IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Page 1 of 2
`
`PATENT
`APPLICATION
`
`1St
`
`---c=
`Commissioner of Patents
`
`
`2, =eV ashington, DC 20231
`
`
`
`o
`Sir:
`
`REQUEST FOR FILING APPLICATION
`Under Rule 53(a), (b) & (f)
`(No Filing Fee or Oath/Declaration)
`(Do NOT use for Provisional or PCT Applications)
`Use for Design or Utility Applications
`RULE 53(f) NO DECLARATION
`P 284943
`Atty. Dkt.
`M#
`
`*00909*
`00909
`
`N/A
`Client Ref
`
`Date:
`
`March 28, 2002
`
`1. This is a Request for filing a new Patent Application(O Design El Utility) entitled:
`
`2. (Complete) Title:
`
`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`a4.44
`
`=cm
`
`.r ---
`C=4 w_—•_-.03
`
`co
`co
`too
`r y
`U
`
`without a filing fee or Oath/Declaration but for which is enclosed the following:
`
`3.
`
`Abstract
`
`
`
`1
`
` page(s).
`
`24
`
`Pages of Specification (only spec. and claims);
`
`5. 0 Specification in non-English language
`
`27
`
`rz.
`
`Drawings:
`
`(1)
`(3)
`(5)
`
` Numbered claim(s); and
`8. O formal of size: LA4 L11"
`sheet(s) D 1 set informal;
`DOMESTIC/INTERNATIONAL priority is claimed under 35 USC 119(e)/120/365(c) based on the following
`rovisional non rovisional and/or PCT international ao lication s :
`Application No.
`Filing Date
`Application No.
`601279,437
`March 29, 2001
`60/279,438
`60/303,806
`June 27, 2001
`60/300,850
`60/348,646
`July 25, 2001
`60/307,358
`
`(2)
`(4)
`(6)
`
`Filing Date
`March 29, 2001
`July 10, 2001
`January 17, 2002
`
`FOREIGN priority is claimed under 35 USC 119(a)-(d)/365(b) based on filing in
`
`Application No.
`
`Filing Date
`
`Application No.
`
`Filing Date
`
`(1)
`(3)
`(5)
`
`(2)
`(4)
`q See 3rd page for additional priorities
`
`11.
`
`0 attached;
`
`0 previously filed (date)
`filed on
`
`(No.) Certified copy (copies):
`in U.S. Application No.
`12. 0 This is a reissue of Patent No.
`See top first page re prior Provisional, National, International application(s) (X box only if info is there and
`q
`13.
`do not complete item 14 or 15.)
`14. This application claims benefit of the following prior US application(s), the contents of which are incorporated
`into this application by this reference:
` filed
`No.
` filed
`No.
`filed
`No.
`filed
`No. PCT/
`designated the U.S. and that International Application 0 was 0 was not
`English
`15.
`
`, which
`published under PCT Acticle 21(2) in
`
`0 See the attached Preliminary Amendment, which amends the specification to claim benefit of the above
`listed US applications
`
`16.
`
`17.
`
`Extension to date:
`
`0 concurrently filed
`
`0 not needed
`
`0 previously filed
`
`0 Small Entity Status is claimed (m -filing confirmation required)
`
`17(a)
`0 Attached:
`(No.) Small Entity Statement(s). (Since 9/8/00 Small Entity Statement not
`essential to make claim)
`17(b) 0 See NONPUBLICATION REQUEST under Rule 213(a) attached (Pat-258)
`
` I-
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`PATENT
`APPLICATION
`
`=Q
`a="
`enn
`Sec
`X=. Commissioner of Patents
`3 ==&/ashington, DC 20231
`
`REQUEST FOR FILING APPLICATION
`Under Rule 53(a), (b) & (f)
`(No Filing Fee or Oath/Declaration)
`(Do NOT use for Provisional or PCT Applications)
`Use for Design or Utility Applications
`
`
`
`
`
`RULE 53(f) NO DECLARATION
`Atty. Dkt.
`
`
`
`P 284943
`Mi#
`
`Page 10f2
`
`-
`
`.
`
`*00909*
`
`0 09 09
`
`|
`|
`
`N/A
`Client Ref
`
`
`
`— wo
`
`=o
`Sir:
`
`
`
`
`
`2. (Complete) Title:
`
`=
`eg
`Bigs =—
`=a
`us =
`oD
`==
`Guanylate Cyclase Receptor Agonists for the Treatment of
`O~—
`EO
`
`Tissue Inflammation and Carcinogenesis
`B=
`2
`
`Date:
`
`March 28, 2002
`
`—_—
`
`1. This is a Request for filing a new Patent Application([_] Design
`
`Utility) entitled:
`
`
`on 1
` without a filing fee or Oath/Declaration but for which is enclosed the following:
`
`3.
`
`Xl Abstract
`
`1
`
`page(s).
`
`_24
`
`Pages of Specification (only spec. and claims);
`
`5. [[] Specification in non-English language
`
`27
`
`Numbered claim(s); and
`
`= [] Drawings:
`
`sheet(s)
`
`[11 set informal;
`
`8. [ formal of size:
`
`[1A4
`
`[1717
`
`
`
`DOMESTIC/INTERNATIONAL priority is claimed under 35 USC 119(e)/120/365(c) based on the following
`-_provisional, nonprovisional and/or PCT international application(s):
` Filing Date
`Application No.
`Filing Date
`Application No.
`
`
`(1)
`60/279,438
`March 29, 2001
`(2)
`60/279,437
`March 29, 2001
`
`(3)
`60/300,850
`June 27, 2001
`(4)
`60/303,806 -
`July 10, 2001
`
`(5)
`60/307,358
`July 25, 2001
`(6)
`60/348,646
`January 17, 2002
`
`
`0.
`EOREIGN priority is claimed under 35 USC 119(a)-(d)/365(b) based on filing in
`
`Application No.
`Filing Date
`Application No.
`Filing Date
`
`(1)
`(2)
`3)
`(4)
`(5)
`[1 See 3" page for additional priorities
`
`11.
`(No.) Certified copy (copies):
`[1 attached;
`[1 previously filed (date)
`in U.S. Application No.
`filed on
`
`
`
`
`
`
`
`
`
`/
`
`
`
`
`
`
`
`12.
`13.
`
`14.
`
`[J]
`[1
`
`Thisis a reissue of Patent No.
`See top first page re prior Provisional, National, International application(s) (X box only if 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.
`/
`filed
`
`No.
`/
`filed
`
`No.
`/
`filed
`
`, which
`No. PCT/
`/
`filed
`
`designated the U.S. and that International Application [| was
`[] was not
`published under PCT Acticle 21(2) in
`English
`15.
`
`[] See the attached Preliminary Amendment, which amends the specification to claim benefit of the above
`listed US applications
`
`16.
`
`17.
`
`17(a)
`
`17(b)
`
`Extension to date:
`
`[1 concurrently filed
`
`[1 not needed
`
`[] previously filed
`
`[_] Small Entity Status is claimed (pre-filing confirmation required)
`
`[1 Attached:
`essential to make claim)
`[1 See NONPUBLICATION REQUEST under Rule 213(a) attached (Pat-258)
`
`(No.} Small Entity Statement(s). (Since 9/8/00 Small Entity Statement not
`
`
`
`
`
`
`
`
`
`Pg.001
`
`MYLAN EXHIBIT - 1004 (Part 1)
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`(1) Inventor Kunwar
`
`Residence
`
`I Blue Bell
`
`First
`
`SHAILUBHAI
`
`Middle Initial
`I PA
`
`Mailing Address
`(include Zip Code)
`
`State/Foreign Country
`City
`600 Wick Lane, Blue Bell, PA 19422, USA
`I
`
`(2) Inventor Gregory
`
`Residence
`
`I St. Louis
`
`First
`
`Middle Initial
`I MO
`
`NIKIFOROVICH
`
`State/Foreign Country
`City
`751 Aramis Drive, St. Louis, MO 63141, USA
`I
`
`Mailing Address
`(include Zip Code)
`
`(3) Inventor Gary
`
`Residence
`
`I Creve Coeur
`
`First
`
`52_
`
`4t
`ci
`w
`z
`Z
`<
`U
`co
`
`18.
`
`[1] Assignee (optional):
`
`19. EZ Attached: Paper copy of Sequence Listing (separately numbered as pages 1-17) and a 3.5 inch
`computer diskette containing a computer readable copy of Sequence Listing. In compliance with
`37 C.F.R. § 1.821(f), Applicants' undersigned attorney hereby states that the content of the paper and
`computer readable copies of the Sequence Listing are the same.
`
`20. This application is made by the following named inventor(s)
`(Listing of inventor(s) not a requirement, but list if known)
`
`(Double check instructions for accuracy.):
`
`Page 2 of 2
`
`Family Name
`F USA
`
`Country of Citizenship
`
`,
`
`Family Name
`I USA
`
`Country of Citizenship
`
`JACOB
`
`S.
`Middle Initial
`I MO
`
`Family Name
`I USA
`
`Mailing Address
`(include Zip Code)
`
`(4) Inventor
`
`Residence
`
`I
`
`Mailing Address
`(include Zip Code)
`
`(5) Inventor
`
`Residence
`
`I
`
`Mailing Address
`(include Zip Code)
`
`State/Foreign Country
`City
`12541 Mason Forest Drive, Creve Coeur, MO 63141, USA
`I
`
`Country of Citizenship
`
`First
`
`City
`
`First
`
`City
`
`Middle Initial
`
`Middle Initial
`
`I
`
`I
`
`I
`
`I
`
`State/Foreign Country
`
`Country of Citizenship
`
`Family Name
`I
`
`State/Foreign Country
`
`Country of Citizenship
`
`Family Name
`I
`
`21. NOTE: FOR ADDITIONAL INVENTORS, "X" box q and list additional
`inventors on attached sheet (incorporated by reference)
`Pillsbury Winthrop LLP
`Intellectual Property Group
`
`*00909*
`00909
`
`By: Atty: Michael A. Sanzo
`
` Reg. No. 36912
`
`Atty/Sec: MAS/AMX
`
`Sig:
`
` Fax: (703) 905-2500
`Tel: (703) 905-2173
`NOTE: File in duplicate with 2 post card receipts (PAT-103) & attachments
`
`/ 1/4
`
`-e,47/
`
`09: fa...g ri p'
`
`-
`
`18.
`
`[| Assignee (optional): CS
`
`—— Page 20f2
`
`19.
`X Attached: Paper copy of Sequence Listing (separately numbered as pages 1-17) and a 3.5 inch
`computer diskette containing a computer readable copy of Sequence Listing. In compliance with
`37C.F.R.§ 1.821(f), Applicants’ undersigned attorney hereby states that the content of the paper and
`computer readable copies of the Sequence Listing are the same.
`
`20. This application is made by the followi
`(Listing of inventor(s) not a requireme
`
`ng named inventor(s)
`nt, but list if known)
`
`(Double check in structions for accuracy.)
`
`
`
` SHAILUBHAI
`
`
`
`
`
`(1) Inventor | Kunwar
`{
`
`First
`Middle Initial
`
`Family Name
`
`Residence
`| Blue Bell
`
`| PA
`| USA
`
`
`
`City
`State/Foreign Country
`Country of Citizenship
`
`600 Wick Lane, Blue Bell, PA 19422, USA
`
`
`
`
`
`Mailing Address
`
`(include Zip Code)
`
`
`
`
`
`(2) Inventor | Gregory
`
`|
`
`{ NIKIFOROVICH
`
`
`
`
`
`
`
`First
`Middie Initial
`Family Name
`
`{| MO
`| USA
`
`Residence
`
`| St. Louis
`
`
`
`
`
`City
`State/Foreign Country
`Country of Citizenship
`
`751 Aramis Drive St. Louis, MO 63141, USA
`
`
`
`
`
`
`
`Mailing Address
`
`(include Zip Code)
`
`
`
`
`SCANNED, # i
`
`
`
`| JACOB
`
`
`
`
`
`(3) Inventor | Gary
`| S.
`
`First
`Middle Initial
`Family Name
`
`
`
`Residence
`| Creve Coeur
`| MO
`| USA
`
`
`
`
`Country of Citizenship
`
`
`
`City
`State/Foreign Country
`
`12541 Mason Forest Drive, Creve Coeur, MO 631441, USA
`
`
`
`
`
`Mailing Address
`(include Zip Code)
`
`
`
`
`
`
`
`
`(4) Inventor |
`
`
`
`
`
`
`
`First
`Middle Initial
`
`Family Name
`
`
`|
`
`|
`
`
`
`|
`
`Residence
`
`
`City
`State/Foreign Country
`Country of Citizenship
`
`
`
`
`
`
`Mailing Address
`(include Zip Code)
`
`
`
`
`
`
`|
`
`
`
`
`
`
`
`
`
`{
`
`|
`
`(5) Inventor |
`
`
`First
`Middle Initial
`
`Family Name
`
`
`Residence
`
`|
`
`|
`
`
`
`\
`
`
`
`
`
`
`
`
`City
`State/Foreign Country
`Country of Citizenship
`
`
`
`
`
`
`
`Mailing Address
`|
`
`(include Zip Code)
`
`
`
`
`
`21. NOTE: FOR ADDITIONAL INVENTORS, "X" pox [] and list additional
`inventors on attached sheet (incorporated by reference)
`pillsbury Winthrop LLP
`Intellectual Property Group
`
`*00909"
`00909
`
`By: Atty:
`
`Michael A. Sanzo
`
`Reg. No.
`
`_36912
`
`Atty/Sec: MAS/AMX
`
`Sig:
`
`Fax: (703) 905-2500
`Tel: (703) 905-2173
`NOTE: File in duplicate with 2 post card receipts (PAT-103) & attachments
`
`.
`
`Pg.002
`
`

`

`ti
`APPLICATION UNDER UNITED STATES PATENT LAWS
`
`Atty. Dkt. No. PW 284943
`
`(M#)
`
`Invention:
`
`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`inventor (s): SHAILUBHAI, Kunwar
`NIKIFOROVICH, Gregory
`JACOB, S. Gary
`
`I=A
`4
`
`•
`
`a
`
`FE 3,
`
`g 4,4 evo
`
`*00909*
`00909
`Pillsbury Winthrop LLP
`
`This is a:
`
`Provisional Application
`
`Regular Utility Application
`
`Continuing Application
`M The contents of the parent are incorporated
`by reference
`
`q PCT National Phase Application
`
`q Design Application
`
`L1 Reissue Application
`
`q Plant Application
`
`LI Substitute Specification
`Sub. Spec Filed
`in App. No.
`
`El Marked up Specification re
`Sub. Spec. filed
`In App. No
`
`SPECIFICATION
`
`30274500_1.DOC
`
`PAT-100CN 10/01
`
`N
`
`4
`
`APPLICATION UNDER UNITED STATES PATENT LAWS
`
`3
`
`*
`
`{
`
`Xx
`
`Atty. Dkt. No.
`
`PW 284943
`
`
`
`(M#)
`
`~ 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
`
`This is a:
`
`Provisional Application
`
`Regular Utility Application
`
`Continuing Application
`[Xl 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 /
`
`Ll
`
`X
`
`[J
`
`OOo onn
`
`Ll
`
`SPECIFICATION
`
`
`
` 30274500_1.DQC
`
`PAT-100CN 10/01
`
`Pg.003
`
`

`

`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`Cross Reference to Related Applications
`
`5
`
`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 March 29, 2001;
`
`60/300,850, filed on June 27, 2001; 60/303,806, filed on July 10, 2001; 60/307,358, filed on
`
`July 25, 2001; and 60/348,646, filed on January 17, 2002.
`
`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
`
`ce 15
`
`RAC
`
`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
`
`c el
`
`Uroguanylin, guanylin and bacterial ST peptides are structurally related peptides that
`
`20
`
`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
`
`25
`
`regulators of CFTR activity, cGMP receptor agonists regulate fluid and electrolyte transport
`
`in the GI tract (1-6; US patent 5,489,670).
`
`The process of epithelial
`
`renewal
`
`involves
`
`the proliferation, migration,
`
`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
`
`1
`
`Guanylate Cyclase Receptor Agonists for the Treatment of
`Tissue Inflammation and Carcinogenesis
`
`Cross Reference to Related Applications
`
`The present
`
`application
`
`claims
`
`the
`
`benefit
`
`of U.S.
`
`provisional
`
`application
`
`nos. 60/279,438,
`
`filed
`
`on March 29,
`
`on March?29,
`2001;
`60/300,850, filed on June 27, 2001; 60/303,806, filed on July 10, 2001; 60/307,358, filed on
`
`2001;
`
`60/279,437,
`
`filed
`
`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
`fo prevent or treat cancerous, pre-cancerous and metastatic growths, particularly in the
`gastrointestinal tract and lungs. In addition, the agonists may be used in the treatment of
`
`inflammatory disorders such as ulcerative colitis and asthma.
`
`Background of the Invention
`
`Uroguanylin, guanylin and bacterial ST peptides are structurally related peptides that
`bind to
`a guanylate cyclase receptor and stimulate intracellular production of cyclic
`guanosine 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).
`
`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
`
`25
`
`30
`
`
`
`Pg.004
`
`

`

`toward the lumen to which they are extruded. As they migrate, the cells lose their capacity
`to divide and become differentiated for carrying out specialized functions of the GI mucosa
`(9). Renewal of GI mucosa is very rapid with complete turnover occurring within a 24-48
`hour period (9). During this process mutated and unwanted cells are replenished with new
`cells. Hence, homeostasis of the GI mucosa is regulated by continual maintenance of the
`balance between proliferation and apoptotic rates (8).
`
`The rates of cell proliferation and apoptosis in the gut epithelium can be increased or
`decreased in a wide variety of different circumstances, e.g., in response to physiological
`stimuli such as aging, inflammatory signals, hormones, peptides, growth factors, chemicals
`
`and dietary habits. In addition, an enhanced proliferation rate is frequently 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.
`
`5
`
`10
`
`15
`
`C.
`
`In addition to a role for uroguanylin and guanylin as modulators of intestinal fluid
`
`.3
`
`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 which this is
`
`claimed to occur is questionable in that WO 01/25266 teaches uroguanylin agonist peptides
`that bind specifically to a guanylate cyclase receptor, termed GC-C, that was first described
`as the receptor for E. coli heat-stable enterotoxin (ST) (4). Knockout mice lacking this
`guanylate cyclase receptor show resistance to ST in intestine, but effects of uroguanylin and
`ST are not disturbed in the 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.
`
`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
`
`25
`
`30
`
`2
`
`toward the lumen to which they are extruded. As they migrate, the cells lose their capacity
`
`to divide and become differentiated for carrying out specialized functions of the GI mucosa
`
`(9). Renewal of GI mucosa is very rapid with complete turnover occurring within a 24-48
`
`hour period (9). During this process mutated and unwanted cells are replenished with new
`
`cells. Hence, homeostasis of the GI mucosa is regulated by continual maintenance of the
`
`balance between proliferation and apoptotic rates (8).
`
`The rates of cell proliferation and apoptosis in the gut epithelium can be increased or
`
`decreased in a wide variety of different circumstances, e.g., in response to physiological
`
`stimuli such as aging, inflammatory signals, hormones, peptides, growth factors, chemicals
`
`10
`
`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.
`
`15
`
`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 which this is
`
`claimed to occur is questionable in that WO 01/25266 teaches uroguanylin agonist peptides
`
`that bind specifically to a guanylate cyclase receptor, termed GC-C, that was first described
`
`as the receptor for E. coli heat-stable enterotoxin (ST) (4). Knockout mice lacking this
`
`guanylate cyclase receptor show resistance to ST in intestine, but effects of uroguanylin and
`
`25
`
`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.
`
`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
`
`30
`
`
`
`Pg.005
`
`

`

`t.
`
`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).
`
`5
`
`10
`
`log),
`
`rrT
`
`15
`
`20
`
`25
`
`30
`
`Uroguanylin and guanylin peptides also appear to promote apoptosis by controlling
`cellular ion flux. Alterations in apoptosis have been associated with tumor progression to
`the metastatic phenotype. While a primary gastrointestinal (GI) cancer is limited to the
`small intestine, colon, and rectum, it may metastasize and spread to such localities as bone,
`lymph nodes, liver, lung, peritoneum, ovaries, brain. By enhancing the efflux of K+ and
`influx of Ca++, uroguanylin and related peptides may promote the death of transformed cells
`and thereby inhibit metastasis.
`
`One of the clinical manifestations of reduced CFTR activity is the inflammation of
`airway passages (21). This effect may be due to CTFR regulating the expression of NF-kB,
`chemokines and cytokines (22-25). Recent reports have also suggested that the CFTR
`channel is involved in the transport and maintenance of reduced glutathione, an antioxidant
`that plays an important role in protecting against inflammation caused by oxidative stress
`(39). Enhancement of intracellular levels of cGMP by way of guanylate cyclase activation
`or by way of inhibition of cGMP-specific phosphodiesterase would be expected to down-
`regulate these inflammatory stimuli. Thus, uroguanylin-type agonists should be useful in the
`prevention and treatment of inflammatory diseases of the lung (e.g., asthma), bowel (e.g.,
`ulcerative colitis and Crohn's disease), pancreas and other organs.
`
`Overall, it may be concluded that agonists of guanylate cyclase receptor such as
`uroguanylin have potential therapeutic value in the treatment of a wide variety of
`inflammatory conditions, cancer (particularly colon cancer) and as anti-metastatic agents.
`The development of new agonists is therefore of substantial clinical importance.
`
`Summary of the Invention
`The present invention is based upon the development of new agonists of guanylate
`cyclase receptor, and new uses of naturally occurring agonists. The agonists are analogs of
`uroguanylin, many of which have superior properties either in terms of improved receptor
`
`3
`
`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.
`
`One of the clinical manifestations of reduced CFTR activity is the inflammation of
`
`airway passages (21). This effect may be due to CTFR
`
`regulating the expression of NF-kB,
`
`chemokines and cytokines (22-25). Recent reports have also suggested that the CFTR
`
`channel is involved in the transport and maintenance of reduced glutathione, an antioxidant
`
`that plays an important role in protecting against inflammation caused by oxidative stress
`
`(39). Enhancement of intracellular levels of cGMP by way of guanylate cyclase activation
`
`or by way of inhibition of cGMP-specific phosphodiesterase would be expected to down-
`
`regulate these inflammatory stimuli. Thus, uroguanylin-type agonists should be useful in the
`
`prevention and treatment of inflammatory diseases of the lung (e.g., asthma), bowel (e.g.,
`
`ulcerative colitis and Crohn's disease), pancreas and other organs.
`
`Overall, it may be concluded that agonists of guanylate cyclase receptor such as
`
`uroguanylin have potential therapeutic value in the treatment of a wide variety of
`
`inflammatory conditions, cancer (particularly colon cancer) and as anti-metastatic agents.
`
`The development of new agonists is therefore of substantial clinical importance.
`
`Summary of the Invention
`
`The present invention is based upon the development of new agonists of guanylate
`
`cyclase receptor, and new uses of naturally occurring agonists. The agonists are analogs of
`
`uroguanylin, many of which have superior properties either in terms of improved receptor
`
`3
`
`25
`
`
`
`30
`
`
`
`Pg.006
`
`

`

`activation, stability, activity at low pH or reduced adverse effects. The peptides may be used
`to treat any condition that responds to enhanced intracellular levels of cGMP. Intracellular
`levels of cGMP can be increased by enhancing intracellular production of cGMP and/or by
`
`inhibition of its degradation by cGMP-specific phosphodiesterases. Among the specific
`
`5
`
`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
`
`10
`
`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 of either structure or function. For the purpose of the
`
`present application, a peptide differs substantially if its structure varies by more than three
`
`amino acids from a peptide of SEQ ID NOs:2-21 or if its activation of cellular cGMP
`
`15
`
`production is reduced or enhanced by more than 50%. Preferably, substantially similar
`
`peptides should differ by no more than two amino acids and not differ by more than about
`
`25% with respect to activating cGMP production. The most preferred peptide is a bicycle
`
`rti
`
`,t!
`..)
`
`having the sequence of SEQ ID NO:20.
`
`20
`
`The peptides may be in a pharmaceutical composition in unit dose form, together
`
`with one or more pharmaceutically acceptable excipients. The term "unit dose form" refers
`to a single drug delivery entity, e.g., a tablet, capsule, solution or inhalation formulation.
`The amount of peptide present should be sufficient to have a positive therapeutic effect
`
`when administered to a patient (typically, between 100 µg and 3 g). What constitutes a
`
`25
`
`"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.
`
`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,
`
`4
`
`activation, stability, activity at low pH or reduced adverse effects. The peptides may be used
`
`to treat any condition that responds to enhanced intracellular levels of cGMP. Intracellular
`
`levels of cGMP can be increased by enhancing intracellular production of cGMP and/or by
`
`inhibition of its degradation by ¢GMP-specific phosphodiesterases. Among the specific
`
`conditions that can be treated or prevented are inflammatory conditions, cancer, polyps, and
`
`metastasis.
`
`In its first aspect, the present invention is directed to a peptide consisting essentially
`
`of the amino acid sequence of any one of SEQ ID NOs:2-21 and to therapeutic
`
`10
`
`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 of either structure or function. For the purpose of the
`
`present application, a peptide differs substantially if its structure varies by more than three
`
`amino acids from a peptide of SEQ ID NOs:2-21 or if its activation of cellular cGMP
`
`production is reduced or enhanced by more than 50%. Preferably, substantially similar
`
`peptides should differ by no more than two amino acids and not differ by more than about
`
`25% with respect to activating cGMP production. The most preferred peptide is a bicycle
`
`having the sequence of SEQ ID NO:20.
`
`The peptides may be in a pharmaceutical composition in unit dose form, together
`
`with one or more pharmaceutically acceptable excipients. The term “unit dose form” refers
`
`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
`
`“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.
`
`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,
`
`4
`
`
`
`Pg