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`IN THE UNITED STATES PATENT AND TRADEMARKOFFICE
`‘
`REQUEST FOR FILING APPLICATION
`PATENT
`Under Rule 53(a), (b) & (f)
`2APPLICATION
`(No Filing Fee or Oath/Declaration)
`eo
`(Do NOTusefor Provisional or PCT Applications)
`==F
`Use for Design orUtility Applications
`a=
`WS
`RULE 53(f) NO DECLARATION
`Dc
`N/A
`P 284943
`Atty. Dkt.
`<2 =Foqn. Commissioner of Patents
`
`S>=washington, DC 20231 M# Client Ref
`
`
`— rw
`ne
`==0
`Date:
`March 28, 2002
`ec =
`Sir:
`a =
`= Sa
`1. This is a Requestforfiling a new Patent Application([_] Design
`a ==0
`
`Page 1 of 2
`
`.
`
`fe
`
`Je
`
`00909
`00909
`
`Utility) entitled:
`
`2. (Complete) Title:
`
`without a filing fee or Oath/Declaration but for which is enclosed the following:
`
`
`
`3.IX]Abstract 1 page(s).
`
`
`
`
`
`PZ
`
`24
`
`Pages of Specification (only spec. and claims);
`
`5. C1 Specification in non-English language
`
`Numbered claim(s); and
`sheet(s)
`
`|] 1 set informal:
`
`8.) formalofsize:
`
`[IJA4
`
`(111’
`
`=
`:
`
`DOMESTIC/INTERNATIONALpriority is claimed under 35 USC 119(e)/120/365(c) based on the following
`
`-_ provisional, nonprovisional and/or PCT international application(s):
`
`ey
`
`July 10, 2001
`
`
`27
`i
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`“7 L] Drawings:
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`
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`
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`
`
`FOREIGNpricrity is claimed under 35 USC 119(a)-(d)/365(b) based on filing in
`
`0.
`
`
`Application No. ApplicationNo.|——_FilingDate—___Filing Date
`
`
`PO
`(GB)“a
`[()CL See 3page for additionalpriorities
`
`11.
`
`12.
`13.
`
`14.
`
`(No.) Certified copy (copies):
`in U.S. Application No.
`
`f
`
`(_] attached;
`
`[_] previouslyfiled (date)
`filed on
`
`(] Thisisa reissue of Patent No.
`(1 Seetopfirst page re prior Provisional, National, International application(s) (X box onlyif info is there and
`do not complete item 14 or 15.)
`This application claims benefit of the following prior US application(s), the contents of which are incorporated
`into this application by this reference:
`No.
`f
`No.
`/
`No.
`/
`
`filed
`filed
`filed
`
`filed
`/
`No. PCT/
`designated the U.S. and that International Application [_] was
`English
`15.
`
`[_] was not
`
`, which
`published under PCT Acticle 21(2) in
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`16.
`17.
`17(a)
`17(b)
`
`make
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`clai
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`] previouslyfiled
`
`[_] See the attached Preliminary Amendment, which amendsthe specification to claim benefit of the above
`listed US applications
`[_] not needed
`Extension to date:
`LJ concurrently filed
`[_] Small Entity Status is claimed (pre-filing confirmation required)
`[1 Attached:
`(No.) Small Entity Statement(s). (Since 9/8/00 Small Entity Statement not
`tial to
`ee
`crSocNONPUBLICATIONREQUEST under Rule 213(a) attached (Pat-258) MSN Exhibit 1004 - Page 1 of 444
`MSN Exhibit 1004 - Page 1 of 444
`MSN v. Bausch - IPR2023-00016
`MSNv. Bausch - IPR2023-00016
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`Page 2 of 2
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`418.
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`xX Attached:PapercopyofSequenceListing(separatelynumberedaspages4-17)and a3.5inch
`(l\Assignee (optional):
`Sequence Listing. In compliance with
`nts’ undersigned attorney hereby statesthatthe contentofthe paper and
`iskette containing a computer ft
`“19.
`computer d
`37 C.F.R.§ 4.821(f), Applica
`f the Sequence Listing are the same-(Double check instructions for accuracy.):
`computer readable copies 9
`madeby the following named inventor(s)
`nt, but list if known)
`20. This application is
`
`Mailing Address
`(include Zip Code)
`
`Mailing Address
`(include Zip
`
`Mailing Address
`(include Zip Code)
`
`Country
`
`of Citizenship
`
`(Listing of inventor(s) nota requireme
`
`Mailing Address
`
`
`*00909*
`21. NOTE: FORADDITIONALINVENTORS, "X" box[_] andlistadditional
`
`(include Zip Code)
`
`ached sheet (incorporated by reference)
`00909
`Pillsbury Winthrop LLP
`Intellectual Property Group
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`MSN Exhibit 1004 - Page 2 of 444 Reg.No._36912N v. Bausch - IPR2023-00016By:Atty: MichaelA.Sanzo
`
`
`MSN Exhibit 1004 - Page 2 of 444
`M
`Le
`MSN v. Bausch - IPR2023-00016
`Sig:
`,
`,
`Fax: (703)905-2500
`Tel: (703) 905-2173
`ith 2 post card receipts (PAT-103) & attachments
`
`inventors on att
`
`Atty/Sec: MAS/AMX
`
`NOTE:File in duplicate w
`
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`4
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`APPLICATION UNDER UNITED STATES PATENT LAWS
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`4
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`4
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`i
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`%
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`Atty. Dkt. No.
`
`_PW 284943
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`(M#)
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`_ Invention:
`
`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`inventor(s):
`
`SHAILUBHAI, Kunwar
`NIKIFOROVICH, Gregory
`JACOB,S. Gary
`
`*00909*
`00909
`Pillsbury Winthrop LLP
`
`
`
`Thisisa:
`
`DO
`
`x]
`
`DoOoouUoOO
`
`L]
`
`Provisional Application
`
`RegularUtility 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
`
`/
`
`SPECIFICATION
`
`30274500_1.D0C
`
`MSN Exhibit 1004 - Page 3 of 444
`MSNExhibit 1004 - Page 3 of 444
`MSN v. Bausch - IPR2023-00016
`MSNv. Bausch - IPR2023-00016
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`PAT-100CN 10/01
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`ad
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`im
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`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`5
`
`Cross Reference to Related Applications
`The present application claims the benefit of U.S. provisional application
`nos. 60/279,438,
`filed on March 29, 2001; 60/279,437,
`filed on March29, 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.
`
`10
`
`‘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.
`
` bind to a guanylate cyclase receptor and stimulate intracellular production of cyclic
`
`Backgroundof the Invention
`
`Uroguanylin, guanylin and bacterial ST peptides are structurally related peptides that
`
`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. Therefore, by serving as paracrine
`regulators of CFTR activity, cGMP receptor agonists regulate fluid and electrolyte transport
`in the GItract (1-6; US patent 5,489,670).
`
`25
`
`the proliferation, migration,
`involves
`renewal
`epithelial
`The process of
`30~—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
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`toward the lumen to which they are extruded. As they migrate, the cells lose their capacity
`
`to divide and becomedifferentiated 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 betweenproliferation and apoptoticrates(8).
`
`Therates 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
`
`10
`
`and dietary habits. In addition, an enhancedproliferation rate is frequently associated with a
`
`reduction in turnover time and an expansion ofthe proliferative zone (10). The proliferation
`
`index has been observed to be much higher in pathological cases of ulcerative colitis and
`
`other GI disorders
`
`(11). Thus,
`
`intestinal hyperplasia is
`
`the major promoter of
`
`gastrointestinal inflammation and carcinogenesis.
`
`15
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`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 suggests a peptide with the active
`
`domain of uroguanylin may function as an inhibitor of polyp development in the colon and
`
`20
`
`may constitute a treatment of colon cancer. However, the mechanism by whichthis 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 wasfirst described
`
`as the receptor for £. coli heat-stable enterotoxin (ST) (4). Knockout mice lacking this
`
`guanylate cyclase receptor show resistance to ST in intestine, but effects of uroguanylin and
`
`25
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`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 uroguanylin was not effected
`
`(12,13). Taken together these data suggest that uroguanylin also binds to a currently
`
`unknown receptor, whichis distinct from GC-C.
`
`30
`
`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 uroguanylin and guanylin have been shown to be localized to
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`regions of the genome frequently 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 mice (15,16).
`
`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.
`
`Oneofthe clinical manifestations of reduced CFTRactivity 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
`channelis 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). Enhancementofintracellular 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
`uroguanylin, many of which have superior properties either in terms of improved receptor
`MSN Exhibit 1004 - Page 6 of 444
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`activation, stability, activity at low pH or reduced adverse effects. The peptides may be used
`
`to treat any condition that responds to enhancedintracellular levels of cGMP. Intracellular
`
`levels of cGMPcan be increased by enhancing intracellular production of cGMP and/or by
`
`inhibition of its degradation by cGMP-specific phosphodiesterases. Among the specific
`
`5
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`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
`
`compositions 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 ofeither structure or function. For the purpose of the
`
`present application, a peptide differs substantially if its structure varies by more than three ©
`
` The peptides may be in a pharmaceutical composition in unit dose form, together
`
`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 similar
`
`peptides should differ by no more than two aminoacids 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.
`
`with one or more pharmaceutically acceptable excipients. The term “unit dose form”refers
`toa 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 ug and 3 g). What constitutes a
`
`25
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`“positive therapeutic effect” will depend upon the particular condition being treated and will
`
`include any significant improvementin 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.
`
`30
`
`The invention also encompasses combination therapy utilizing a guanylate cyclase
`
`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 present in amounts known in the art to be therapeutically effective when administered to
`
`a patient. Anti-neoplastic agents may include alkylating agents, epipodophyllotoxins,
`MSN Exhibit 1004 - Page 7 of 444
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`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 regimentailoredto 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 cancerofepithelial 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 methodsoftreating primary cancers, other than primary colon
`cancer, by administering an effective dosage of a peptide selected from the groupconsisting
`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.
`
`10
`
` mae
`
`
`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 accordingto the invention. In a preferred embodiment, the
`targeted receptor is found oncells of gastrointestinal (GI) cancers and on metastasizedcells
`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 ofaction,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.
`
`25
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`30
`
`Peptides may be administeredeither alone or together with one or more inhibitors of
`cGMP dependent phosphodiesterase. Examples of cGMP dependent phosphodiesterase
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`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.
`
`10
`
`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 Gl 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 and3, 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 inventionis directed to a method of treating an
`inflammatory disorder in a mammalian gastrointestinal tract. The inflammatory disorder
`maybeclassified 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 enterocytes.
`
`25
`
`30
`
`In a broadersense, the invention includes methods of inducing apoptosis in a patient
`by administering an effective amountof a peptide having the sequence of any one of SEQ
`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 a
`target tissue. For example, sufficient peptide may be given to induce an increased rate of
`cell death in a neoplastic growth.
`
`The mostpreferred peptide for use in the methods described aboveis the peptide
`defined by SEQ ID NO:20. The sequenceis as follows (see also Table 3):
`Asn! Asp” Gl’ Cys* Glu’ Leu® Cys’ Val® Asn? Val'° Ala!! Cys” Thr? Gly* CysLeu!®
`*
`#%
`*
`2k
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`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 cGMPproduction dueto its enhanced binding
`constant for the guanylate cyclase receptor, and is superior to uroguanylin with regard to
`
`the
`temperature and protease stability and with regard to its biological activity at
`physiologically favorable pH range (pH6to 7) in the large intestine.
`
`10
`
`
`
`25
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`30
`
`The guanylate cyclase receptor agonists used in the methods described above may
`be administered either orally, systemically or locally. Dosage forms include preparations for
`inhalation or injection, solutions, suspensions, emulsions, tablets, capsules, topical salves
`and lotions, transdermal compositions, other known peptide formulations and pegylated
`peptide analogs. An effective dosage of the composition will typically be between about 1
`ug and about 10 mg per kilogram body weight, preferably between about 10 1g to 5 mg of
`the compound per kilogram body weight. Adjustments in dosage will be made using
`methodsthat are routine in the art and will be based uponthe 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, 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 betweencellular proliferation and
`apoptosis and that a reduction in cGMPlevels, due to a deficiency of uroguanylin/guanylin
`and/or due to the activation 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 cCPLA2, 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
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`may be used as a means oftreating and 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 mayproveto be an important regulator of the balance between
`cell proliferation and apoptosis that will be affected by compositions altering cGMP
`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 inflammatory diseases of the
`GI tract and general organ inflammation (e.g., asthma, nephritis, hepatitis, pancreatitis,
`bronchitis, cystic fibrosis).
`
`10
`
`
`
`25
`
`30
`
`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 ofa 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 methodsof 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 cGMP-dependent mechanism that regulates the balance between cellular
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`proliferation and apoptosis in metastatic tumorcells may serve as a mechanism fortargeting
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`and treating metastatic tumors. The liver is the most commonsite of metastasis from a
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`primary colorectal cancer. Toward later stages of disease, colorectal metastatic cells may
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`5_.also invade other parts of the body. It is important to note that metastatic cells originating
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`from the primary site in the gastrointestinal tract typically continue to express guanylate
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`cyclase receptors and therefore,
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`these cells should be sensitive to apoptosis therapy
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`mediated by intestinal guanylate cyclase receptors. Peptides having uroguanylin activity,
`
`when used either
`
`alone or
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`in combination with specific
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`inhibitors of cGMP-
`
`10
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`phosphodiesterase, also retard the onset of carcinogenesis in gut epithelium by restoring a
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`healthy balance between cell proliferation and apoptosis via a cGMP-mediated mechanism.
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` equivalent to at least a portion of the binding domain comprising amino acid residues 3-15
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`Asused herein, the term “guanylate cyclase receptor” refers to the class of guanylate
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`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
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`and/or other compounds that bind to a guanylate cyclase receptor and stimulate cGMP
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`production. The term also includesall peptides that have amino acid sequences substantially
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`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 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
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`25
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`peptide’s ability to bind to a guanylate cyclase receptor and stimulate cGMP production.
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`Strategy and design of novel guanylate cyclase receptor agonists
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`Uroguanylin is a peptide secreted by the goblet and other epithelial cells lining the
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`gastrointestinal mucosa as pro-uroguanylin, a functionally inactive form. The human pro-
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`30_—peptide is subsequently converted to the functionally active 16 amino acid peptide set forth
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`in SEQ ID NO:1 (human uroguanylin sequence, see Table 2) in the lumen of the intestine
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`by endogenous proteases. Since uroguanylin is a heat-resistant, acid-resistant, and
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`proteolysis-resistant peptide, oral or systemic administration of this peptide and/or other
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`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
`
`5
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`some which produce superior cGMP enhancing properties and/or other beneficial
`
`characteristics (e.g., improved temperature stability, enhanced protease stability, or superior
`
`activity at preferred pH’s) compared to previously known uroguanylin peptides. The
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`peptides may be usedto inhibit GI inflammation and for treating or preventing the onset of
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`polyp formation associated with gut inflammation. Epithelial tissues susceptible to cancer
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`10—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.
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`Bot
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`
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`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
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`compounds known to interact with guanylate cyclase receptors, namely for human
`uroguanylin, bicyclo [4,12; 7,15]Asn!-Asp’-Asp*-Cys'-Glu’-Leu®-Cys’-Val®-Asn?-Val'°-
`Ala'!-Cys'?-Thr'?-Gly'*-Cys'*-Leu’® (UG, SEQ ID NO:1); human guanylin, bicyclo [4,12;
`7,15|Pro'-Gly?-Thr’-Cys*-Glu’-lle*-Cys’-Ala®-Tyr?-Ala!°-Ala"!-Cys!*-Thr'3-Gly"*-Cys!5
`(GU, SEQ ID NO:22); and £. coli small heat-stable enterotoxin, tricyclo [6,10; 7,15; 11-18]
`a Asn’-Ser’-Ser’-Asn*-Tyr’-Cys°-Cys’-Glu®-Lew’-Cys!?-Cys!"-Asn!?-Pro?-Ala!*-Cys!5-
`Thr'®-Gly'’-Cys'*-Tyr'? (ST, SEQ ID NO:23). Geometrical comparisons of all possible
`low-energy conformations for these three compounds were used to reveal the common 3D
`
`structures that served as the “templates” for the bioactive conformation,
`
`i.e., for the
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`25
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`conformation 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
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`individual substitutions for various amino acid residues.
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`30
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`Energy calculations were performed by use of build-up procedures (30). The
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`ECEPP/2 potential field (31,32) was used assuming rigid valence geometry with planar
`trans-peptide bonds, including that for Pro’? in ST. The @ angle in Pro! was allowed to
`
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`vary. Aliphatic and aromatic hydrogens were generally included in united atomic centers of
`
`CHn type; H%-atoms and amide hydrogens were described explicitly.
`
`The main calculation scheme involved several successive steps. First, the sequences
`5.of the two monocyclic model fragments (three fragments for ST), Ac-cyclo (Cys'-...-Cys’) -
`NMe, were considered, where all residues except Cys, Gly and Pro were replaced by
`
`alanines; the i and j values correspondedto the sequences of GU, UG and ST.Atthis step,
`
`all possible combinations of local minima for the peptide backbone for each amino acid
`
`residue were considered,i.e., the minima in the Ramachandran mapof E, F, C, D, A and A *
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`10
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`types (accordingto the notation in (33)) for the Ala residue; of E*, F*, C* D* A,E, FE. CD
`
`
`
`
`15
`:
`
`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 cycle employing the parabolic potential
`
`functions, intrinsic to the ECEPP force field, was found by checking an energy profile of
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`rotation around the dihedral angle 1 for the D-Cys residue.
`
`Totally, as many as ca. 180,000 conformations for each of the cyclic moie