No. 60/348,646, filed on Jan.
`17, 2002.
`
`(51) Int. Cl.
`A61K 38/12
`
`(2006.01)
`
`(52) U.S. Cl.
`
` 530/317; 530/300; 530/326;
`514/10; 514/13
` 530/317,
`(58) Field of Classification Search
`530/300, 326; 514/10, 13
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,489,670 A
`5,518,888 A
`5,601,990 A
`5,731,159 A
`5,879,656 A
`5,928,873 A
`5,969,097 A
`2002/0128176 Al *
`2005/0032684 Al
`
`2/1996 Currie et al.
`5/1996 Waldman
`2/1997 Waldman
`3/1998 Waldman
`3/1999 Waldman
`7/1999 Waldman
`10/1999 Wiegand et al.
`9/2002 Forssmann et al.
`2/2005 Cetin et al.
`
`FOREIGN PATENT DOCUMENTS
`
`WO
`WO
`
`WO 02/098912 A2
`WO 02/098912 A3
`
`12/2002
`12/2002
`
` 514/2
`
`Primary Examiner Stephen L. Rawlings
`(74) Attorney, Agent, or Firm Mintz, Levin, Cohn, Ferris
`Glovsky and Popeo, P.C.; Ivor R. Elrifi
`
`(57)
`
`ABSTRACT
`
`A method of treatment of inflamed, pre-cancerous or can-
`cerous 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 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 degra-
`dation of cGMP. Without requiring a particular mechanism
`of action, this treatment may restore a healthy balance
`between proliferation and apoptosis in the subject's popu-
`lation of epithelial cells, and also suppress carcinogenesis.
`Thus, the method may be used to treat, inter alia, inflam-
`mation, including gastrointestinal inflammatory disorders,
`general organ inflammation and asthma, and carcinogenesis
`of the lung, gastrointestinal tract, bladder, testis, prostate and
`pancreas, or polyps.
`
`6 Claims, No Drawings
`
`MYLAN - EXHIBIT 1005
`
`

`

`US 7,041,786 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Shailubhai K. Curr. Opin. Drug Discov. Devel. Mar. 2002;
`5 (2): 261-8.*
`Shailubhai et al., "Uroguanylin Treatment Suppresses Polyp
`Formation in the Apc Mini+ Mouse and Induces Apoptosis
`in Human Colon Adenocarcinoma Cells via Cyclic GMP"
`Cancer Research 60 (Sep. 15, 2000) 5151-5157.
`Carrithers et al., "Guanylyl cyclase C is a selective marker
`for metastatic colorectal tumors in human extraintestinal
`tissues" Proc. Natl. Acad. Sci. USA 93 (Dec. 1996) 14827-
`14832.
`Hill et al., "Analysis of the human guanylin gene and the
`processing and cellular localization of the peptde" Proc.
`Natl. Acad. Sci. USA 92 (Mar. 1995) 2046-2050.
`
`Hamra et al., "Uroguanylin: Structure and activity of a
`second endogenous peptide
`that stimulates
`intestinal
`guanylate cyclase" Proc. Natl. Acad. Sci. USA 90 (Nov.
`1993) 10464-10468.
`De Sauvage et al., "Precursor structure, expression and
`tissue distribution of human guanylin" Proc. Natl. Acad. Sci.
`USA 89 (Oct. 1992) 9089-9093.
`Currie et al., "Guanylin: An endogenous activator of
`intestinal guanylate cyclase" Proc. Natl. Acad. Sci. USA 89
`(Feb. 1992) 947-951.
`Sindice, et al., Journal of Biological Chemistry, 277:17758-
`17764 (2002).
`
`* cited by examiner
`
`

`

`US 7,041,786 B2
`
`1
`GUANYLATE CYCLASE RECEPTOR
`AGONISTS FOR THE TREATMENT OF
`TISSUE INFLAMMATION AND
`CARCINOGENESIS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`The present application claims the benefit of U.S. provi-
`sional application No. 60/348,646, filed on Jan. 17, 2002.
`
`FIELD OF THE INVENTION
`
`The present invention relates to the therapeutic use of
`guanylate cyclase receptor agonists as a means for enhanc-
`ing 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 can-
`cerous, 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 monophosphate (cGMP) (1-6). This results in the
`activation of the cystic fibrosis 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. There-
`fore, by serving as paracrine regulators of CFTR activity,
`cGMP receptor agonists regulate fluid and electrolyte trans-
`port in the GI tract (1-6; U.S. Pat. No. 5,489,670).
`The process of epithelial renewal involves the prolifera-
`tion, 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 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 mutated and unwanted
`cells are replenished with new cells. Hence, homeostasis of
`the GI mucosa is regulated by continual maintenance of the
`balance between proliferation and apoptotic rates (8 ).
`The rates of cell proliferation and apoptosis in the gut
`epithelium can be increased or 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 dietary habits. In
`addition, an enhanced proliferation rate is frequently asso-
`ciated with a reduction in turnover time and an expansion of
`the proliferative zone (10). The proliferation index has been
`observed to be much higher in pathological cases of ulcer-
`ative colitis and other GI disorders (11). Thus, intestinal
`hyperplasia is the major promoter of gastrointestinal inflam-
`mation and carcinogenesis.
`
`30
`
`2
`In addition to a role for uroguanylin and guanylin as
`modulators of intestinal fluid and ion secretion, these pep-
`tides may also be involved in the continual renewal of GI
`mucosa. Previously published data in WO 01/25266 sug-
`5 gests a peptide with the active domain of uroguanylin may
`function as an inhibitor of polyp development in the colon
`and may constitute a treatment of colon cancer. However,
`the mechanism by which this is claimed to occur is ques-
`tionable in that WO 01/25266 teaches uroguanylin agonist
`10 peptides that bind specifically to a guanylate cyclase recep-
`tor, 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
`15 ST in intestine, but effects of uroguanylin and ST are not
`disturbed in the kidney in vivo (3). These results were
`further supported by the fact that membrane depolarization
`induced by guanylin was blocked by genistein, a tyrosine
`kinase inhibitor, whereas hyperpolarization induced by
`20 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.
`Other papers have reported that production of uroguanylin
`and guanylin is dramatically decreased in pre-cancerous
`25 colon polyps and tumor tissues (14-17). In addition, genes
`for both uroguanylin and guanylin have been shown to be
`localized to regions of the genome frequently associated
`with loss of heterozygosity in human colon carcinoma
`(18-20). Taken together, these findings indicate that urogua-
`nylin, 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 dem-
`onstrating oral administration of uroguanylin inhibits polyp
`formation in mice (15,16).
`Uroguanylin and guanylin peptides also appear to pro-
`mote apoptosis by controlling cellular ion flux. Alterations
`in apoptosis have been associated with tumor progression to
`the metastatic phenotype. While a primary gastrointestinal
`40 (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 I(' and influx of Ca++, urogua-
`nylin and related peptides may promote the death of trans-
`45 formed 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
`50 have also suggested that the CFTR channel is involved in the
`transport and maintenance of reduced glutathione, an anti-
`oxidant 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
`55 activation or by way of inhibition of cGMP-specific phos-
`phodiesterase would be expected to down-regulate these
`inflammatory stimuli. Thus, uroguanylin-type agonists
`should be useful in the prevention and treatment of inflam-
`matory diseases of the lung (e.g., asthma), bowel (e.g.,
`60 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 thera-
`peutic value in the treatment of a wide variety of inflam-
`65 matory conditions, cancer (particularly colon cancer) and as
`anti-metastatic agents. The development of new agonists is
`therefore of substantial clinical importance.
`
`35
`
`

`

`US 7,041,786 B2
`
`3
`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
`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 of cGMP can be
`increased by enhancing intracellular production of cGMP
`and/or by inhibition of its degradation by cGMP-specific
`phosphodiesterases. Among the specific conditions that can
`be treated or prevented are inflammatory conditions, cancer,
`polyps, and metastasis.
`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 composi-
`tions 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, a
`peptide differs substantially if its structure varies by more
`than three amino acids from a peptide of SEQ ID NOs:2-21
`or if its activation of cellular cGMP production is reduced or
`enhanced by more than 50%. Preferably, substantially simi-
`lar peptides should differ by no more than two amino acids
`and not differ by more than about 25% with respect to
`activating cGMP production. The most preferred peptide is
`a bicycle having the sequence of SEQ ID NO:20.
`The peptides may be in a pharmaceutical composition in
`unit dose form, together with one or more pharmaceutically
`acceptable excipients. The term "unit dose form" refers to a
`single drug delivery entity, e.g., a tablet, capsule, solution or
`inhalation formulation. The amount of peptide present
`should be sufficient to have a positive therapeutic effect
`when administered to a patient (typically, between 100 µg
`and 3 g). What constitutes a "positive 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 example, it may
`constitute a reduction in inflammation, a shrinkage of polyps
`or tumors, a reduction in metastatic lesions, etc.
`The invention also encompasses combination therapy
`utilizing a guanylate cyclase receptor agonist administered
`either alone or together with an inhibitor of cGMP-depen-
`dent phosphodiesterase, an anti-inflammatory agent or an
`anticancer agent. These agents should be present in amounts
`known in the art to be therapeutically effective when admin-
`istered to a patient. Anti-neoplastic agents may include
`alkylating agents, epipodophyllotoxins, nitrosoureas, anti-
`metabolites, vinca alkaloids, anthracycline antibiotics, nitro-
`gen mustard agents, and the like. Particular anti-neoplastic
`agents may include tamoxifen, TAXOLTM, 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, par-
`ticularly 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 syn-
`thetic guanylate cyclase receptor agonist. The term "effec-
`tive amount" refers to sufficient agonist to measurably
`
`30
`
`4
`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 sub-
`stitutions not present in known endogenous guanylate
`5 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
`10 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
`15 treating tumor metastasis from a primary tumor mass. Meta-
`static 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
`20 cells derived from those cancers. Such receptors are typi-
`cally 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
`25 mechanism of action, it is believed that the peptides will act
`by binding to these cellular receptors and inducing apopto-
`sis. Metastatic tumors may also be treated by administering
`any known form of uroguanylin or guanylin (preferably
`human) or by administering E. coli ST peptide.
`Peptides may be administered either alone or together
`with one or more inhibitors of cGMP dependent phosphodi-
`esterase. Examples of cGMP dependent phosphodiesterase
`inhibitors include suldinac sulfone, zaprinast, and motapi-
`zone. Treatable forms of cancer include breast cancer, col-
`35 orectal cancer, lung cancer, ovarian cancer, pancreatic can-
`cer, 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
`40 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 inflam-
`45 mation (e.g., inflammation associated with the GI tract,
`asthma, nephritis, hepatitis, pancreatitis, bronchitis, or cystic
`fibrosis) of a subject by administering a composition com-
`prising an agonist of a guanylate cyclase receptor that
`enhances intracellular production of cGMP. Preferred pep-
`s() tide 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 option-
`ally be administered with one or more inhibitors of cGMP
`dependent phosphodiesterase, e.g., suldinac sulfone, zapri-
`55 oast, or motapizone. In a preferred embodiment, the inven-
`tion is directed to a method of treating an inflammatory
`disorder in a mammalian gastrointestinal tract. The inflam-
`matory disorder may be classified as an inflammatory bowel
`disease, and more particularly may be Crohn's disease or
`60 ulcerative colitis. Administration may be enteric, and
`employ formulations tailored to target enterocytes.
`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
`65 ID 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
`
`

`

`US 7,041,786 B2
`
`5
`a target tissue. For example, sufficient peptide may be given
`to induce an increased rate of cell death in a neoplastic
`growth.
`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):
`
`6
`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
`5 envisioned that ion transport across the plasma membrane
`may prove to be an important regulator of the balance
`
`Asn1 Asp2 G111.3 Cys 4 G111.5 Leu 6 Cy's' Val 8 Asn9 Va110 Ala11 Cys12 Thr13 Gly14 Cys15 Leu16
`
`and wherein there is one disulfide linkage between the
`cysteine at position 4 and the cysteine at position 12; and a
`second disulfide linkage between the cysteine at position 7
`and the cysteine at position 15 (SEQ ID NO:20). This
`peptide has been found to have enhanced biological activity
`as an agonist of cGMP production due to its enhanced
`binding constant for the guanylate cyclase receptor, and is
`superior to uroguanylin with regard to temperature and
`protease stability and with regard to its biological activity at
`the physiologically favorable pH range (pH 6 to 7) in the
`large intestine.
`The guanylate cyclase receptor agonists used in the meth-
`ods described above may be administered either orally,
`systemically or locally. Dosage forms include preparations
`for inhalation or injection, solutions, suspensions, emul-
`sions, tablets, capsules, topical salves and lotions, transder-
`mal compositions, other known peptide formulations and
`pegylated peptide analogs. An effective dosage of the com-
`position will typically be between about 1 µg and 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 are
`routine in the art and will be based upon the particular
`composition being used and clinical considerations. Ago-
`nists 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, additional drugs
`should be administered at a dosage that is therapeutically
`effective using the 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 and that a reduction in cGMP levels, due to a
`deficiency of uroguanylin/guanylin and/or due to the acti-
`vation of cGMP-specific phosphodiesterases, is an early and
`critical step in neoplastic transformation. A second concept
`is that the release of arachidonic acid from membrane
`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, 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-dependent mechanism, is
`thought to be involved in the control of proinflammatory
`processes. Therefore, elevating intracellular levels of cGMP
`may be used as a means of treating and controlling inflam-
`matory bowel diseases such as ulcerative colitis and Crohn's
`
`between cell proliferation and apoptosis that will be affected
`by compositions altering cGMP concentrations. Uroguany-
`15 lin has been shown to stimulate I(' 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
`20 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
`25 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 inflammatory diseases of the GI
`30 tract and general organ inflammation (e.g., asthma, nephri-
`tis, hepatitis, pancreatitis, bronchitis, cystic fibrosis).
`In another aspect, the invention is directed to a method for
`preventing, treating or retarding the onset of cancer, par-
`35 ticularly cancer of epithelial cells, in a subject by adminis-
`tering 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
`40 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
`45 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 effec-
`tive 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 cGMP-dependent mechanism that regulates the bal-
`55 ance between cellular proliferation and apoptosis in meta-
`static tumor cells may serve as a mechanism for targeting
`and treating metastatic tumors. The liver is the most com-
`mon site of metastasis from a primary colorectal cancer.
`Toward later stages of disease, colorectal metastatic cells
`60 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 gua-
`nylate cyclase receptors and therefore, these cells should be
`sensitive to apoptosis therapy mediated by intestinal gua-
`65 nylate cyclase receptors. Peptides having uroguanylin activ-
`ity, when used either alone or in combination with specific
`inhibitors of cGMP-phosphodiesterase, also retard the onset
`
`50
`
`

`

`US 7,041,786 B2
`
`7
`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 pro-
`duction. 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:1. This term also covers fragments and
`pro-peptides that bind to guanylate cyclase receptor and
`stimulate cGMP production. The term "substantially equiva-
`lent" 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-resistant pep-
`tide, oral or systemic administration of this peptide and/or
`other peptides similar to the functionally active 16 amino
`acid peptide sequence of SEQ ID NO:1 may be effectively
`employed in treatment methods.
`Peptides similar to, but distinct from, uroguanylin are
`described below, including some which produce superior
`cGMP enhancing properties and/or other beneficial charac-
`teristics (e.g., improved temperature stability, enhanced pro-
`tease stability, or superior activity at preferred pH's) com-
`pared to previously known uroguanylin peptides. The
`peptides may be used to inhibit GI inflammation and for
`treating or preventing the onset of polyp formation associ-
`ated with gut inflammation. Epithelial tissues susceptible to
`cancer cell formation may also be treated. The guanylate
`cyclase receptor agonists described have the amino acid
`sequences shown in Tables 2 and 3. The "binding domain"
`for agonist-receptor interaction includes the amino acid
`residues from 3-15 of SEQ ID NO:1.
`Molecular modeling was applied to the design of novel
`guanylate cyclase receptor agonists using methods detailed
`in (30). It consisted of energy calculations for three com-
`pounds known to interact with guanylate cyclase receptors,
`namely for human uroguanylin, bicyclo [4,12; 7,15]Asn -
`2
`Asp -Asp3-Cys4-Glus-Leu6Cys7-Val$-Asn9-Val10-Ala"_
`Cys'2-Thr13-Gly14-Cysts-Leu16
`(UG, SEQ ID NO:1);
`human guanylin, bicyclo [4,12; 7,15]Pro -Gly2-Thr3-Cys4-
`Glu -Ile6-Cys7-Alas-Tyr9-Alal °-Ala" -Cys12-Thr13-Gly14-
`Cys15 (GU, SEQ ID NO:22); and E. coli small heat-stable
`enterotoxin, tricyclo [6,10; 7,15; 11-18] Asn -Ser2-Ser3-
`Asn -Tyr-5 -Cys6-Cys7-Glus -Leu9-Cys 1° -Cys" -Asn12-Pro 3 -
`Alam-Cys15-Thr16
`-Gly"-Cys"-Tyr'
`(ST, SEQ
`ID
`NO:23). Geometrical comparisons of all possible low-en-
`ergy conformations for these three compounds were used to
`reveal the common 3D structures that served as the "tem-
`
`5
`
`8
`plates" for the bioactive conformation, i.e., for the confor-
`mation presumably adopted by GU, UG and ST during
`interaction with receptor. It allowed designing novel analogs
`with significantly increased conformational population of
`the bioactive conformation at the expense of other low-
`energy conformations by selecting individual substitutions
`for various amino acid residues.
`Energy calculations were performed by use of build-up
`10 procedures (30). The ECEPP/2 potential field (31,32) was
`used assuming rigid valence geometry with planar trans-
`peptide bonds, including that for Pro13 in ST. The w angle in
`Pro13 was allowed to vary. Aliphatic and aromatic hydrogens
`were generally included in united atomic centers of CH,
`15 type; H"-atoms and amide hydrogens were described explic-
`itly.
`The main calculation scheme involved several successive
`steps. First, the sequences of the two monocyclic model
`fragments (three fragments for ST), Ac-cyclo (Cys- . . .
`20 -Cys')-NMe, were considered, where all residues except
`Cys, Gly and Pro were replaced by alanines; the i and j
`values corresponded to the sequences of GU, UG and ST. At
`this step, all possible combinations of local minima for the
`peptide backbone for each amino acid residue were consid-
`25 ered, 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, C D and A* types
`for the Gly residue; and of F, C and A types for Pro. For each
`backbone conformation, one optimal possibility to close a
`30 cycle employing the parabolic potential functions, intrinsic
`to the ECEPP force field, was found by checking an energy
`profile of rotation around the dihedral angle xi for the D-Cys
`residue.
`Totally, as many as ca. 180,000 conformations for each of
`35 the cyclic moieties were considered. Then, the conformers
`satisfying the E-E„„,<AE-15 kcal/mol criterion and differ-
`ing by more than 40° in at least one value of any backbone
`dihedral angle were selected (from ca. 3,000 to 8,000
`conformations for different model fragments). At the next
`step, the selected conformations of the matching monocyclic
`fragments were overlapped to create possible conformations
`of the bicyclic model fragments (the tricyclic fragments in
`the case of ST). Typically, this procedure yielded ca.
`45 20,000-30,000 conformations. All these conformations
`were submitted for a new