Europaisches
`Patenta mt
`European
`Patent Office
`Office europeen
`des brevets
`
`Beschwerdekammern
`Boards of Appeal
`Chambres de recours
`
`European Patent Office
`80298 MUNICH
`GERMANY
`Tel +49 (0)89 2399 -0
`Fax +49 (0)89 2399 -4465
`
`I 111111 11111 11111 11111 11111 11111 11111 11111 1111 1111
`
`Adams, Harvey Vaughan John
`Mathys & Squire LLP
`120 Holborn
`London
`EC1N 2SQ
`ROYAUME UNI
`
`Anmeldung Nr./Application No./Demande n°//Patent Nr./Patent No./Brevet n°
`Zeichen/Reference/Reference
`/ 1379224
`OPPO01 02721604.3
`117839
`Anmelder/Applicant/Demandeur//Patentinhaber/Proprietor/Titulaire
`Synergy Pharmaceuticals, Inc.
`
`Datum/Date
`11.06.13
`
`Appeal number:
`
`T1366/12-3.3.04
`
`Please find enclosed a copy of the decision dated 04.06.13.
`
`The Registrar P. Cremona
`Tel.: 089 / 2399 - 3341
`
`ud rd kem
`
`941743
`
`Annex(es):
`
`Acknowledgement of receipt - EPO Form 2936
`
`Registered letter with advice of delivery
`
`EPO Form 3032
`
`01/11
`
`Pg. 001
`
`MYLAN EXHIBIT - 1067
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd.
`IPR2022-00722
`
`

`

`BESCHWERDEKAMMERN BOARDS OF APPEAL OF CHAMBRES DE RECOURS
`DES EUROPAISCHEN
`THE EUROPEAN PATENT DE L'OFFICE EUROPEEN
`OFFICE
`PATENTAMTS
`DES BREVETS
`
`Internal distribution code:
`(A) [ ] Publication in OJ
`(B) [ ] To Chairmen and Members
`(C) [ ] To Chairmen
`(D) [X] No distribution
`
`
`Datasheet for the decision
`of 4
`June 2013
`
`Case Number:
`
`T 1366/12 - 3.3.04
`
`Application Number:
`
`02721604.3
`
`Publication Number:
`
`1379224
`
`IPC:
`
`C07K 7/00, C07K 7/64,
`A61K 38/04, A61K 38/12,
`A61P 35/00, A61P 29/00
`
`Language of the proceedings:
`
`EN
`
`Title of invention:
`Guanylate cyclase receptor agonists for the treatment of
`tissue inflammation and carcinogenesis
`
`Patent Proprietor:
`Synergy Pharmaceuticals, Inc.
`
`Opponents:
`Hill, Christopher Michael
`CombiMab, Inc.
`
`Headword:
`Guanylate cyclase receptor agonists/SYNERGY
`
`Relevant legal provisions:
`EPC Art. 108
`EPC R. 101(1)
`
`Keyword:
`"Admissibility of appeal (no) - missing statement of grounds"
`
`Decisions cited:
`
`Catchword:
`
`C9597.D
`EPA Form 3030
`
`This datasheet is not part of the Decision.
`It can be changed at any time and without notice.
`
`Pg. 002
`
`

`

`Europaisches
`Patentamt
`
`European
`Patent Office
`
`Office europeen
`des brevets
`
`Beschwerdekammern
`
`Boards of Appeal
`
`Chambres de recours
`
`Case Number: T 1366/12 3.3 04
`
`DECISION
`of the Technical Board of Appeal 3.3.04
`of 4 June 2013
`
`Appellant:
`(Opponent 1)
`
`Representative:
`
`Respondent:
`(Patent Proprietor)
`
`Representative:
`
`Hill, Christopher Michael
`Page White & Farrer
`Bedford House
`John Street
`London WC1N 2BF (GB)
`
`Adams, Harvey Vaughan John
`Mathys & Squire LLP
`120 Holborn
`London EC1N 2SQ (GB)
`
`Synergy Pharmaceuticals, Inc.
`Suite 450
`2 Executive Drive
`Somerset, NJ 08873 (US)
`
`Mintz Levin Cohn Ferris Glovsky and Popeo LLP
`Alder Castle
`10 Noble Street
`London EC2V 7JX (GB)
`
`Party as of right:
`(Opponent 2)
`
`CombiMab, Inc.
`9700 Great Seneca Highway
`Rockville, MD 20850 (US)
`
`Representative:
`
`Goodfellow, Hugh Robin
`Carpmaels & Ransford
`One Southampton Row
`London ?C1B 5HA (GB)
`
`Decision under appeal:
`
`Interlocutory decision of the Opposition
`Division of the European Patent Office posted
`on 29 March 2012 concerning maintenance of the
`European Patent No. 1379224 in amended form.
`
`Composition of the Board:
`
`Chairman:
`Members:
`
`C. Rennie-Smith
`G. Alt
`B. Claes
`
`C 9 5 9 '7 . D
`
`Pg. 003
`
`

`

`- 1 -
`
`T 1366/12
`
`Summary of Facts and Submissions
`
`I.
`
`The appeal is against the decision of the Opposition
`
`Division of the European Patent Office dated
`
`29 March 2012 according to which the European patent
`
`No. 1 379 224 in amended form complies with the
`
`requirements of the EPC.
`
`II.
`
`The opponent I (hereinafter "appellant") filed a notice
`
`of appeal on 8 June 2012 and paid the fee for appeal on
`
`the same day.
`
`III.
`
`No statement setting out the grounds of appeal pursuant
`
`to Article 108 EPC was filed by the appellant. The
`
`notice of appeal contains nothing that could be
`
`regarded as a statement of grounds.
`
`IV.
`
`By a communication dated 18 December 2012, sent by
`
`registered letter with advice of delivery, the Registry
`
`of the board informed the appellant that no statement
`
`setting out the grounds of appeal had been filed and
`
`that the appeal could be expected to be rejected as
`
`inadmissible. The appellant was invited to file
`
`observations within two months.
`
`V.
`
`The appellant filed no observations in response to said
`
`communication.
`
`C9597.D
`
`Pg. 004
`
`

`

`- 2 -
`
`T 1366/12
`
`Reasons for the decision
`
`As no statement setting out the grounds of appeal has been
`
`filed, the appeal has to be rejected as inadmissible
`
`(Article 108 EPC, third sentence, in conjunction with
`
`Rule 101(1) EPC).
`
`Order
`
`For these reasons it is decided that:
`
`The appeal is rejected as inadmissible.
`
`The Registrar:
`
`The Chairman:
`
`eschwerde
`.sEucopa.,„.
`
`17
`
`4tt
`
`Cremona
`
`Rennie-Smith
`
`'pi a,
`etz?
`CO
`)
`(5
`
`ttA eNe.6'4
`
`'/
`
`/3 + leau
`
`tP
`
`C9597.D
`
`Pg. 005
`
`

`

`100 YEARS OF PROTECTING INTELLECTUAL PROPERTY
`
`European Patent Office
`Erhardtstrasse 27
`D-80298 Munich
`Germany
`
`Ref:
`
`117839
`
`MATHYS &SQUIRE
`cn Y
`IN I LLLtt iUtiL
`London I Cambridge I Manchester I Reading I York
`
`Mathys & Squire LLP
`120 Holborn
`London EC I N 2SQ
`T*44(0)20 7830 0000
`F:+44(0)20 7830 0001
`mail@mathys-squire.com
`www.mathys-squire.com
`
`_ atE
`
`08 June 2012
`
`E-Filing
`
`Dear Sirs
`
`European Patent No. 1 379 224 (Synergy Pharmaceuticals, Inc.)
`"Guanylate cyclase receptor agonists for the treatment of tissue inflammation and
`carcinogenesis"
`Opposition thereto by HILL, Christopher Michael et al
`
`We refer to the Decision of the Opposition Division dated 29 March 2012.
`
`In accordance with the provisions of Article 108 EPC, on behalf of Opponent 1:
`
`HILL, Dr. Christopher Michael
`c/o Page White & Farrer
`Bedford House
`John Street
`London
`WC1N 2BF
`United Kingdom
`
`(a natural person having British nationality who is resident in the UK)
`
`we hereby give Notice of Appeal against the above-mentioned Decision of the Opposition
`Division.
`
`We request cancellation of the Decision of the Opposition Division to the extent that it decided
`to maintain the patent in suit, in an amended form, on the basis of Patentee's Auxiliary
`Request 2. We further request: (i) revocation of the patent in suit in its entirety and for all
`designated states; and (ii) Oral Proceedings in the event that the Board of Appeal forms any
`intention not to revoke the patent in suit in its entirety on the basis of the written procedure.
`
`We confirm our request on the attached EPO Form 1038E for the Appeal fee of EUR 1,240 to
`be debited from our deposit account no. 2805.0049. Any shortfall in the fee should be debited
`from our deposit account no. 2805.0049 and any refund made to that same account.
`
`EUROPEAN PATENT ATTORNEYS I CHARTERED PATENT ATTORNEYS I EUROPEAN TRADEMARK ATTORNEYS I REGISTERED TRADEMARK AGENTS
`Mathys & Squire is a limited liability partnership registered in England & Wales under registered number 0C335375, and is regulated by IPReg. Its registered office is 120 Holborn, London EC1N 2SQ.
`A list of the members of the LLP is open to Inspection at the registered office. We use the word "partner to refer to a member of the LLP or an employee or consultant of similar seniority.
`
`Pg. 006
`
`

`

`European Patent Office
`08 June 2012
`
`Opponent l's representative is:
`
`ADAMS; Harvey Vaughan John, et al
`Mathys & Squire LLP
`120 Holborn
`London EC1N 2SQ
`United Kingdom
`
`(a natural person having British nationality and a principal place of business in the UK).
`
`A written statement of grounds of appeal will be submitted separately within the appropriate
`term.
`
`Yours faithfully
`
`ADAMS, Harvey Vaughan John
`For and on behalf of
`MATHYS & SQUIRE LLP
`Association No 171
`
`/lh
`
`Pg. 007
`
`

`

`Europaisches
`Patentamt
`
`European
`Patent Office
`
`Office europeen
`des brevets
`
`Sender:
`Mr. Harvey ADAMS
`Mathys & Squire LLP
`171
`120 Holborn
`London EC1N 2SQ
`United Kingdom
`
`Phone: +44 (0)20 7830 0000
`Fax: +44 (0)20 7830 0001
`E-mail: mail@mathys-squire.com
`
`Letter accompanying subsequently filed items
`
`80298 Munich
`Germany
`Tel. +49(0)89 2399-0 I Fax -4465
`
`P.O. Box 5818
`NL-2280 HV Rijswijk
`Netherlands
`Tel. +31(0)70 340-2040 I Fax -3016
`
`10958 Berlin
`Germany
`Tel. +49(0)30 25901-0 I Fax -840
`
`The document(s) listed below is (are) subsequently filed documents pertaining to the following application:
`
`Application number
`
`Applicant's or representative's reference
`
`02721604.3
`
`117839
`
`Description of document
`1 Maintenance of the application
`
`Original file name
`117839.pdf
`
`Assigned file name
`MEPA-1.pdf
`
`Signatures
`
`Place:
`
`Date:
`
`Signed by:
`
`Association:
`
`Capacity:
`
`London
`
`03 April 2012
`
`GB, Mathys & Squire LLP, I. Kazi 15911
`
`171
`
`(Representative)
`
`Pg. 008
`
`

`

`5.7. A
`411
`
`•
`
`rbrirtb,w,7,0-% w="•,.-;'?‘
`
`„, r
`
`.
`
`EP 1 379 224 B1
`
`Description
`
`Field of the Invention
`
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`[0001] The present invention relates to the therapeutic use of guanylate cyclase receptor agonists as a means for
`enhancing the intracellular production of cGMP. The agonists may be used either alone or in combination with inhibitors
`of cGMP-specific phosphodiesterase to prevent or treat cancerous, pre-cancerous and metastatic growths, particularly
`in the gastrointestinal tract and lungs. In addition, the agonists may be used in the treatment of inflammatory disorders
`such as ulcerative colitis and asthma.
`
`Background of the Invention
`
`[0002] 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.
`Therefore, by serving as paracrine regulators of CFTR activity, cGMP receptor agonists regulate fluid and electrolyte
`transport in the GI tract (1-6; US patent 5,489,670),
`[0003] The process of epithelial renewal involves the proliferation, migration, differentiation, senescence, and eventual
`loss of GI cells in the lumen (7,8). The GI mucosa can be divided into three distinct zones based on the proliferation
`index of epithelial cells. One of these zones, the proliferative zone, consists of undifferentiated stem cells responsible
`for providing a constant source of new cells. The stem cells migrate upward toward the lumen to which they 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, pep-
`tides, growth factors, chemicals and dietary habits. In addition, an enhanced proliferation rate is frequently associated
`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 ulcerative colitis and other GI disorders (11). Thus, intestinal
`hyperplasia is the major promoter of gastrointestinal inflammation and carcinogenesis.
`In addition to a role for uroguanylin and guanylin as modulators of intestinal fluid and ion secretion, these
`[0004]
`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 may constitute a treatment of colon cancer. However, the mechanism by which this is claimed to occur is questionable
`in that WO 01/25266 teaches uroguanylin agonist peptides that bind specifically to a guanylate cyclase receptor, termed
`GC-C, that was first described as the receptor for E. coil heat-stable enterotoxin (ST) (4). Knockout mice lacking this
`guanylate cyclase receptor show resistance to ST in intestine, but effects of uroguanylin and ST are not disturbed in
`the 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, which is distinct
`from GC-C.
`[0005] 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 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).
`[0006] 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.
`[0007] One of the clinical manifestations of reduced CFTR activity is the inflammation of airway passages (21). This
`effect may be due to CTFR regulating the expression of NF-KB, chemokines and cytokines (22-25). Recent reports
`have also suggested that the CFTR channel is involved in the transport and maintenance of reduced glutathione, an
`
`3
`
`Pg. 009
`
`

`

`EP 1 379 224 81
`
`antioxidant that plays an important role in protecting against inflammation caused by oxidative stress (39). Enhancement
`of intracellular levels of cGMP by way of guanylate cyclase activation or by way of inhibition of cGMP-specific phos-
`phodiesterase would be expected to down-regulate these inflammatory stimuli. Thus, uroguanylin-type agonist 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.
`[0008] 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.
`[0009] Shailubhai et al. (2000) Cancer Research, vol.60, pages 5151-5157 reports that uroguanylin treatment sup-
`presses polyp formation in the Apc../. mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic
`GMP, WO01/25266 discloses the use of uroguanylin as an intestinal cancer inhibiting agent.
`
`Summary of the Invention
`
`ca
`[0010] The present disclosure is based upon thigeZepment ofgew agonistt of guanylate cyclase receptor and new
`uses of naturally out..., it iy ay,,„;,.1.r). The agonistieriranalog of .ar .04_1,02
`r
`uroguanylin, as defined in the appended claims
`activation, stability, activity-at low pH or roducod advc, so cffccta% he peptide" 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 bydIOMP-specific phosphodiesterases. Among the specific
`conditions that can be treated or prevented are inflammatory conditions, cancer, polyps, and metastatis.
`[0011]
`1In its first
`
`ect, the present invention is directed to a peptideafined in claim 1 (or to a conjugate thereof
`peptide" Th., ,,,u.t iJi their etcpepttle Is a
`as defined in claim
`and to therapeutic compositions which contain
`-bicycle having the coqucricc, ,:,f SEQ ID NO:20r,
`[0012] The peptide/ may be in a pharmaceutical composition in unit dose form, together with one or more pharma-
`ceutically 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
`affect 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.
`[0013] Combination therapy utilizing a guanylate cyclase receptor agonists administered either alone or together with
`an inhibitor of cGMP-dependent phosphodiesterase, an anti-inflammatory agent or an anticancer agent is also contem-
`plated. 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, nitrosoureas, antimetabolites,
`vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, and the like. Particular anti-neoplastic agents may
`include tamoxifen, taxol, etoposide and 5-fluorouracil, Antiviral and monoclonal antibody therapies may be combined
`with chemotherapeutic compositions comprising at least one guanylate cyclase receptor agonist in devising a treatment
`regimen tailored to a patient's specific needs.
`[0014] Another aspect, herein described is the use of a peptide or peptide conjugate as defined in the appended
`claims in the manufacture of a medicament for preventing, treating or retarding the onset of cancer, particularly cancer
`of epithelial cells, or polyps in a subject.
`[0015] The disclosure also relates to medicaments for preventing or treating tumor metastasis from a primary tumor
`mass. Metastatic tumor cells having guanylate cyclase receptors may be targeted by peptides generated according to
`the invention. In a preferred embodiment, the targeted receptor is found on cells of gastrointestinal (GI) cancers and on
`metastasized cells derived from those cancers. Such receptors are typically transmembrane proteins with an extracellular
`ligand-binding domain, a membrane-spanning domain, and an intracellular domain with guanylate cyclase activity.
`Although the disclosure is not bound by any particular mechanism of action, it is believed that the peptides will act by
`binding to these cellular receptors and inducing apoptosis. Metastatic tumors may also be treated by administering any
`known form of uroguanylin or guanylin (preferably human) or by administering E. coil ST peptide.
`[0016] Peptides may be administered either alone or together with one or more inhibitors of cGMP dependent phos-
`phodiesterase. Examples of cGMP dependent phosphodiesterase inhibitors include suldinac sulfone, zaprinast, and
`motapizone. Treatable forms of cancer include breast cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic
`cancer, prostate cancer, renal cancer, and testicular cancer. Colon carcinogenesis may be prevented by inhibiting pre-
`cancerous colorectal polyp development via administration of a composition according to the invention. It is believed
`that the peptides should be especially effective with respect to the treatment of colon cancer and in preventing the
`metastasis of colon tumors.
`[0017]
`In a broader sense, the disclosure relates to medicaments for inducing apoptosis in a patient by administering
`
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`

`

`EP 1 379 224 61
`
`an effective amount of a peptide as defined in claim 1. 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 neoplastic growth.
`[0018] The inoat prefcm,dpeptide for use in the methods described above is the peptide defined by SEQ ID NO:20.
`The sequence is as follows (see also Table 3):
`
`Asn1 Asp2 Glu3 Cys4 Glu, Leu, Cys7 Val8 Asn9 Va110 Alatt Cys72 Thr13 Gty14 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.
`
`[0019] 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 1µg 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. 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 and of the Disclosure
`
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`[0020] The present disclosure 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 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 cPLA2, COX-2 and possibly 5-lipoxygenase during the process of inflam-
`mation, is down-regulated by a cGMP-dependent mechanism, leading to reduced levels of prostaglandins and leukot-
`rienes, 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, ele-
`vating intracellular levels of cGMP may be used as a means of treating 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).
`[0021] Without intending to be bound by any theory, it is envisioned that ion transport across the plasma membrane
`40 may prove to 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 k.. acted to induce apoptosis in target cells. Therefore, administration of
`the novel peptide? defined by SEQ ID NO?:&X,
`', as shown in Tablei ao€1.3 kor uroguanylin, or guanylin or E. coli ST
`-poptidc.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).
`[0022]
`In another aspect, the disclosure relates to preventing, treating or retarding the onset of cancer, particularly
`cancer of epithelial cells, in a subject to which a composition comprising an effective amount of a guanylate cyclase
`receptor agonist, preferably a synthetic a guanylate cyclase receptor agonist is abministered. 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 sequen s substitutions not present in
`known endogenous guanylate c326se agonista such as uroguanylin, The agonist
`a peptide selcctcd from-
`thace.defined by SEQ ID NC4:2-21 and which-A listed in Tablet 2 and 3. Also included in the disclosure are methods
`of treating primary and metastatic cancers, other than primary colon cancer, by administering an effective dosage of a
`peptide selected from the group consisting of: urognanylin; guanylin; and E. coil ST peptide. Any known form of uro-
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`EP 1 379 224 B1
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`guanylin or guanylin can be used for this purpose, although the human peptides are preferred.
`[0023] The cGMP-dependent mechanism that regulates the balance between cellular proliferation and apoptosis in
`metastatic tumor cells may serve as a mechanism for targeting and treating metastatic tumors. The liver is the most
`common site of metastasis from a primary colorectal cancer. Toward later stages of disease, colorectal metastatic cells
`may also invade other parts of the body. It is important to note that metastatic cells originating from the primary site in
`the gastrointestinal tract typically continue to express guanylate cyclase receptors and therefore, these cells should be
`sensitive to apoptosis therapy mediated by intestinal guanylate cyclase receptors. Peptides having uroguanylin activity,
`when used either alone or in combination with specific inhibitors of cGMP-phosphodiesterase, also retard the onset of
`carcinogenesis in gut epithelium by restoring a healthy balance between cell proliferation and apoptosis via a cGMP-
`10 mediated mechanism.
`[0024] 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.
`[0025] As used herein, the term "guanylate cyclase receptor-agonist" refers to peptides and/or other compounds that
`bind to a guanylate cyclase receptor and stimulate cGMP production. The term also includes all peptides that have
`amino acid sequences substantially equivalent to at least a portion of the binding domain comprising amino acid residues
`3-15 of SEQ ID NO: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 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
`
`[0026] 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 peptide, 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: may be effectively employed in treatment methods.
`[0027] Peptides similar to, but distinct from, uroguanylin are described below, including some which produce superior
`cGMP enhancing properties and/or other beneficial characteristics (e.g., improved temperature stability, enhanced pro-
`tease stability, or superior activity at preferred pH's) compared to previously known uroguanylin peptides. The peptides
`may be used to inhibit GI inflammation and for treating or preventing the onset of polyp formation associated 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.
`[0028] 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 compounds known to interact with guanylate cyclase
`receptors, namely for human uroguanylin, bicyclo [4,12; 7,15]Asnl-Asp2-Asp3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-
`[4,12; 7,15]
`ID NO:1); human guanylin, bicyclo
`Vali 0-Alal 1-Cys12-Thr13-Gly14-Cys15-Leul 6
`(UG, SEQ
`
`Prol -Gly2-Thr3-Cys4-Glu5-IIe6-Cys7-Ala8-Tyrg-Alalo-Alall-Cys12-Thr13- Giym_c- si5 (GU, SEQ ID NO:22); and E. coli y
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`small heat-stable enterotoxin, tricyclo [6,10; 7,15; 11-18] Asni-Ser2-Ser3-Asn4-Tyrs-Cys6-Cys7-Glu8-Leu9-Cysl 0-
`cysii_Asni 2_proi3_Alai 4_ Cys15-Thr16-Gly17-Cys18-Tyr19 (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 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 individual substitutions for
`various amino acid residues.
`[0029] Energy calculations were performed by use of build-up 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 co angle
`in Pro13 was allowed to vary. Aliphatic and aromatic hydrogens were generally included in united atomic centers of CHn
`type; Ha-atoms and amide hydrogens were described explicitly.
`[0030] The main calculation scheme involved several successive steps. First, the sequences of the two monocyclic
`model fragments (three fragments for ST), Ac-cyclo (Cysi -...-Cysi) -NMe, were considered, where all residues except
`Cys, Gly and Pro were replaced by alanines; the i and j values corresponded to the sequences of GU, UG and ST. At
`this step, all possible combinations of local minima for the peptide b