(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2005/0020811 A1
`(43) Pub. Date:
`Jan. 27, 2005
`Currie et al.
`
`US 2005.0020811A1
`
`(54) METHODS AND COMPOSITIONS FOR THE
`TREATMENT OF GASTRONTESTINAL
`DSORDERS
`(76) Inventors: Mark G. Currie, Sterling, MA (US);
`Shalina Mahajan-Miklos, Needham,
`MA (US)
`Correspondence Address:
`FISH & RICHARDSON PC
`225 FRANKLIN ST
`BOSTON, MA 02110 (US)
`(21) Appl. No.:
`10/796,719
`(22) Filed:
`Mar. 9, 2004
`Related U.S. Application Data
`(63) Continuation-in-part of application No. 10/766,735,
`filed on Jan. 28, 2004.
`(60) Provisional application No. 60/443,098, filed on Jan.
`28, 2003. Provisional application No. 60/471,288,
`filed on May 15, 2003. Provisional application No.
`60/519,460, filed on Nov. 12, 2003.
`
`Publication Classification
`
`(51) Int. Cl. ............................... C07K 7/08; CO7K 7/06
`(52) U.S. Cl. ............................................ 530/327; 530/328
`
`(57)
`
`ABSTRACT
`
`The present invention features compositions and related
`methods for treating IBS and other gastrointestinal disorders
`and conditions (e.g., gastrointestinal motility disorders,
`functional gastrointestinal disorders, gastroesophageal
`reflux disease (GERD), Crohn's disease, ulcerative colitis,
`Inflammatory bowel disease, functional heartburn, dyspep
`sia (including functional dyspepsia or nonulcer dyspepsia),
`gastroparesis, chronic intestinal pseudo-obstruction (or
`colonic pseudo-obstruction), and disorders and conditions
`asSociated with constipation, e.g., constipation associated
`with use of opiate pain killers, post-Surgical constipation
`(post-operative ileus), and constipation associated with neu
`ropathic disorders as well as other conditions and disorders
`using peptides and other agents that activate the guanylate
`cyclase C (GC-C) receptor.
`
`MYLAN - EXHIBIT 1032
`
`

`

`Patent Application Publication Jan. 27,2005 Sheet 1 of 19
`
`
`
`
`
`
`
`SINVINVALNVNISWOODSY40SISATVYNYACILdadSWO1
`
`US 2005/0020811 Al
`
` rbb968Loly6BEOzLEPLE59.
`
`
`
`
`
`
`ealy
`
`£286bESLL+S0E2
`
`+SSWSOL++
`
`
`ge0e sreeseBF697
`Zzé020
`
`ANGE
`
`ezeLOE
`
`Lbe
`
`(xz‘9S)WS6LLELINrEOVOODGW
`
`OOL
`
`%
`
`y:ONGI03S
`
`ealy
`
`+954OL‘+
`
`eel1Z¥0b+E602
`
`
`
`(exz'9S)WSBLLLINreOvVOODGW
`
`OOF
`
`%
`
`G:ONGI03S
`
`
`
`
`
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 2 of 19
`
`US 2005/0020811 A1
`
`EL '91-'
`
`
`OILBHLN?S JO SISATVNV SWOT
`
`
`
`((OLI) HdVH9OIVINOHHO NOI TVIOL) c :ON CII DES
`
`
`
`
`
`
`
`00’09 00'9900'0900:gv 00:0w00,9€.00'0900,9200’0200:gl00’0||0079
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 3 of 19
`
`US 2005/0020811 A1
`
`ol ela
`
`
`
`
`
`
`
`(SISATvNwº :ON di Das Nid=sn»Nvia - 001
`
`
`
`
`
`Ho HdWM9OIVINOHHO NOI TWLOI) SISATvNw SWOT
`
`
`
`
`
`
`
`
`
`EWL1 ----- -- - -+-+-+-+-+-+-+--- *- -
`
`
`
`00:09 , 00:gg , 00:09 . 00.9v , 00:07 ) 00:92 ' 000€ 00.sz , 00:02| 00 91 , 00:01 , 09:9
`
`9%
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 4 of 19
`
`US 2005/0020811 A1
`
`N
`
`
`
`
`
`O-OS) TVN||SNB LNI EHL NI SEC]|LdBd CJEZISEHLN?S ATVOIVNEHO
`
`
`
`
`
`
`
`Å\/SSW ALIAILOV »JOLdBOBH
`
`z 914
`
`(u/Ioud) dNOO
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 5 of 19
`
`US 2005/0020811 A1
`
`i
`
`
`
`35
`a
`ges 9. E CD
`
`o
`9.
`CD
`D
`
`o o do o do o do o
`O O CO. N. CO LO w cy) CN V
`v
`.
`(eol%) peomeleouesia
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 6 of 19
`
`US 2005/0020811 A1
`
`
`
`s
`s
`m Š
`S
`
`s
`No
`
`d d d d d d d d d d
`O) oo N. CO LO w
`() (N V -
`(IWLOL 96) OT3AWLONWISIO
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 7 of 19
`
`US 2005/0020811 A1
`
`
`
`:
`
`(eio L%) peene. Leouesia
`
`

`

`
`
`= .
`KE
`©
`
`
`
`uu
`a»
`ir
`oO
`Q
`
`N
`uw
`=a
`>
`
`==
`
`7
`
`<O
`
`I
`
` Patent A
`
`
`
`blication Jan. 27,2005 Sheet 8 of 19
`
`2
`
`
`
`
`
`
`
`US 2005/0020811 Al
`
`<
`
`4
`
`oe
`
`.
`
`|
`
`L
`9
`
`.
`
`oO
`
`. *
`DG
`
`|
`
`.
`
`<
`
`|
`
`%
`
`Oo
`
`I.Y
`Z = —_
`HY
`S- ‘
`|
`%
`FIG.4B
`
`
`
`OODOmearR OU TON
`
`sess0ee8ge8ge0o
`V (leio. Jo 9.) poieaeleouesia
`“(ero Jo %) pajaneay aoue}sig
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 9 of 19
`
`US 2005/0020811 A1
`
`
`
`O2
`
`55
`
`LL
`
`:
`
`O O. O. O. O. O. O. O.
`V
`
`(e)ol%) pelemeLeouesld
`
`

`

`US 2005/0020811 Al
`
`WS‘Old
`
`
`
`
`
`—(By/6wz'0)—(g-9px6y/Bw)922917-ININ
`
`
`
`@UJOUlEZcp4»Ge¢gt2.GbFFGO
`
`0
`
`0
`
`Z0'0
`
`wT
`
`©o
`
`O 3
`
`90°0A800—
`
`©
`
`ey SSCOIED/SIN
`
`wt
`~~
`
`© O
`
`80
`
`910
`
`Patent Application Publication Jan. 27,2005 Sheet 10 of 19
`
`
`
`
`
`
`
`TAGOWNOILAYOASTWNILSALNISSNOWVNI,WYONTAZSA922917-WN
`
`
`
`
`
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 11 of 19
`
`US 2005/0020811 A1
`
`2.
`
`
`
`N SNS
`s
`
`%
`
`o |
`
`S
`N
`
`CD
`.9
`C
`CD
`
`

`

`Z|---- ? V9 '91-'
`
`
`
`§ (c-01 X 6x/6u) osoa
`
`Patent Application Publication Jan. 27, 2005 Sheet 12 of 19
`
`
`
`
`
`TEGOW NOLLBHOHS TVNLISHINI
`
`
`
`=SnOWN NI 91/9LV-NW GNW G?6-QW QBIVAJ?N?9 XT1NWNIGWOO=8 |
`
`Ole OIS)
`
`5Oz
`
`G?0||G 0 P----——–|——| 0
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 13 of 19
`
`US 2005/0020811 A1
`
`
`
`
`
`·00' L-GW---
`
`
`
`(6×6n) osoa
`
`89,914
`
`
`
`
`3.Sf?OWN NI SECII LaBd CJEZISEHLN?S WTTWOIWNEHO
`
`
`T?COW NOLIBHOHS TVNLISHINI
`
`
`
`
`
`(10\u00+) IS eußis 6x/6nOG-:- (Ionuoo -) ap?uea –o–
`
`92/9!!-WW-o–/G?6-C]|W|----
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 14 of 19
`
`US 2005/0020811 A1
`
`
`
`(ulug)
`suopoenuoo euluopae Jo JequnN
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 15 of 19
`
`US 2005/0020811 A1
`
`
`
`M i
`
`V
`
`Vm
`
`i
`
`to O Lo O Lo O Lo o
`co
`(Y
`N N v-
`w
`sBujuum Jouequnn
`
`

`

` |
`
`
`FIG8Btt—
`ie oi Ss
`
`-2/5
`
`@
`
`-
`
`2o>
`
`SBuiujiuM jo JaquNN |
`
`13mg/kg.mg/kgog
`
`gy
`
`m x|83aio
`
`o
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 17 of 19
`
`US 2005/0020811 A1
`
`
`
`s
`t;
`s
`
`O
`
`D
`N
`
`s s
`('g/9%).9ll-WW-Isz
`
`GN
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 18 of 19
`
`US 2005/0020811 A1
`
`
`
`
`
`+O NOLLd'HOS8V OIWELSAS WnWINIW
`
`(SWOT NO GESVE) OOL L-GW
`
`SWOT uO pºseq
`
`Owz 00z 09: OzL 08 0v 0
`
`(uffin) uoeupueouoo
`
`

`

`Patent Application Publication Jan. 27, 2005 Sheet 19 of 19
`
`US 2005/0020811 A1
`
`
`
`00? L-GW HO NOLLd'HOSavoIWELSAS WnWINIW
`
`
`(\/SITE NO CIESVG)
`
`\/SITE uO pºseq) {
`
`
`
`| 091, 00|| OG| 0
`
`(u?br) uopenueouoo
`
`

`

`US 2005/0020811 A1
`
`Jan. 27, 2005
`
`METHODS AND COMPOSITIONS FOR THE
`TREATMENT OF GASTRONTESTINAL
`DISORDERS
`
`CLAIM OF PRIORITY
`0001. This application is a continuation in part of U.S.
`Utility patent application Ser. No. 10/766,735, filed Jan. 28,
`2004, which claims priority under 35 USC S 119(e) to U.S.
`Provisional Patent Application Ser. No. 60/443,098, filed on
`Jan. 28, 2003; U.S. Provisional Patent Application Ser. No.
`60/471,288, filed on May 15, 2003 and U.S. Provisional
`Patent Application Ser. No. 60/519,460, filed on Nov. 12,
`2003, the entire contents of which are hereby incorporated
`by reference.
`
`TECHNICAL FIELD
`0002 This invention relates to methods and compositions
`for treating various disorders, including gastrointestinal dis
`orders, obesity, congestive heart failure and benign prostatic
`hyperplasia.
`
`BACKGROUND
`0003 Irritable bowel syndrome (IBS) is a common
`chronic disorder of the intestine that affects 20 to 60 million
`individuals in the US alone (Lehman Brothers, Global
`Healthcare-Irritable bowel syndrome industry update, Sep
`tember 1999). IBS is the most common disorder diagnosed
`by gastroenterologists (28% of patients examined) and
`accounts for 12% of Visits to primary care physicians
`(Camilleri 2001, Gastroenterology 120:652-668). In the US,
`the economic impact of IBS is estimated at S25 billion
`annually, through direct costs of health care use and indirect
`costs of absenteeism from work (Talley 1995, Gastroenter
`ology 109:1736-1741). Patients with IBS have three times
`more absenteeism from work and report a reduced quality of
`life. Sufferers may be unable or unwilling to attend social
`events, maintain employment, or travel even short distances
`(Drossman 1993, Dig Dis Sci 38:1569-1580). There is a
`tremendous unmet medical need in this population Since few
`prescription options exist to treat IBS.
`0004 Patients with IBS suffer from abdominal pain and
`a disturbed bowel pattern. Three subgroups of IBS patients
`have been defined based on the predominant bowel habit:
`constipation-predominant (c-IBS), diarrhea-predominant
`(d-IBS) or alternating between the two (a-IBS). Estimates of
`individuals who suffer from c-IBS range from 20-50% of the
`IBS patients with 30% frequently cited. In contrast to the
`other two Subgroups that have a similar gender ratio, c-IBS
`is more common in women (ratio of 3:1) (Talley et al. 1995,
`Am J Epidemiol 142:76-83).
`0005. The definition and diagnostic criteria for IBS have
`been formalized in the “Rome Criteria” (Drossman et al.
`1999, Gut 45:Suppl II: 1-81), which are well accepted in
`clinical practice. However, the complexity of Symptoms has
`not been explained by anatomical abnormalities or meta
`bolic changes. This has led to the classification of IBS as a
`functional GI disorder, which is diagnosed on the basis of
`the Rome criteria and limited evaluation to exclude organic
`disease (Ringel et al. 2001, Annu Rev Med 52: 319-338).
`IBS is considered to be a “biopsychosocial disorder result
`ing from a combination of three interacting mechanisms:
`altered bowel motility, an increased Sensitivity of the intes
`
`tine or colon to pain stimuli (visceral sensitivity) and psy
`chosocial factors (Camilleri 2001, Gastroenterology
`120:652-668). Recently, there has been increasing evidence
`for a role of inflammation in etiology of IBS. Reports
`indicate that Subsets of IBS patients have small but signifi
`cant increases in colonic inflammatory and mast cells,
`increased inducible nitric oxide (NO) and synthase (iNOS)
`and altered expression of inflammatory cytokines (reviewed
`by Talley 2000, Medscape Coverage of DDW week).
`
`SUMMARY
`0006 The present invention features compositions and
`related methods for treating IBS and other gastrointestinal
`disorders and conditions (e.g., gastrointestinal motility dis
`orders, functional gastrointestinal disorders, gastroesoph
`ageal reflux disease (GERD), Crohn's disease, ulcerative
`colitis, Inflammatory bowel disease, functional heartburn,
`dyspepsia (including functional dyspepsia or nonulcer dys
`pepsia), gastroparesis, chronic intestinal pseudo-obstruction
`(or colonic pseudo-obstruction), and disorders and condi
`tions associated with constipation, e.g., constipation associ
`ated with use of opiate pain killers, post-Surgical constipa
`tion, and constipation associated with neuropathic disorders
`as well as other conditions and disorders. The compositions
`feature peptides that activate the guanylate cyclase C (GC
`C) receptor.
`0007. The present invention also features compositions
`and related methods for treating obesity, congestive heart
`failure and benign prostatic hyperplasia (BPH).
`0008. Without being bound by any particular theory, in
`the case of IBS and other gastrointestinal disorders the
`peptides are useful because they can increase gastrointesti
`nal motility.
`0009. Without being bound by any particular theory, in
`the case of IBS and other gastrointestinal disorders the
`peptides are useful, in part, because they can decrease
`inflammation.
`0010 Without being bound by any particular theory, in
`the case of IBS and other gastrointestinal disorders the
`peptides are also useful because they can decrease gas
`trointestinal pain or visceral pain.
`0011. The invention features pharmaceutical composi
`tions comprising certain peptides that are capable of acti
`vating the guanylate-cyclase C (GC-C) receptor. Also within
`the invention are pharmaceutical compositions comprising a
`peptide of the invention as well as combination composi
`tions comprising a peptide of the invention and a Second
`therapeutic agent, e.g., an agent for treating constipation
`(e.g., a chloride channel activator such as SPI-0211;
`Sucampo Pharmaceuticals, Inc., Bethesda, Md., a laxative
`such as MiraLax; Braintree Laboratories, Braintree Mass.)
`or Some other gastrointestinal disorder. Examples of a Sec
`ond therapeutic agent include: acid reducing agents Such as
`proton pump inhibitors (e.g. omeprazole, esomeprazole,
`lansoprazole, pantorazole and rabeprazole) and H2 receptor
`blockers (e.g. cimetidine, ranitidine, famotidine and nizati
`dine), pro-motility agents Such as motilin agonists (e.g
`GM-611 or mitemcinal fumarate), and 5HT receptor ago
`nists (e.g. 5HT4 receptor agonists such as ZelnormoE); 5HT3
`receptor agonists such as MKC-733), 5HT receptor antago
`nists (e.g. 5HT1, 5HT2, 5HT3 (e.g. alosetron), and 5HT4
`
`

`

`US 2005/0020811 A1
`
`Jan. 27, 2005
`
`receptor antagonists, muscarinic receptor agonists, anti
`inflammatory agents, antispasmodics, antidepressants, cen
`trally-acting analgesic agents. Such as opiod receptor ago
`nists, opiod receptor antagonists (e.g. naltrexone), agents for
`the treatment of Inflammatory bowel disease, Crohn's dis
`ease and ulcerative colitis (e.g., Traficet-ENTM (ChemoCen
`tryx, Inc.; San Carlos, Calif.) agents that treat gastrointes
`tinal or visceral pain and coMP phosphodiesterase
`inhibitors (motapizone, Zaprinast, and Suldinac Sulfone). The
`peptides of the invention can also be used in combination
`with agents Such a tianeptine (StablonE) and other agents
`described in U.S. Pat. No. 6,683,072; (E)-4 (1.3bis(cyclo
`hexylmethyl)-1,2,34-tetrahydro-2,6-diono-9H-purin-8-yl
`)cinnamic acid nonaethylene glycol methyl ether ester and
`related compounds described in WO 02/067942. The pep
`tides can also be used in combination with treatments
`entailing the administration of microorganisms useful in the
`treatment of gastrointestinal disorders such as IBS. Probac
`trix(R) (The BioBalance Corporation; New York, N.Y.) is one
`example of a formulation that contains microorganisms
`useful in the treatment of gastrointestinal disorders. In
`addition, the pharmaceutical compositions can include an
`(OK) agent Selected from the group consisting of Ca
`channel blockers (e.g., Ziconotide), 5HT receptor agonists
`(e.g. 5HT1, 5HT2, 5HT3 and 5HT4 receptor agonists) 5HT
`receptor antagonists (e.g. 5HT1, 5HT2, 5HT3 and 5HT4),
`opioid receptor agonists (e.g., loperamide, fedotozine, and
`fentanyl, naloxone, naltrexone, methyl naloZone, nalmefene,
`cypridime, beta funaltrexamine, naloxonazine, naltrindole,
`and nor-binaltorphimine, morphine, diphenyloxylate,
`enkephalin pentapeptide, and trimebutine), NK1 receptor
`antagonists (e.g., ezlopitant and SR-14033, SSR-241585),
`CCK receptor agonists (e.g., loxiglumide), NK1 receptor
`antagonists, NK3 receptor antagonists (e.g., talnetant, osan
`etant SR-142801, SSR-241585), norepinephrine-serotonin
`reuptake inhibitors (NSR1; e.g., milnacipran), Vanilloid and
`cannabanoid receptor agonists (e.g., arVanil), Sialorphin,
`Sialorphin-related peptides comprising the amino acid
`sequence QHNPR (SEQ ID NO: 111) for example, VOH
`NPR (SEQ ID NO: 112); VROHNPR (SEQ ID NO:113);
`VRGOHNPR (SEQ ID NO: 114); VRGPQHNPR (SEQ ID
`NO:115); VRGPROHNPR (SEQ ID NO: 116); VRG
`PRROHNPR (SEQ ID NO: 117); and ROHNPR (SEQ ID
`NO: 118), compounds or peptides that are inhibitors of
`neprilysin, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH;
`WO 01/019849 A1), loperamide, Tyr-Arg (kyotorphin),
`CCK receptor agonists (caerulein), conotoxin peptides, pep
`tide analogs of thymulin, loxiglumide, dexloxiglumide (the
`R-isomer of loxiglumide) (WO 88/05774) and other anal
`gesic peptides or compounds can be used with or linked to
`the peptides of the invention.
`0012. The invention includes methods for treating vari
`ous gastrointestinal disorders by administering a peptide that
`acts as a partial or complete agonist of the GC-C receptor.
`The peptide includes at least Six cysteines that form three
`disulfide bonds. In certain embodiments the disulfide bonds
`are replaced by other covalent croSS-links and in Some cases
`the cysteines are substituted by other residues to provide for
`alternative covalent cross-links. The peptides may also
`include at least one trypsin or chymotrypsin cleavage Site
`and/or a carboxy-terminal analgesic peptide or Small mol
`ecule, e.g., Asp?he or Some other analgesic peptide. When
`present within the peptide, the analgesic peptide or Small
`molecule may be preceded by a chymotrypsin or trypsin
`
`cleavage Site that allows release of the analgesic peptide or
`Small molecule. The peptides and methods of the invention
`are also useful for treating pain and inflammation associated
`with various disorders, including gastrointestinal disorders.
`Certain peptides include a functional chymotrypsin or
`trypsin cleavage Site located So as to allow inactivation of
`the peptide upon cleavage. Certain peptides having a func
`tional cleavage Site undergo cleavage and gradual inactiva
`tion in the digestive tract, and this is desirable in Some
`circumstances. In certain peptides, a functional chymot
`rypsin Site is altered, increasing the Stability of the peptide
`in vivo.
`0013 The invention includes methods for treating other
`disorderS Such as congestive heart failure and benign pros
`tatic hyperplasia by administering a peptide or Small mol
`ecule (parenterally or orally) that acts as an agonist of the
`GC-C receptor. Such agents can be used in combination with
`natriuretic peptides (e.g., atrial natriuretic peptide, brain
`natriuretic peptide or C-type natriuretic peptide), a diuretic,
`or an inhibitor of angiotensin converting enzyme.
`0014. The invention features methods and compositions
`for increasing intestinal motility. Intestinal motility involves
`Spontaneous coordinated dissentions and contractions of the
`Stomach, intestines, colon and rectum to move food through
`the gastrointestinal tract during the digestive process.
`0015. In certain embodiments the peptides include either
`one or two or more contiguous negatively charged amino
`acids (e.g., Asp or Glu) or one or two or more contiguous
`positively charged residues (e.g., Lys or Arg) or one or two
`or more contiguous positively or negatively charged amino
`acids at the carboxy terminus. In these embodiments all of
`the flanking amino acids at the carboxy terminus are either
`positively or negatively charged. In other embodiments the
`carboxy terminal charged amino acids are preceded by a
`Leu. For example, the following amino acid Sequences can
`be added to the carboxy terminus of the peptide: Asp; ASp
`Lys; Lys Lys Lys Lys Lys Lys(SEQ ID NO:123); Asp Lys
`Lys Lys Lys Lys Lys (SEQ ID NO: 124); Leu Lys Lys; and
`Leu Asp. It is also possible to Simply add Leu at the carboxy
`terminus.
`0016. In a first aspect, the invention features a peptide
`comprising, consisting of, or consisting essentially of the
`amino acid Sequence (I): Xaa, Xaa, Xaa- Xaa, Xaas CySg
`CyS7Xaas Xaao CySo CyS11 Xaa12 Xaa13 Xaa14 CyS1s Xaa16
`Xaa17 CyS Xaao Xaao Xaa (SEQ ID NO:119) wherein:
`Xaa, Xaa, Xaa, Xaa, Xaas is ASn Ser Ser ASn Tyr (SEQ ID
`NO:121) or is missing or Xaa, Xaa, Xaa, Xaa is missing.
`In certain embodiments Xaas, Xaao, Xaa12, Xaa, Xaa,
`Xaaz, and Xaa, can be any amino acid. In certain embodi
`ments Xaas is ASn, Trp, Tyr, Asp, or Phe. In other embodi
`ments, Xaas can also be Thr or Ile. In other embodiments
`Xaa, is Tyr, Asp or Trp. In some embodiments Xaas is Glu,
`Asp, Gln, Gly or Pro. In other embodiments Xaas is Glu; in
`Some embodiments Xaa, is Leu, Ile, Val, Ala, LyS, Arg, Trp,
`Tyr or Phe in some embodiments Xaa, is Leu, Ile, Val, Lys,
`Arg, Trp, Tyr or Phe.
`0017. In certain embodiments, the peptide includes dis
`ulfide bonds between CyS and Cys, between CyS., and
`Cys, and between Cysio and Cysis. In other embodiments,
`the peptide is a reduced peptide having no disulfide bonds.
`In still other embodiments the peptide has one or two
`disulfide bonds Selected from the group consisting of a
`
`

`

`US 2005/0020811 A1
`
`Jan. 27, 2005
`
`disulfide bond between Cys and Cys, a disulfide bond
`between CyS, and Cyss and a disulfide bond between CySo
`and CYS.
`0.018. In certain embodiments, an amino acid can be
`replaced by a non-naturally occurring amino acid or a
`naturally or non-naturally occurring amino acid analog. For
`example, an aromatic amino acid can be replaced by 3,4-
`dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothy
`ronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine
`(norTyr). Phg and norTyr and other amino acids including
`Phe and Tyr can be Substituted by, e.g., a halogen, -CH,
`-OH, -CH-NH, -C(O)H, -CHCH, -CN,
`-CHCHCH, -SH, or another group. Further examples
`of unnatural amino acids include: an unnatural analogue of
`tyrosine; an unnatural analogue of glutamine; an unnatural
`analogue of phenylalanine; an unnatural analogue of Serine;
`an unnatural analogue of threonine; an alkyl, aryl, acyl,
`azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl,
`alkynl, ether, thiol, Sulfonyl, Seleno, ester, thioacid, borate,
`boronate, phospho, phosphono, phosphine, heterocyclic,
`enone, imine, aldehyde, hydroxylamine, keto, or amino
`Substituted amino acid, or any combination thereof; an
`amino acid with a photoactivatable croSS-linker; a spin
`labeled amino acid; a fluorescent amino acid; an amino acid
`with a novel functional group; an amino acid that covalently
`or noncovalently interacts with another molecule; a metal
`binding amino acid; a metal-containing amino acid; a radio
`active amino acid; a photocaged and/or photoisomerizable
`amino acid; a biotin or biotin-analogue containing amino
`acid; a glycosylated or carbohydrate modified amino acid; a
`keto containing amino acid; amino acids comprising poly
`ethylene glycol or polyether; a heavy atom Substituted
`amino acid (e.g., an amino acid containing deuterium,
`tritium, C, N, or O); a chemically cleavable or pho
`tocleavable amino acid; an amino acid with an elongated
`Side chain; an amino acid containing a toxic group; a Sugar
`Substituted amino acid, e.g., a Sugar Substituted Serine or the
`like; a carbon-linked Sugar-containing amino acid; a redox
`active amino acid; an O.-hydroxy containing acid; an amino
`thio acid containing amino acid; an O., C. disubstituted amino
`acid; a 3-amino acid; a cyclic amino acid other than proline;
`an O-methyl-L-tyrosine; an L-3-(2-naphthyl)alanine; a
`3-methyl-phenylalanine; a p-acetyl-L-phenylalanine; an
`0-4-allyl-L-tyrosine; a 4-propyl-L-tyrosine; a tri-O-acetyl
`GlcNAcB-Serine; an L-Dopa; a fluorinated phenylalanine;
`an isopropyl-L-phenylalanine; a p-azido-L-phenylalanine; a
`p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine; an
`L-phosphoSerine; a phosphonoSerine; a phosphonotyrosine;
`a p-iodo-phenylalanine; a 4-fluorophenylglycine; a p-bro
`mophenylalanine; a p-amino-L-phenylalanine, a isopropyl
`L-phenylalanine, L-3-(2-naphthyl)alanine; an amino-, iso
`propyl-, or O-allyl-containing phenylalanine analogue; a
`dopa, O-methyl-L-tyrosine; a glycosylated amino acid; a
`p-(propargyloxy)phenylalanine, dimethyl-Lysine, hydroxy
`proline, mercaptopropionic acid, methyl-lysine, 3-nitro-ty
`rosine, norleucine, pyro-glutamic acid, Z (Carbobenzoxyl),
`e-Acetyl-Lysine, B-alanine, aminobenzoyl derivative, ami
`nobutyric acid (Abu), citrulline, aminohexanoic acid, ami
`noisobutyric acid, cyclohexylalanine, d-cyclohexylalanine,
`hydroxyproline, nitro-arginine, nitro-phenylalanine, nitro
`tyrosine, norvaline, octahydroindole carboxylate, omithine,
`penicillamine, tetrahydroisoquinoline, acetamidomethyl
`protected amino acids and a pegylated amino acid. Further
`
`examples of unnatural amino acids can be found in U.S.
`20030108885, U.S. 20030082575, and the references cited
`therein.
`0019 Methods to manfacture peptides containing
`unnatural amino acids can be found in, for example, U.S.
`20030108885, U.S. 20030082575, Deiters et al., JAm Chem
`Soc. (2003) 125:11782-3, Chin et al., Science (2003)
`301:964-7, and the references cited therein.
`0020. The peptides of the invention can be modified
`using Standard modifications. Modifications may occur at
`the amino (N-), carboxy (C-) terminus, internally or a
`combination of any of the preceeding. In one aspect of the
`invention, there may be more than one type of modification
`on the peptide. Modifications include but are not limited to:
`acetylation, amidation, biotinylation, cinnamoylation, farne
`Sylation, formylation, myristoylation, palmitoylation, phos
`phorylation (Ser, Tyr or Thr), Stearoylation, Succinylation,
`Sulfurylation and cyclisation (via disulfide bridges or amide
`cyclisation), and modification by Cy3 or Cy5. The peptides
`of the invention may also be modified by 2, 4-dinitrophenyl
`(DNP), DNP-lysin, modification by 7-Amino-4-methyl-cou
`marin (AMC), flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-
`Diazole), p-nitro-anilide, rhodamine B, EDANS (5-((2-ami
`noethyl)amino)naphthalene-1-Sulfonic
`acid),
`dabcyl,
`dabsyl, dansyl, texas red, FMOC, and Tamra (Tetramethyl
`rhodamine). The peptides of the invention may also be
`conjugated to, for example, BSA or KLH (Keyhole Limpet
`Hemocyanin).
`0021. In some embodiments Xaa is ASn, Tyr, Asp or
`Ala. In other embodiments Xaa is ASn. In Some embodi
`ments Xaa is Ala, Pro or Gly, and in other embodiments it
`is Pro. In Some embodiments Xaa is Ala, Leu, Ser, Gly,
`Val, Glu, Gln, Ile, Leu, LyS, Arg, or Asp, and in other
`embodiments it is Ala or Gly, and in still other embodiments
`it is Ala. In Some embodiments Xaa, is Thr, Ala, ASn, LyS,
`Arg, Trp, Xaa, is Gly, Pro or Ala, Xaao is Selected from
`Trp, Tyr, Phe, ASn and Leu or Xaao is Selected from Trp,
`Tyr, and Phe or Xaa, is selected from Leu, Ile and Val; or
`Xaao is His or Xaao is Selected from Trp, Tyr, Phe, ASn, Ile,
`Val, His and Leu, and Xaao Xaa is Asp?he or is missing
`or Xaao is ASnor Glu and Xaa- is missing or XaalgXaao
`Xaa- is missing. The invention also features methods for
`treating a gastrointestinal disorder (e.g., a gastrointestinal
`motility disorder, a functional gastrointestinal disorder, gas
`troesophageal reflux disease, functional heartburn, dyspep
`sia, functional dyspepsia, nonulcer dyspepsia, gastroparesis,
`chronic intestinal pseudo-obstruction, colonic pseudo-ob
`Struction), obesity, congestive heart failure or benign pros
`tatic hyperplasia by administering a composition comprising
`an aforementioned peptide
`0022. When Xaa, is Trp, Tyr or Phe or when Xaa, is Trp
`the peptide has a potentially functional chymotrypsin cleav
`age Site that is located at a position where cleavage will
`inactivate GC-C receptor binding by the peptide. When Xaa,
`is LyS or Arg or when Xaa, is LyS or Arg, the peptide has
`a potentially functional trypsin cleavage Site that is located
`at a position where cleavage will inactivate GC-C receptor
`binding by the peptide.
`0023. When Xaac is Trp, Tyr or Phe, the peptide has a
`chymotrypsin cleavage Site that is located at a position
`where cleavage will liberate the portion of the peptide
`carboxy-terminal to Xaao. When Xaao is Leu, Ile or Val,
`
`

`

`US 2005/0020811 A1
`
`Jan. 27, 2005
`
`the peptide can have a chymotrypsin cleavage Site that is
`located at a position where cleavage will liberate the portion
`of the peptide amino-terminal to Xaao. At relatively high
`pH the same effect is seen when Xaa, is His. When Xaac
`is LyS or Arg, the peptide has a trypsin cleavage site that is
`located at a position where cleavage will liberate portion of
`the peptide carboxy-terminal to Xaao. Thus, if the peptide
`includes an analgesic peptide carboxy-terminal to Xaao, the
`peptide will be liberated in the digestive tract upon exposure
`to the appropriate protease. Among the analgesic peptides
`which can be included in the peptide are: AspPhe (as
`Xaa-oxaa), endomorphin-1, endomorphin-2, nocistatin,
`dalargin, lupron, and Substance P and other analgesic pep
`tides described herein. These peptides can, for example, be
`used to replace Xaa-oxaa2.
`0024. When Xaa, or the amino-terminal amino acid of
`the peptide of the invention (e.g., Xaa- or Xaa) is Trp, Tyr
`or Phe, the peptide has a chymotrypsin cleavage site that is
`located at a position where cleavage will liberate the portion
`of the peptide amino-terminal to Xaa (or Xaa- or Xaa-)
`along with Xaa, Xaa- or Xaas. When Xaa, or the amino
`terminal amino acid of the peptide of the invention (e.g.,
`Xaa, or Xaas) is Lys or Arg, the peptide has a trypsin
`cleavage Site that is located at a position where cleavage will
`liberate portion of the peptide amino-terminal to Xaa, along
`with Xaa, Xaa- or Xaa). When Xaa, or the amino-terminal
`amino acid of the peptide of the invention is Leu, Ile or Val,
`the peptide can have a chymotrypsin cleavage Site that is
`located at a position where cleavage will liberate the portion
`of the peptide amino-terminal to Xaa. At relatively high pH
`the same effect is seen when Xaa is His. Thus, for example,
`if the peptide includes an analgesic peptide amino-terminal
`to Xaa, the peptide will be liberated in the digestive tract
`upon exposure to the appropriate protease. Among the
`analgesic peptides which can be included in the peptide are:
`Asplphe, endomorphin-1, endomorphin-2, nocistatin, dalar
`gin, lupron, and Substance p and other analgesic peptides
`described herein.
`0025. When fully folded, disulfide bonds are present
`between: CyS and CyS, CyS7 and CySs, and CySo and
`CySs. The peptides of the invention bear Some Sequence
`similarity to ST peptides. However, they include amino acid
`changes and/or additions that improve functionality. These
`changes can, for example, increase or decrease activity (e.g.,
`increase or decrease the ability of the peptide to Stimulate
`intestinal motility), alter the ability of the peptide to fold
`correctly, the stability of the peptide, the ability of the
`peptide to bind the GC-C receptor and/or decrease toxicity.
`In Some cases the peptides may function more desirably than
`wild-type ST peptide. For example, they may limit undesir
`able Side effects Such as diarrhea and dehydration.
`0026. In some embodiments one or both members of one
`or more pairs of CyS residues which normally form a
`disulfide bond can be replaced by homocysteine, 3-mercap
`toproline (Kolodziej et al. 1996 Int J Pept Protein Res
`48:274); B, B dimethylcysteine (Hunt et al. 1993 Int J Pept
`Protein Res 42:249) or diaminopropionic acid (Smith et al.
`1978 J. Med Chem 21:117) to form alternative internal
`croSS-links at the positions of the normal disulfide bonds.
`0027. In addition, one or more disulfide bonds can be
`replaced by alternative covalent croSS-links, e.g., an amide
`bond, an ester linkage, an alkyl linkage, a thioester linkage,
`
`a lactam bridge, a carbamoyl linkage, a urea linkage, a
`thiourea linkage, a phosphonate ester linkage, an alkyl
`linkage, and alkenyl linkage, an ether, a thioether linkage, or
`an amino linkage. For example, Ledu et al. (Proceedings
`Natl Acad. Sci. 100:11263-78, 2003) described methods for
`preparing lactam and amide cross-linkS. Schafineister et al.
`(J. Am. Chem. Soc. 122:5891, 2000) describes stable, all
`carbon croSS-linkS. In Some cases, the generation of Such
`alternative croSS-linkS requires replacing the CyS residues
`with other residues Such as LyS or Glu or non-naturally
`occurring amino acids.
`0028. In the case of a peptide comprising the Sequence
`(I): Xaa, Xaa-Xaas Xaa, Xaas CyS. CyS7 Xaas Xaao CySo
`CyS11 Xaa12 Xaa1a Xaa14 CyS1s Xaa16 Xaa17 CyS1s Xaa19
`Xaa-oxaa wherein: Xaa, Xaa, Xaa, Xaa, Xaas is missing
`and/or the Sequence Xaao Xaao Xaa- is missing, the
`peptide can Still contain additional carboxyterminal or
`amino terminal amino acids or both. For example, the
`peptide can include an amino terminal Sequence that facili
`tates recombinant production of the peptide and is cleaved
`prior to administration of the peptide to a patient. The
`peptide can also include other amino terminal or carboxy
`terminal amino acids. In Some cases the additional amino
`acids protect the peptide, Stabilize the peptide or alter the
`activity of the peptide. In Some cases Some or all of these
`additional amino acids are removed prior to administration
`of the peptide to a patient. The peptide can include 1, 2, 3,
`4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 7080,90, 100 or more
`amino acids at its amino terminus or carboxy terminus or
`both. The number of flanking amino acids need not be the
`Same. For example, there can be 10 additional amino acids
`at the amino terminus of the peptide and none at the carboxy
`terminus.
`0029. In one embodiment the peptide comprises the
`amino acid Sequence (I): Xaa, Xaa, Xaa, Xaa, Xaas Cys,
`CyS7Xaas Xaao CySo CyS11 Xaa12 Xaa13 Xaa14 CyS1s Xaa16
`Xaa17 CyS Xaao Xaao Xaa (SEQID NO: 144) wherein:
`Xaa, Xaa-Xaas Xaa, Xaas is missing, Xaas is Glu, Xaao is
`Leu, Ile, LyS, Arg, Trp, Tyr or Phe, Xaa is ASn; Xaa, is
`Pro; Xaa, is Ala; Xaa, is Thr, Ala, LyS, Arg, Trp, Xaa, is
`Gly, Xaao is Tyr or Leu, and