pRc,v 0
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`REQUEST FOR FILING PROVISIONAL PATENT APPLICATION
`Under 35 USC 111(b)
`(Not for DESIGN cases)
`
`;:1
`
`Aka =e,
`m m.
`=•=r.
`woo
`row. — 0
`u
`Box:
`PROVISIONAL'_'
`APPLICATION
`
`r.
`
`
`Ca
`
`
`
` Commissioner of Patents
`76 (ashington, D.C. 20231
`
`PROVISIONAL APPLICATION
`Under Rule 53(c)
`
`Sir:
`
`IIIIIIII tll IIIIIIIIIIIIIII II
`
`
`00909
`
`Herewith is a PROVISIONAL APPLICATION
`Title: GUANYLATE CYCLASE RECEPTOR AGONISTS FOR
`THE TREATMENT OF TISSUE INFLAMMATION AND
`CARCINOGENESIS
`Including:
`
`Client Ref
`
`Atty. Dkt. PW 284936
`M#
`Date: January 17, 2002
`3. D Drawings:
` sheet(s)
`
` pages 2. O Specification in non-English language
`. Specification: 31
`:24. The invention O was IM was not made by, or under a contract with, an agency of the U.S. Government.
`If yes, Government agency/contact # =
`5 D Attached is an assignment and cover sheet. Please return the recorded assignment to the undersigned.
`[21 is Not claimed
`O is claimed (pre-filing confirmation required)
`6.
`Small Entity Status m
`NOTE: Do NOT File IDS!
`Attached:
`
`0
`
`This application is made by the following named inventor(s) (Double check instructions for accuracy.):
`
`\(1) Inventor Kunwar
`
`Residence
`
`I Somerset
`
`(2) inventor Gregory
`
`Residence
`
`I St. Louis
`
`(3) Inventor Gary
`
`Residence
`
`I St. Louis
`
`(4) Inventor
`
`Residence
`
`I
`
`(5) Inventor
`
`Residence
`
`I
`
`First
`
`City
`
`First
`
`City
`
`First
`
`City
`
`First
`
`City
`
`First
`
`SHAILUBHAI
`
`Middle Initial
`New Jersey
`State/Foreign Cauhtry
`
`,
`
`NIKIFOROVICH
`
`V.
`Middle Initial „
`I Missouri
`
`State/Foreign CPuntry
`
`Family Nartie
`I USA
`
`Country of Citizenship
`
`FamilyNarne „
`I USA
`
`Country of Citizenship
`
`JACOB
`
`S.
`Middle Initial
`I Missouri
`
`Family Naine ,
`I USA
`
`State/Foreign Country
`
`,Country of Citizenship'
`
`Middle Initial
`
`I
`
`Middle Initial
`
`I
`
`Family Name
`
`State/Foreign Country
`
`CoUntry.of Citizenship:
`
`Family Name
`I
`
`Country of ;Citizenship.
`City
`State/Foreigh Country
`9. NOTE: FOR ADDITIONAL INVENTORS, check box q and attach sheet (PAT102A) with same information
`regarding additional inventors.
`
`30255518_1.DOC
`
`P AT-1 02NCN 10101
`
`a.
`
`A /PRoV
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`REQUEST FOR FILING PROVISIONAL PATENT APPLICATION
`Under 35 USC 111(b)
`(Not for DESIGN cases)
`
`
`
`
`
`| PROVISIONAL APPLICATION
`
`
`Under Rule 53(c)
`
`
`
`
`
`=:
`=o
`=_
`=u
`og
`
`20/11/10
`
`=n Commissioner of Patents
`
`ashingon. 0.0. 2020
`
`Sir:
`
`= =x
`sS=°
`rS=a
`T="
`Box:
`PROVISIONAL" —
`APPLICATION
`| I
`|
`|
`|
`
`IMUM
`
`|
`
`|
`
`00909
`
`|
`
`a PROVISIONAL APPLICATION
`Herewith is
`Title:
`GUANYLATE CYCLASE RECEPTOR AGONISTS FOR
`THE TREATMENT OF TISSUE INFLAMMATION AND
`CARCINOGENESIS
`Including:
`
`Atty. Dkt.
`
`PW 284936
`M#
`
`
`
`Client Ref
`
`Date: January 17, 2002
`
`sheet(s)
`
`3. [] Drawings:
`2. [] Specification in non-English language
`pages
`31
`. Specification:
`. The invention [_] was IX] was not made by, or under a contract with, an agency of the U.S. Government.
`If yes, Government agency/contact # =
`Attached is an assignment and cover sheet. Please return the recorded assignment to the undersigned.
`Small Entity Status @
`is Not claimed
`[J is claimed (pre-filing confirmation required)
`NOTE: Do NOT File IDS!
`Attached:
`
`.[1
`
`.[0
`
`
`
`
`
`This application is made by the following named inventor(s) (Double check instructions for accuracy.):
`
`
`
`|
`
`
`
`
`| SHAILUBHAI
`“R(1) Inventor | Kunwar
`
`___Famiy Name
`Middle Inifal_____
`i EEE
`First
`
`
`| USA
`_[ New Jersey
`J Residence | Somerset
`"Country of Citizenship
`
`City
`Ln
`)
`StatefForeign Country
`CL
`
`
`
`@1r Inventor | Gregory
`
`
`
`| NIKIFOROVICH
`
`Co
`First
`:
`Middle Inifial
`. Family Name
`
`| Missouri
`| USA
`Residence
`| St. Louis
`ER
`_
`City
`State/Forsign Couritry
`Country of Citizenship
`.
`
`
`
`|v.
`
`
`La) Inventor | Gary
`|s.
`JACOB
`
`First
`Middle Initial
`Family Name, .
`
`| Missouri
`| usa
`Residence
`| St. Louis
`
`:
`:
`StatefForeign County >...
`City
`"_Coimtry of Citizenship"
`
`
`
`(4) Inventor |
`1
`|
`hE
`First
`Middle Initial
`Family Name:
`
`
`Residence
`|
`
`.
`City
`-State/Foreign Country
`Country.of Citizenship:
`~
`
`
`
`(5) Inventor |
`|
`First
`Middle Initial
`Family Namé
`
`
`\
`
`:
`
`y
`
`Residence
`
`[
`
`|
`
`|
`
`. Country of Citizenship.
`. StatefForeign Country
`City
` 9. NOTE: FOR ADDITIONAL INVENTORS, check box [_] and attach sheet (PAT102A) with same information
`regarding additional inventors.
`
`30255518_1.D0C
`
`PAT-102A/CN 10/01
`
`
`
`
`
`
`Pg.001
`
`MYLAN EXHIBIT - 1059
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

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`PAT-102A/CN 10/01
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`Pg.002
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`

`

`APPLICATION UNDER UNITED STATES PATENT LAWS
`
`Atty. Did. No. PW 284936
`
`(M#)
`
`Invention:
`
`GUANYLATE CYCLASE RECEPTOR AGONISTS FOR THE TREATMENT OF TISSUE
`INFLAMMATION AND CARCINOGENESIS
`
`Inventor (s): Kunwar SHAILUBHAI
`Gregory V. NIKIFOROVICH
`Gary S. JACOB
`
`ie
`
`00909
`Pillsbury Winthrop LLP
`
`This is a:
`
`Provisional Application
`
`111
`
`Regular Utility Application
`
`Continuing Application
`lE1 The contents of the parent are incorporated
`by reference
`
`q PCT National Phase Application
`
`q Design Application
`q Reissue Application
`
`q Plant Application
`
`q Substitute Specification
`Sub. Spec Filed
`in App. No.
`
`q Marked up Specification re
`Sub. Spec. filed
`In App. No
`
`SPECIFICATION
`
`Document2
`
`PAT-100CN 10/01
`
`Es
`
`v
`
`APPLICATION UNDER UNITED STATES PATENT LAWS
`
`Atty. Dkt. No.
`
`_PW 284936
`(M#)
`
`Invention:
`
`GUANYLATE CYCLASE RECEPTOR AGONISTS FOR THE TREATMENT OF TISSUE
`INFLAMMATION AND CARCINOGENESIS
`
`Inventor (s).
`
`Kunwar SHAILUBHAI
`Gregory V. NIKIFOROVICH
`Gary S. JACOB
`
`HII
`00909
`Pillsbury Winthrop LLP
`
`This is a:
`
`PJ
`
`Provisional Application
`
`Regular Utility Application
`
`Continuing Application
`The contents of the parent are incorporated
`by reference
`
`PCT National Phase Application
`
`Design Application
`
`Reissue Application
`
`Plant Application
`
`Substitute Specification
`Sub. Spec Filed
`
`
` in App. No. /
`
`
`
`
`
`Marked up Specification re
`Sub. Spec. filed
`
`In App. No
`/
`
`
`
`[1]
`
`[1
`
`O0o0Odnd
`
`[1]
`
`SPECIFICATION
`
`Document?
`
`
`
`PAT-100CN 10/01
`
`Pg.003
`
`

`

`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`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 and pre-cancerous growths, particularly in the gastrointestinal tract,
`pancreas and lungs. In addition, the agonists may be used in the treatment of inflammatory
`disorders and asthma.
`
`5
`
`10
`
`Background of the Invention
`Uroguanylin, guanylin and bacterial ST peptides are structurally related agonist
`peptides that bind to a guanylate cyclase receptor and stimulate intracellular production of
`cyclic guanosine monophosphate (cGMP) (8-13). This can result in activation of the cystic
`fibrosis transmembrane conductance regulator (CFTR), an apical membrane channel for efflux
`of chloride from enterocytes lining the intestinal tract (8-13). Activation of CFTR and the
`subsequent enhancement of transepithelial secretion of chloride leads to stimulation of sodium
`(Na+) and water secretion into the intestinal lumen. Therefore, one of the physiological
`functions of these hormones is the regulation of fluid and electrolyte transport in the GI tract
`by serving as paracrine regulators of CFTR activity (8-13).
`
`25
`
`30
`
`One of the major clinical manifestations of defective mutations in CFTR is excessive
`inflammation in airway passages (19), implying that CFTR is involved in control of
`proinflammatory signaling pathways, particularly via regulation of expression of NF-kB,
`chemokines and cytokines (20-22). Several lines of evidence suggest that CFTR dysfunction
`influences production of some of these molecules (23). Exactly how CFTR dysfunction
`contributes to the activation of and nuclear localization of NF-KB is unclear. One of the
`possible mechanisms could be via regulation of intracellular levels of K+/Na+ and via
`regulation of acidity (pI-I) of specific intracellular compartments affecting relevant kinase and
`phosphatase activities (24). Recent reports have noted that the CFTR channel is also involved
`in transport and maintenance of reduced glutathione, an antioxidant that plays an important
`role in protection against oxidative stress and free-radical mediated cell damage. It is also very
`
`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`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 and pre-cancerous growths, particularly in the gastrointestinal tract,
`
`pancreas and lungs. In addition, the agonists may be used in the treatment of inflammatory
`
`10
`
`disorders and asthma.
`
`Background of the Invention
`
`Uroguanylin, guanylin and bacterial ST peptides are structurally related agonist
`
`peptides that bind to a guanylate cyclase receptor and stimulate intracellular production of
`
`cyclic guanosine monophosphate (cGMP) (8-13). This can result in activation of the cystic
`
`fibrosis transmembrane conductance regulator (CFTR), an apical membrane channel for efflux
`
`of chloride from enterocytes lining the intestinal tract (8-13). Activation of CFTR and the
`
`subsequent enhancement of transepithelial secretion of chloride leads to stimulation of sodium
`
`(Na") and water secretion into the intestinal lumen. Therefore, one of the physiological
`
`functions of these hormones is the regulation of fluid and electrolyte transport in the GI tract
`
`by serving as paracrine regulators of CFTR activity (8-13).
`
`One of the major clinical manifestations of defective mutations in CFTR is excessive
`
`inflammation in airway passages (19), implying that CFTR is involved in control of
`
`25
`
`proinflammatory signaling pathways, particularly via regulation of expression of NF-kB,
`
`chemokines and cytokines (20-22). Several lines of evidence suggest that CFTR dysfunction
`
`influences production of some of these molecules (23). Exactly how CFTR dysfunction
`
`contributes to the activation of and nuclear localization of NF-KB is unclear. One of the
`
`possible mechanisms could be via regulation of intracellular levels of K+/Nat+ and via
`
`30
`
`regulation of acidity (pH) of specific intracellular compartments affecting relevant kinase and
`
`phosphatase activities (24). Recent reports have noted that the CFTR channel is also involved
`
`in transport and maintenance of reduced glutathione, an antioxidant that plays an important
`
`role in protection against oxidative stress and free-radical mediated cell damage. It is also very
`
`
`
`Pg.004
`
`

`

`well established that excessive oxidative stress is one of the primary reasons for activation of
`NF-KB and in pathogenesis of inflammatory diseases and cancer (25).
`
`In addition to a role for uroguanylin and guanylin as modulators of intestinal fluid and
`ion secretion, these peptides may also be involved in the continual renewal of GI mucosa.
`
`5
`
`Previously published data in WO 01/25266 Al suggests 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 questionable, as WO 01/25266 Al teaches uroguanylin agonist peptides that bind
`
`to
`
`specifically to a guanylate cyclase receptor, termed GC-C, that was first described as the
`
`receptor for E. coli heat-stable enterotoxin (ST) (26). In contrast, uroguanylin was shown to
`
`activate K+ conductance via a so far unknown receptor distinct from GC-C (27). This
`
`conclusion was further supported by the fact that membrane depolarization induced by
`
`guanylin was blocked by genistein, a tyrosine kinase inhibitor, whereas hyperpolarization
`
`induced by uroguanylin was not. Taken together these data suggest that uroguanylin binds to a
`
`currently unknown receptor, which is distinct from GC-C (27).
`
`Several lines of evidence have implicated efflux of K+ and influx of Ca++ in the
`
`induction of apoptosis. First, staphylococcal a -toxin has been shown to induce apoptosis by
`
`selectively decreasing the intracellular concentrations of monovalent cations. Second, apoptotic
`and shrunken cells have been shown to contain reduced levels of intracellular K+ as compared
`to those in normal cells. Third, an intracellular concentration of K+ in excess of 150 mM has
`been shown to inhibit apoptosis by inhibiting proapoptotic nucleases such as caspase-3.
`Finally, activation of K+ efflux and Ca++ influx has been shown to stimulate enzymatic
`activities of several nucleases that play critical roles in the induction of apoptosis (28-32).
`
`The therapeutic benefits of non-steroidal anti-inflammatory drugs (NSAIDs) in the
`treatment of familial adenomatous polyposis, certain other cancers and in inflammatory
`diseases are well documented and impressive. However, the mechanisms by which NSAIDs
`act to reduce inflammatory signals and tumorigenesis remain unclear. NSAIDs are known to
`bind and inhibit the cyclooxygenase (COX) enzymes, COX-1 and COX-2, which produce
`prostaglandins (PGs). A role for PGs in promoting inflammation and tumorigenesis is very
`well supported by the observations that, relative to normal tissue, inflamed tissues and tumor
`
`:35
`
`20
`
`25
`
`30
`
`2
`
`well established that excessive oxidative stress is one of the primary reasons for activation of
`
`NF-KB and in pathogenesis of inflammatory diseases and cancer (25).
`
`In addition to a role for uroguanylin and guanylin as modulators of intestinal fluid and
`
`ion secretion, these peptides may also be involved in the continual renewal of GI mucosa.
`
`Previously published data in WO 01/25266 Al suggests 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 questionable, as WO 01/25266 Al teaches uroguanylin agonist peptides that bind
`
`10
`
`specifically to
`
`a guanylate cyclase receptor, termed GC-C, that was first described as the
`
`receptor for E.coli heat-stable enterotoxin (ST) (26). In contrast, uroguanylin was shown to
`
`activate K* conductance via a
`
`so
`
`far unknown receptor distinct from GC-C (27). This
`
`conclusion was further supported by the fact that membrane depolarization induced by
`
`guanylin was blocked by genistein, a tyrosine kinase inhibitor, whereas hyperpolarization
`
`induced by uroguanylin was not. Taken together these data suggest that uroguanylin binds to a
`
`currently unknown receptor, which is distinct from GC-C (27).
`
`Several lines of evidence have implicated efflux of K* and influx of Ca’™ in the
`
`induction of apoptosis. First, staphylococcal a-toxin has been shown to induce apoptosis by
`
`selectively decreasing the intracellular concentrations of monovalent cations. Second, apoptotic
`
`and shrunken cells have been shown to contain reduced levels of intracellular K™ as compared
`
`to those in normal cells. Third, an intracellular concentration of K' in excess of 150 mM has
`
`been shown to inhibit apoptosis by inhibiting proapoptotic nucleases such as caspase-3.
`
`Finally, activation of K' efflux and Ca" influx has been shown to stimulate enzymatic
`
`25
`
`activities of several nucleases that play critical roles in the induction of apoptosis (28-32).
`
`The therapeutic benefits of non-steroidal anti-inflammatory drugs (NSAIDs) in the
`
`treatment of familial adenomatous polyposis, certain other cancers and in inflammatory
`
`diseases are well documented and impressive. However, the mechanisms by which NSAIDs
`
`30
`
`act to reduce inflammatory signals and tumorigenesis remain unclear. NSAIDs are known to
`
`bind and inhibit the cyclooxygenase (COX) enzymes, COX-1 and COX-2, which produce
`
`prostaglandins (PGs). A role for PGs in promoting inflammation and tumorigenesis is very
`
`well supported by the observations that, relative to normal tissue, inflamed tissues and tumor
`
`
`
`Pg.005
`
`

`

`tissues contain higher levels of both PGs and COX-2 (32-36). Further evidence of the
`importance of the COX-2 pathway, in particular, is provided by anti-tumorigenic activities of
`selective COX-2 inhibitors and NSAIDs (37,38). This is consistent with a recent report
`demonstrating that the deletion of cytosolic phospholipase A2 (cPLA2) suppressed tumor
`formation in Apcmini+ mouse (39). However, disruption of the mouse group IIA secretory
`
`5
`
`phospholipase A2 (sPLA2) locus, another potential source of arachidonic acid for COX-2,
`
`promoted tumor formation, suggesting the possibility of a PG independent mechanism (39).
`
`We believe that activation of cPLA2, COX-2 and possibly 5-lipoxygenase (5-LO)
`
`10
`
`during the processes of inflammation and carcinogenesis might be regulated by a cGMP-
`
`dependent mechanism. Therefore, enhancement of intracellular levels of cGMP by way of
`
`guanylate cyclase activation and/or by way of inhibition of cGMP-specific phosphodiesterase
`
`is expected to downregulate inflammatory stimuli. A cGMP-dependent mechanism, which
`
`regulates the balance between proliferation and apoptosis, is also thought to be involved in the
`
`5
`
`control of proinflammatory processes. Therefore, elevating intracellular levels of cGMP is
`
`expected to be a promising approach to the treatment and control of inflammatory bowel
`
`diseases such as UC and Crohn's disease and to treat other organ inflammation.
`
`:20
`
`25
`
`30
`
`Asthma is also an inflammatory disease of lung tissue, which is primarily due to airway
`microvascular leakage and excessive synthesis of leukotrienes (40). The asthmatic response
`has been observed to be inhibited by treatment with the leukotriene receptor antagonist ONO-
`1078 (41), suggesting that leukotrienes might be involved in the etiology of the disease.
`Leukotrienes are products of 5-lipoxygenase, an enzyme that is up-regulated in a variety of
`inflammatory diseases (42). Several lines of evidence have suggested that excessive oxidative
`stress, reduced intracellular levels of glutathione and activation of NF-kB might be key factors
`promoting enhanced production of 5-lipoxygenase (43, 44). We propose that enhancement of
`intracellular production of cGMP is an attractive approach to suppress production of 5-
`lipoxygenase and thereby, this approach can be used to develop therapeutic agents for
`treatment of asthma and bronchitis. Recent research articles have suggested that uroguanylin
`inhibits leukotriene synthesis in airway epithelial cells, presumably via downregulation of 5-
`lipoxygenase (41, 42). The inventive peptides listed in Table 2 and 3 (SEQ. ID NOs.: 2-18) are
`proposed to be efficacious in the treatment of asthma, bronchitis and other organ inflammation.
`Recent experiments demonstrate that production of uroguanylin and guanylin is
`dramatically decreased in pre-cancerous colon polyps and tumor tissues (45-48). In addition,
`
`3
`
`tissues contain higher levels of both PGs and COX-2 (32-36). Further evidence of the
`
`importance of the COX-2 pathway, in particular, is provided by anti-tumorigenic activities of
`
`selective COX-2 inhibitors and NSAIDs (37,38).
`
`This is consistent with a recent report
`
`demonstrating that the deletion of cytosolic phospholipase A, (cPLA) suppressed tumor
`
`formation in Apc
`
`min mouse (39).
`
`However, disruption of the mouse group IIA secretory
`
`phospholipase A; (sPLA;) locus, another potential source of arachidonic acid for COX-2,
`
`promoted tumor formation, suggesting the possibility of a PG independent mechanism (39).
`
`We believe that activation of cPLA,, COX-2 and possibly 5-lipoxygenase (5-LO)
`
`10
`
`during the processes of inflammation and carcinogenesis might be regulated by a ¢cGMP-
`
`dependent mechanism. Therefore, enhancement of intracellular levels of cGMP by way of
`
`guanylate cyclase activation and/or by way of inhibition of cGMP-specific phosphodiesterase
`
`is expected to downregulate inflammatory stimuli.
`
`A cGMP-dependent mechanism, which
`
`regulates the balance between proliferation and apoptosis, is also thought to be involved in the
`
`control of proinflammatory processes.
`
`Therefore, elevating intracellular levels of cGMP is
`
`expected to be a promising approach to the treatment and control of inflammatory bowel
`
`diseases such as UC and Crohn's disease and to treat other organ inflammation.
`
`Asthma is also an inflammatory disease of lung tissue, which is primarily due to airway
`
`microvascular leakage and excessive synthesis of leukotrienes (40). The asthmatic response
`
`has been observed to be inhibited by treatment with the leukotriene receptor antagonist ONO-
`
`1078 (41), suggesting that leukotrienes might be involved in the etiology of the disease.
`
`Leukotrienes are products of 5-lipoxygenase, an enzyme that is up-regulated in a variety of
`
`inflammatory diseases (42). Several lines of evidence have suggested that excessive oxidative
`
`25
`
`stress, reduced intracellular levels of glutathione and activation of NF-kB might be key factors
`
`promoting enhanced production of 5-lipoxygenase (43, 44). We propose that enhancement of
`
`intracellular production of cGMP is an attractive approach to suppress production of 5-
`
`lipoxygenase and thereby, this approach can be used to develop therapeutic agents for
`
`treatment of asthma and bronchitis. Recent research articles have suggested that uroguanylin
`
`30
`
`inhibits leukotriene synthesis in airway epithelial cells, presumably via downregulation of 5-
`
`lipoxygenase (41, 42). The inventive peptides listed in Table 2 and 3 (SEQ. ID NOs.: 2-18) are
`
`proposed to be efficacious in the treatment of asthma, bronchitis and other organ inflammation.
`
`Recent experiments demonstrate that production of uroguanylin and guanylin is
`
`dramatically decreased in pre-cancerous colon polyps and tumor tissues (45-48). In addition,
`
`
`
`Pg.006
`
`

`

`genes for both uroguanylin and guanylin have been shown to be localized on the mouse
`chromosome 4 and to a syntenic position on human chromosome 1p34-35. This region, also
`known as the `cancer modifier region', is frequently associated with loss of heterozygosity in
`human colon carcinoma (49-51). Taken together, these findings suggest that uroguanylin and
`
`5
`
`guanylin might be regulators of the continual renewal process of the GI tract.
`
`These findings raise the possibility that the reduction in the production of these peptides
`
`might lead to disruption of the renewal of GI mucosa resulting in the formation of polyps and
`
`growth of tumors. Because production of uroguanylin and guanylin is essentially lost in polyps,
`
`10
`
`it is tempting to speculate that these peptides might be involved at an early stage of colon
`
`carcinogenesis. This possibility is consistent with a more recent study demonstrating that oral
`
`administration of uroguanylin inhibited polyp formation in nude mice (46, 47). Although the
`
`biochemical mechanism(s) for this chemopreventative activity is not known, it is plausible that
`
`these peptides might participate in a cGMP-mediated signaling mechanism in enterocytes that
`
`regulates mucosal cell turnover by initiating programmed cell death (apoptosis).
`
`The guanylate cyclase receptor-ligand system is also localized in the epithelium of
`
`other, non-intestinal mammalian organs, indicating that this system may be an appropriate
`
`target for interrupting carcinogenesis and tumor formation, and to treat inflammatory diseases
`
`2-0
`
`of a variety of tissues.
`
`Summary of the Invention
`
`The present invention is based upon the concept that the cGMP-dependent mechanism
`
`that regulates the balance between proliferation and apoptosis is an early step in the process of
`
`25
`
`carcinogenesis. Therefore, elevating intracellular levels of cGMP may be used to treat or
`
`prevent cancer and inflammatory diseases. Enhancement of intracellular production of cGMP
`
`may also be used to suppress the production of 5-lipoxygenase and to thereby treat organ
`
`inflammation such as that associated with asthma and bronchitis.
`
`30
`
`Intracellular levels of cGMP can be increased by enhancing intracellular production of
`
`cGMP and/or by inhibiting its degradation by phosphodiesterase. Uroguanylin and other
`
`guanylate cyclase receptor agonists enhance cGMP production by activating the guanylate
`
`cyclase enzyme. Therefore, peptides and other cGMP receptor agonist molecules have been
`
`developed as agents for treating or preventing cancer and inflammatory disease in mammals.
`
`4
`
`genes for both uroguanylin and guanylin have been shown to be localized on the mouse
`
`chromosome 4 and to a syntenic position on human chromosome 1p34-35. This region, also
`
`known as the ‘cancer modifier region’, is frequently associated with loss of heterozygosity in
`
`human colon carcinoma (49-51). Taken together, these findings suggest that uroguanylin and
`
`guanylin might be regulators of the continual renewal process of the GI tract.
`
`These findings raise the possibility that the reduction in the production of these peptides
`
`might lead to disruption of the renewal of GI mucosa resulting in the formation of polyps and
`
`growth of tumors. Because production of uroguanylin and guanylin is essentially lost in polyps,
`
`10
`
`it
`
`is tempting to speculate that these peptides might be involved at an early stage of colon
`
`carcinogenesis. This possibility is consistent with a more recent study demonstrating that oral
`
`administration of uroguanylin inhibited polyp formation in nude mice (46, 47). Although the
`biochemical mechanism(s) for this chemopreventative activity is not known, it is plausible that
`these peptides might participate in a cGMP-mediated signaling mechanism in enterocytes that
`
`regulates mucosal cell turnover by initiating programmed cell death (apoptosis).
`
`The guanylate cyclase receptor-ligand system is also localized in the epithelium of
`
`other, non-intestinal mammalian organs, indicating that this system may be an appropriate
`
`target for interrupting carcinogenesis and tumor formation, and to treat inflammatory diseases
`
`of a variety of tissues.
`
`Summary of the Invention
`The present invention is based upon the concept that the cGMP-dependent mechanism
`that regulates the balance between proliferation and apoptosis is an early step in the process of
`
`25
`
`carcinogenesis. Therefore, elevating intracellular levels of cGMP may be used to treat or
`
`prevent cancer and inflammatory diseases. Enhancement of intracellular production of cGMP
`
`may also be used to suppress the production of 5-lipoxygenase and to thereby treat organ
`
`inflammation such as that associated with asthma and bronchitis.
`
`30
`
`Intracellular levels of cGMP can be increased by enhancing intracellular production of
`
`¢GMP and/or by inhibiting its degradation by phosphodiesterase. Uroguanylin and other
`
`guanylate cyclase receptor agonists enhance ¢cGMP production by activating the guanylate
`
`cyclase enzyme. Therefore, peptides and other cGMP receptor agonist molecules have been
`
`developed as agents for treating or preventing cancer and inflammatory disease in mammals.
`
`
`
`Pg.007
`
`

`

`The agonists may be used either alone or in combination with inhibitors of cGMP-dependent
`
`phosphodiesterase, to treat or prevent cancer and inflammation of the pancreas, lung, GI tract
`
`and other tissues.
`
`5
`
`In one 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. The term
`
`"effective amount" refers to sufficient agonist to measurably increase intracellular levels of
`
`cGMP. In a preferred embodiment the agonist is a peptide selected from those shown as SEQ
`
`10
`
`ID NOs: 2-18 and which are listed in Tables 2 and 3. As noted above, these peptides may be
`
`administered either alone or together with one or more inhibitors of cGMP dependent
`
`:.n
`
`phosphodiesterase. Examples of cGMP dependent phosphodiesterase inhibitors include
`
`suldinac sulfone, zaprinast, and motapizone. Forms of cancer treatable by the method include
`
`breast cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, prostatic
`
`5
`
`cancer, renal cancer, and testicular cancer. Colon carcinogenesis may be prevented by
`
`inhibiting pre-cancerous colorectal polyp development via administration of a composition
`
`rri
`
`according to the invention.
`
`In another aspect, the invention is directed to a method for treating, preventing, or
`
`retarding the onset of general organ inflammation (e.g., inflammation associated with asthma,
`
`nephritis, hepatitis, pancreatitis, bronchitis, 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: 2-18 shown in Tables 2 and 3. These peptides may optionally be administered with
`
`25
`
`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 an inflammatory bowel disease, and more particularly may be Crohn's disease
`
`or ulcerative colitis. Administration may be enteric, and employ formulations tailored to target
`
`30
`
`enterocytes.
`
`One particularly preferred peptide fo