(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`4 September 2008 (04.09.2008)
`
`(51) International Patent Classification:
`C07K 14/00 (2006.01)
`
`41 14
`1 41 40liff0.
`TIA I I
`"AN-T.74#
`PCT
`
`(10) International Publication Number
`WO 2008/106429 A2
`(74) Agent: MEIKLEJOHN, Anita L.; Fish & Richardson
`P.C., P.O. Box 1022, Minneapolis, MN 55440-1022 (US).
`
`
`
`111111111111110111111101111111111110I01111111111111111111111111111111111111111IIIIIII
`
`(21) International Application Number:
`PCT/US2008/054972
`
`(22) International Filing Date:
`26 February 2008 (26.02.2008)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/891,626
`
`26 February 2007 (26.02.2007) US
`
`(71) Applicants (for all designated States except US): MICRO-
`BIA, INC. [US/US]; 320 Bent Street, Cambridge, MA
`02141 (US). 1VHLNE, G. Todd [US/US]; 320 Bent Street,
`Cambridge, MA 02141 (US).
`
`=
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): ZEVIMER, Daniel
`P. [US/US]; 391 Broadway Street, Apt. 501, Somerville,
`MA 02145 (US). CURRIE, Mark G. [US/US]; 18 Hall
`Avenue, Sterling, MA 01564 (US). FRETZEN, Angelika
`[DE/US]; 155R Summer Street, #1, Somerville, MA 02143
`(US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
`EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
`IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC,
`LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN,
`MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH,
`PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV,
`SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
`ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL,
`NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`— without international search report and to be republished
`upon receipt of that report
`
`=
`
`(54) Title: METHODS AND COMPOSITIONS FOR THE TREATMENT OF HEART FAILURE AND OTHER DISORDERS
`
`Figure 1. EC50 of peptides in in vitro cGMP assay
`
`1
`a.
`
`Seq ID NO:5-
`
`Seq ID NO:4-
`
`Seq ID NO:2-
`
`Seq ID NO:1-
`
`Seq ID NO:3-
`
`Seq ID NO:6 (B)-
`
`Seq ID NO:6 (A)-
`
`II
`
`100
`1000
`O
`O
`EC50 (nM)
`O (57) Abstract: Peptides that act as GC-C receptor agonists and contain at least one D-cys and are useful for the treatment of diuresis
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`10
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`10000
`
`and heart disease as well as other disorders are described.
`
`MYLAN - EXHIBIT 1011
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`WO 2008/106429
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`PCT/US2008/054972
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`Methods and Compositions for the Treatment of Heart
`
`Failure and other Disorders
`
`TECHNICAL FIELD
`
`5
`
`This disclosure relates to methods and compositions for the treatment of heart failure,
`
`gastrointestinal disorders and other disorders.
`
`BACKGROUND
`
`Heart failure is a hemodynamic disorder resulting from impairment of the ability of the
`
`10
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`ventricle to fill with and/or eject blood. The disorder is commonly characterized by
`
`shortness of breath, fatigue, limited exercise tolerance, and fluid retention (both
`
`pulmonary congestion and peripheral edema). Heart failure is generally progressive and
`
`can result in Class IV heart failure (NYHA Heart Failure Classification) in which any
`
`physical activity brings on symptoms such as shortness of breath, and symptoms can
`occur even when the patient is at rest. Patients with symptoms of advanced heart failure
`
`15
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`are treated by tightly controlling fluid status and are often administered intravenous
`
`peripheral vasodilators and/or positive inotropic agents. Patients suffering Class IV heart
`
`failure should be at complete rest (confined to a bed or chair). Among the agents that are
`
`intravenously administered for treatment of advanced heart failure are dobutamine (beta
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`20
`
`receptor antagonist), milrinone (phosphodiesterase inhibitor), and nesiritide. Nesiritide is
`a cardiac derived peptide hormone (human natriuretic peptide B) that is thought to bind to
`and activate guanylate cyclase A (GC-A) receptor.
`
`The guanylate cyclase-C (GC-C) receptor (reviewed by Lucas et al. 2000 Phannacol. Rev
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`25
`
`52:375-414 and Vaandragcr et al. 2002 Molecular and Cellular Biochemistry 230:73-83)
`
`is a key regulator in mammals of intestinal function (although low levels of GC-C have
`been detected in other tissues). GC-C responds to the endogenous hormones, guanylin
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`and uroguanylin, and to enteric bacterial peptides from the heat stable enterotoxin family
`(ST peptides). When agonists bind to GC-C, there is an elevation of the second
`messenger, cyclic GMP, and an increase in chloride and bicarbonate secretion, resulting
`in an increase in intestinal fluid secretion.
`
`SUMMARY
`
`Described herein are methods for treating other disorders such as congestive heart failure
`and benign prostatic hyperplasia by administering a peptide or small molecule
`(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.
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`5
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`10
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`The peptides described herein can be used alone or in combination therapy to prevent
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`15
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`and/or treat disorders associated with fluid and sodium retention,, e.g., diseases of the
`
`electrolyte-water/electrolyte transport system within the kidney, gut and urogenital
`
`system, heart failure (e.g., congestive heart failure or acute heart failure), hypertension,
`
`salt dependent forms of high blood pressure, hepatic edema, and liver cirrhosis. In
`
`addition they can be used to facilitate diuresis or control intestinal fluid. The peptides
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`20
`
`and agonists described herein can also be used to treat disorders where there is abnormal
`
`proliferation of epithelial cells within the kidney (e.g. as in the case of renal cancer).
`
`The peptides and agonists described herein can be used alone or in combination therapy
`to prevent and/or treat kidney disease. "Kidney disease" includes renal failure (including
`
`25
`
`acute renal failure), renal insufficiency, nephrotic edema, glomerulonephritis,
`pyelonephritis, kidney failure, chronic renal failure, nephritis, nephrosis, azotemia,
`uremia, immune renal disease, acute nephritic syndrome, rapidly progessive nephritic
`
`syndrome, nephrotic syndrome, Berger's Disease, chronic nephritic/proteinuric syndrome,
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`tubulointerstital disease, nephrotoxic disorders, renal infarction, atheroembolic renal
`disease, renal cortical necrosis, malignant nephroangiosclerosis, renal vein thrombosis,
`renal tubular acidosis, renal glucosuria, nephrogenic diabetes insipidus, Bartter's
`Syndrome, Liddle's Syndrome, polycystic kidney disease, medullary cystic disease,
`5 medullary sponge kidney, hereditary nephritis, and nail-patella syndrome, along with any
`disease or disorder that relates to the renal system and related disorders, as well as
`symptoms indicative of, or related to, renal or kidney disease and related disorders.
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`10
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`The peptides and agonists described herein can be used alone or in combination therapy
`to prevent or treat polycystic kidney disease. Polycystic kidney disease" "PKD" (also
`called "polycystic renal disease") refers to a group of disorders characterized by a large
`
`number of cysts distributed throughout dramatically enlarged kidneys. The resultant cyst
`
`development leads to impairment of kidney function and can eventually cause kidney
`
`failure. "PKD" specifically includes autosomal dominant polycystic kidney disease
`
`15
`
`(ARPKD) and recessive autosomal recessive polycystic kidney disease (ARPKD), in all
`
`stages of development, regardless of the underlying cause.
`
`The peptides and agonists described herein can be used for treating heart failure,
`
`including heart failure at any of stages I-IV according to New York Heart Association
`
`20
`
`(NYHA) Functional Classification.
`
`The peptides can also be used for treating IBS and other gastrointestinal disorders and
`
`conditions (e.g., gastrointestinal motility disorders, chronic intestinal pseudo-obstruction,
`
`colonic pseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, functional
`
`dyspepsia, nonulcer dyspepsia, a functional gastrointestinal disorder, functional
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`25
`
`heartburn, gastroesophageal reflux disease (GERD), gastroparesis, irritable bowel
`
`syndrome (IBS, e.g., constipation predominant-IBS, diarrhea predominat-IBS, and/or
`
`alternating-IBS)), post-operative ileus, ulcerative colitis, chronic constipation, and
`
`disorders and conditions associated with constipation (e.g. constipation associated with
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`use of opiate pain killers, post-surgical constipation, and constipation associated with
`neuropathic disorders as well as other conditions and disorders are described herein
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`5
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`Without being bound by any particular theory, in the case of heart failure, salt retention,
`fluid retention disorders and combinations thereof the peptides are also useful because
`they may elicit one or more of diuresis, naturesis and/or kaliuresis. Thus, the peptides
`
`described herein may be diuretics.
`
`Without being bound by any particular theory, in the case of IBS and other
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`10
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`gastrointestinal disorders the peptides are useful because they may increase
`
`gastrointestinal motility. The peptides may also decrease inflammation and may decrease
`
`gastrointestinal pain, visceral pain, chronic visceral hypersensitivity, or hypersensitivity
`
`to colorectal distension.
`
`15 Described herein are pharmaceutical compositions comprising certain peptides that are
`
`capable of activating the guanylate-cyclase C (GC-C) receptor. Also described herein are
`
`pharmaceutical compositions comprising a peptide or GC-C agonist described herein and
`
`one or more additional therapeutic agents including, without limitation, the agents
`
`described herein. The other agents can be administered with the peptides described
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`20
`
`herein (simultaneously or sequentially). They can also be linked to a peptide described
`
`herein to create therapeutic conjugates.
`
`Described herein are methods for treating various disorders by administering a peptide
`that acts as a partial or complete agonist of the GC-C receptor. In certain embodiments,
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`25
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`the peptide includes at least six cysteines that can 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
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`site and/or an amino or carboxy-terminal analgesic peptide or small molecule, e.g.,
`AspPhe 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. 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 functional cleavage site undergo
`cleavage and gradual inactivation in the digestive tract, and this is desirable in some
`circumstances. In certain peptides, a functional chymotrypsin site is altered, increasing
`the stability of the peptide in vivo.
`
`The methods described herein include a method for increasing intestinal motility
`comprising administering a GC-C receptor agonist, e.g., a peptide described herein, to a
`patient in need thereof; a method for treating a disorder associated with reduced
`gastrointestinal transit rates or reduced gastrointestinal motility comprising administering
`a GC-C receptor agonist, e.g., a peptide described herein, to a patient in need thereof; a
`method for treating a gastrointestinal hypomotility disorder comprising administering a
`GC-C receptor agonist, e.g., a peptide described herein, to a patient in need thereof; a
`method for treating a non-inflammatory gastrointestinal disorder comprising
`administering a GC-C receptor agonist, e.g., a peptide described herein, to a patient in
`need thereof; a method for treating a gastrointestinal disorder other than Crohn's disease
`and ulcerative colitis comprising administering a GC-C receptor agonist to a patient in
`need thereof; and methods and compositions for increasing intestinal motility comprising
`administering a GC-C receptor agonist to a patient in need thereof. The disorders which
`can be treated by administering a GC-C receptor agonist include, for example,
`constipation, constipation dominant irritable bowel syndrome and pelvic floor
`dyssynergia. In certain embodiments the patient has been diagnosed as suffering from
`IBS according to the Rome criteria. In certain embodiments the patient is female.
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`5
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`5
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`10
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`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, any of the following amino acid sequences can be added to the
`carboxy terminus of the peptide: Asp; Asp Lys; Lys Lys Lys Lys Lys Lys; Asp Lys Lys
`Lys Lys Lys Lys; Leu Lys Lys; and Leu Asp. It is also possible to simply add Leu at the
`carboxy terminus.
`
`Described herein is a peptide or a pharmaceutically acceptable salt thereof comprising the
`amino acid sequence:
`
`Xaai Xaa, Xaa3 Cys Glu Xaa6 Xaa7 Cys Xaa9 Pro Ala Cys Thr Gly Xaa15 Xaa16 (SEQ ID
`NO:7) or a pharmaceutically acceptable salt thereof,
`
`15
`
`wherein
`
`Xaal is any amino acid or is missing;
`
`Xaa2 is Ala, Gly, Lys, Ser, Val or is missing;
`
`Xaa3 is Cys or D-Cys;
`
`Xaa6 is any amino acid;
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`20
`
`Xaa7 is Cys or D-Cys;
`
`Xaa9 is Asn or Thr;
`
`Xaa15 is Cys or D-Cys;
`
`Xaa16 is Lys, Tyr or is missing;
`
`provided that:
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`25
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`(a) one or more of Xaa3, Xaa7 and Xaa15 is D-Cys when Xaa16 is other than Lys; and
`(b) the peptide does not consist of the sequence D-Cys Cys Glu Leu Cys Cys Asn Pro
`
`Ala Cys Thr Gly Cys.
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`In various embodiments: Xaa3 is D-Cys; Xaa7 is D-Cys; Xaa15 is D-Cys; Xaa3 is D-Cys;
`Xaa7 is Cys; Xaa15 is D-Cys; Xaa6 is Val, Ile, Leu Tyr, Phe, or Trp; Xaa6 is Val, Ile, or
`
`Leu; Xaa6 is Val; Xaa6 is Ile; Xaa6 is Lcu; Xaa6 is Tyr, Phe, Trp; Xaa1 is any amino acid;
`
`Xaa l is Gly or Ala; Xaa1 is Gly; Xaal is Ala; Xaal is missing; Xaa6 is Tyr; Xaa6 is Phe;
`
`5 Xaa6 is Trp; at least one of Xaa3, Xaa7 and Xaa15 is D-Cys; at least two of Xaa3, Xaa7 and
`
`Xaa15 are D-Cys; Xaa3, Xaa7 and Xaals arc all D-Cys; Xaa9 is Asn; Xaa9 is Thr; Xaa16 is
`
`Lys; Xaa16 is Tyr; Xaa16 is missing; the peptide is a peptide in any of Figures 3a and 3b;
`and the peptide is purified.
`
`10 Also described is a pharmaceutical composition comprising any of the aforementioned
`
`peptides and a pharmaceutically acceptable carrier.
`
`15
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`20
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`25
`
`Also described is a method for reducing fluid retention, the method comprising
`administering the pharmaceutical composition comprising any of the aforementioned
`peptides and a pharmaceutically acceptable carrier or a pharmaceutical composition
`comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys
`
`Asn Pro Ala Cys Thr Gly Cys.
`
`Also described is a method of treating a disorder selected from: heart failure,
`hypertension, salt dependent forms of high blood pressure, hepatic edema, or liver
`cirrhosis comprising administering a pharmaceutical composition comprising any of the
`aforementioned peptides and a pharmaceutically acceptable carrier or a pharmaceutical
`composition comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu
`Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
`
`The peptides can be used to treat chronic or acute heart failure. In acute heart failure the
`patient appears to be in good health, but suddenly develops a large myocardial infarction
`or rupture of a cardiac valve. The acute heart failure is usually largely systolic and the
`sudden reduction in cardiac output often results in systemic hypotension without
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`peripheral edema. Chronic heart failure is typically observed in patients with dilated
`cardiomyopathy or multivalvular heart disease that develops or progresses slowly. In
`chronic heart failure, arterial pressure tends to be well maintained until very late in the
`course, but there is often accumulation of peripheral edema
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`5
`
`Also described is a method for increasing naturesis comprising administering the
`pharmaceutical composition comprising any of the aforementioned peptides and a
`pharmaceutically acceptable carrier or a pharmaceutical composition comprising a
`peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala
`io Cys Thr Gly Cys.
`
`Also described is a method for increasing diuresis comprising administering the
`
`pharmaceutical composition comprising any of the aforementioned peptides and a
`
`pharmaceutically acceptable carrier or a pharmaceutical composition comprising a
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`15
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`peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala
`
`Cys Thr Gly Cys.
`
`Also described is a method of treating a gastrointestinal disorder comprising
`
`administering the pharmaceutical composition comprising any of the aforementioned
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`20
`
`peptides and a pharmaceutically acceptable carrier or a pharmaceutical composition
`
`comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys
`
`Asn Pro Ala Cys Thr Gly Cys.
`
`In various embodiments the gastrointestinal disorder is selected from: a gastrointestinal
`25 motility disorder, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction,
`Crohn's disease, duodenogastric reflux, dyspepsia, functional dyspepsia, nonulcer
`dyspepsia, a functional gastrointestinal disorder, functional heartburn, gastroesophageal
`reflux disease (GERD), gastroparesis, irritable bowel syndrome, post-operative ileus,
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`inflammatory bowel disorder, ulcerative colitis, constipation, chronic constipation,
`chronic idiopathic constipation.
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`5
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`Also described is a method for treating obesity comprising administering the
`pharmaceutical composition comprising any of the aforementioned peptides and a
`pharmaceutically acceptable carrier or a pharmaceutical composition comprising a
`peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala
`Cys Thr Gly Cys.
`
`10 Also described is a method for treating benign prostatic hyperplasia comprising
`administering the pharmaceutical composition comprising any of the aforementioned
`peptides and a pharmaceutically acceptable carrier or a pharmaceutical composition
`comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys
`Asn Pro Ala Cys Thr Gly Cys.
`
`15
`
`Also described is a method for treating constipation comprising administering the
`pharmaceutical composition comprising any of the aforementioned peptides and a
`pharmaceutically acceptable carrier or a pharmaceutical composition comprising a
`peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala
`20 Cys Thr Gly Cys.
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`25
`
`In various embodiments: the constipation is idiopathic constipation; the constipation is
`chronic idiopathic constipation; the gastrointestinal disorder is irritable bowel syndrome;
`the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome; the
`irritable bowel syndrome is constipation-predominant irritable bowel syndrome; the
`irritable bowel syndrome is alternating-irritable bowel syndrome; the gastrointestinal
`disorder is inflammatory bowel disorder; the gastrointestinal disorder is Crohn's disease;
`and the gastrointestinal disorder is ulcerative colitis.
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`Also described is a method for increasing gastrointestinal motility comprising
`administering the pharmaceutical composition comprising any of the aforementioned
`peptides and a pharmaceutically acceptable carrier or a pharmaceutical composition
`comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys
`5 Asn Pro Ala Cys Thr Gly Cys.
`
`Also described is a method for decreasing gastrointestinal pain or visceral pain
`comprising administering the pharmaceutical composition comprising any of the
`aforementioned peptides and a pharmaceutically acceptable carrier or a pharmaceutical
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`10
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`composition comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu
`
`Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys.
`
`Also described is a method of preventing or treating a side-effect associated with opioid
`
`administration, the method comprising administering to a patient that is being treated
`
`15 with an opioid a pharmaceutical composition comprising any of the aforementioned
`
`peptides and a pharmaceutically acceptable carrier or a pharmaceutical composition
`
`comprising a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys
`
`Asn Pro Ala Cys Thr Gly Cys. In various embodiments: the patient is being treated with
`
`an opioid selected from the group consisting of alfentanil, buprenorphine, butorphanol,
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`20
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`codeine, dezocine, dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levorphanol,
`
`meperidine (pethidine), methadone, morphine, nalbuphine, oxycodone, oxymorphone,
`
`pentazocine, propiram, propoxyphene, sufentanil and tramadol; the patient is being
`treated with an opioid selected from the group consisting of: morphine, codeine,
`oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl and tramadol; the side
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`25
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`effect is selected from the group consisting of constipation, nausea and vomiting; and the
`
`method further comprises administering an opioid antagonist (e.g., naloxone or
`
`naltrexone).
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`Also described is a pharmaceutical composition comprising an opioid and any forgoing
`peptide or a peptide consisting of the amino acid sequence D-Cys Cys Glu Leu Cys Cys
`Asn Pro Ala Cys Thr Gly Cys. In various embodiments: the opioid is selected from the
`group consisting of alfentanil, buprenorphine, butorphanol, codeine, dezocine,
`dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levorphanol, meperidine
`(pethidine), methadone, morphine, nalbuphine, oxycodone, oxymorphonc, pentazocine,
`
`propiram, propoxyphene, sufentanil and tramadol; and the opioid is selected from the
`
`goup consisting of: morphine, codeine, oxycodone, hydrocodone, dihydrocodeine,
`
`propoxyphene, fentanyl and tramadol.
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`5
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`10
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`Also described is a pharmaceutical kit comprising:
`
`(a)
`
`a first container containing pharmaceutical dosage units comprising an effective
`
`amount of an opioid; and
`
`(b)
`
`a second container containing pharmaceutical dosage units comprising an
`
`15
`
`effective amount of a forgoing peptide or a peptide consisting of the amino acid sequence
`
`D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys. In various embodiments: the
`
`opioid is selected from the group consisting of alfentanil, buprenorphine, butorphanol,
`codeine, dezocine, dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levorphanol,
`
`meperidine (pethidine), methadone, morphine, nalbuphine, oxycodone, oxymorphone,
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`20
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`pentazocine, propiram, propoxyphene, sufentanil and tramadol; and the opioid is selected
`
`from the group consisting of: morphine, codeine, oxycodone, hydrocodone,
`
`dihydrocodeine, propoxyphene, fentanyl and tramadol.
`
`Also described is a method for preparing a pharmaceutical composition comprising
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`25
`
`admixing a forgoing peptide and a pharmaceutically acceptable carrier.
`
`Also described herein are purified peptides comprising, consisting of, or consisting
`essentially of the amino acid sequence of SEQ ID NO: 7 and those peptides depictured in
`
`Figures 3a and Figure 3b.
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`Also described herein are pharmaceutical compositions comprising peptides comprising,
`consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:7 and
`those peptides depicted in Figure 3a and Figure 3b.
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`5
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`15
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`In certain embodiments, for example, when fully folded, the peptide includes disulfide
`bonds between Cys3 and Cyss, between Cys4 and Cys12 and between Cys7 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 chosen from: a disulfide
`bond between Cys3 and Cys8, a disulfide bond between Cys4 and Cysi2 and a disulfide
`bond between Cys7 and Cysts. In other embodiments, one or more of Cys3, Cys7, or Cysts
`is a D-Cys residue and the D-Cys residues can form disulfide bonds in the same manner
`as the Cys residues. Thus, the peptide may include, for example, one or more disulfide
`bonds between D-Cys3 and Cyss, between Cys4 and Cys12i between D-Cys7 and Cysi5,
`between Cys7 and D-Cysi 5, between D-Cys7 and D-Cysis.
`
`In some embodiments the peptide is 13, 14, 15, or 16 amino acids long.
`
`In certain embodiments, one or more amino acids can be replaced by a non-naturally
`
`20
`
`occurring amino acid or a naturally or non-naturally occurring amino acid analog. In
`
`certain embodiments, one or more L-amino acids can be substituted with a D-amino acid.
`
`There are many amino acids beyond the standard 20 amino acids (Ala, Arg, Asn, Asp,
`
`Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val). Some
`are naturally-occurring others are not (see, for example, Hunt, The Non-Protein Amino
`
`25 Acids: In Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman and Hall,
`
`1985). For example, an aromatic amino acid can be replaced by 3,4-dihydroxy-L-
`
`phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, 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, -C1-13, -OH, -CH2NH3, -C(O)H, -CH2CH3, -CN, -
`
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`CH2CH2CH3, -SH, or another group. Any amino acid can be substituted by the D-form
`of the amino acid. Thus, for example, a cysteine residue can be substituted by a D-
`cysteine residue.
`
`5 With regard to non-naturally occurring amino acids or naturally and non-naturally
`occurring amino acid analogs, a number of substitutions in the peptide of SEQ ID NO:7
`or the peptides of Figure 3a and Figure 3b arc possible alone or in combination.
`
`Glu can be replaced by gamma-Hydroxy-Glu or gamma-Carboxy-Glu.
`
`10
`
`Ala can be replaced by an alpha substituted amino acid such as L-alpha-
`
`methylphenylalanine or by analogues such as: 3-Amino-Tyr; Tyr(CH3); Tyr(PO3(CH3)2);
`
`Tyr(SO3H); beta-Cyclohexyl-Ala; beta-(1-Cyclopenteny1)-Ala; beta-Cyclopentyl-Ala;
`
`beta-Cyclopropyl-Ala; beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-I -yI)-
`
`15 Ala; beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe; Cyclohexyl-
`
`Gly; tBu-Gly; beta-(3-benzothienyl)-Ala; beta-(2-thieny1)-Ala; 5-Methyl-Trp; and 4-
`
`Methyl-Trp.
`
`Pro can be an N(alpha)-C(alpha) cyclized amino acid analogues with the structure:
`
`20
`
`N —R
`H
`
`(CH2)n
`
`n = 0, 1, 2, 3 . Pro can also be homopro (L-pipecolic acid); hydroxy-Pro; 3,4-Dehydro-
`
`Pro; 4-fluoro-Pro; or alpha-methyl-Pro.
`
`Val or Leu can also be an alpha-substitued or N-methylated amino acid such as alpha-
`
`25
`
`amino isobutyric acid (aib), L/D-alpha-ethylalanine (LID-isovaline), L/D-methylvaline,
`
`or L/D-alpha-methylleucine or a non-natural amino acid such as beta-fluoro-Ala.
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`Gly can be alpha-amino isobutyric acid (aib) or L/D-alpha-ethylalanine (L/D-isovaline).
`
`5
`
`10
`
`15
`
`20
`
`25
`
`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;
`an amino acid that is amidated at a site that is not naturally amidated, a metal-containing
`amino acid; a radioactive 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 polyethylene
`glycol or polyether; a heavy atom substituted amino acid (e.g., an amino acid containing
`deuterium, tritium, 13C, 15N, or 18O); a chemically cleavable or photocleavable 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 a.-hydroxy containing acid;
`an amino thio acid containing amino acid; an a, a disubstituted amino acid; a n-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-phenylalaninc; an 0-4-all yl-L-
`tyrosine; a 4-propyl-L-tyrosine; a tri-O-acetyl-G1cNAcp-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-
`bromophenylalanine; a p-amino-L-phenylalanine; an isopropyl-L-phenylalanine; L-3-(2-
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`naphthyl)alanine; an amino-, isopropyl-, 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-
`
`tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl); E-Acetyl-Lysine; 13-alanine;
`
`5
`
`aminobenzoyl derivative; aminobutyric acid (Abu); citrulline; aminohexanoic acid;
`
`aminoisobutyric acid; cyclohexylalanine; d-cyclohexylalaninc; hydroxyproline; nitro-
`
`arginine; nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindole carboxylate;
`
`ornithine; penicillamine; tetrahydroisoquinoline; acetamidomethyl protected amino acids
`
`and pegylated amino acids. Further examples of unnatural amino acids and amino acid
`
`10
`
`analogs can be found in U.S. 20030108885, U.S. 20030082575, US20060019347
`
`(paragraphs 410-418) and the references cited therein. The peptides described herein can
`
`include further modifications including those described in US20060019347, paragraph
`
`589.
`
`15
`
`In some embodiments, an amino acid can be replaced by a naturally-occurring, non-
`
`essential amino acid, e.g., taurine.
`
`Methods to manfacture peptides containing unnatural amino acids can be found in, for
`example, US20030108885, US20030082575, US20060019347, Deiters et al., J Am
`
`20 Chem Soc. (2003) 125:11782-3, Chin et al., Science (2003) 301:964-7, and the references
`
`cited the