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US008779090B2

(12) United States Patent (10) Patent No.: US 8,779,090 B2 Zimmer et al. (45) Date of Patent: Jul. 15, 2014

(54) METHODS AND COMPOSITIONS FOR THE (56) References Cited TREATMENT OF HEART FAILURE AND U.S. PATENT DOCUMENTS OTHERDISORDERS M 5,518,888 A * 5/1996 Waldman ...... 435/723 (75) Inventors: Daniel Zimmer, Somerville, MA (US); 7,304,036 B2 12/2007 Currie et al. Angelika Fretzen, Somerville, MA 7,371,727 B2 5/2008 Currie et al. (US); Mark Currie, Sterling, MA (US); 225, 33 SE: G. Todd Milne, Brookline, MA (US) 2004/0266989 Al 12/2004 Curtie et al. 2006/0281682 A1 12/2006 Currie et al. (73) Assignee: Ironwood Pharmaceuticals, Inc., 2009,019 1611 A1 7, 2009 Currie et al. Cambridge, MA (US) 2009,0253634 A1 10, 2009 Currie et al. (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 WO WO99/09416 A2 2?1999 U.S.C. 154(b) by 1147 days. WO WO2004O71436 A2 2/2004 WO WO 2004O71436 A2 * 8, 2004 (21) Appl. No.: 12/528,737 WO WO2005087797 A1 9, 2005 OTHER PUBLICATIONS (22) PCT Filed: Feb. 26, 2008 The Mayo Clinic Colon Cancer Symptoms http://www. (86). PCT No.: PCT/US2008/054972 mayoclinic.com/health/colon-cancer/DS00035/ DSECTION=symptoms, last visited Jun. 24, 2013.* S371 (c)(1), McMillan, Assessing and Managing -Induced Constipation in (2), (4) Date: Sep. 28, 2010 Adults with Cancer, Cancer Control, vol. 11, Supp. 1, No. 3, May/ Jun. 2004. (87) PCT Pub. No.: WO2008/106429 Lussier Adjuvant Analgesics in Cancer Pain, The Oncologist, vol. 9, pp. 571-591, 2004.* PCT Pub. Date: Sep. 4, 2008 Wolfe, H.R. et al., “A comparative molecular field analysis (COMFA) of the structural determinants of heat-stable enterotoxins (65) Prior Publication Data mediating activation of guanylyl cyclase C. J. of Med. Chem. Vol. 45(8): 1731-1734, Apr. 11, 2002. US 2011 FOO21419 A1 Jan. 27, 2011 Kubota, H., et al., “A Long-acting Heat-stable Enterotoxin Analog of US 2012/0040892 A9 Feb. 16, 2012 Enterotoxigenic Escherichia coli with a Single D-”, Biochem. and BioPhys. Research Communications vol. 161(1):229 235, May 30, 1989. Related U.S. Application Data * cited by examiner (60) Provisional application No. 60/891,626, filed on Feb. 26, 2007. Primary Examiner — James H Alstrum Acevedo Assistant Examiner — Jeanette Lieb (51) Int. Cl. (74) Attorney, Agent, or Firm — Honigman Miller Schwartz A6 IK38/04 (2006.01) & Cohn; Kelly T. Murphy; Jonathan P. O'Brien serie Cool) (57) ABSTRACT USPC ...... 530/327, 514/16.4: 514/215 Peptides that act as GC-C receptor agonists and contain at (58) Field of Classification Search least one D-cys and are useful for the treatment of diuresis and None heart disease as well as other disorders are described. See application file for complete search history. 1 Claim, 64 Drawing Sheets

U.S. Patent Jul. 15, 2014 Sheet 14 of 64 US 8,779,090 B2

Gly Cys Cys Glu Tyr D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Gly Cys Cys Glu Tyr D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Lys Gly Cys Cys Glu Tyr D-Cys Cys Asn Pro Ata Cys Thr Gly D-Cys Tyr Gly Cys Cys Glu Tyr D-Cys Cys Thr Pro Ata Cys Thr Gly Cys - Gly Cys Cys Giu Tyr D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Gly Cys Cys Glu Tyr D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Gly Cys Cys Glu Tyr D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Gly Cys Cys Glu Tyr D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Gly Cys Cys Glu Tyr D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys yr O Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys thr Gly Cys - Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr u- Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys thr Gly D-Cys - -- Gly Cys. Cys. Gu Phe Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys OD Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Tyr aA Gly Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys Thr Gly Cys - Gly Cys Cys Glu Phe Cys Cys. Thir Pro Aa Cys Thr Gly Cys Lys Gly Cys Cys Glu Phe Cys Cys Thur Pro Ala Cys Thr Gly Cys Tyr reur Gly Cys Cys Glu Phe Cys Cys Thur Pro Aa Cys Thr Gly DeCys - area Gly Cys Cys Glu Phe Cys Cys Thur Pro Ala Cys Thr Gly D-Cys Lys Kaw Gly Cys Cys Giu Phe Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys -- Ap Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Aa Cys Thr Gly Cys lys r Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr MD Gly Cys Cys. Gu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly O-Cys - r Gly Cys Cys. Gu Phe D-Cys Cys Asn Pro Ala are Gly Cys Cys. Gu Phe D-Cys Cys Asn Pro Ala OO Gly Cys. Cys. Gu Pha D-Cys Cys Thr Pro Ala Gly Cys Cys Giu Phe D-Cys Cys Thr Pro Ala Thr Gly Cys Cya Glu Phe D-Cys Cys Thr Pro Aa Cys. Thr Gly Cys Cys Glu Phe D-Cys Cys Thr Pro Ala r Gly Cys. Cys Glu Phe D-Cys Cys Thr Pro As r Gly Cys Cys Glu Phe D-Cys Cys Thr Pro Aa T DO Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Tr ana Gly Cys Cys Glu Trp Cys Cys Asn. Pro Ala Cys. Thr Gly Cys Lys ar Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr o Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr -- Gly Cys Cys Glu Trip Cys. Cys Asn. Pro Ala Cys. Thr Gly Cys Cys Giu Tp Cys Cys Asr Pro Ala Cys Thr up Gly Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys. Thr per Gly Cys Cys Glu Tp Cys Cys Thr Pro Al Cys Thr Gly CyS Lys Gly Cys Cys. Gu Trp Cys Cys Thr Pro Ala Cys. Thr Gly Cys Tyr aga Gly Cys Cys GEu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys Cys. Gu Trp Cys Cys Thr Pro Ala Cys Thr Gly D-CyS Lys Gly Cys Cys. Gu Trp Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr Gly Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys - Gly Cys Cys Giu Trp C-Cys Cys Asr Pro Ala Cys. Thir Gly CyS Lys area Gly Cys Cys Glu Tip D-Cys Cys As Pro Ala Cys Thr Gly Cys Tyr p Gly Cys Cys Gu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys as Gly Cys Cys Giu Trp O-Cys Cys Asn Pro Ala Cys. Thr Gly C-Cys Lys a Gly Cys Cys Giu Tip D-Cys Cys Asn Pro Ala Cys Thr Gly d-Cys Tyr wns Gly Cys Cys Glu Tip D-Cys Cys Thr Pro Aa Cys Thr Gly Cys ess Gly Cys Cys Glu Trp D-Cys. Cys tr Pro Ala Cys Thr Gly Cys Lys area Gly Cys Cys Giu Trp D-Cys. Cys Thr Pro Aa Cys Thr Gly Cys Tyr Gly Cys Cys Glu Trp O-Cys Cys Thr Pro Ala Cys Thr Gly (D-Cys - r Gly Cys Cys Glu Trp D-Cys. Cys Thr Pro Aa Cys Thr Gly O-Cys Lys --- Gly Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr e Gly D-Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys - re- Gly D-Cys Cys Giu Wall Cys Cys Asn Pro As Cys Thr Gly Cys lys Gly D-Cys Cys Gu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

FIG. 3A-12

U.S. Patent Jul. 15, 2014 Sheet 17 of 64 US 8,779,090 B2

-- Gly D-CyS Cys Giu Trp Cys Cys Asn Pro Ala Cys Thr Gly D-Cys -- --- Gly D-Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Gly D-Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Tyr --- Gly O-Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys - --- Gly O-Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys am Gly D-Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr on- Gly D-Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly D-Cys - - Gly D-Cys. Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys arra Gly D-Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr - Gly D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Gly D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Gly D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Gly D-Cys Cys Glu Trip D-Cys Cys Asn Pro Ala Cys thr Gly D-Cys re Gly D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Lys --ro Gly D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr area Gly D-Cys Cys Glu Tip D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - on- Gly D-Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys --- Gly D-Cys Cys Glu Tip D-Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr -- Gly (D-Cys Cys Glu Tip D-Cys Cys Thr Pro Ala Cys Thr Gly (D-Cys - -- Gly D-Cys Cys Glu Trp D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Gly D-Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr --- Lys Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys - -- Lys. Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly CyS Lys --- Lys Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr - Lys Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - a- Lys Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys -- Lys Cys Cys Glu Wai Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Tyr Lys. Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly Cys - s Lys Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Lys Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr d Lys Cys Cys Glu Val Cys Cys Thr Pro Aa Cys. Thr Gly D-Cys - Kame Lys Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys s Lys Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr -- Lys Cys Cys Glu Wal D-Cys Cys Asn Pro Aa Cys. Thr Gly Cys are arer ty's Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys r Lys Cys Cys Glu Val O-Cys Cys Asn Pro Ala Cys. Thr Gly Cys Tyr as a Lys Cys Cys Glu Wal D-Cys Cys Asn Pro Aa Cys. Thir Gly D-Cys - Lys Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys. Thir Gly D-CyS Lys Lys Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr -- Lys Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - se lys Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Lys Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu Wal D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys -- Lys Cys Cys Glu Wal D-Cys Cys Thr Pro Ala Cys. Thr Gly D-Cys Lys - Lys Cys Cys Glu Wal D-Cys Cys Thr Pro Aa Cys. Thr Gly D-Cys Tyr -- Lys Cys Cys Glu ille Cys Cys Asn Pro Ala Cys. Thr Gly Cys - -- Lys Cys Cys Glu Ike Cys Cys Asn Pro Ala Cys. Thr Gly Cys Lys w Lys Cys Cys Giu le Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr as a Lys Cys Cys Glu e Cys Cys Asn Pro Ala Cys Thr Gly D-Cys -

-- Lys Cys Cys Glu ite cys cys Thr Pro

ur Lys Cys Cys Glu ite Cys Cys Thr Pro Cys Gly D-C is on - Lys. Cys Cys Glu le Cys Cys Thr Pro Lys Cys Cys Giu le D-Cys Cys Asn Pro Lys Cys Cys Glu like D-Cys Cys Asn Pro Lys Cys Cys Glu le D-Cys Cys Asn Pro

FIG. 3A-15

U.S. Patent Jul. 15, 2014 Sheet 23 of 64 US 8,779,090 B2

Ser Cys Cys Glu Leu Cys Cys Asn Pro - Ser Cys Cys Glu Leu Cys Cys Asn Pro D Ser Cys Cys Glu eu Cys Cys Asn Pro re Ser Cys Cys Gu Leu Cys Cys. Thir Fro WMO Ser Cys Cys Glu Leu Cys Cys thr Pro --- Ser Cys Cys Gu Leu Cys Cys Thr Pro Ser Cys Cys Glu leu Cys Cys Thr Pro ar Ser Cys Cys Giu Leu D-Cys Cys Asn Pro re Ser Cys Cys. Gu let D-Cys Cys Asni Pro r Ser Cys Cys Giu Leu D-Cys Cys Asn Pro Ser Cys Cys Glu Leu D-Cys Cys Asn Pro va Ser Cys Cys Glu eu D-Cys Cys Asn Pro u- Ser Cys Cys Gu Leu D-Cys Cys Asn Pro ar Ser Cys Cys Giu Leu D-Cys Cys. Thir Pro a- Ser Cys Cys Giu Leu D-Cys Cys Thr Pro Ser Cys Cys Glu Leu D-Cys Cys Thr Pro a- Ser Cys Cys Gu Leu (D-Cys Cys Thr P Ser Cys Cys Glu Leu D-Cys Cys Thur Pro -- Ser Cys Cys Glu Leu D-Cys Cys Thr Pro a Ser Cys Cys Glu Tyr Cys Cys Asn Pro Ser Cys Cys Glu Tyr Cys Cys Asn Pro -- Ser Cys Cys Glu Tyr Cys Cys Asn Pro - Ser Cys Cys Gu Tyr Cys Cys Asn Pro Cys r Gly sCys XM Ser Cys Cys Glu Tyr Cys Cys Asn Pro Ser Cys Cys Glu Tyr Cys Cys Asn Pro wo- Ser Cys Cys Glu Tyr Cys Cys Thr Pro Cys Thr Gly Cys - Ser Cys Cys Giu Tyr Cys Cys Thr Pro Cys Thr Gly Cys Lys ana Ser Cys Cys Glu Tyr Cys Cys Thr Pro Cys Thr Gly Cys Tyr ra-ar Ser Cys Cys Glu Tyr Cys Cys Thr Pro Cys Thr Gly (D-Cys - - Ser Cys Cys Glu Tyr Cys Cys Thr Pro Cys thr Gly D-Cys tyrs ar Ser Cys Cys Glu Tyr Cys Cys Thr Pro Cys Thr Gly D-Cys Tyr xx Ser Cys Cys Glu Tyr C-Cys Cys Asn. Pro Cys Thr Gly Cys - Ser Cys Cys Glu Tyr D-Cys Cys Asr Pro Cys Thr Gly Cys Lys Ser Cys Cys Giu Tyr D-Cys Cys Asn Pro Cys Thr Gly Cys Tyr --- Ser Cys Cys Glu lyr D-Cys Cys Asn Pro Cys Thr Gly D-Cys - Ser Cys Cys Glu Tyr C-Cys Cys Asn. Pro Cys Thr Gly D-Cys Lys rves Ser Cys Cys Glu Tyr C-Cys Cys Asn. Pro Cys Thr Gly D-Cys Tyr 4-Y- Ser Cys Cys Glu Tyr C-Cys. Cys Thr Pro Cys thr Gly Cys - area Ser Cys Cys Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly Cys lys aka- Ser Cys Cys Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly Cys Tyr wn Ser Cys Cys Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly D-Cys - X Ser Cys Cys Glu Tyr C-Cys Cys Thur Pro Cys Thr Gly D-Cys Lys area Ser Cys Cys Giu Tyr D-Cys Cys Thr Pro Cys Thr Gly D-Cys Tyr Ser Cys Cys. Gu Pe Cys Cys Asn Pro Cys Thr Gly Cys - MO Ser Cys Cys Giu Phe Cys Cys Asn Pro Cys Thr Gly Cys Lys Ser Cys Cys. Gu Phe Cys Cys Asn Pro Cys Thr Gly Cys yr Ser Cys Cys. Gu Phe Cys Cys Asn Pro Ala Cys Thr Gly O-Cys -- r Ser Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly D-Cys ty's va Ser Cys Cys Gu Phe Cys Cys ASn Pro Ala Cys Thr Gly O-Cys Tyr u- Ser Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys Thr Gly Cys - -a Ser Cys Cys Giu Phe Cys Cys Thr Pro At Cys Thr Gly Cys Lys re Ser Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr ar Ser Cys Cys Glu Phe Cys Cys Thr Pro A Cys Thr Gly D-Cys - rer Ser Cys Cys. Gu Phe Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys a- Ser Cys Cys Glu Phe Cys Cys. Ther Pro Aa Cys Thr Gly D-Cys Tyr - Ser Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys re Ser Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys M- Ser Cys Cys Glu Pha D-Cys Cys Asr Pro Ala Cys Thr Gly Cys Tyr

FIG. 3A-21

U.S. Patent Jul. 15, 2014 Sheet 28 of 64 US 8,779,090 B2

Wal Cys Cys Glu Leu D-CyS Cys Asn Pro Wa Cys Cys Glu leu D-Cys Cys Asn Pro Wall Cys Cys Glu Leu D-Cys Cys ASn Pro Wa Cys Cys Glu leu D-Cys Cys Thr Pro Val Cys Cys Glu Leu D-Cys Cys Thr Pro Val Cys. Cys Glu leu D-Cys Cys Thy Pro Wa Cys Cys Giu Leu d-Cys Cys Thy Pro We Cys Cys Glu Leu (D-Cys Cys Thr Pro Val Cys Cys Glu Leu DCys Cys Thr Pro Wal Cys Cys Glu Tyr Cys Cys Asn Pro Wal Cys Cys Glu Tyr Cys Cys Asn Pro Wal Cys Cys Giu Tyr Cys Cys Asn Pro -- Wall Cys Cys Gru Tyr Cys Cys Asn Pro Wal Cys Cys Glu Tyr Cys Cys Asn Pro Val Cys Cys Glu Tyr Cys Cys Asn Pro Wa Cys Cys Glu Tyr Cys Cys Thr Pro Wal Cys Cys Glu Tyr Cys Cys Thr Pro - Va. Cys Cys Giu Tyr Cys Cys Thr Pro w Wa Cys Cys Glu Tyr Cys Cys Thr Pro uw- Wa Cys Cys Glu Tyr Cys Cys Thur Pro Wai Cys Cys Glu Tyr Cys Cys Thr Pro Wai Cys Cys Glu Tyr D-Cys Cys Asn Pro --- Wa Cys Cys Glu Tyr O-Cys Cys Asn Pro Wall Cys Cys Glu Tyr D-Cys Cys Asn Pro n-na Wat Cys Cys Giu Tyr D-Cys Cys Asn Pro -- Val Cys Cys Glu Tyr D-Cys Cys Asn Pro --- Val Cys Cys Glu Tyr D-Cys Cys Asn Pro -- Wal Cys Cys Glu Tyr D-Cys Cys Thr Pro Val Cys Cys Glu Tyr D-Cys Cys Thr Pro Val Cys Cys Glu Tyr D-Cys Cys Thr Wal Cys Cys Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly -Cys Val Cys Cys Glu Tyr D-Cys Cys Thr Pro Wal Cys Cys Glu Tyr D-Cys Cys Pro Val Cys Cys Glu Phe Cys Cys Asn Wall Cys Cys Glu Phe Cys Cys Asn Pro Wa Cys Cys Giu Phe Cys Cys Asn Pro Val Cys Cys Glu Phe Cys Cys Asn Pro Wa Cys Cys Glu Phe Cys Cys Asn Pro Wa Cys Cys Glu Phe Cys Cys Asn Pro Wa Cys Cys Glu Phe Cys Cys Thy Pro Wa Cys Cys Glu Phe Cys Cys Thr Pro Wat Cys Cys Glu Pha Cys Cys Thr Pro Wal Cys Cys Glu Phe Cys Cys Thr Pro Wa Cys Cys Glu Pha Cys Cys Thr Pro Wa Cys Cys Glu Phe Cys Cys Thr Pro Waf Cys Cys Glu Phe D-Cys Cys Asn Pro We Cys Cys Glu Pha D-Cys Cys Asn Pro Wal Cys Cys Glu Phe D-Cys Cys Asn Pro Wai Cys Cys Glu Phe D-Cys Cys Asn Pro Wa Cys Cys Glu Phe D-Cys Cys Asn Pro Wa Cys Cys Gu Phe O-Cys Cys Asin Pro Wa Cys Cys Glu Phe D-Cys Cys Thr - Wa Cys Cys Glu Phe D-Cys Cys Thr Pro -va Val Cys. Cys Glu Phe D-Cys Cys Thr Pro -- Wall Cys Cys Glu Phe D-Cys Cys Thr Pro uses Val Cys Cys Glu Phe D-Cys Cys Thir s Wal Cys. Cys Glu Phe D-Cys Cys Tr Pro Wal Cys Cys Glu Trp Cys Cys Asn Pro wamp Val Cys Cys Glu Trip Cys Cys Asn Pro wrur Val Cys Cys Giu Trp Cys Cys Asn Pro

FIG. 3A-26

U.S. Patent Jul. 15, 2014 Sheet 33 of 64 US 8,779,090 B2

Ala mo Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala spuru Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala - Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Giy O-Cys Tyr Ala r Cys Cys Glu Tyr Cys Cys Thr Pro Ala Cys. Thir Gly Cys so Ala - Cys Cys Glu Tyr Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Ala -- Cys Cys Glu Tyr Cys Cys Thy Pro Aa Cys Thr Gly Cys Tyr Ala -- Cys Cys Giu Tyr Cys Cys sh Pro Aa Cys Thr Gly D-Cys - Ala Cys Cys Gifu Tyr Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Aa o Cys Cys Glu Tyr Cys Cys thr Pro Ala Cys Thr Gly O-Cys Tyr Aa Cys Cys Gru Tyr D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala - Cys Cys Glu Tyr D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Cys Cys Glu Tyr D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys Tyr Ala - Cys Cys Giu Tyr D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Aa unru Cys Cys Glu Tyr O-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala - Cys Cys Glu Tyr D-Cys Cys Asn Pro Ala Cys. Thir Gly D-Cys Tyr Ala r Cys Cys Giu Tyr D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala --- Cys Cys Gifu Tyr D-Cys Cys Thur Pro Ala Cys Thr Gly Cys Lys Ala - Cys Cys Gu Tyr D-Cys. Cys Thr Pro Aa Cys Thur Gly Cys Tyr Ala an Cys Cys Glu Tyr D-Cys Cys Thr Pro Ala Cys thr Gly D-Cys - Ala Cys Cys Glu Tyr D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala -- Cys Cys Glu Tyr D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Ala ar Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ale - Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys lys Ala --- Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala -- Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys - Ala - Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala vosa Cys Cys Giu Phe Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala r Cys Cys Glu Pha Cys Cys Thr Pro Aa Cys Thr Gly Cys - Aa wrwr. Cys Cys Glu Phe Cys Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala -- Cys Cys Glu Phe Cys. Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala - Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys Thr Gly 0-Cys eas Ala - Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys Thr Gly (D-Cys Lys Ala --- Cys Cys Glu Phe Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Ala ass Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala -- Cys Cys Giu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys. Thir Gly Cys Tyr Aa Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala - Cys Cys Giu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Aa Cys Cys Glu Phe D-Cys Cys thr Pro Aa Cys Thr Gly Cys - Aa Cys Cys Glu Phe D-Cys Cys Thr Pro Ale Cys Thr Gly Cys Lys Ala Cys Cys Glu Phe D-Cys Cys Thy Pro Aa Cys Thr Gly Cys Tyr Ala - Cys Cys Glu Phe D-Cys. Cys Thur Pro Aa Cys Thr Gly D-Cys - Ala --- Cys Cys Glu Phe D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Aa -- Cys Cys Glu Phe D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr Ala - Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala r Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala re Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala - Cys Cys Glu Trp Cys Cys ASn Pro Ala Cys Thr Gly (D-Cys - Aa Cys Cys Glu Trip Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala Cys Cys Giu Tip Cys Cys Asn Pro Ala Cys Thr Gly O-Cys Tyr Ala -- Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala --- Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Ala arr Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Ot Cys Cys Giu Trp Cys Cys Thr Pro Ala Cys Thr Gly (D-Cys - Ala warp Cys Cys Glu Trp Cys Cys. Thi? Pro Aa Cys Thr Gly D-Cys Lys Ala -- Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr As Cys Cys Glu Tap D-Cys Cys Asn Pro Ala Cys thr Gly Cys - 3. Cys Cys Glu Trp D-Cys Cys Asn Pro A. Cys Thr G y CyS L ys 8 Cys Cys Glu Trp D-cys Cys Asn Pro A. a. Cys thr G y Cy S Ty

FIG. 3A-31

U.S. Patent Jul. 15, 2014 Sheet 37 of 64 US 8,779,090 B2

Ala Ala Cys Cys Glu ite Cys Cys Asn Pro Ala C tr G D-CYs see Ala Aa Cys Cys Gu ite Cys. Cys Asn Pro Ala 3. Thr y 5. Lys Ala Aa Cys Cys Giu le Cy’s Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ala Cys Cys Giu ls Cys. Cys Thr Pro A Cys Thr Gly Cys - Ala Aa Cys Cys Glu le Cys Cys Thr Pro A Cys. Thr Gly Cys Lys Ala Ala Cys Cys Glu Ie Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Ala Cys Cys Glu ite Cys Cys Thr Pro Ale Cys Thr Gly D-Cys - Ala Ala Cys Cys Giu te Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Aa Cys Cys Glu Ike Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Ala Ala Cys Cys Stu Ile D-Cys Cys Asn Pro Ala cys Thr Gly cs - Ala Aa Cys Cys Glu e D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ala Cys Cys Glu (le D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ala Cys Cys Glu Ile DCys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Aa CyS Cys Glu Ee DeCys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala Aa Cys. Cys Glu lie D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ala Cys Cys GEu e D-Cys Cys Thr Pro Ala Cys. Thr Gly Cys - Ala Ala Cys Cys Glu ite D-Cys Cys Thr Pro Ala Cys. Thr Gly Cys lys Aa Aa Cys Cys Giu le D-Cys Cys Thr Pro Ala Cys Thr Gly Cys tyr Ala A. Cys Cys Giu le D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Ala Aa Cys Cys Glu ite D-Cys Cys Thr Pro Ala Cys thr Gly (D-Cys Lys Ala Ala Cys Cys Glu ille D-Cys Cys thr Pro Ala Cys Thr Gly -Cys Tyr Ala Ala Cys Cys Glu eu Cys Cys Asn Pro Ala Cys Thr Gly Cys - Aa Ala Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys. Thir Gly Cys Lys Ala Ala Cys Cys Glu eu Cys Cys Asn Pro Ata Cys Thr Gly Cys Tyr Aa Aa Cys Cys Glu leu Cys Cys Asn Pro Ala Cys Thr Gy D-Cys - Ala Ala Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Aa Ala Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Aa Ala Cys Cys Glu leU Cys Cys Thr Pro Ala Cys. Thr Gly Cys - Aa Aa Cys Cys Glu Leu Cys Cys Thr Pro Ala Cys. Thr Gly Cys Lys Ala Ala Cys Cys Glu teu Cys Cys Thr Pro Ala Cys. Thr Gly Cys Tyr Aa As Cys Cys Glu Leu Cys Cys Thr Pro Aa Cys. Thr Gly D-Cys - Ala Ala Cys Cys. Gu Leu Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Ala Cys Cys Giu leu Cys Cys Thr Pro Aa Cys thr Gly D-Cys Tyr Ala Ala Cys Cys Glu eu D-Cys. Cys Asn Pro Ala Cys Thr Gly Cys - Ala Aa Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Aa Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ala Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly O-Cys - Ala Ala Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys. Ther Gly D-Cys Lys Ala Aa Cys Cys Glu Leu DeCys Cys Ash Pro Ala Cys Thr Gly D-Cys Tyr Ale Ala Cys. Cys Glu Leu (D-Cys Cys Thr Pro Ale Cys Thr Gly Cys see Ala Ala Cys Cys Giu leu D-Cys Cys Thr Pro Ala Cys Thr Gly Cys lys Ala Ala Cys Cys Glu leu D-Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala Aa Cys Cys. Glu Leu D-Cys Cys The Pro Aa Cys Thr Gly D-Cys - Ala Aa Cys Cys Glu e D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Aa Cys Cys Glu Leu D-Cys Cys Thr Pro At Cys Thr Gly D-Cys Tyr Aa Aa Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ala Cys Cys Giu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ala Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Aa Aa Cys. Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly DeCys sa Ala Aa Cys Cys Glu Tyr Cys Cys Ash Pro Ala Cys Thr Gly D-Cys Lys Ala Aa Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ala Cys. Cys Glu Tyr Cys Cys thr Pro Aa Cys Thr Gly Cys - Ala Ala Cys Cys. Gu Tyr Cys Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala Aa Cys Cys. Gu Tyr Cys Cys thr Pro Ale Cys Thr Gly Cys Tyr Ala Aa Cys Cys Gu Tyr Cys Cys Thr Pro Ala Cys Thr Gly C-Cys - Aa Aa Cys Cys Gu Tyr Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Aa As Cys Cys. Gu Tyr Cys Cys Thr Pro Ala Cys Thur Gly D-Cys Tyr Ala Ala Cys Cys. Gu Tyr D-Cys Cys Asn Pro Ala Cys Thr Gy Cys - Ala Ala Cys Cys Gu Tyr D-Cys Cys Asn Pro Ala Cys Thr Gy Cys Lys Aa Aa Cys Cys Giu Tyr D-Cys Cys Asn Pro Ala Cys. Thir Gly Cys TyT

FIG. 3A-35

U.S. Patent Jul. 15, 2014 Sheet 41 of 64 US 8,779,090 B2

As Ala D-Cys Cys Glu Trp Cys Cys Asr Pro Ala Cys Thr Gly D-Cys - Ala Aa D-Cys Cys Giu Trp Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Lys Ala Aa D-Cys Cys Glu Tp Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Aa D-Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys - Aa Ala D-Cys Cys Gu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Aa Ala D-Cys Cys Gru Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala As D-Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Aa Aa D-Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Aa Ala D-Cys Cys Glu Tip Cys Cys Thr Pro Aa Cys. Thr Gly D-Cys Tyr Aa Ala D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gy Cys - Ala Ala OsCys Cys Glu Trip D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ala D-Cys Cys Glu Trip D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ala D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Ala D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly DeCys Lys Ala Aa D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Tyr Ala Aa D-Cys Cys Glu Trip D-Cys Cys Thr Pro Ala Cys. Thr Gly Cys - Ala Ala D-Cys Cys Glu Trp D-Cys Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala AE D-Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Aa Aa O-Cys Cys Glu Trp D-Cys. Cys Thr Pro Ala Cys thr Gly D-Cys as Aa Aa O-Cys Cys Giu Trp D-Cys Cys Thr Pr Ala Cys Thr Gly D-Cys Lys Ala Ala D-Cys Cys Giu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr Ala Gly Cys Cys Glu Val Cys Cys Asr Pro Ala Cys Thr Gly Cys see Ala Gly Cys Cys Giu Val Cys Cys Asn Pro Ala Cys. Thr Gly Cys Lys Ala Gly Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Giu was Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Gly Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala Gly Cys Cys Giu Val Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Gly Cys Cys Gtu Val Cys Cys Thr Pro Ala Cys Thr Gly Cys - Aa Gly Cys Cys Glu Val Cys Cys Thr Pro At Cys Thr Gly Cys Lys Ala Gly Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Val Cys Cys Thr Pro Aa Cys Thr Gly D-Cys as Ala Gly Cys Cys Glu Val Cys Cys Thr Pro Aa Cys Thr Gly D-Cys lys Ala Gly Cys Cys Giu Val Cys. Cys Thr Pro A Cys. Thr Gly D-Cys Tyr Ala Gly Cys Cys Glu Wal D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - As Gly Cys Cys Glu Wad D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Gly Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Aa Gly Cys Cys Giu Wal D-Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys (ys Ala Gly Cys Cys Giu Wa D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Gly Cys Cys Giu Val D-Cys Cys Thr Pro Aa Cys Thr Gly Cys - Ala Gly Cys Cys Glu Wad D-Cys Cys Thr Pro As Cys Thr Gly Cys Lys Aa Gly Cys Cys Glu Me D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Val D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Ala Gly Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys As Gly Cys Cys Glu Wall D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr As Gly Cys Cys Glu tle Cys Cys Asn Pro Ala Cys Thr Gly Cys ros Ala Gly Cys Cys Glu le Cys Cys Asn Pro Ala Cys Thr Gy Cys Lys Ala Gly Cys Cys Glu le Cys Cys Asn Pro As Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu ite Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Gly Cys Cys Glu ite Cys Cys Asn Pro Ala Cys Thr Gly D-Cys lys Ala Gly Cys Cys Glu te Cys Cys Agn Pro Ala Cys Thr Gly O-Cys Tyr Ala Gly Cys Cys Glu the Cys Cys Thr Pro Aa Cys Thr Gly Cys - Ala Gly Cys Cys Glu le Cys Cys Thr Pro Ala Cys Thr Gly Cys lys Ala Gly Cys Cys Glu le Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu e Cys Cys Thr Pro Ala Cys Thr Gly D-Cys - Ala Gly Cys Cys Glu ite Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys As Gly Cys Cys Glu e Cys Cys Thr Pro Aa Cys Thr Gly O-Cys Tyr Ala Gly Cys Cys Glu Ee D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Gly Cys Cys Git e D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Gly Cys Cys Glu le D-Cys Cys Asr Pro Ala Cys. Thr Gly Cys. Tyr

FG, 3A-39

U.S. Patent Jul. 15, 2014 Sheet 43 of 64 US 8,779,090 B2

Aa Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys - Ala Gly Cys Cys Glu Phe Cys Cys Asn Pro Aa Cys. Thr Gly D-Cys Lys Aa Gly Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Giy D-Cys Tyr Ala Gly Cys Cys Glu Phe Cys Cys Thr Pro Aa Cys Thr Gly Cys - Ala Gly Cys Cys Glu Phe Cys Cys. Thir Pro Aa Cys Thr Gly CyS Lys Ala Gly Cys Cys Giu Phe Cys Cys Thur Po Ala Cys. Thr Gy Cys Tyr Ala Gly Cys Cys Glu Phe Cys Cys Thr Pro Ala cys. Thr Gly D-cys -. Ala Gly Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Aa Gly Cys Cys Glu Phe Cys Cys. Ther Pro Ala Cys Thr Gly D-Cys Tyr As Gly Cys Cys Giu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys as Aa Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly CyS Lys Ala Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly C-Cys - Ala Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-cys Lys Ala Gly Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Gly Cys Cys Gtu Phe D-Cys Cys Thr Pro Aa Cys Thr Gly Cys - Ala Gly Cys Cys Glu Phe D-Cys Cys Thur Pro Aa Cys Thr Gly Cys lys Ala Gly Cys Cys Glu Phe D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Phe D-Cys Cys Thr Pre Aa Cys Thr Gly D-Cys - Ala Gly Cys Cys Glu Phie D-Cys Cys Thr Pro Aa Cys Thr Gly (D-Cys Lys Aa Gly Cys Cys Glu Phe D-Cys Cys Thr Pro Aa Cys Thr Gly C-Cys Tyr Ala Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thir Gly Cys - Ala Gly Cys Cys Giu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Aa Gly Cys Cys Glu Trip Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly O-Cys Lys Ala Gly Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Gly Cys Cys Giu Trp Cys Cys Thr Pro At Cys Thr Gly Cys - Ala Gly Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala Gly Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly O-Cys - Ala Gly Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly O-Cys Lys Ala Gly Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly O-Cys Tyr As Gly Cys Cys Glu Trp DeCys Cys Asn Pro Ala Cys. Thr Gly Cys - Ala Gly Cys Cys Glu Trp d-Cys Cys Asn Pro Ala Cys Thr Gly Cys lys Ala Gly Cys Cys Glu Trp D-Cys Cys Ash Pro Ala Cys Thr Gly Cys Tyr Ala Gly Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys - Aa Gly Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Giy D-Cys Lys Ala Gly Cys Cys Glu Trip D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Gly Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gily Cys - Aa Gly Cys Cys Glu Trip D-Cys Cys Thr Pro Ala Cys. Thr Gly Cys Lys Ala Gly Cys Cys Glu Trp D-Cys Cys hir Pro Ala Cys Thr Gy Cys Tyr Ala Gly Cys Cys Glu Trp D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys s Ala Gly Cys Cys Glu Trp D-Cys Cys thr Pro Ala Cys Thr Giy D-Cys Lys Aa Gly Cys Cys Glu Trp O-Cys Cys Thr Pro Aa Cys thr Gly D-Cys Tyr Aa Gly (D-Cys Cys Glu Wal Cys Cys Asin Pro Ata Cys Thr Gly Cys -- Ala Gly D-Cys Cys Glu Val Cys Cys Asn Pro Ata Cys Thr Gly Cys Lys Ala Gly D-Cys Cys Glu Wal Cys Cys Asin Pro Ata Cys. Thr Gly Cys Tyr Ala Gly D-Cys Cys Glu Val Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys - Aa Gly D-Cys Cys Glu Val Cys Cys Asn Pro Ala Cys. Thir Gly C-Cys lys Ala Gly (D-Cys Cys Glu Val Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Tyr Ala Gly D-Cys Cys Glu Val Cys. Cys Thr Pre Aa Cys Thur Gly Cys - Aa Gly O-Cys Cys Glu Val Cys Cys Thr Pre Aa Cys Thr Gly Cys Lys Ala Gly D-Cys Cys Glu Wal Cys Cys Thr Pro As Cys Thr Gly Cys Tyr Aa Gly (D-Cys Cys Glu Val Cys Cys Thr Pro Aa Cys. Thr Gly D-Cys - Ala Gly D-Cys Cys Glu Wal Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys As Gly D-Cys Cys Glu Val Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Ala Gly D-Cys Cys Gifu Val D-Cys Cys Asn Pro Ala Cys Thr Gly Cys re As Gly D-Cys Cys Glu Vel D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Aa Gly D-Cys Cys Glu Val D-Cys Cys Asn Pro Aa Cys. Thr Gly Cys Tyr

FIG. 3A-41

U.S. Patent Jul. 15, 2014 Sheet 51 of 64 US 8,779,090 B2

Ala Ser Cys Cys Glu Val Cys Cys Asr Pro Ala Cys thr Gly DeCys - Ala Ser Cys Cys Giu Val Cys Cys Asn Pro Ala Cys. Thir Gly D-Cys Lys Aa Ser Cys Cys Glu Wal Cys Cys Asn Pro Ala Cys Ther Gly D-Cys Tyr Ala Ser Cys Cys Giu Wai Cys Cys Thr Pro Ala Cys. Thir Gly Cys - Ala Ser Cys Cys GEu Val Cys Cys Thr Pro Aa Cys. Thr Gly Cys tys Ala Ser Cys Cys GEu Val Cys Cys Ther Pro Aa Cys Thr Gly Cys Tyr Ala Ser Cys Cys Glu Val Cys Cys Thr Pro Ala cys. Thr Gly D-Cys re Ala Ser Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Giy D-Cys Lys Ala Ser gys Cys Glu Val Cys cys Thr Pro Ala cys Thr Giy D-cys Tyr Aa Ser Cys Cys Glu Wal DCys Cys Asn Pro Ala Cys Thr Gly Cys -- Ala Ser Cys Cys Glu Val D-Cys Cys Asn Pro Ala cys. Thr Gly cys Lys Ala Ser Cys Cys Glu Val DCys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ser Cys Cys Gu Wall D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys su Ala Ser Cys Cys Glu Wat O-Cys. Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala Ser Cys Cys Gtu Val D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Tyr Ala Ser Cys Cys Glu Val D-Cys. Cys Thr Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys Thr Gly CyS Lys Aa Ser Cys Cys Glu Wa D-Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Aa Ser Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys. Thr Gly D-Cys - Ala Ser Cys Cys Glu wal D-Cys Cys Thr Pro Aa Cys. Thr Giy D-Cys Lys As Ser Cys Cys Glu Wall D-Cys. Cys Ther Pro Ala Cys. Thr Gly D-Cys Tyr Ala Ser Cys Cys Glu e Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys. Gu le Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ser Cys Cys GEu lie Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ser Cys Cys GEu le Cys Cys Asn Pro Ata Cys Thr Gly D-Cys - Ala Ser Cys Cys. Gu le Cys Cys Asn Pro Ala Cys Thr Gly D-Cys lys Ala Ser Cys Cys Giu le Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser Cys Cys Giu e Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys Glu le Cys. Cys Thr Pro Ala Cys Thr Gly Cys Lys Ala Ser Cys Cys Giu Ele Cys. Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala Ser Cys Cys Giu le Cys Cys Thr Pro Ala Cys Thr Gly D-Cys - Ala Ser Cys Cys Glu le Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Aa Ser Cys Cys Glu le Cys Cys Thr Pro Aa Cys Thr Gly D-Cys tyr Ala Ser Cys Cys Glu le D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys Giu le D-Cys Cys Asn Pro Ala Cys Thr Gly CyS Lys Ala Ser Cys Cys Giu e D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ser Cys Cys Giu e D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Ser Cys Cys Giu le D-Cys Cys Asn Pro Ala Cys. Ther Gly D-Cys Lys Aa Ser Cys Cys Gru lie D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Aa Ser Cys Cys Glu Ike D-Cys Cys Thr Pro Aa Cys Thr Gly Cys - Ala Ser Cys Cys Glu Ile D-Cys. Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala Ser Cys Cys Giu le D-Cys. Cys Thr Pro A Cys Thr Gly Cys Tyr Ala Ser Cys Cys Glu Ile D-Cys Cys Thr Pro At Cys Thr Gly D-Cys - Ala Ser Cys Cys Glu e D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys lys Aa Se Cys Cys Glu ite O-Cys Cys. Thr Pro Aa Cys thr Gly O-Cys Tyr Ala Ser Cys Cys Gu eu Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys Gu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ser Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys y Ala Ser Cys Cys Glu eu Cys Cys Asn Pro Ala Cys thr Gly D-Cys - Aa Sea Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly O-Cys Lys Aa Ser Cys Cys Glu lieu Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Aa Ser Cys Cys Glu Lau Cys. Cys Thr Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys Glu Leu Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Ala Ser Cys Cys Glu Leu Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala Ser Cys Cys Gu Leu Cys Cys thr Pro Aa Cys Thr Gly D-Cys - Ala Se Cys Cys Gu Leu Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Ser Cys Cys Glu Leu Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr Aa Ser Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys. Gu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ser Cys Cys. Gu Leu (D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

FIG. 3A-49 U.S. Patent Jul. 15, 2014 Sheet 52 of 64 US 8,779,090 B2

Ser Cys Cys Gu leu d-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ser Cys Cys Giu Leu D-Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Lys Ser Cys Cys Glu Leu D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - Sar Cys Cys Gu Leu D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Ser Cys Cys Gru leu D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ser Cys Cys Gru Leu D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Ser Cys Cys Glu Leu D-Cys Cys Thr Pro Cys. Thr Gly O-Cys Lys Cys Thr Gly D-Cys Tyr Ser Cys Cys Gu Tyr Cys Cys Asn Pro Cys. Thr Gly Cys - Ser Cys Cys Glu Tyr Cys Cys Asn Pro Cys. Thr Gly Cys ty's Ser Cys Cys Glu Tyr Cys Cys Asn Pro Cys thr Gly Cys Tyr Ser Cys Cys Glu Tyr Cys Cys Asn Pro Cys thr Gly D-Cys - Pro Cys Thr Gly D-Cys Lys Pro Cys Thr Gly D-Cys Tyr Pro Cys Thr Gly Cys - Cys Thr Gly Cys Lys Pro Cys Tr Gly Cys Tyr Pro Cys Thr Gly DeCys as Pro Cys Thr Gly D-Cys Lys Pro Cys Thr Gly O-Cys Tyr Ser Cys Cys Gu Tyr D-Cys Cys Asn Pro Cys Thr Gly Cys -- Ser Cys Cys Glu Tyr D-Cys Cys Asn Pro Cys Thr Gly Cys Lys Ser Cys Cys Giu Tyr DeCys Cys Ash Pro Cys Thr Gly Cys Tyr Ser Cys Cys Glu Tyr D-Cys Cys Asn Pro Cys Thr Gly D-Cys - Ser Cys Cys GEu Tyr D-Cys Cys Asn Pro Cys Thr Gly D-Cys Lys Cys Thr Gly D-Cys Tyr Ser Cys Cys Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly Cys as Ser Cys Cys Gu OCys r Pro Cys Thr Gly Cys Lys Cys Thr Gly Cys Tyr Ser Cys Cys. Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly DeCys see Ser Cys Cys Glu Tyr O-Cys Cys Thr Pro Cys Thr Gly Cys Lys Ser Cys Cys Glu Tyr D-Cys Cys Thr Pro Cys Thr Gly (D-Cys Tyr Ser Cys Cys GEu Phe Cys Cys Asn Pro Cys Thr Gly Cys - Ser Cys. Cys Giu Phe Cys Cys Asn Pro Cys Thr Gly Cys Lys Ser Cys Cys Glu Phe Cys Cys Asn Pro Cys r Gly Cys Tyr Ser Cys. Cys. Gu Phe Cys Cys Asn Pro Cys thr Gly D-Cys - Ser Cys Cys Giu Phe Cys Cys Asn Pro Cys Thr Gly D-Cys Lys Ser Cys Cys Glu Phe Cys Cys Asn Pro Cys thr Gly D-Cys Tyr Ser Cys Cys Giu Phe Cys Cys Thr Pro Cys Thr Gly Cys - Ser Cys Cys Giu Phe Cys Cys Thr Pro Cys Thr Gly Cys Lys Cys Thr Gly Cys Tyr Cys Thr Gly D-Cys - Ser Cys Cys Giu Phe Cys Cys Thr pro Cys Thr Gly D-Cys Lys Ser Cys Cys Gfu Phe Cys Cys Thr Pro Cys Thr Gly D-Cys Tyr Ser Cys Cys Glu Phe D-Cys Cys Asn Pro Cys Thr Gly Cys - Ser Cys. Cys Glu Phe DCys. Cys Asn Pro Cys Thr Gly Cys Lys Ser Cys Cys Glu Phe DLCys. Cys Asn Pro Cys Thr Gly Cys Tyr Ser Cys Cys. Gu Phie D-Cys Cys Asn Pro Cys Thr Gly D-Cys - Ser Cys Cys Giu Phe D-Cys Cys Asn Pro Cys Thr Gly O-Cys Lys Ser Cys Cys Glu Phe D-Cys Cys Asn Pro Cys. Thr Gly D-Cys Tyr Ser Cys Cys Gtu Phe D-Cys Cys Thr Pro Cys Thr Gly Cys - Ser Cys Cys Glu Phe D-Cys Cys Thr Pro Cys Thr Gly Cys Lys Ser Cys Cys Glu Phe D-Cys. Cys Thr Pro Cys. Thr Gly Cys tyr Cys Thr Gly D-Cys - Ser Cys Cys Glu Phe DCys Cys Thr Pro Cys Thr Gly -Cy& Lys Ser Cys Cys. Gu Phe D-Cys. Cys Thr Pro Cys Thr Gly D-Cys Tyr Cys Thr Gly Cys rer Ser Cys Cys Glu Trp Cys Cys Asn Pro Cys Thr Gly Cys Lys Ser Cys Cys Glu Trp Cys Cys Asn Pro Cys Thr Gly Cys Tyr

FIG. 3A-50 U.S. Patent Jul. 15, 2014 Sheet 53 of 64 US 8,779,090 B2

Ala Ser Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly O-Cys - Aa Ser Cys Cys Glu Trp Cys Cys Asn Pro Aa Cys Thr Gly O-Cys Lys Ala Ser Cys Cys Glu Trp Cys Cys Asr Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser Cys Cys Glu Tp Cys. Cys Thr Pro Ala Cys. Thr Gly Cys a Ala Ser Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala Ser Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala Ser Cys Cys Glu Trp Cys Cys Thr Pro Aa Cys Thr Gly (D-Cys - Ala Ser Cys Cys Glu Trp Cys Cys Thr Prs. As Cys Thr Gly D-Cys Lys Ala Ser Cys Cys Glu Trp Cys Cys Thir Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys - Ala Ser Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Llys Ala Ser Cys Cys Giu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ser Cys Cys Glu Trp O-Cys Cys Asn Pro Ala Cys Thr Gly O-Cys - Ala Ser Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Ala Ser Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys Tyr Ala Ser Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala Ser Cys Cys Glu Tip D-Cys Cys Thr Pro Aa Cys thr Gly Cys (ys Ala Ser Cys Cys Giu Tip D-Cys Cys Thr Pro Aa Cys thr Gly Cys Tyr Ala Ser Cys Cys Glu Trip D-Cys Cys Thr Pro Aa Cys Thr Gly (D-Cys - Aa Ser Cys Cys Giu Tip D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Ser Cys Cys Glu Trip D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Ala Se D-Cys Cys Glu Wal Cys Cys Asn Pro Ala Cys Thr Giy Cys - Ala Ser D-Cys Cys Glu Wal Cys Cys Asn Pro Ala Cys Thr Gly Cys lys Ala Ser D-Cys Cys Giu Wat Cys Cys Asn Pro Aa Cys Thr Gly Cys Tyr Ala Set D-Cys Cys Glu Wal Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys - Ala Ser D-Cys Cys Glu Wal Cys Cys Asr Pro Ala Cys thr Gly D-Cys lys Ala Ser D-Cys Cys. Gu Wall Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Tyr Aa Se D-Cys Cys Giu Wa Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala Ser D-Cys Cys. Gu Val Cys. Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala Ser D-Cys Cys Glu Wal Cys. Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Ser D-Cys Cys Glu Val Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Ala Ser D-Cys Cys Glu Val Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Ala Ser D-Cys Cys Glu Wal Cys Cys Thr Pro Ala Cys Thr Gly D-Cys ty Ala Ser D-Cys Cys Gu Wal D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ser D-Cys Cys Glu Wal D-Cys Cys Asn Pro Ala Cys. Thr Gly CyS Lys Ala Ser D-Cys Cys Glu Wal D-Cys Cys Asn Pro Aa Cys Thr Gly Cys Tyr Ala Ser D-Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys - Ala Ser D-Cys Cys Glu Wal D-Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys lys Ala Ser D-Cys Cys Glu Wal D-Cys Cys Asn Pro Aa Cys Thr Gly D-Cys Tyr Ala Ser D-Cys Cys Glu Wal D-Cys Cys Thr Pro Aa Cys thr Gly Cys re Aa Ser D-Cys Cys Glu Wal D-Cys Cys Thy Pro Ala Cys thr Gly CyS Lys Aa Ser D-Cys Cys Gtu Val D-Cys Cys Thr Pro Aa Cys thr Gly Cys Tyr Ala Ser D-Cys Cys Glu Val D-Cys. Cys Thr Pro Aa Cys thr Gly (D-Cys - Ala Ser D-Cys Cys Glu Val D-Cys Cys Thr Pro Aa Cys Thr Gly (D-Cys Lys Ala Ser D-Cys Cys Gu Val D-Cys Cys Thr Pru Aa Cys Thr Gly D-Cys Tyr Aa Ser D-Cys Cys Giu e Cys. Cys Asn Pro Aa Cys Thr Gly Cys - Ala Ser D-Cys Cys. Gu e Cys. Cys Asn Pro Ala Cys thr Gly Cys Lys Ala Set D-Cys Cys Glu ille Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Ser D-Cys Cys Giu le Cys Cys Asn Pro Ala Cys. Thr Gly O-Cys as Ala Ser D-Cys Cys GFu le Cys Cys Asn Pro Ala Cys Thr Gly O-Cys lys Ala Ser D-Cys Cys Glu Ele Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Tyr Aa Ser D-Cys Cys Glu ille Cys Cys Thr Pro Aa Cys Thr Gly Cys - As Ser D-Cys Cys Glu (le Cys Cys Thr Pro Aa Cys. Thr Gly Cys Lys Ala Ser D-Cys Cys Glu Efe Cys. Cys. This Pro Aa Cys. Thr Gly Cys Tyr Aa Ser D-Cys Cys Glu ta Cys Cys thr Pro Aa Cys Thr Gly D-Cys as Aa Ser D-Cys Cys Glu lie Cys Cys Thr Pro Aa Cys Thr Gly C-Cys Lys Aa Ser D-Cys Cys Glu e Cys Cys thr Pro Aa Cys Thr Gly D-Cys Tyr Aa Ser D-Cys Cys Giu Ele D-Cys Cys Asn Pro Ata Cys Thr Gly Cys - Ala Ser D-Cys Cys Glu fle D-Cys Cys Asn Pro Ata Cys Thr Gly Cys lys Ala Ser D-Cys Cys Giu ite D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

FIG. 3A-51

U.S. Patent Jul. 15, 2014 Sheet 55 of 64 US 8,779,090 B2

a Ser D-Cys Cys Glu Phe Cys Cys Asn Pro Ala cys Thr Gly D-cys - a Ser D-Cys Cys Su Phe Cys Cys Asn Pro Ala cys Thr Giy D-cs Lys A Ser P9ys Sys Su Phe Cys Cys Asn Pro Ala cys Thr Giy D-cys Tyr Aa Ser D-Cys Cys Glu Phe Cys Cys Thr Pro Ala Cys. Thr Gly Cys - Ala Ser D-Cys Cys Glu Phe Cys Cys Thr Pro Aa Cys Thr Gly Cys ty's Ala Ser D-Cys Cys Glu Phe Cys Cys Thr Pro Aa Cys Thr Gly Cys Tyr Ala Ser D-Cys Cys Glu Phe Cys Cys. Thr Pro Ala Cys Thr Gly D-Cys - Ala Ser D-Cys Cys Glu Phe Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Ser D-Cys Cys Glu Phe cys cys. Thir Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser D-Cys Cys Glu Phe D-Cys. Cys Asn Pro Ala Cys Thr Gly Cys - Ala Ser D-Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ser D-Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr As Ser D-Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Aa Ser D-Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys lys Aa Ser D-Cys Cys Glu Phe D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser D-Cys Cys Glu Phe D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - Aa Ser D-Cys Cys Glu Phe D-Cys Cys Thr Pro Aa Cys Thr Gly Cys Lys Ala Ser D-Cys Cys Glu Phe D-Cys Cys Thr Pro A Cys Thr Gly Cys Tyr Aa Ser D-Cys Cys Gtu Phe D-Cys Cys Thr Pro At Cys. Thr Gly D-Cys - Aa Ser D-Cys Cys Glu Phe D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Aa Ser D-Cys Cys Gifu Phie D-Cys Cys Thr Pro Aa Cys Thr Gly D-Cys Tyr Ala Ser D-Cys Cys Glu Trp Cys Cys Asr Pro Ala Cys Thr Gly Cys - Aa Ser D-Cys Cys Glu Trp Cys. Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ser D-Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys. Thr Gly Cys Tyr Ala Ser D-Cys Cys Giu Trp Cys Cys Asn. Pro Ala Cys Thur Gly D-Cys - Ala Ser D-Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Lys Ala Ser D-Cys Cys Giu Trp Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser D-Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala Ser D-Cys Cys Glu Trp Cys Cys Thr Pro Ala Cys Thr Gly Cys Lys Aa Ser D-Cys Cys GEu Trp Cys Cys Thr Pro Ale Cys Thr Gly Cys Tyr As Ser D-Cys Cys Glu Trip Cys Cys Thr Pro Ala Cys Thr Gly D-Cys re Ala Ser D-Cys Cys Glu Trip Cys Cys Thr Pro Ala Cys Thur Gly D-Cys Lys Ala Ser D-Cys Cys Glu Trp Cys Cys Thr Pro A Cys. Thr Gly D-Cys Tyr Ala Sef D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys - Aa Ser D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Lys Ala Ser D-Cys Cys GEu Trp D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys Tyr Ala Ser D-Cys Cys GEu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly D-CyS - Ala Sar D-Cys Cys Glu Trp D-Cys Cys Asn Pro Ala Cys Thr Gly D-CyS Lys Ala Ser D-Cys Cys Gtu Trp D-Cys. Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Ser D-Cys Cys Glu Trp D-Cys Cys Thr Pro Aa Cys Thr Gly Cys - Aa Ser D-Cys Cys Glu Trp C-Cys Cys Thr Pro Ala Cys. Thr Gly Cys Lys Aa Ser D-Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Ser D-Cys Cys Glu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys - Ala Ser D-Cys Cys Giu Trp C-Cys. Cys Thr Pro Aa Cys Thr Gly O-CyS Lys Ala Ser D-Cys Cys GEu Trp D-Cys Cys Thr Pro Ala Cys Thr Gly (D-Cys Tyr Ala Wat Cys Cys Glu Wad Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Val Cys Cys Glu Wai Cys. Cys Asn Pro Ala Cys Thr Gly Cys Lys Aa Va. Cys Cys Gu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Aa Wall Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly (D-Cys - Ala Wal Cys Cys Glu Wal Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Aa Val Cys Cys Giu Wal Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Val Cys Cys Giu Val Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala Wall Cys Cys Gil Val Cys. Cys Thr Pro Ala Cys Thr Gly CyS -ys Ala Wall Cys Cys Glu Wal Cys. Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Wall Cys Cys Glu Wal Cys Cys Thr Pro Aa Cys Thr Gly O-Cys - Ala Val Cys Cys Glu Wal Cys Cys Thr Pro Aa Cys Thr Gly (D-Cys Lys Ala Wel Cys Cys Gu Val Cys Cys Thr Pro Aa Cys. Thr Gly D-Cys Tyr Ala W Cys Cys. Gu Wall D-Cys Cys Asn Pro Ala Cys thr Gly Cys - Ala Wal Cys Cys Gus Val O-Cys Cys Asn Pro Ale Cys. Thr Gly Cys lys Ala Val Cys Cys Glu Val D-Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

FIG. 3A-53 U.S. Patent Jul. 15, 2014 Sheet 56 of 64 US 8,779,090 B2

Ala Wall Cys Cys Glu Val C-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys ss Aa Wall Cys Cys Glu Wal D-Cys Cys Asn Pro Ata Cys Thr Gly D-Cys Lys Ala W Cys Cys Glu Wai DeCys Cys Asn Pro Ata Cys thr Gly D-Cys Tyr Ala Wat Cys Cys Glu Wai C-Cys Cys Thr Pro As Cys. Thr Gly Cys - Aa Wal Cys Cys Glu Wal D-Cys Cys Thr Pro Ala Cys. Thr Gly Cys Lys Ala Val Cys Cys Glu Wall D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Wall Cys Cys Giu W D-Cys Cys Thr Pro Ala Cys Thr Gly O-Cys - Ala Wall Cys Cys Glu Wall D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Ala Wat Cys Cys Glu Val D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr As Wa Cys Cys Giu Ife Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Wall Cys Cys Glu Efe Cys Cys Asn Pro Ala Cys Thr Gly Cys tys Ala Wel Cys. Cys Glu lie Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Wall Cys. Cys Glu e Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys - Ala Wal Cys Cys Giu e Cys Cys Asn Pro Ala Cys. Thr Gly D-Cys Lys Aa Wal Cys Cys Glu le Cys. Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Wall Cys. Cys Giu le Cys. Cys Thr Pro Ala Cys Thr Gly Cys -- Ala Wal Cys Cys Glu te Cys Cys Thir Pro Ala Cys Thr Gly Cys Llys Ala Wall Cys Cys Giu ille Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Aa Wall Cys Cys Giu e Cys. Cys Thr Pro Aa Cys Thr Gly D-Cys - Ala Val Cys Cys Glu le Cys. Cys Thr Pro Aa Cys Thr Gly D-Cys Lys Ala Wal Cys Cys Gius e Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Tyr Ala Wall Cys Cys Glu ille D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Wa Cys Cys Glu ite D-Cys Cys Asn Pro Ala Cys. Thr Gly Cys Lys Ala Wai Cys Cys Giu le OCys Cys Asn. Pro Ala Cys Thr Gly Cys Tyr Aa Wa Cys Cys Glu ite D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys - Ala Val Cys Cys Giu Ile Cys Cys Asn Pro Ala Cys Thr Giy D-Cys Lys Ala Wa Cys Cys Glu le D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Val Cys Cys Glu ite D-Cys Cys Thr Pro Ala Cys Thr Gly Cys - Ala Val Cys Cys Glu ille D-Cys Cys Thr Pro Aa Cys thr Gly CyS Lys Ala Wal Cys Cys Glu Ike DeCys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Wal Cys Cys Glu ille D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys - Ala Val Cys Cys Glu ite D-Cys Cys Thr Pro Ala Cys thr Gly D-Cys Lys As Wa Cys Cys Glu le D-Cys Cys Thr Pro Ala Cys. Thr Gly D-Cys Tyr Aa Wai Cys Cys GEu Leu. Cys Cys Asn. Pro Ala Cys Thr Gly Cys - Ala Wall Cys Cys Glu leu. Cys Cys Asn Pro Aa Cys Thr Gly Cys Lys Ala Wall Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Ala Val Cys Cys Gu Leu Cys Cys Asn Pro Aa Cys Thr Gly D-Cys - Ala wa Cys Cys Giu Leu. Cys Cys. As Pro Aa Cys Thr Gly D-Cys Lys Ala Wel Cys Cys Giu leu Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Tyr Ala Wal Cys Cys Glu Leu Cys Cys Thr Pro Ata Cys Thr Gly Cys - Ala Val Cys Cys Glu eu Cys Cys Thr Pre Aa Cys. Thr Gly Cys Lys Ala Val Cys Cys Glu leu Cys Cys Thr Pro Ae Cys Thr Gly Cys Tyr Ala Wall Cys Cys Glu eu. Cys Cys Thr Pro Aa Cys Thr Gly D-Cys - Ala Val Cys Cys Giu leu Cys Cys Thr Pro Ala Cys. Thr Gly D-Cys Lys Ala We Cys Cys Glu leu Cys Cys Thr Pro Ale Cys Thr Gly D-Cys Tyr Ala Val Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly Cys - Ala Wall Cys Cys Giu Leu C-Cys Cys Asn Pro Ata Cys ther Gly Cys Lys Aa Wall Cys Cys Giu Leu -Cys Cys Asn Pro Ata Cys Thr Gly Cys Tyr Aa Wall Cys Cys Glu Leu D-Cys Cys ASn Pro Ata Cys Thr Gly D-Cys - Aa Wall Cys Cys Glu Leu D-Cys Cys Asn Pro Ala Cys Thr Gly D-Cys Lys Aa Wall Cys Cys Glu Leu D-Cys Cys Asr Pro Ala Cys Thr Gly D-Cys Tyr Ala Val Cys Cys Glu Leu D-Cys Cys Thr Pro Aa Cys Thr Gly Cys - Ala Val Cys Cys Glu leu D-Cys Cys Thr Pro Aa Cys. Thr Gly Cys Lys Ala Val Cys Cys Glu Leu D-Cys Cys Thr Pro Ala Cys Thr Gly Cys Tyr Ala Wal Cys Cys Glu, Leu D-Cys. Cys Thr Pro Ale Cys. Thr Gly D-Cys - Ala Val Cys Cys Glu leu D-Cys Cys Thr Pro Ala Cys Thr Gly D-Cys Lys Ala Val Cys Cys Glu Leu O-Cys Cys Thr Pro Aa Cys Thr Gly O-Cys Tyr Aa Wal Cys Cys Gu Tyr Cys Cys Asn Pro Ata Cys Thr Gly Cys - Ala Wal Cys Cys Giu Tyr Cys Cys Asn Pro Ata Cys. Thr Gly Cys Lys Ala Wal Cys Cys Glu Tyr Cys Cys Asn Pro Ata Cys Thr Gly Cys Tyr

FG. 3A-54

US 8,779,090 B2 1. 2 METHODS AND COMPOSITIONS FOR THE uretic peptide or C-type natriuretic peptide), a diuretic, or an TREATMENT OF HEART FAILURE AND inhibitor of angiotensin converting enzyme. OTHERDISORDERS The peptides described herein can be used alone or in combination therapy to prevent and/or treat disorders associ CLAIM OF PRIORITY ated with fluid and Sodium retention, e.g., diseases of the electrolyte-water/electrolyte transport system within the kid This application is the U.S. National Phase of PCT Appli ney, gut and urogenital system, heart failure (e.g., congestive cation No. PCT/US2008/054972 filed Feb. 26, 2008, which heart failure or acute heart failure), hypertension, salt depen claims priority, of U.S. Application No. 60/891,626, which dent forms of high blood pressure, hepatic edema, and liver 10 cirrhosis. In addition they can be used to facilitate diuresis or was filed Feb. 26, 2007. Both of these applications are incor control intestinal fluid. The peptides and agonists described porated herein in their entirety. herein can also be used to treat disorders where there is TECHNICAL FIELD abnormal proliferation of epithelial cells within the kidney (e.g. as in the case of renal cancer). This disclosure relates to methods and compositions for the 15 The peptides and agonists described herein can be used treatment of heart failure, gastrointestinal disorders and other alone or in combination therapy to prevent and/or treat kidney disorders. disease. "Kidney disease' includes renal failure (including acute renal failure), renal insufficiency, nephrotic edema, SEQUENCE LISTING glomerulonephritis, pyelonephritis, kidney failure, chronic renal failure, nephritis, nephrosis, azotemia, uremia, immune This application incorporates by reference in its entirety renal disease, acute nephritic syndrome, rapidly progressive the Sequence Listing entitled IW045PCT1US1 nephritic syndrome, nephrotic syndrome, Berger's Disease, 052113 ST25.txt (1,294 kilobytes) which was created May chronic nephritic/proteinuric syndrome, tubulointerstital dis 21, 2013 and filed with the application on May 24, 2013. ease, nephrotoxic disorders, renal infarction, atheroembolic 25 renal disease, renal cortical necrosis, malignant nephroangio BACKGROUND Sclerosis, renal vein thrombosis, renal tubular acidosis, renal glucosuria, nephrogenic diabetes insipidus, Bartter's Syn Heart failure is a hemodynamic disorder resulting from drome, Liddle's Syndrome, polycystic kidney disease, med impairment of the ability of the ventricle to fill with and/or ullary cystic disease, medullary sponge kidney, hereditary eject blood. The disorder is commonly characterized by 30 nephritis, and nail-patella syndrome, along with any disease shortness of breath, fatigue, limited exercise tolerance, and or disorder that relates to the renal system and related disor fluid retention (both pulmonary congestion and peripheral ders, as well as symptoms indicative of, or related to, renal or edema). Heart failure is generally progressive and can result kidney disease and related disorders. in Class IV heart failure (NYHA Heart Failure Classification) The peptides and agonists described herein can be used in which any physical activity brings on symptoms such as 35 alone or in combination therapy to prevent or treat polycystic shortness of breath, and symptoms can occur even when the kidney disease. Polycystic kidney disease” “PKD (also patient is at rest. Patients with symptoms of advanced heart called “polycystic renal disease') refers to a group of disor failure are treated by tightly controlling fluid status and are ders characterized by a large number of cysts distributed often administered intravenous peripheral vasodilators and/ throughout dramatically enlarged kidneys. The resultant cyst or positive inotropic agents. Patients suffering Class IV heart 40 development leads to impairment of kidney function and can failure should be at complete rest (confined to a bed or chair). eventually cause kidney failure. “PKD’specifically includes Among the agents that are intravenously administered for autosomal dominant polycystic kidney disease (ADPKD) treatment of advanced heart failure are dobutamine (beta and recessive autosomal recessive polycystic kidney disease receptor antagonist), milrinone (phosphodiesterase inhibi (ARPKD), in all stages of development, regardless of the tor), and nesiritide. Nesiritide is a cardiac derived peptide 45 underlying cause. hormone (human natriuretic peptide B) that is thought to bind The peptides and agonists described herein can be used for to and activate guanylate cyclase A (GC-A) receptor. treating heart failure, including heart failure at any of stages The guanylate cyclase-C (GC-C) receptor (reviewed by I-IV according to New York Heart Association (NYHA) Lucas et al. 2000 Pharmacol. Rev 52:375-414 and Vaandrager Functional Classification. et al. 2002 Molecular and Cellular Biochemistry 230:73-83) 50 The peptides can also be used for treating IBS and other is a key regulator in mammals of intestinal function (although gastrointestinal disorders and conditions (e.g., gastrointesti low levels of GC-C have been detected in other tissues). nal motility disorders, chronic intestinal pseudo-obstruction, GC-C responds to the endogenous hormones, guanylin and colonic pseudo-obstruction, Crohn's disease, duodenogastric uroguanylin, and to enteric bacterial peptides from the heat reflux, dyspepsia, functional dyspepsia, nonulcer dyspepsia, stable enterotoxin family (ST peptides). When agonists bind 55 a functional gastrointestinal disorder, functional heartburn, to GC-C, there is an elevation of the second messenger, cyclic gastroesophageal reflux disease (GERD), gastroparesis, irri GMP, and an increase in chloride and bicarbonate secretion, table bowel syndrome (IBS, e.g., constipation predominant resulting in an increase in intestinal fluid secretion. IBS, diarrhea predominat-IBS, and/or alternating-IBS)), post-operative ileus, ulcerative colitis, chronic constipation, SUMMARY 60 and disorders and conditions associated with constipation (e.g. constipation associated with use of opiate pain killers, Described herein are methods for treating other disorders post-Surgical constipation, and constipation associated with Such as congestive heart failure and benign prostatic hyper neuropathic disorders as well as other conditions and disor plasia by administering a peptide or Small molecule ders are described herein (parenterally or orally) that acts as an agonist of the GC-C 65 Without being bound by any particular theory, in the case receptor. Such agents can be used in combination with natri of heart failure, salt retention, fluid retention disorders and uretic peptides (e.g., atrial natriuretic peptide, brain natri combinations thereof the peptides are also useful because US 8,779,090 B2 3 4 they may elicit one or more of diuresis, naturesis and/or In certain embodiments the peptides include either one or kaliuresis. Thus, the peptides described herein may be diuret two or more contiguous negatively charged amino acids (e.g., ics. Asp or Glu) or one or two or more contiguous positively Without being bound by any particular theory, in the case charged residues (e.g., Lys or Arg) or one or two or more of IBS and other gastrointestinal disorders the peptides are contiguous positively or negatively charged amino acids at useful because they may increase gastrointestinal motility. the carboxy terminus. In these embodiments all of the flank The peptides may also decrease inflammation and may ing amino acids at the carboxy terminus are either positively decrease gastrointestinal pain, visceral pain, chronic visceral or negatively charged. In other embodiments the carboxy hypersensitivity, or hypersensitivity to colorectal distension. terminal charged amino acids are preceded by a Leu. For 10 example, any of the following amino acid sequences can be Described herein are pharmaceutical compositions com added to the carboxy terminus of the peptide: Asp; Asp Lys; prising certain peptides that are capable of activating the Lys Lys Lys Lys Lys Lys; Asp Lys Lys Lys Lys Lys Lys; Leu guanylate-cyclase C (GC-C) receptor. Also described herein Lys Lys; and Leu Asp. It is also possible to simply add Leu at are pharmaceutical compositions comprising a peptide or the carboxy terminus. GC-C agonist described herein and one or more additional 15 Described herein is a peptide or a pharmaceutically accept therapeutic agents including, without limitation, the agents able salt thereof comprising the amino acid sequence: described herein. The other agents can be administered with Xaa, Xaa-Xaa-. CyS Glu Xaa, Xaa, CyS Xaa, Pro Ala Cys the peptides described herein (simultaneously or sequen Thr Gly Xaas Xaa, (SEQID NO:7) or a pharmaceutically tially). They can also be linked to a peptide described herein acceptable salt thereof, to create therapeutic conjugates. wherein Described herein are methods for treating various disor Xaa is any amino acid or is missing: ders by administering a peptide that acts as a partial or com Xaa- is Ala, Gly, Lys, Ser, Val or is missing: plete agonist of the GC-C receptor. In certain embodiments, Xaa- is Cys or D-Cys: the peptide includes at least six cysteines that can form three Xaa is any amino acid: disulfide bonds. In certain embodiments the disulfide bonds 25 Xaa, is Cys or D-Cys: are replaced by other covalent cross-links and in some cases Xaao is ASn or Thr, the cysteines are substituted by other residues to provide for Xaas is CyS or D-Cys, alternative covalent cross-links. The peptides may also Xaa is Lys, Tyr or is missing: include at least one trypsin or chymotrypsin cleavage site provided that: and/or an amino or carboxy-terminal analgesic peptide or 30 (a) one or more of Xaa, Xaa, and Xaas is D-Cys when Xaa, Small molecule, e.g., AspPhe or some other analgesic peptide. is other than Lys; and When present within the peptide, the analgesic peptide or (b) the peptide does not consist of the sequence D-Cys Cys Small molecule may be preceded by a chymotrypsin or Glu Leu Cys Cys ASn Pro Ala Cys Thr Gly Cys (SEQ ID trypsin cleavage site that allows release of the analgesic pep NO:33). tide or Small molecule. Certain peptides include a functional 35 In various embodiments: Xaa is D-Cys, Xaa, is D-Cys: chymotrypsin or trypsin cleavage site located so as to allow Xaas is D-CyS:Xaa is D-Cys, Xaa, is Cys; Xaas is D-Cys, inactivation of the peptide upon cleavage. Certain peptides Xaa is Val, Ile, Leu Tyr, Phe, or Trp; Xaa is Val, Ile, or Leu: having a functional cleavage site undergo cleavage and Xaa is Val; Xaa is Ile: Xaa is Leu; Xaa is Tyr, Phe, Trp; gradual inactivation in the digestive tract, and this is desirable Xaa is any amino acid; Xaa is Gly or Ala; Xaa is Gly; Xaa, in Some circumstances. In certain peptides, a functional chy 40 is Ala; Xaa is missing: Xaa is Tyr, Xaa is Phe: Xaa is Trp. motrypsin site is altered, increasing the stability of the peptide at least one of Xaa, Xaa, and Xaas is D-Cys; at least two of in vivo. Xaa, Xaa, and Xaas are D-Cys, Xaa, Xaa, and Xaas are all The methods described herein include a method for D-Cys, Xaa, is ASn; Xaa, is Thr, Xaa, is Lys; Xaa is Tyr; increasing intestinal motility comprising administering a Xaa is missing; the peptide is a peptide in any of FIGS. 3a GC-C receptor agonist, e.g., a peptide described herein, to a 45 and 3b; and the peptide is purified. patient in need thereof; a method for treating a disorder asso Also described is a pharmaceutical composition compris ciated with reduced gastrointestinal transit rates or reduced ing any of the aforementioned peptides and a pharmaceuti gastrointestinal motility comprising administering a GC-C cally acceptable carrier. receptor agonist, e.g., a peptide described herein, to a patient Also described is a method for reducing fluid retention, the in need thereof; a method for treating a gastrointestinal hypo 50 method comprising administering the pharmaceutical com motility disorder comprising administering a GC-C receptor position comprising any of the aforementioned peptides and agonist, e.g., a peptide described herein, to a patient in need a pharmaceutically acceptable carrier or a pharmaceutical thereof a method for treating a non-inflammatory gas composition comprising a peptide consisting of the amino trointestinal disorder comprising administering a GC-C acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys receptor agonist, e.g., a peptide described herein, to a patient 55 Thr Gly Cys (SEQID NO:33). in need thereof; a method for treating a gastrointestinal dis Also described is a method of treating a disorder selected order other than Crohn's disease and ulcerative colitis com from: heart failure, hypertension, salt dependent forms of prising administering a GC-C receptoragonist to a patient in high blood pressure, hepatic edema, or liver cirrhosis com need thereof, and methods and compositions for increasing prising administering a pharmaceutical composition com intestinal motility comprising administering a GC-C receptor 60 prising any of the aforementioned peptides and a pharmaceu agonist to a patient in need thereof. The disorders which can tically acceptable carrier or a pharmaceutical composition be treated by administering a GC-C receptoragonist include, comprising a peptide consisting of the amino acid sequence for example, constipation, constipation dominant irritable D-Cys CysGlu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys bowel syndrome and pelvic floor dyssynergia. In certain (SEQ ID NO:33). embodiments the patient has been diagnosed as Suffering 65 The peptides can be used to treat chronic or acute heart from IBS according to the Rome criteria. In certain embodi failure. In acute heart failure the patient appears to be in good ments the patient is female. health, but Suddenly develops a large myocardial infarction or US 8,779,090 B2 5 6 rupture of a cardiac valve. The acute heart failure is usually bowel syndrome is alternating-irritable bowel syndrome; the largely systolic and the Sudden reduction in cardiac output gastrointestinal disorder is inflammatory bowel disorder; the often results in Systemic hypotension without peripheral gastrointestinal disorder is Crohn's disease; and the gas edema. Chronic heart failure is typically observed in patients trointestinal disorder is ulcerative colitis. with dilated cardiomyopathy or multivalvular heart disease Also described is a method for increasing gastrointestinal that develops or progresses slowly. In chronic heart failure, motility comprising administering the pharmaceutical com arterial pressure tends to be well maintained until very late in position comprising any of the aforementioned peptides and the course, but there is often accumulation of peripheral a pharmaceutically acceptable carrier or a pharmaceutical edema. composition comprising a peptide consisting of the amino Also described is a method for increasing naturesis com 10 acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys prising administering the pharmaceutical composition com Thr Gly Cys (SEQID NO:33). prising any of the aforementioned peptides and a pharmaceu Also described is a method for decreasing gastrointestinal tically acceptable carrier or a pharmaceutical composition pain or visceral pain comprising administering the pharma comprising a peptide consisting of the amino acid sequence ceutical composition comprising any of the aforementioned D-Cys CysGlu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys 15 peptides and a pharmaceutically acceptable carrier or a phar (SEQ ID NO:33). maceutical composition comprising a peptide consisting of Also described is a method for increasing diuresis com the amino acid sequence D-Cys CysGlu Leu Cys Cys AsnPro prising administering the pharmaceutical composition com Ala Cys Thr Gly Cys (SEQID NO:33). prising any of the aforementioned peptides and a pharmaceu Also described is a method of preventing or treating a tically acceptable carrier or a pharmaceutical composition side-effect associated with administration, the method comprising a peptide consisting of the amino acid sequence comprising administering to a patient that is being treated D-Cys CysGlu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys with an opioid a pharmaceutical composition comprising any (SEQ ID NO:33). of the aforementioned peptides and a pharmaceutically Also described is a method of treating a gastrointestinal acceptable carrier or a pharmaceutical composition compris disorder comprising administering the pharmaceutical com 25 ing a peptide consisting of the amino acid sequence D-Cys position comprising any of the aforementioned peptides and Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys (SEQID a pharmaceutically acceptable carrier or a pharmaceutical NO:33). In various embodiments: the patient is being treated composition comprising a peptide consisting of the amino with an opioid selected from the group consisting of alfenta acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys nil, , , , , dihydro Thr Gly Cys (SEQID NO:33). 30 codeine, , , , levorpha In various embodiments the gastrointestinal disorder is nol, meperidine (), , , selected from: a gastrointestinal motility disorder, chronic malbuphine, , , , propi intestinal pseudo-obstruction, colonic pseudo-obstruction, ram, propoxyphene, and ; the patient is Crohn's disease, duodenogastric reflux, dyspepsia, func being treated with an opioid selected from the group consist tional dyspepsia, nonulcer dyspepsia, a functional gas 35 ing of morphine, codeine, oxycodone, hydrocodone, dihy trointestinal disorder, functional heartburn, gastroesophageal drocodeine, propoxyphene, fentanyl and tramadol; the side reflux disease (GERD), gastroparesis, irritable bowel syn effect is selected from the group consisting of constipation, drome, post-operative ileus, inflammatory bowel disorder, nausea and Vomiting; and the method further comprises ulcerative colitis, constipation, chronic constipation, chronic administering an opioid antagonist (e.g., or naltr idiopathic constipation. 40 exone). Also described is a method for treating obesity comprising Also described is a pharmaceutical composition compris administering the pharmaceutical composition comprising ing an opioid and any forgoing peptide or a peptide consisting any of the aforementioned peptides and a pharmaceutically of the amino acid sequence D-Cys Cys Glu Leu Cys Cys Asn acceptable carrier or a pharmaceutical composition compris Pro Ala Cys Thr Gly Cys (SEQ ID NO:33). In various ing a peptide consisting of the amino acid sequence D-Cys 45 embodiments: the opioid is selected from the group consist Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys (SEQID ing of , buprenorphine, butorphanol, codeine, dezo NO:33). cine, , fentanyl, hydrocodone, hydromor Also described is a method for treating benign prostatic phone, , meperidine (pethidine), methadone, hyperplasia comprising administering the pharmaceutical morphine, , oxycodone, oxymorphone, pentaZo composition comprising any of the aforementioned peptides 50 cine, , propoxyphene, Sufentanil and tramadol; and and a pharmaceutically acceptable carrier or a pharmaceuti the opioid is selected from the group consisting of morphine, cal composition comprising a peptide consisting of the amino codeine, oxycodone, hydrocodone, dihydrocodeine, pro acid sequence D-Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys poxyphene, fentanyl and tramadol. Thr Gly Cys (SEQID NO:33). Also described is a pharmaceutical kit comprising: Also described is a method for treating constipation com 55 (a) a first container containing pharmaceutical dosage units prising administering the pharmaceutical composition com comprising an effective amount of an opioid; and prising any of the aforementioned peptides and a pharmaceu (b) a second container containing pharmaceutical dosage tically acceptable carrier or a pharmaceutical composition units comprising an effective amount of a forgoing peptide or comprising a peptide consisting of the amino acid sequence a peptide consisting of the amino acid sequence D-CyS Cys D-Cys CysGlu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys 60 Glu Leu Cys Cys ASn Pro Ala Cys Thr Gly Cys (SEQ ID (SEQ ID NO:33). NO:33). In various embodiments: the opioid is selected from In various embodiments: the constipation is idiopathic the group consisting of alfentanil, buprenorphine, butorpha constipation; the constipation is chronic idiopathic constipa nol, codeine, dezocine, dihydrocodeine, fentanyl, hydroc tion; the gastrointestinal disorder is irritable bowel syndrome; odone, hydromorphone, levorphanol, meperidine (pethi the irritable bowel syndrome is diarrhea-predominant irri 65 dine), methadone, morphine, nalbuphine, oxycodone, table bowel syndrome; the irritable bowel syndrome is con oxymorphone, pentazocine, propiram, propoxyphene, Sufen stipation-predominant irritable bowel syndrome; the irritable tanil and tramadol; and the opioid is selected from the group US 8,779,090 B2 7 8 consisting of morphine, codeine, oxycodone, hydrocodone, Cyclohexyl-Gly; thBu-Gly; beta-(3-benzothienyl)-Ala; beta dihydrocodeine, propoxyphene, fentanyl and tramadol. (2-thienyl)-Ala; 5-Methyl-Trp.; and 4-Methyl-Trp. Also described is a method for preparing a pharmaceutical Pro can be an N(alpha)-C(alpha) cyclized amino acid ana composition comprising admixing a forgoing peptide and a logues with the structure: pharmaceutically acceptable carrier.

Also described herein are purified peptides comprising, consisting of, or consisting essentially of the amino acid sequence of SEQID NO: 7 and those peptides depictured in FIG. 3a and FIG. 3b (SEQ ID NOs: 60-3511 and SEQ ID NOs: 1-6). Also described herein are pharmaceutical compositions comprising peptides comprising, consisting of, or consisting essentially of the amino acid sequence of SEQID NO:7 and those peptides depicted in FIG.3a and FIG.3b (SEQID NOs: 15 60-3511 and SEQ ID NOs: 1-6). Pro can also be homopro (L-pipecolic acid); hydroxy-Pro; In certain embodiments, for example, when fully folded, 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro. the peptide includes disulfide bonds between Cys and Cyss, Val or Leu can also be an alpha-substituted or N-methy between CyS and Cys and between Cys, and Cysis. In lated amino acid Such as alpha-amino isobutyric acid (aib), other embodiments, the peptide is a reduced peptide having L/D-alpha-ethylalanine (L/D-isovaline), L/D-methylvaline, no disulfide bonds. In still other embodiments the peptide has or L/D-alpha-methyleucine or a non-natural amino acid Such one or two disulfide bonds chosen from: a disulfide bond as beta-fluoro-Ala. between Cys and Cyss, a disulfide bond between Cys and Gly can be alpha-amino isobutyric acid (aib) or L/D-alpha Cys and a disulfide bond between Cys, and Cysis. In other ethylalanine (L/D-isovaline). embodiments, one or more of Cys, CyS7, or Cysis is a D-Cys 25 Further examples of unnatural amino acids include: an residue and the D-Cys residues can form disulfide bonds in unnatural analogue of tyrosine; an unnatural analogue of the same manner as the Cys residues. Thus, the peptide may ; an unnatural analogue of phenylalanine; an include, for example, one or more disulfide bonds between unnatural analogue of serine; an unnatural analogue of threo D-CyS and Cyss, between Cys and Cys, between D-Cys, nine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, and Cyss, between Cys, and D-Cyss, between D-Cys, and 30 hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, Sulfonyl, D-Cysis. Seleno, ester, thioacid, borate, boronate, phospho, In some embodiments the peptide is 13, 14, 15, or 16 amino phosphono, phosphine, heterocyclic, enone, imine, aldehyde, acids long. hydroxylamine, keto, or amino Substituted amino acid, or any In certain embodiments, one or more amino acids can be combination thereof, an amino acid with a photoactivatable replaced by a non-naturally occurring amino acid or a natu 35 cross-linker, a spin-labeled amino acid; a fluorescent amino rally or non-naturally occurring amino acid analog. In certain acid; an amino acid with a novel functional group; an amino embodiments, one or more L-amino acids can be substituted acid that covalently or noncovalently interacts with another with a D-amino acid. There are many amino acids beyond the molecule; a metal binding amino acid; an amino acid that is standard 20 amino acids (Ala, Arg, ASn, Asp, Cys, Gln, Glu, amidated at a site that is not naturally amidated, a metal Gly. His, Ile, Leu, Lys, Met, Phe, Pro, Ser. Thr, Trp, Tyr, and 40 containing amino acid; a radioactive amino acid; a photo Val). Some are naturally-occurring others are not (see, for caged and/or photoisomerizable amino acid; a biotin or example, Hunt, The Non-Protein Amino Acids: In Chemistry biotin-analogue containing amino acid; a glycosylated or car and Biochemistry of the Amino Acids, Barrett, Chapman and bohydrate modified amino acid; a keto containing amino Hall, 1985). For example, an aromatic amino acid can be acid; amino acids comprising polyethylene glycol or poly replaced by 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-ty 45 ether; a heavy atom Substituted amino acid (e.g., an amino rosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or acid containing deuterium, tritium, C, N, or 'O); a nor-tyrosine (norTyr). Phg and norTyr and other amino acids chemically cleavable orphotocleavable amino acid; an amino including Phe and Tyr can be substituted by, e.g., a halogen, acid with an elongated side chain; an amino acid containing a -CH3, -OH, -CH-NH –C(O)H, -CHCH –CN, toxic group; a Sugar Substituted amino acid, e.g., a Sugar —CH2CH2CH, -SH, or another group. Anyamino acid can 50 Substituted serine or the like; a carbon-linked Sugar-contain be substituted by the D-form of the amino acid. Thus, for ing amino acid; a redox-active amino acid; an O.-hydroxy example, a cysteine residue can be substituted by a D-cysteine containing acid; an amino thio acid containing amino acid; an residue. C. C. disubstituted amino acid; a 3-amino acid; a cyclic amino With regard to non-naturally occurring amino acids or acid other than proline; an O-methyl-L-tyrosine; an L-3-(2- naturally and non-naturally occurring amino acid analogs, a 55 naphthyl)alanine; a 3-methyl-phenylalanine; a p-acetyl-L- number of substitutions in the peptide of SEQID NO:7 or the phenylalanine; an 0-4-allyl-L-tyrosine; a 4-propyl-L-ty peptides of FIG.3a and FIG. 3b (SEQID NOs: 60-3511 and rosine; a tri-O-acetyl-GlcNAc3-serine; an L-Dopa; a SEQID NOs: 1-6) are possible alone or in combination. fluorinated phenylalanine; an isopropyl-L-phenylalanine; a Glu can be replaced by gamma-Hydroxy-Glu or gamma p-azido-L-phenylalanine; a p-acyl-L-phenylalanine; a p-ben Carboxy-Glu. 60 Zoyl-L-phenylalanine; an L-phosphoserine; a phospho Ala can be replaced by an alpha Substituted amino acid noSerine; a phosphonotyrosine; a p-iodo-phenylalanine; a Such as L-alpha-methylphenylalanine or by analogues such 4-fluorophenylglycine; a p-bromophenylalanine; a p-amino as: 3-Amino-Tyr; Tyr(CH); Tyr(PO(CH)); Tyr(SOH): L-phenylalanine; an isopropyl-L-phenylalanine; L-3-(2- beta-Cyclohexyl-Ala; beta-(1-Cyclopentenyl)-Ala; beta-Cy naphthyl)alanine; an amino-, isopropyl-, or O-allyl-contain clopentyl-Ala; beta-Cyclopropyl-Ala; beta-Quinolyl-Ala; 65 ing phenylalanine analogue; a dopa, O-methyl-L-tyrosine; a beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala; beta-(2- glycosylated amino acid; a p-(propargyloxy)phenylalanine; Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe, Fluoro-Phe: dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; US 8,779,090 B2 10 methyl-lysine; 3-nitro-tyrosine; norleucine; pyro-glutamic cally adding sugar moieties (WO 88/02756; WO 89/09786; acid; Z (Carbobenzoxyl); e-Acetyl-Lysine; B-alanine; ami DE 3910667 A1, EPO 374 089 A2; and U.S. Pat. No. 4,861, nobenzoyl derivative; aminobutyric acid (Abu); citrulline; 755), adding cationic anchors (EP0363589), lipid moieties aminohexanoic acid; aminoisobutyric acid; cyclohexylala (WO91/09837; U.S. Pat. No. 4,837,303) or the substituents nine; d-cyclohexylalanine, hydroxyproline; nitro-arginine; described as compounds I, II, and III in U.S. Pat. No. 5,552, nitro-phenylalanine; nitro-tyrosine; ; octahydroin 520. dole carboxylate; ornithine; penicillamine; tetrahydroiso The peptides described herein bear some sequence simi quinoline; acetamidomethyl protected amino acids and pegy larity to ST peptides. However, they include amino acid lated amino acids. Further examples of unnatural amino acids changes and/or additions that improve functionality. These and amino acid analogs can be found in U.S. 20030.108885, 10 changes can, for example, increase or decrease activity (e.g., U.S. 20030082575, US20060019347 (paragraphs 410-418) increase or decrease the ability of the peptide to stimulate and the references cited therein. The peptides described intestinal motility), alter the ability of the peptide to fold herein can include further modifications including those correctly, alter the stability of the peptide, alter the ability of described in US20060019347, paragraph 589. the peptide to bind the GC-C receptor and/or decrease toxic In some embodiments, an amino acid can be replaced by a 15 ity. In some cases the peptides may function more desirably naturally-occurring, non-essential amino acid, e.g., . than wild-type ST peptide. For example, they may limit unde Methods to manufacture peptides containing unnatural sirable side effects such as diarrhea and dehydration. amino acids can be found in, for example, US20030.108885, In some embodiments one or both members of one or more US20030082575, US20060019347, Deiters et al., J. Am pairs of Cys residues (including where a Cys residue has been Chem. Soc. (2003) 125:11782-3, Chin et al., Science (2003) substituted with a D-cys residue) which normally form a 301:964-7, and the references cited therein. disulfide bond can be replaced by homocysteine, penicil Peptides that include non-natural amino acids can also be lamine, 3-mercaptoproline (Kolodzie et al. 1996 Int J Pept prepared using the methods described in WO02086075. Protein Res 48:274); B, B dimethylcysteine (Hunt et al. 1993 The peptides described herein can have one or more con hit J Pept Protein Res 42:249) or diaminopropionic acid ventional peptide bonds replaced by an alternative bond. Such 25 (Smith et al. 1978 J. Med Chem 21:117) to form alternative replacements can increase the stability of the peptide. For internal cross-links at the positions of the normal disulfide example, replacement of the peptide bond between D-Cysis bonds. or Cyss and Xaa, with an alternative bond can reduce cleav In addition, one or more disulfide bonds can be replaced by age by carboxypeptidases and may increase half-life in the alternative covalent cross-links, e.g., an amide linkage digestive tract. Bonds that can replace peptide bonds include: 30 (-CHCH(O)NHCH or -CH-NHCH(O)CH ), an a retro-inverso bonds (C(O)—NH instead of NH CO); a ester linkage, a thioester linkage, a lactambridge, a carbam reduced amide bond (NH CH); a thiomethylene bond oyl linkage, a urea linkage, a thiourea linkage, a phosphonate (S CH or CH S); an oxomethylene bond (O CH or ester linkage, an alkyl linkage (—CH2CH2CH2CH2—), an CH-O); an ethylene bond (CH, CH); a thioamide bond alkenyl linkage( CH-CH=CHCH ), an ether linkage (C(S) NH); a trans-olefine bond (CH=CH); an fluoro sub 35 (—CHCHOCH or —CHOCHCH ), a thioether stituted trans-olefine bond (CF=CH); a ketomethylene bond linkage ( CHCH-SCH or —CHSCHCH ), an (C(O) CHR or CHR C(O) wherein R is H or CH; and a amine linkage ( CHCH-NHCH- O fluoro-ketomethylene bond (C(O) CFR or CFR C(O) —CH-NHCHCH ) or a thioamide linkage (—CHCH(S) wherein R is H or For CH. HNHCH-or-CHNHCH(S)CH, ). For example, Ledu The peptides described herein can be modified using stan 40 et al. (Proc Natl Acad. Sci. 100:11263-78, 2003) describe dard modifications. Modifications may occur at the amino methods for preparing lactam and amide cross-links. Schafi (N—), carboxy (C ) terminus, internally or a combination neister et al. (J. Am. Chem. Soc. 122:5891, 2000) describes of any of the preceeding. In one aspect described herein, there stable, hydrocarbon cross-links. Hydrocarbon cross links can may be more than one type of modification of the peptide. be produced via metathesis (or methathesis followed by Modifications include but are not limited to: acetylation, ami 45 hydrogenation in the case of Saturated hydrocarbons cross dation, biotinylation, cinnamoylation, farnesylation, formy links) using one or another of the Grubbs catalysts (available lation, myristoylation, palmitoylation, phosphorylation (Ser, from Materia, Inc. and Sigma-Aldrich and described, for Tyr or Thr), Stearoylation, Succinylation, Sulfurylation and example, in U.S. Pat. Nos. 5,831,108 and 6,111,121). In some cyclisation (via disulfide bridges or amide cyclisation), and cases, the generation of Such alternative cross-links requires modification by Cy3 or Cy5. The peptides described herein 50 replacing the Cys residues with other residues such as Lys or may also be modified by 2,4-dinitrophenyl (DNP). DNP Glu or non-naturally occurring amino acids. In addition the lysin, modification by 7-Amino-4-methyl-coumarin (AMC), lactam, amide and hydrocarbon cross-links can be used to flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro stabilize the peptide even if they link amino acids at positions anilide, rhodamine B, EDANS (5-((2-aminoethyl)amino) other than those occupied by CyS. Such cross-links can occur naphthalene-1-sulfonic acid), dabcyl, dabsyl, dansyl, Texas 55 between two amino acids that are separated by two amino Red, FMOC, and Tamra (Tetramethylrhodamine). The pep acids or between two amino acids that are separated by six tides described herein may also be conjugated to, for amino acids (see, e.g., Schafineister et al. (J. Am. Chem. Soc. example, polyethylene glycol (PEG); alkyl groups (e.g., 122:5891, 2000)). C1-C20 straight or branched alkyl groups); fatty acid radi The peptide may contain additional carboxyterminal or cals; combinations of PEG, alkyl groups and fatty acid radi 60 amino terminal amino acids or both. For example, the peptide cals (see U.S. Pat. No. 6,309,633; Soltero et al., 2001 Inno can include an amino terminal sequence that facilitates vations in Pharmaceutical Technology 106-110); BSA and recombinant production of the peptide and is cleaved prior to KLH (Keyhole Limpet Hemocyanin). The addition of PEG administration of the peptide to a patient. The peptide can also and other polymers which can be used to modify peptides include other amino terminal or carboxyterminal amino described herein is described in US2006.019347 section IX. 65 acids. In some cases the additional amino acids protect the The peptides and agonists described herein can be chemi peptide, stabilize the peptide or alter the activity of the pep cally modified to increase therapeutic activity by syntheti tide. In some cases some or all of these additional amino acids US 8,779,090 B2 11 12 are removed prior to administration of the peptide to a patient. Described herein is a method for treating congestive heart The peptide can include 1,2,3,4,5,10, 15, 20, 25, 30, 40, 50, failure, the method comprising administering to a patient a 60, 7080, 90, 100 or more amino acids at its amino terminus pharmaceutical composition comprising a purified peptide or carboxy terminus or both. The number of flanking amino comprising, consisting of or consisting essentially of the acids need not be the same. For example, there can be 10 amino acid sequence of SEQID NO:7 (e.g., a peptide in FIG. additional amino acids at the amino terminus of the peptide 3a or FIG. 3b; (SEQ ID NOs: 60-3511 and SEQ ID NOs: and none at the carboxy terminus. 1-6)). The peptide can be administered alone or in combina The peptides can be co-administered with or linked, e.g., tion with another agent for treatment of congestive heart covalently linked to any of a variety of other peptides or failure, for example, a natriuretic peptide Such as atrial natri compounds including analgesic peptides or analgesic com 10 uretic peptide, brain natriuretic peptide or C-type natriuretic pounds including, without limitation, the agents described peptide, an inhibitor of angiotensin converting enzyme, a herein. diuretic (e.g. furesomide (Lasix), bumetanide (Bumex), Amino acid, non-amino acid, peptide and non-peptide ethacrynic acid (Edecrin), torsemide (Demadex), spacers can be interposed between a peptide that is a GC-C (Midamor), spironolactone (Aldactone), chorthiazide (Di receptor agonist and a peptide that has some other biological 15 uril), metolaZone (Zaroxylyn)), an Angiotension-Converting function, e.g., an analgesic peptide or a peptide used to treat Enzyme (ACE) inhibitor (e.g. captopril (Capoten), enalopril obesity. The linker can be one that is cleaved from the flanking (Vasotec), lisinopril (Prinivil, Zestril), ramipril (Altace)), a peptides in Vivo or one that remains linked to the flanking Beta blocker (e.g. carvedilol (Coreg) or an inotropes (e.g. peptides in vivo. For example, , beta-alanine, glycyl digoxin, dobutaimine, dopamine Milrinone). In various glycine, glycyl-beta-alanine, gamma-aminobutyric acid, embodiments the congestive heart failure is categorized as 6-aminocaproic acid, L-phenylalanine, L-tryptophan and Class II congestive heart failure; the congestive heart failure glycil-L-Valil-L-phenylalanine can be used as spacers (Chal is categorized as Class III congestive heart failure; and the tin et al. 2003 Helvetica Chimica Acta 86:533-547; Calicetiet congestive heart failure is categorized as Class W congestive al. 1993 FARMCO 48:919-32) as can polyethylene glycols heart failure. The New York Heart Association (NYHA) func (Butterworth et al. 1987 J. Med. Chem. 30:1295-302) and 25 tional classification system relates congestive heart failure maleimide derivatives (King et al. 2002 Tetrahedron Lett. symptoms to everyday activities and the patient’s quality of 43:1987-1990). Various other linkers are described in the life. The NYHA defines the classes of patient symptoms literature (Nestler 1996 Molecular Diversity 2:35-42: Finnet relating to congestive heart failure as: Class II-slight limita al. 1984 Biochemistry 23:2554-8; Cook et al. 1994 Tetrahe tion of physical activity, comfortable at rest, but ordinary dron Lett. 35:6777-80; Brokx et al. 2002 Journal of Con 30 physical activity results in fatigue, palpitation, or dyspnea; trolled Release 78:115-123; Griffin et al. 2003 J. Am. Chem. Class III-marked limitation of physical activity, comfortable Soc. 125:6517-6531; Robinson et al. 1998 Proc. Natl. Acad. at rest, but less than ordinary activity causes fatigue, palpita Sci. USA 95:5929-5934). Linkers are also described in tion, or dyspnea and Class IV-unable to carry out any physical US20050171014, for example, amino acid linkers such as activity without discomfort, symptoms of cardiac insuffi FALA, VLALA, ALAL, ALALA, 2-cyclohexyl-L-alanine 35 ciency at rest, if any physical activity is undertaken, discom LALA, 2-cyclohexyl-L-alanine-2-cyclohexyl-L-alanine fort is increased. Heart failure treatment using the peptides LAL, 1-naphtyl-alanine-Chal AL and 1-naphtyl-alanine and methods described herein can also be classified according LALA. Peptides and agonists described herein can also be to the ACC/AHA guidelines (Stage A. At risk for developing conjugated to: an affinity tag (such as (histidine 6) H6), a HIV heart failure without evidence of cardiac dysfunction; Stage tat peptide residues 49-57, HIV tat peptide residues 49-56, the 40 B: Evidence of cardiac dysfunction without symptoms; Stage tat sequenceYGRKKRRQRRR (SEQID NO:34), a polyargi C: Evidence of cardiac dysfunction with symptoms; and nine peptide having from 6 to 20 residues (such as R6) and the Stage D: Symptoms of heart failure despite maximal following peptide sequences: YARKARROARR (SEQ ID therapy). NO:35), YARAAARQARA (SEQ ID NO:36), YARAAR Described herein is a method for treating a gastrointestinal RAARR (SEQ ID NO:37) YARAARRAARA (SEQ ID 45 disorder, the method comprising administering a peptide NO:38), ARRRRRRRRR (SEQ ID NO:39), and YAAAR comprising, consisting essentially of or consisting of the RRRRRR (SEQ ID NO:40), which are disclosed in WO amino acid sequence of SEQID NO:7, e.g., a peptide in FIG. 99/29721 and in U.S. Pat. No. 6,221,355. 3a or FIG.3b (SEQID NOs: 60-3511 and SEQID NOs: 1-6)). The peptides described herein can be attached to one, two In various embodiments: the patient is suffering from a dis or more different moieties each providing the same or differ 50 order selected from the group consisting of gastrointestinal ent functions. For example, the peptide can be linked to a motility disorders, chronic intestinal pseudo-obstruction, molecule that is an analgesic and to a peptide that is used to colonic pseudo-obstruction, Crohn's disease, duodenogastric treat obesity. The peptide and various moieties can be ordered reflux, dyspepsia, functional dyspepsia, nonulcer dyspepsia, in various ways. For example, a peptide described herein can a functional gastrointestinal disorder, functional heartburn, have an analgesic peptide linked to its amino terminus and an 55 gastroesophageal reflux disease (GERD), gastroparesis, irri anti-obesity peptide linked to its carboxy terminus. The addi table bowel syndrome, post-operative ileus, ulcerative colitis, tional moieties can be directly covalently bonded to the pep chronic constipation, and disorders and conditions associated tide or can be bonded via linkers. with constipation (e.g. constipation associated with use of The peptides described herein can be a cyclic peptide or a opiate pain killers, post-Surgical constipation, and constipa linear peptide. In addition, multiple copies of the same pep 60 tion associated with neuropathic disorders as well as other tide can be incorporated into a single cyclic or linear peptide. conditions and disorders are described herein); the patient is The peptides can include the amino acid sequence of a Suffering from a gastrointestinal motility disorder, chronic peptide that occurs naturally in a vertebrate (e.g., mamma intestinal pseudo-obstruction, colonic pseudo-obstruction, lian) species or in a bacterial species. In addition, the peptides Crohn's disease, duodenogastric reflux, dyspepsia, func can be partially or completely non-naturally occurring pep 65 tional dyspepsia, nonulcer dyspepsia, a functional gas tides. Also within the disclosure are peptidomimetics corre trointestinal disorder, functional heartburn, gastroesophageal sponding to the peptides described herein. reflux disease (GERD), gastroparesis, inflammatory bowel US 8,779,090 B2 13 14 disease, irritable bowel syndrome (e.g. d-IBS, c-IBS, and/or the amino acid sequence of SEQID NO:7, e.g., a peptide in a-IBS), post-operative ileus, ulcerative colitis, chronic con FIG.3a or FIG.3b (SEQID NOs: 60-3511 and SEQID NOs: stipation, and disorders and conditions associated with con 1-6). stipation (e.g. constipation associated with use of opiate pain Also described is a method for treating benign prostatic killers, post-Surgical constipation, and constipation associ 5 hyperplasia, the method comprising administering to a ated with neuropathic disorders as well as other conditions patient a pharmaceutical composition comprising a purified and disorders are described herein); the patient has been peptide comprising, consisting of or consisting essentially of diagnosed with a functional gastrointestinal disorder accord the amino acid sequence of SEQID NO:7, e.g., a peptide in ing to the Rome Criteria (e.g. Rome II), the patient has been FIG.3a or FIG.3b (SEQID NOs: 60-3511 and SEQID NOs: 10 1-6). The peptide can be administered alone or in combina diagnosed with irritable bowel syndrome (e.g. (e.g. diarrhea tion with another agent for treatment of BPH, for example, a predominant-IBS, constipation predominant-IBS, and/or 5-alpha reductase inhibitor (e.g., finasteride) or an alpha adr alternating-IBS), according to the Rome Criteria (e.g. Rome energic inhibitor (e.g., doxazosine). II); the composition is administered orally; the peptide com Also described is a method for treating or reducing pain, prises 30 or fewer amino acids, the peptide comprises 20 or 15 including visceral pain, pain associated with a gastrointesti fewer amino acids, the peptide comprises no more than 5 nal disorder or pain associated with some other disorder, the amino acids prior to Cys, the peptide comprises 14 amino method comprising administering to a patient a pharmaceu acids, the peptide comprises 13 amino acids; the peptide tical composition comprising or consisting essentially of a comprises 150, 140,130, 120, 110, 100,90,80, 70, 60, 50, 40, purified peptide comprising, consisting of or consisting or 30 or fewer amino acids. In other embodiments, the peptide essentially of the amino acid sequence of SEQID NO:7, e.g., comprises 20 or feweramino acids. In other embodiments the a peptide in FIG. 3a or FIG. 3b (SEQ ID NOs: 60-3511 and peptide comprises no more than 20, 15, 10, or 5 peptides SEQID NOs: 1-6). Subsequent to Cyss. In certain embodiments Xaao is a chy Also described is a method for treating inflammation, motrypsin or trypsin cleavage site and an analgesic peptide is including inflammation of the gastrointestinal tract, e.g., present immediately following Xaao. 25 inflammation associated with a gastrointestinal disorder or Described herein is a method for treating a patient suffering infection or some other disorder, the method comprising from constipation. Clinically accepted criteria that define administering to a patient a pharmaceutical composition constipation include the frequency of bowel movements, the comprising a purified peptide comprising, consisting of or consistency of feces and the ease of bowel movement. One consisting essentially of the amino acid sequence of SEQID common definition of constipation is less than three bowel 30 NO: 7, e.g., a peptide in FIG. 3a or FIG. 3b (SEQ ID NOs: 60-3511 and SEQID NOs: 1-6). movements per week. Other definitions include abnormally In prevention and/or treatment disorders associated with hard stools or defecation that requires excessive straining fluid and Sodium retention (e.g., heart failure, congestive (Schiller 2001, Aliment Pharmacol Ther 15:749-763). Con heart failure, kidney disease, etc), the agents described herein stipation may be idiopathic (functional constipation or slow 35 can be administered, for example, via a parenteral route, transit constipation) or secondary to other causes including intravenously, and/or Subcutaneously. Intravenous adminis neurologic, metabolic or endocrine disorders. These disor tration may comprise, for example, (1) one or more succes ders include diabetes mellitus, hypothyroidism, hyperthy sive rounds of a bolus followed by an infusion or an infusion roidism, hypocalcaemia, Multiple Sclerosis, Parkinson's dis followed by a bolus, (2) infusion, and (3) bolus administra ease, spinal cord lesions, Neurofibromatosis, autonomic 40 tion. The dosage may vary depending on the administration neuropathy, Chagas disease, Hirschsprung's disease and Cys schedule. Thus, bolus administrations may involve dosing tic fibrosis. Constipation may also be the result of Surgery from about 0.1-1000 ug/kg, from about 1-100 ug/kg, or from (postoperative ileus) or due to the use of drugs such as anal about 10, 15, 20, 25, or 30 ug/kg. Infusion administrations gesics (like ), antihypertensives, anticonvulsants, may involve dosing from about 0.1-1000 ug/kg/hour, from antidepressants, antispasmodics and antipsychotics. Also dis 45 about 1-100 ug/kg/hour, about 10 ug/kg/hour. During the closed are methods for increasing gastrointestinal motility in duration of an infusion administration, the dosage may vary a patient, the method comprising administering to a patient a (for example, decreasing overtime, increasing over time, and pharmaceutical composition comprising a purified peptide combinations thereof) or may remain constant. Subcutaneous comprising, consisting of or consisting essentially of the administration may involve dosing from about 0.1-1000 amino acid sequence of SEQID NO:7, e.g., a peptide in FIG. 50 ug/kg, from about 1-100 ug/kg, from about 10, 15, 20, 25, or 3a or FIG.3b (SEQID NOs: 60-3511 and SEQID NOs: 1-6). 30 ug/kg. Also disclosed are methods for increasing the activity of As noted above, isolated nucleic acid molecules compris (activating) an intestinal guanylate cyclase (GC-C) receptor ing a sequence encoding a peptide described herein are in a patient, the method comprising administering to a patient described. Also described are vectors, e.g., expression vectors a pharmaceutical composition comprising a purified peptide 55 that include Such nucleic acid molecules and can be used to comprising, consisting of or consisting essentially of the express a peptide described herein in a cultured cell (e.g., a amino acid sequence of SEQID NO:7, e.g., a peptide in FIG. eukaryotice cell or a prokaryotic cell). The vector can further 3a or FIG.3b (SEQID NOs: 60-3511 and SEQID NOs: 1-6). include one or more regulatory elements, e.g., a heterologous Also disclosed is an isolated nucleic acid molecule com promoter or elements required for translation operably linked prising a nucleotide sequence encoding a peptide comprising, 60 to the sequence encoding the peptide. In some cases the consisting of or consisting essentially of SEQID NO:7, e.g., nucleic acid molecule will encode an amino acid sequence a peptide in FIG. 3a or FIG. 3b (SEQ ID NOs: 60-3511 and that includes the amino acid sequence of a peptide described SEQID NOs: 1-6). herein. For example, the nucleic acid molecule can encode a Also described is a method for treating obesity, the method preprotein or a preproprotein that can be processed to produce comprising administering to a patient a pharmaceutical com 65 a peptide described herein. In cases where unnatural amino position comprising or consisting essentially of a purified acids are present in the peptides described herein, selector peptide comprising, consisting of or consisting essentially of codons can be utilized in the synthesis of Such peptides simi US 8,779,090 B2 15 16 lar to that described in US20060019347 (for example, para tical composition comprising, consisting essentially of, or graphs 398-408,457-499, and 576-588) herein incorporated consisting of a peptide or agonist described herein and a by reference. pharmaceutically acceptable carrier. The composition can be A vector that includes a nucleotide sequence encoding a administered in combination with another agent for treatment peptide described herein or a peptide or peptide comprising a of hypertension, for example, a diuretic, an ACE inhibitor, an peptide described herein may be either RNA or DNA, single angiotensin receptor blocker, a beta-blocker, or a calcium or double-stranded, prokaryotic, eukaryotic, or viral. Vectors channel blocker. can include transposons, viral vectors, episomes, (e.g., plas Also described is a method for treating secondary hyperg mids), chromosomes inserts, and artificial chromosomes (e.g. BACs orYACs). Suitable bacterial hosts for expression of the 10 lycemias in connection with pancreatic diseases (chronic encode peptide or peptide include, but are not limited to, E. pancreatitis, pancreasectomy, hemochromatosis) or endo coli. Suitable eukaryotic hosts include yeast Such as S. Cer crine diseases (acromegaly, Cushing's syndrome, pheochro evisiae, other fungi, Vertebrate cells, invertebrate cells (e.g., mocytoma or hyperthyreosis), drug-induced hyperglycemias insect cells), plant cells, human cells, human tissue cells, and (benzothiadiazine saluretics, diazoxide or glucocorticoids), whole eukaryotic organisms. (e.g., a transgenic plant or a 15 pathologic glucose tolerance, hyperglycemias, dyslipopro transgenic animal). Further, the vector nucleic acid can be teinemias, adiposity, hyperlipoproteinemias and/or hypoten used to transfect a virus Such as vaccinia or baculovirus (for sions is described. The method comprises: administering to example using the Bac-to-Bac R. Baculovirus expression sys the patient a pharmaceutical composition comprising, con tem (Invitrogen Life Technologies, Carlsbad, Calif.)). sisting essentially of, or consisting of a peptide or agonist As noted above the disclosure includes vectors and genetic described herein and a pharmaceutically acceptable carrier. constructs suitable for production of a peptide described The peptides described herein can be present with a coun herein or a peptide or peptide comprising Such a peptide. terion. Useful counterions include salts of acetate, benzene Generally, the genetic construct also includes, in addition to Sulfonate, benzoate, calcium edetate, camsylate, carbonate, the encoding nucleic acid molecule, elements that allow 25 citrate, edetate (EDTA), edisylate, embonate, esylate, fuma expression, such as a promoter and regulatory sequences. The rate, gluceptate, gluconate, glutamate, glycolylarsanilate, expression vectors may contain transcriptional control hexylresorcinate, iodide, , chloride, hydroxynaph sequences that control transcriptional initiation, Such as pro thoate, isethionate, lactate, lactobionate, estolate, maleate, moter, enhancer, operator, and repressor sequences. A variety malate, mandelate, mesylate, mucate, napsylate, nitrate, pan of transcriptional control sequences are well known to those 30 tothenate, phosphate, Salicylate, Stearate. Succinate, Sulfate, in the art and may be functional in, but are not limited to, a tartarate, tartrate, hydrochlorate, theoclate, acetamidoben bacterium, yeast, plant, or animal cell. The expression vector Zoate, adipate, alginate, aminosalicylate, anhydromethyl can also include a translation regulatory sequence (e.g., an enecitrate, ascorbate, aspartate, camphorate, caprate, untranslated 5' sequence, an untranslated 3' sequence, a poly caproate, caprylate, cinnamate, cyclamate, dichloroacetate, A addition site, or an internal ribosome entry site), a splicing 35 formate, gentisate, glucuronate, glycerophosphate, glycolate, sequence or splicing regulatory sequence, and a transcription hippurate, fluoride, malonate, napadisylate, nicotinate, ole termination sequence. The vector can be capable of autono ate, orotate, oxalate, oxoglutarate, palmitate, pectinate, pec mous replication or it can integrate into host DNA. tinate polymer, phenylethylbarbiturate, picrate, propionate, Also described are isolated host cells harboring one of the 40 pidolate, sebacate, rhodanide, tosylate, and tannate. forgoing nucleic acid molecules and methods for producing a Also described are methods for producing any of the for peptide by culturing Such a cell and recovering the peptide or going peptides comprising providing a cell harboring a a precursor of the peptide. Recovery of the peptide or precur nucleic acid molecule encoding the peptide, culturing the cell Sor may refer to collecting the growth solution and need not under conditions in which the peptide is expressed, and iso involve additional steps of purification. Proteins of the 45 lating the expressed peptide. present disclosure, however, can be purified using standard Also described are methods for producing any of the for purification techniques, such as, but not limited to, affinity going peptides comprising chemically synthesizing the pep chromatography, thermaprecipitation, immunoaffinity chro tide and then purifying the synthesized peptide. Also matography, ammonium sulfate precipitation, ion exchange described are pharmaceutical compositions comprising the chromatography, filtration, electrophoresis and hydrophobic 50 forgoing peptides. Also described are nucleic acid molecules interaction chromatography. encoding any of the forgoing peptides, vectors (e.g., expres The peptides can be purified. Purified peptides are peptides sion vectors) containing Such nucleic acid molecules and host separated from other proteins, lipids, and nucleic acids or cells harboring the nucleic acid molecules or vectors. from the compounds from which is it synthesized. The pep Metabolites of Asparagine tide can constitute at least 10, 20, 5070, 80 or 95% by dry 55 In some cases an asparagine (Asn) of a peptide described weight of the purified preparation. hereincan be metabolized to have a different structure and the Also described is a method of increasing the level of cyclic GC receptoragonist containing Such a metabolite of ASn may guanosine 3'-monophosphate (cGMP) in an organ, tissue retain activity. Peptides where one or more ASn, e.g., one or (e.g., the intestinal mucosa), or cell (e.g., a cell bearing GC-A 60 more ASn of an embodiment of SEQID NO:7, e.g., a peptide receptor) by administering to a patient a composition com in FIG. 3a or FIG. 3b (SEQID NOs: 60-3511 and SEQ ID prising or consisting essentially of a purified peptide com NOs: 1-6) described herein are replaced by a metabolite of prising, consisting of or consisting essentially of the amino Asn can be useful in the methods described herein and can be acid sequence of SEQID NO:7 (e.g., a peptide in FIG. 3a or present in a pharmaceutical composition that optionally con FIG. 3b (SEQID NOs: 60-3511 and SEQID NOs: 1-6)). 65 tains one or more additional active ingredients. Also described is a method for treating hypertension. The For example, one or more ASn of a peptide and the peptide method comprises: administering to the patient a pharmaceu bond carboxy terminal thereto having the structure: US 8,779,090 B2 17 18 -continued O or iso ASn:

HN 5 NH HN A-C1-x O 10 The Asp can be L-Asp or D-Asp. The iso Asn can be can replaced by a group having a structure selected from: D-isoASn or L-isoASn. Considering the asparagine only, one or more ASn having the structure: 15 y N 1.

25 can be optionally replaced by a group having a structure selected from (a), (b) and (c):

30 (a) O

35 y HO N 1. i O

AC-XO 40 (b)

Thus, the Asn and the peptide bond carboxy terminal there HO to can be replaced by a cyclic imide: 45 X,H

50

(c) y N HO O

th 55 x,H Asp:

60 provided that an ASn at the carboxy terminus is not replaced by structure (a) or structure (c). When the ASn is at the car boxy terminus of the peptide, structure (a) cannot form. Since structure (c) is formed through structure (a), structure (c) 65 cannot be formed when the ASn is at the carboxy terminus. The formation of the various metabolites of Asp is depicted below. US 8,779,090 B2 20

O

O O R Fragments N OH H2N O

O

R

O direct hydrolysis: pH 1-2 O D.L HN R Aspartyl peptide

N N

O cyclicCs imide formation: O slow at acidic pH R maximum stability at pH 3-4 more prevalent at pH 7-11; Subject to buffer catalysis N

O

D.L cyclic imide

R O

O

OH N : I L-iso Aspartyl peptide

50 The details of one or more embodiments described herein DETAILED DESCRIPTION are set forth in the accompanying description. All of the publications, patents and patent applications are hereby The peptides described herein bind to the intestinal guany incorporated by reference. late cyclase (GC-C) receptor, a regulator of fluid and electro 55 lyte balance. The apical membrane of the intestinal epithelial FIGURES Surface is a major site of GC-C receptor expression. Activa tion of the GC-C receptor in the intestine leads to an increase FIG. 1 depicts the results of studies on certain peptides in intestinal epithelial cyclic GMP (cGMP). This increase in tested in the intestinal GC-C receptor activity assay. 60 cGMP is believed to cause a decrease in water and sodium FIG. 2 depicts the results of studies on certain peptides absorption and an increase in chloride and potassium ion tested in the rat diuresis assay. secretion, leading to changes in intestinal fluid and electrolyte FIGS. 3a and 3b depict certain peptides within SEQ ID transport and increased intestinal motility. The intestinal NO:7. GC-C receptor possesses an extracellular ligand binding FIG. 4 depicts various pre, pro, N-terminal non-core, and 65 region, a transmembrane region, an intracellular protein C-terminal non-core sequences that can be included in a kinase-like region and a cyclase catalytic domain. Proposed peptide comprising SEQID NO:7. functions for the GC-C receptor are fluid and electrolyte US 8,779,090 B2 21 22 homeostasis, the regulation of epithelial cell proliferation and acid sequence Ser Asp Trp Cys CysGluVal Cys Cys Asn Pro the induction of apoptosis (Shalubhai 2002 Curr Opin Drug Ala Cys Ala Gly Cys(SEQID NO:16); V cholerae non-01 ST Dis Devel 5:261-268). peptide (Takao et al. (1985) FEBS lett. 193:250) having the In addition to being expressed in the intestine by gas mature amino acid sequence Ile Asp CyS CyS Glu Ile CyS Cys trointestinal epithelial cells, GC-C is expressed in extra-in ASn Pro Ala Cys Phe Gly Cys Leu Asn (SEQID NO:17); and testinal tissues including kidney, lung, pancreas, pituitary, V mimicus ST peptide (Arita et al. 1991 FEMS Microbiol. adrenal, developing liver and gallbladder (reviewed in Vaan Lett. 79:105) having the mature amino acid sequence Ile Asp drager 2002 Mol Cell Biochem 230:73-83, Kulaksiz et al. Cys CysGlu Ile Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu 2004, Gastroenterology 126:732-740) and male and female Asn (SEQID NO:18). reproductive tissues (reviewed in Vaandrager 2002 Mol Cell 10 The immature (including pre and pro regions) form of E. Biochem 230:73-83). This suggests that the GC-C receptor coli ST-1A (ST-P) protein has the sequence: mkklmlaifisvls agonists can be used in the treatment of disorders outside the fpsfsqstesldsskekitletkkcdv GI tract, for example, congestive heart failure and benign vknnsekksenmnntfy.ccelccnpacagcy (SEQ ID NO:19; see prostatic hyperplasia. GenBank R. Accession No. PO1559 (gi:123711). The pre In humans, the GC-C receptor is activated by guanylin 15 sequence extends from aa 1-19. The pro sequence extends (Gn) (U.S. Pat. No. 5,969,097), uroguanylin (Ugn) (U.S. Pat. from aa 20-54. The mature protein extends from 55-72. The No. 5,140,102) and lymphoguanylin (Forte et al. 1999 Endo immature (including pre and pro regions) form of E. coli crinology 140:1800-1806). Interestingly, these agents are ST-1B (ST-H) protein has the sequence: mkksilfiflsvlsfsp 10-100 fold less potent than a class of bacterially derived faqdakpVesskekitleskkc peptides, termed ST (reviewed in Gianella 1995 J Lab Clin niakkSnksgpesmns.Snyccelccnpactgcy (SEQ ID NO:20; see Med 125:173-181). ST peptides are considered super ago GenBank R. Accession No. P07965 (gi:3915589)). The imma nists of GC-C and are very resistant to proteolytic degrada ture (including pre and pro regions) form of Y. enterocolitica tion. ST protein has the sequence: mkkivfvlvmlssfgafgqetv.sgqfs ST peptide is capable of stimulating the enteric nervous dalstpitaevykdacdpplppaevssawdccdvccnpacagc (SEQ ID system (Rolfe et al., 1994, J Physiolo 475: 531-537; Rolfe et 25 NO:21; see GenBank R. Accession No. 525659 (gi:282047)). al. 1999 Gut 44:615-619; Nzegwu et al. 1996Exp Physiol81: The peptides described herein, e.g., a peptide comprising 313-315). Also, c0MP has been reported to have antinocice SEQID NO:7 (e.g., a peptide of FIG.3a or FIG.3b (SEQID ptive effects in multiple animal models of pain (Lazaro Ibanez NOs: 60-3511 and SEQID NOs: 1-6)) can include all or part et al. 2001 Eur J Pharmacol 426: 39-44: Soares et al. 2001 of such pre and/or pro sequences. British J Pharmacol 134: 127-131; Jain et al. 2001 Brain Res 30 FIG. 4 depicts various pre, pro, N-terminal non-core, and 909: 170-178: Amarante et al. 2002 EurJ Pharmacol 454:19 C-terminal non-core sequences that can be included in a 23). Thus, GC-C agonists may have both an analgesic as well peptide comprising SEQID NO:7 (e.g., a peptide of FIG.3a an anti-inflammatory effect. or FIG. 3b (SEQID NOs: 60-3511 and SEQID NOs: 1-6)). In bacteria, ST peptides are derived from a preproprotein Thus, a peptide can include the amino acid sequence: that generally has at least 70 amino acids. The pre and pro 35 A-B-C-D'-E' wherein: regions are cleaved as part of the secretion process, and the A' is an amino acid sequence comprising a pre sequence resulting mature protein, which generally includes fewer than depicted in FIG. 4 or is missing: 20 amino acids, is biologically active. B' is an amino acid sequence comprising a pro sequence Among the known bacterial ST peptides are: E. coli ST Ib depicted in FIG. 4 or is missing: (Moseley et al. 1983 Infect. Immun. 39:1167) having the 40 C" is an amino acid sequence comprising an N-terminal mature amino acid sequence Asn Ser Ser Asn Tyr Cys CysGlu non-core sequence depicted in FIG. 4 or is missing: Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID D' is an amino acid sequence comprising a GC-C receptor NO:8); E. coli ST la (So and McCarthy 1980 Proc. Natl. agonist peptide amino acid sequence (e.g., SEQ ID Acad. Sci. USA 77:4011) having the mature amino acid NO:7, a peptide in FIG.3a or a peptide in FIG.3b (SEQ sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys ASn Pro 45 ID NOs: 60-3511 and SEQID NOs: 1-6)); and Ala Cys Ala Gly Cys Tyr (SEQIDNO:9). E. coli ST I* (Chan E" is an amino acid sequence comprising a C-terminal and Giannella 1981 J. Biol. Chem. 256: 7744) having the non-core sequence depicted in FIG. 4 or is missing: mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu The peptides described herein, like the bacterial ST pep Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys Asn (SEQ ID tides, have six Cys (or D-Cys) residues. These six residues NO:10): C. freundii ST peptide (Guarino et al. 1989b Infect. 50 form three disulfidebonds in themature and active form of the Immun. 57:649) having the mature amino acid sequence ASn peptide. If the six Cys (or D-Cys) residues are identified, from Thr Phe Tyr Cys Cys Glu Leu Cys Cys ASn Pro Ala Cys Ala the amino to carboxy terminus of the peptide, as A, B, C, D, Gly Cys Tyr (SEQID NO:11); Y enterocolitica ST peptides, E, and F, then the disulfide bonds form as follows: A-D, B-E, Y-ST(Y-STa), Y-STb, and Y-STc (reviewed in Huang et al. and C F. The formation of these bonds is thought to be 1997 Microb. Pathog. 22:89) having the following pro-form 55 important for GC-C receptor binding. amino acid sequences: Gln Ala Cys Asp Pro Pro Ser Pro Pro Certain of the peptides described herein may include anal Ala Glu Val Ser Ser Asp Trp Asp Cys Cys Asp Val Cys Cys gesic or antinociceptive tags such as the carboxy-terminal ASn Pro Ala Cys Ala Gly Cys (SEQID NO:12) (as well as a sequence AspPhe immediately following a Trp, Tyr or Phe Ser-7 to Leu-7 variant of Y-STa (SEQIDNO:13), (Takao et al. that creates a functional chymotrypsin cleavage site or fol 1985 Eur. J. Biochem. 152:199)); Lys Ala Cys Asp Thr Gln 60 lowing Lys or Arg that creates a functional trypsin cleavage ThrPro SerPro Ser Glu Glu Asn Asp-Asp Trp Cys CysGluVal site. Chymotrypsin in the intestinal tract can potentially Cys Cys ASn Pro Ala Cys Ala Gly Cys (SEQID NO:14); Gln cleave such peptides immediately carboxy terminal to the Glu Thr Ala Ser Gly Gln Val Gly Asp Val Ser Ser Ser Thr Ile Trp, Phe or Tyr residue, releasing the dipeptide, AspPhe. This Ala Thr GluVal Ser Glu Ala Glu Cys GlyThr Gln Ser Ala Thr dipeptide has been shown to have analgesic activity in animal Thr Gln Gly Glu Asn Asp Trp Asp Trp Cys Cys Glu Leu Cys 65 models (Abdikkahi et al. 2001 Fundam Clin Pharmacol Cys ASn Pro Ala Cys Phe Gly Cys (SEQID NO:15), respec 15:117-23; Nikfar et al 1997, 29:583-6; Edmundson et al tively; Y kristensenii ST peptide having the mature amino 1998 Clin Pharmacol Ther 63:580-93). In this manner such US 8,779,090 B2 23 24 peptides can treat both pain and inflammation. Other analge Production of Peptides sic peptides can be present at the amino or carboxy terminus Useful peptides can be produced either in bacteria includ of the peptide (e.g., following a functional cleavage site) ing, without limitation, E. coli, or in other existing systems for including: endomorphin-1, endomorphin-2, nocistatin, dalar peptide or protein production (e.g., Bacillus subtilis, bacu gin, lupron, and Substance P. 5 lovirus expression systems using Drosophila Sf9 cells, yeast In some cases, the peptides described herein are produced or filamentous fungal expression systems, mammalian cell as a prepro protein that includes the amino terminal leader expression systems), or they can be chemically synthesized. Sequence: mkksilfiflsvlsfspfaqdak If the peptide or variant peptide is to be produced in bac pvesskekitleskkcniakkSnksgpesmn (SEQID NO:41). Where teria, e.g., E. coli, the nucleic acid molecule encoding the 10 peptide will preferably also encode a leader sequence that the peptide is produced by a bacterial cell, e.g., E. coli, the permits the secretion of the mature peptide from the cell. forgoing leader sequence will be cleaved and the mature Thus, the sequence encoding the peptide can include the pre peptide will be efficiently secreted from the bacterial cell. sequence and the pro sequence of for example, a naturally U.S. Pat. No. 5,395,490 describes vectors, expression sys occurring bacterial ST peptide. The secreted, mature peptide tems and methods for the efficient production of ST peptides 15 can be purified from the culture medium. in bacterial cells and methods for achieving efficient secretion The sequence encoding a peptide described herein is pref of mature ST peptides. The vectors, expression systems and erably inserted into a vector capable of delivering and main methods described in U.S. Pat. No. 5,395,490 can be used to taining the nucleic acid molecule in a bacterial cell. The DNA produce the ST peptides and variant ST peptides of the molecule may be inserted into an autonomously replicating present disclosure vector (suitable vectors include, for example, pGEM37 and Variant Peptides pcDNA3, and derivatives thereof). The vector nucleic acid The disclosure includes variant peptides which can include may be a bacterial or bacteriophage DNA such as bacterioph one, two, three, four, five, six, seven, eight, nine, or ten (in age lambda or M13 and derivatives thereof. Construction of a some embodiments fewer than 5 or fewer than 3 or 2 or fewer) vector containing a nucleic acid described herein can be fol amino acid Substitutions and/or deletions compared to the 25 lowed by transformation of a host cell such as a bacterium. sequences of SEQ ID NO:7 (e.g., a sequence in FIG. 3a or Suitable bacterial hosts include but are not limited to, E. coli, FIG.3b (SEQID NOs: 60-3511 and SEQID NOs: 1-6)) The B. subtilis, Pseudomonas, Salmonella. The genetic construct Substitution(s) can be conservative or non-conservative. The also includes, in addition to the encoding nucleic acid mol naturally-occurring amino acids can be substituted by D-iso ecule, elements that allow expression, such as a promoter and mers of any amino acid, non-natural amino acids, natural and 30 regulatory sequences. The expression vectors may contain natural amino acid analogs and other groups. A conservative transcriptional control sequences that control transcriptional amino acid substitution results in the alteration of an amino initiation, such as promoter, enhancer, operator, and repressor acid for a similar acting amino acid, or amino acid of like sequences. A variety of transcriptional control sequences are charge, polarity, or hydrophobicity. At some positions, even well known to those in the art. The expression vector can also conservative amino acid substitutions can alter the activity of 35 include a translation regulatory sequence (e.g., an untrans the peptide. A conservative Substitution can Substitute a natu lated 5' sequence, an untranslated 3' sequence, or an internal rally-occurring amino acid for a non-naturally-occurring ribosome entry site). The vector can be capable of autono amino acid. The amino acid Substitutions among naturally mous replication or it can integrate into host DNA to ensure occurring amino acids are listed in Table II. stability during peptide production. 40 The protein coding sequence that includes a peptide TABLE II described herein can also be fused to a nucleic acid encoding a peptide affinity tag, e.g., glutathione S-transferase (GST), For Amino Acid Code Replace with any of maltose E binding protein, protein A, FLAG tag, hexa-histi Alanine Ala Gly, Cys, Ser dine, myc tag or the influenza HA tag, in order to facilitate Arginine Arg Lys, His 45 purification. The affinity tag or reporter fusion joins the read Asparagine ASn Asp, Glu, Gln, ing frame of the peptide of interest to the reading frame of the Aspartic Acid Asp ASn, Glu, Gln gene encoding the affinity tag such that a translational fusion Cysteine Cys Met, Thr, Ser Glutamine Gln ASn, Glu, Asp is generated. Expression of the fusion gene results in transla Glutamic Acid Glu Asp, ASn, Gln tion of a single peptide that includes both the peptide of Glycine Gly Ala 50 interest and the affinity tag. In some instances where affinity Histidine His Lys, Arg tags are utilized, DNA sequence encoding a protease recog Isoleucine Ile Val, Leu, Met Leucine Leu Val, Ile, Met nition site will be fused between the reading frames for the Lysine Lys Arg, His affinity tag and the peptide of interest. Methionine Met Ile, Leu, Val Genetic constructs and methods suitable for production of Phenylalanine Phe Tyr, His, Trp 55 Proline Pro immature and mature forms of the peptides and variants Serine Ser Thr, Cys, Ala described herein in protein expression systems other than Threonine Thr Ser, Met, Val bacteria, and well knownto those skilled in the art, can also be Tryptophan Trp Phe, Tyr used to produce peptides in a biological system. Tyrosine Tyr Phe, His Mature peptides and variants thereof can be synthesized by Wall Leu, Ile, Met 60 the Solid-phase chemical synthesis. For example, the peptide can be synthesized on Cyc(4-CH.Bxl)-OCH-4-(oxym In some circumstances it can be desirable to treat patients ethyl)-phenylacetamidomethyl resin using a double coupling with a variant peptide that binds to and activates intestinal program. Protecting groups must be used appropriately to GC-C receptor, but is less active than the non-variant form the create the correct disulfide bond pattern. For example, the peptide. This reduced activity can arise from reduced affinity 65 following protecting groups can be used: t-butyloxycarbonyl for the receptor or a reduced ability to activate the receptor (alpha-amino groups); acetamidomethyl (thiol groups of Cys once bound or reduced stability of the peptide. residues B and E): 4-methylbenzyl (thiol groups of Cys resi US 8,779,090 B2 25 26 dues C and F); benzyl (y-carboxyl of glutamic acid and the Cells are collected by centrifugation at 5,000 g for 10 hydroxyl group of threonine, if present); and bromobenzyl minutes. The cell pellet is discarded and the Supernatant is (phenolic group of tyrosine, if present). Coupling is effected passed through a 50 Kd ultrafiltration unit. The 50 Kd filtrate with symmetrical anhydride oft-butoxylcarbonylamino acids (0.6 liters) is loaded onto a 110 ml Q-Sepharose fast Flow or hydroxybenzotriazole ester (for asparagine or glutamine column (Amersham Pharmacia, equilibrated with 20 mM residues), and the peptide is deprotected and cleaved from the Tris-HCl pH 7.5) at a flow rate of 400 ml/hour. The column is Solid Support in hydrogen fluoride, dimethyl sulfide, anisole, washed with six volumes of 20 mM Tris-HCl pH 7.5 and and p-thiocresol using 8/1/1/0.5 ratio (v/v/v/w) at 0°C. for 60 proteins are eluted with 50 mMacetic acid collecting 50 ml min. After removal of hydrogen fluoride and dimethylsulfide fractions. Fractions containing peptide are pooled and the by reduced pressure and anisole and p-thiocresol by extrac 10 solvent is removed by rotary evaporation. The dried proteins tion with ethyl ether and ethyl acetate sequentially, crude are resuspended in 10 ml of 8% acetic acid, 0.1% trifluoro peptides are extracted with a mixture of 0.5M sodium phos acetic acid (TFA) and loaded onto a Varian Polaris C18-A phate buffer, pH 8.0 and N,N-dimethylformamide using 1/1 column (250x21.2 mm 10 TM, equilibrated in the same buffer) at a flow rate of 20 ml/min. The column is washed with 100 ml ratio, V/v. The disulfide bond for Cys residues B and E is the 15 of 8% methanol, 0.1% TFA and developed with a gradient formed using dimethyl sulfoxide (Tam et al. (1991) J. Am. (300 ml) of 24 to 48% methanol, 0.1% TFA, collecting 5-ml Chem. Soc. 113:6657-62). The resulting peptide is the puri fractions. Fractions containing peptide are pooled and the fied by reverse-phase chromatography. The disulfide bond solvent is removed by rotary evaporation. The peptides are between Cys residues Cand F is formed by first dissolving the dissolved in 0.1% TFA and lyophilized. peptide in 50% acetic acid in water. Saturated iodine solution Peptide fractions are analyzed by standard LCMS and in glacial acetic acid is added (1 ml iodine solution per 100 ml HPLC. Peptides can also be chemically synthesized by a Solution). After incubation at room temperature for 2 days in commercial peptide synthesis company. an enclosed glass container, the solution is diluted five-fold with deionized water and extracted with ethyl ether four times Example 2 for removal of unreacted iodine. After removal of the residual 25 amount of ethyl ether by rotary evaporation the solution of Activation of the Intestinal GC-C Receptor by crude product is lyophilized and purified by successive Peptides reverse-phase chromatography. Peptides can also be synthesized by many other methods The ability of peptides to activate the intestinal GC-C including Solid phase synthesis using traditional FMOC pro 30 receptor was assessed in an assay employing the T84 human tection (i.e., coupling with DCC-HOBt and deprotection with colon carcinoma cell line (AmericanType Culture Collection piperidine in DMF). Cys thiol groups can be trityl protected. (Bethesda, Md.)). For the assays cells were grown to conflu Treatment with TFA can be used for final deprotection of the ency in 24-well culture plates with a 1:1 mixture of Ham's peptide and release of the peptide from the solid-state resin. In F12 medium and Dulbecco's modified Eagle's medium many cases air oxidation is sufficient to achieve proper dis 35 (DMEM), supplemented with 5% fetal calf serum and were ulfide bond formation. used at between passages 54 and 60. Briefly, monolayers of T84 cells in 24-well plates were Example 1 washed twice with 1 ml/well DMEM, then incubated at 37°C. for 10 min with 0.45 ml DMEM containing 1 mM isobutyl Preparation of Peptides 40 methylxanthine (IBMX), a cyclic nucleotide phosphodi esterase inhibitor. Test peptides (500) were then added and Peptides can be recombinantly produced in bacteria as incubated for 30 minutes at 37°C. The media was aspirated follows. T7 expression vectors, pFT26b(+) (Novagen) and the reaction was then terminated by the addition of ice expressing the peptide of interest are constructed using stan cold 0.5 ml of 0.1N HC1. The samples were heldon ice for 20 dard molecular biology techniques and are transformed into 45 minutes and then evaporated to dryness using a heat gun or E. coli bacterial host BL21 DE3 (Invitrogen). A single vacuum centrifugation. The dried samples were resuspended colony is innoculated and grown shaking overnight at 30°C. in 0.5 ml of phosphate buffer provided in the Cayman Chemi in L broth-i-25 mg/l kanamycin. The overnight culture is cal Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, added to 3.2 L of batch medium (Glucose 25 g/l. Casamino Mich.). Cyclic GMP was measured by EIA according to Acids 5 g/l. Yeast Extract 5 g/l. KHPO 13.3 g/l. (NH) 50 procedures outlined in the Cayman Chemical Cyclic GMP HPO, 4 g/1, MgSO.7H2O1.2 ul, Citric Acid 1.7 g/l. EDTA EIA kit. FIG. 1 shows the activity of chemically synthesized 8.4 mg/l, CoCl-6HO 2.5 mg/l, MnC1-4H2O 15 mg/l, peptide variants (depicted below) in the GC-C receptor activ CuCl-4H2O1.5 mg/l, HBO 3 mg/l, Na MoO-2HO 2.5 ity assay. ECso is defined as the concentration by which 50% mg/l, Zn Acetate-2HO 13 mg/l, Ferric Citrate 100 mg/l, of the maximal activity is seen. Maximum coMP level in Kanamycin 25 mg/l, Antifoam DFO 1 ml/l) and fermented 55 assay is determined as the activity of the positive ST control, using the following process parameters: pH 6.7-control with Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly base only (28% NH-OH), 30°C., aeration: 5 liters perminute. Cys-Tyr SEQID NO:6 and set to 100%. The positive control After the initial consumption of batch glucose (based on was tested twice in this assay. monitoring dissolved oxygen (DO) levels), 1.5 L of feed medium (Glucose 700 g/l. Casamino Acids 10 g/l. Yeast 60 Example 3 Extract 10 g/l, MgSO-7HO 4 g/l. EDTA 13 mg/l, CoCl 6H2O4 mg/l, MnC1-4H2O 23.5 mg/l, CuCl2-4H2O2.5 mg/l, Diuresis and Naturesis Assays HBO, 5 mg/l, NaMoO-2HO 4 mg/l, ZnAcetate-2HO 16 mg/l, Ferric Citrate 40 mg/l, Antifoam DFO 1 ml/l) is added Effect on Diuresis and Natriuresis at a feed rate controlled to maintain 20% DO. IPTG is added 65 The effect of peptides/GC-agonists described herein on to 0.2 mM 2 hours post feed start. The total run time is diuresis and natriuresis can be determined using methodol approximately 40-45 hours (until feed exhaustion). ogy similar to that described in WO06/001931 (examples 6 US 8,779,090 B2 27 28 (p. 42) and 8 (p. 45)). Briefly, a peptide described herein and radioactivity is measured. Giannella et al. (Am. J. Physiol. (180-pmol) is infused for 60 min into a group of 5 anesthe 245: G492-G498) observed that the Kd for wild-type ST tized rats. Given an estimated rat plasma volume of 10 mL. peptide in this same assay is ~13 nm. the infusion rate is approximately 3 umol/mL/min. Blood Similar competition binding assays can be performed in pressure, urine production, and sodium excretion are moni 5 intestinal epithelial cells from wild-type and guanylate tored for approximately 40 minutes prior to the infusion, cyclase C knockout (GC-C KO: Mann et al. 1997 Biochem during the infusion, and for approximately 50 minutes after and Biophysical Research Communications 239:463) mice. the infusion to measure the effect of the peptide/GC-Cagonist Mouse intestinal epithelial cells are prepared as described on diuresis and natriuresis. For comparison, a control group above for rat intestinal epithelial cells except the cells are of five rats is infused with regular saline. Urine and sodium 10 homogenized with an Omni homogenizer for 20 seconds on excretion can be assessed. Dose response can also be deter the maximum setting to make a Suspension of cells. mined. A peptide/GC-C agonist described herein is infused The binding of peptides to GC-C receptors on the cell intravenously into rats over 60 minutes. Urine is collected at surface of human colonic cells (T84 cells; ATCC Catalog No. 30 minute intervals up to 180 minutes after termination of CCL-248) can be characterized in a competitive radioligand peptide/GC-C agonist infusion, and urine Volume, sodium 15 binding assay at pH conditions of 5, 7 and 8. The radiolabeled excretion, and potassium excretion are determined for each tracer used in these experiments is 'I-labeled control pep collection interval. Blood pressure is monitored continu tide. To determine binding constants, competitive inhibition ously. For each dose a dose-response relationship for urine of binding is used. T84 cells are cultured in T-150 plastic Volume, sodium and potassium excretion can be determined. flasks in DMEM and Ham's F-12 medium containing 5% Plasma concentration of the peptide/GC-agonist is also deter fetal bovine serum. Monolayers at 60-70% confluency (ap mined before and after iv infusion. proximately 107 cells) are collected by gentle scraping fol Rat Diuresis Experiment lowed by centrifugation, and washed twice in 50 mL of phos Female Sprague-Dawley rats (>170g, 2-8 per group) are phate-buffered saline (PBS). The cells are resuspended in 1 given 3.0 mL ofiosotonic saline perorally, and then anesthe mL DMEM containing 20 mMN-(2-hydroxymethyl)pipera tized with isoflurane/oxygen. Once an appropriate level of 25 Zine-N'-(2-ethanesulfonic acid) (Hepes), pH 7.0 and 0.5% anesthesia has been achieved, a sterile polyurethane catheter bovine serum albumin (BSA). T84 cells are incubated with a (~1.6 cm, 0.6 mm ID, 0.9 mm OD) is inserted 1.5-2.0 cm into constant amount of ''I-control peptide containing various the urethra and secured using 1-2 drops of veterinary bond concentrations of cold competitor. Free 'I-control peptide adhesive applied to urethra/catheter junction. Rats are then is separated from boundtracerby rapid suction filtration. The dosed with either vehicle or testarticle via the intravenous or 30 binding reactions are carried out in 1.5 mL microfuge tubes in intraperitoneal route. Rats are then placed in appropriately 0.24 mL of DMEM/20 mM Hepes pH 7.0/0.5% BSA con sized rat restraint tubes, with the catheter protruding out of the taining: 2.5x10T84 cells (0.25 mg protein), 200,000 cpm restraint tube into a 10 mL graduated cylinder. Rats are 'I-control peptide (41 fmol, 170 uM), and 0.01 to 1,000 nM allowed to regain consciousness, and the Volume of urine competitor. Binding assays at pH 5.0 are done in DMEM/20 excreted over a 1-5 hour duration is recorded periodically for 35 mM 2-(N-morpholino) ethanesulfonic acid (Mes), pH 5.0. each rat. FIG. 2 shows the results of several peptides Binding assays in pH 8.0 are done in DMEM/20 mM Hepes/ described herein tested in the rat diuresis assay. 50 mM sodium bicarbonate pH 8.0. One sample contains no competitor (Bo) and another contained no cells. After incu Example 4 bation at 37°C. for 1 h, the reaction mixtures are applied to 40 Whatman GF/B glass-fiber filters by suction filtration. The Kd Determination and Binding Assays filters are then rinsed with 10 mL ice-cold PBS buffer, inserted into plastic tubes, and added to 2 mL Scintillation A competition binding assay can be performed using rat fluid. Radioactivity is measured in a LS 6500 liquid scintil intestinal epithelial cells to determine the affinity of a peptide lation counter (Beckman-Coulter). The percent bound in each for intestinal GC-G receptor. Epithelial cells from the small 45 sample is calculated by the equation: intestine of rats are obtained as described by Kessler et al. (J. Biol. Chem. 245: 5281-5288 (1970)). Briefly, animals are % B/Bo (sample cpm-no cells cpm)x100. (Bo cpm-no sacrificed and their abdominal cavities exposed. The small cells cpm) intestine is rinsed with 300 ml ice cold saline or PBS. 10 cm Nonlinear regression analysis of the binding data is used to of the small intestine measured at 10 cm from the pylorus is 50 calculate the concentration of competitor that resulted in 50% removed and cut into 1 inch segments. Intestinal mucosa is radioligand bound (ICs). The apparent dissociation equilib extruded from the intestine by gentle pressure between a rium constant (K) for each competitor is obtained from the piece of parafilm and a P-1000 pipette tip. Intestinal epithelial ICso values and the previously reported estimate of the dis cells are placed in 2 ml PBS and pipetted up and down with a sociation constant for the radioligand, Kas 15 nM (Hamra et 5 ml pipette to make a Suspension of cells. Protein concen 55 al. 1997 PNAS 2705-10) and the method of Cheng and Pru tration in the Suspension is measured using the Bradford soff 1973 Biochem Pharmacol 22:3099-108. Using a two site method (Anal. Biochem. 72: 248-254 (1976)). model, high and low affinity-binding sites are identified on A competition binding assay can be performed based on T84 cells (K, and K) for all the test agents. the method of Giannella et al. (Am. J. Physiol. 245: G492 G498) between III labeled peptide. The assay mixture 60 Example 5 contains: 0.5 ml of DME with 20 mM HEPES-KOH pH 7.0, 0.9 mg of the cell suspension listed above, 21.4 fmol 'II Pharmacokinetic Properties of Peptides SEQ ID NO:4 (42.8 pM), and different concentrations of competitor peptide (0.01 to 1000 nM). The mixture is incu To study the pharmacokinetics of peptides, absorbability bated at room temperature for 1 hour, and the reaction is 65 studies in mice are performed by administering a peptide stopped by applying the mixture to GF/B glass-fiber filters intravaneously via tail vein injection or orally by gavage to (Whatman). The filters are washed with 5 ml ice-cold PBS 8-week-old CD1 mice. Serum is collected from the animals at US 8,779,090 B2 29 30 various time points and tested for the presence of peptide saline (PBS) containing 0.1% bovine serum albumin (BSA), using a competitive enzyme-linked immunoabsorbent assay. 1:40,000 final dilution, 0.0022 ug), and incubated 1 to 4 hat A similar bioavailability study can be performed in which 4°C. One tube contains the Zero standard (B) and another no LCMS rather than ELISA is used to detect peptide. Initially, standard and no antibody (non-specific binding, NSB). serum samples are extracted from the whole blood of exposed Labeled tracer (0.018 uCi diluted in RIA washbuffer) is then and control mice, then injected directly (10 mL) onto an added and incubated at 4°C. for 12 to 18 h. The antibody in-line solid phase extraction (SPE) column (Waters Oasis bound fraction containing peptide is collected by magnetic HLB 25 um column, 2.0x15 mm direct connect) without separation using 10 uL of sheep anti-mouse IgG beads pre further processing. The sample on the SPE column is washed viously washed twice in 10 volumes RIA assay buffer. The with a 5% methanol, 95% dHO solution (2.1 mL/min, 1.0 10 beads are then washed twice with 1 mL of RIA wash buffer, minute), then loaded onto an analytical column using a valve collected by magnetic separation, resuspended in 0.1 mL of switch that places the SPE column in an inverted flow path RIA washbuffer, and added to 2 mL scintillation fluid. Radio onto the analytical column (Waters Xterra MS C85 um IS activity is measured in a LS 6500 scintillation counter (Beck column, 2.1 x20 mm). The sample is eluted from the analyti man-Coulter). The binding efficiency is defined as the percent cal column with a reverse phase gradient (Mobile Phase A: 10 15 radioactivity in the Bo sample compared to the input counts. mMammonium hydroxide in dHO, Mobile Phase B: 10 mM The percent bound in each sample is calculated by the equa ammonium hydroxide in 80% acetonitrile and 20% metha tion: nol; 20% B for the first 3 minutes then ramping to 95% B over 4 min. and holding for 2 min., all at a flow rate of 0.4 % B/Bo (sample cpm-NSB cpm)x100/(Bo cpm-NSB mL/min.). At 9.1 minutes, the gradient returns to the initial cpm) conditions of 20% B for 1 min. Peptide is eluted from the A standard curve is prepared by plotting % B/Bo as a analytical column, and it is detected by triple-quadrapole function of the 10 g peptide concentration. A concentration mass spectrometry. Instrument response is converted into vs. time plot is generated from the data in GraphPad Prism or concentration units by comparison with a standard curve Summit Software PKSolutions 2.0 to generate oral and i.v. using known amounts of chemically synthesized peptide pre 25 PK curves. The area under the curve from T=0 to 4 hours pared and injected in mouse serum using the same procedure. (AUC) is calculated by the software for both p.o. and i.v. Similarly, oral bioavailabity is determined in rats using dosed animals. If the values are below the lower limit of LCMS methodology. Rat plasma samples containing peptide detection (LOD) than the LOD is used to estimate the value are extracted using a Waters Oasis MAX 96 well solid phase (in this experiment 2 nM). Oral Bioavailabilty (F) is calcu extraction (SPE) plate. A 200 uL volume of rat plasma is 30 lated using the equation: mixed with 200 uL of 'C, 'N peptide in the well of a prepared SPE plate. The samples are drawn through the sta F.(AUC (0 AD.)(AUC (0-4) tionary phase with 15 mm Hg vacuum. All Samples are rinsed where D, and D. equal the intravenous and oral dose, with 200 uL of 2% ammonium hydroxide in water followed respectively. by 200 uL of 20% methanol in water. The samples are eluted 35 with consecutive 100 uL volumes of May 20, 1975 formic Example 6 acid/water/methanol and 100 uL May 15, 1980 formic acid/ water/methanol. The samples are dried under nitrogen and In Vitro Proteolytic Stability of Peptides resuspended in 100 uL of 20% methanol in water. Samples are analyzed by a Waters Quattro Micro mass spectrometer 40 The stability of peptides in the presence of several mam coupled to a Waters 1525 binary pump with a Waters 2777 malian digestive enzymes can be determined. Peptides are autosampler. A 40 L Volume of each sample is injected onto exposed to a variety of in vitro conditions including digestive a Thermo Hypersil GOLD C18 column (2.1x50mm, 5 Tm). enzymes and low phenvironments designed to simulate gas Peptides are eluted by a gradient over 3 minutes with aceto tric fluid. Peptides are incubated with chymotrypsin, trypsin, nitrile and water containing 0.05% trifluoroacetic acid. 45 pepsin, aminopeptidase, carboxypeptidase A, or simulated Oral bioavailability can also be determined using a radio gastric fluid (sgf) at ph 1.0. Samples are collected at 0, 3, and immunoassay (RIA) detection method. Female CD-1 mice 24h for all conditions except pepsin digestion and the SGF. (Charles River, Wilmington, Mass.) weighing approximately For the latter two conditions, samples are obtained at 0, 1, and 25g (7-8 weeks old) or female CD rats (Charles River, Wilm 3 h. Negative control samples are prepared for initial and final ington, Mass.) weighing approximately 153 g are included in 50 time points. A separate, positive activity control is run in this study. Monoclonal antibody recognizing the peptide and parallel to test peptide. All samples are analyzed by LC/MS 'I labeled-peptide, a labeled tracer, are used in these experi Chymotrypsin ments. Animals are fasted overnight before administration of 500 ul samples of 0.01 mg/mL peptide and guanylin compounds. Blood is drawn from all dosed animals by retro (Sigma-Aldrich, G116; positive control) are prepared in the orbital eye bleeding at specific intervals and test compound 55 chymotrypsin reaction buffer (100 mM Tris-HCl, 10 mM levels are analyzed by radioimmunoassay (RIA). Samples CaCl, pH 7.5) in 2 mL eppendorf tubes. Zero and 24 h. (80 uL) are first diluted to 0.5 mL with start buffer (8% control samples are prepared by adding 5 uL of a 10 mM methanol, 0.095% TFA in water) and applied to C18 columns chymostatin (Sigma-Alrich, C7268; a chymotrypsin inhibi previously conditioned with 1 mL methanol and equilibrated tor) stock for a final concentration of 100 uM. All samples are with 2 mL of start buffer. Afterwashing with 1 mL start buffer, 60 incubated at 37° C. for 5 min. 20 uL of a 0.01 mg/mL chy peptide is eluted with 0.8 mL of 80% methanol, 0.05% TFA motrypsin Stock (C-chympotrypsin from bovine pancreas: and dried down in a centrifugal evaporator. Samples are Sigma-Aldrich, C6423) are added to each sample for a 0.0004 reconstituted in 0.194 mL assay buffer (PBS buffer, pH 7.4, mg/mL final concentration. Samples are returned to the 37 containing 10% fetal bovine serum). Standard dilutions of C. water bath. The reaction is quenched with 5uL of a 10 mM peptide are made in rat plasma. To perform RIA analysis, 65 chymostatin stock at each time point for a final concentration samples from dosed animal and standards are mixed with 5 of 100 uM. No extra chymostatin is added to the control uL diluted antibody (in RIA washbuffer: phosphate-buffered samples as they already had inhibitor. Samples are Subse US 8,779,090 B2 31 32 quently flash frozen in liquid nitrogen, and stored at -80° C. Bestatin hydrochloride (BioChemika, 08170; an aminopep until analysis. Upon analysis, samples are thawed and trans tidase inhibitor) stock is added to each control sample (0 and ferred to a 1 mL 96-well plate. Standards of peptide and 24 h), for a final concentration of 0.05 mg/mL. All control and guanylin are prepared in chymotrypsin reaction buffer at test samples (0.3, and 24 h) are incubated at 37°C. for 5 min. 0.625, 1.25, 2.50, 5.00, and 10.00 ug/mL concentrations. 5 0.02 U. aminopeptidase (Aminopeptidase M. amino acid aryl These standards are used to generate a standard curve for amidase (Roche, 102768; U-hydrolysis of 1.0 umol of quantification of samples. When necessary, the standard L-leucinamide to leucine and NH3 per min at pH 8.5 at 25° curves are also used to calculate the concentration of the C.) are added to each sample. Samples are returned to the 37 corresponding digestion product. 10 LIL injections are made C. water bath. The reaction is quenched with 5 uL of a 5 of each sample and standard. 10 mg/mL Bestatin hydrochloride stock at the propertime point. Trypsin No extra Bestatin hydrochloride is added to the control 500 uL samples of 0.01 mg/mL peptide and BAEE (N, samples since they already had inhibitor present. Samples are Benzoyl-L-arginine ethyl ester hydrochloride: Sigma-Ald Subsequently flash frozen in liquid nitrogen, and stored at rich, B4500; positive control) are prepared with trypsin reac -80°C. until analysis. Upon analysis, samples are thawed and tion buffer (100 mM Tris-HCl, pH 7.5) in 2 mL eppendorf 15 transferred to a 1 mL 96-well plate. Standards of peptide are tubes. Zero and 24 h time point control samples are prepared prepared in aminopeptidase reaction buffer at 0.625, 1.25, (N=1) with 5uL of a 100 mg/mL AEBSF (4-(2-aminoethyl) 2.50, 5.00, and 10.00 ug/mL concentrations. These standards benzenesulfonyl fluoride hydrochloride; a trypsin inhibitor) are used to generate a standard curve for quantification of stock for a final concentration of 1 mg/mL. All control and samples. When necessary, the standard curves are also used to test samples (0.3, and 24 h) are incubated at 37°C. for 5 min. calculate the concentration of the corresponding digestion Twenty (20) pull of a 0.01 mg/mL trypsin (Sigma-Aldrich, product. Ten (10) uL injections are made of each sample and T6467) stock are added to each sample for a final concentra standard. tion of 0.0004 mg/mL. Samples are returned to the 37° C. Carboxypeptidase A water bath. The reaction is quenched with 5 uL of a 100 500 uL samples of 0.01 mg/mL peptide and N-CBZ-Gly mg/mL AEBSF stock, which is added to each sample at the 25 cine-Glycine-Leucine (Z-Gly-Gly-Leu, Sigma-Aldrich, indicated timepoint, for a final concentration of 1 mg/mL. No C8501; positive control) are prepared in the carboxypeptidase extra AEBSF is added to the control samples as they already A reaction buffer (25 mM Tris-HCl, 500 mMNaCl, pH 7.5) in had inhibitor. Samples are subsequently flash frozen in liquid 2 mL eppendorf tubes. Five (5) L of a 40 g/mL carbox nitrogen, and stored at -80°C. until analysis. Upon analysis, ypeptidase inhibitor (carboxypeptidase inhibitor from potato samples are thawed and transferred to a 1 mL 96-well plate. 30 tuber (Sigma-Aldrich, C0279) stock is added to each control Standards of peptide and BAEE are prepared in trypsin reac sample (0 and 24 h), for a final concentration of 0.4 ug/mL. tion buffer at 0.625, 1.25, 2.50, 5.00, and 10.00 ug/mL con All control and test (0, 3 and 24 h) samples are incubated at centrations. These standards are used to generate a standard 37°C. for 5 min. Twenty (20) of a 0.01 mg/mL carboxypep curve for quantification of samples. When necessary, the stan tidase A (Carboxypeptidase A from human pancreas; Sigma dard curves are also used to calculate the concentration of the 35 Aldrich, C5358) stock is added to each sample. The samples corresponding digestion product. Ten (10) uL injections are are returned to the 37°C. water bath. The reaction is quenched made of each sample and standard. with 5 L of a 40 g/mL carboxypeptidase inhibitor at the Pepsin proper time point. No extra carboxypeptidase inhibitor is 500 uL samples of 100 U/mL pepsin (Pepsin porcine gas added to the control samples since there is already inhibitor tric mucosa; Sigma-Aldrich, P68871; U-release of 0.01 40 present. Samples are Subsequently flash frozen in liquid nitro absorbance at 280 nM (A280) as TCA soluble hydrolysis gen, and stored at -80° C. until analysis. Upon analysis, products perminat 37°C. of hemoglobin) are prepared in the samples are thawed and transferred to a 1 mL 96-well deep pepsin reaction buffer (100 mM HCl-KCl pH 2.0) in 5 mL microtiter plate. Standards of peptide and Z-Gly-Gly-Leu are polystyrene round bottom tubes. To the control samples (0 prepared in carboxypeptidase Areaction buffer at 0.625, 1.25, and 24 h), 500 uL of a 1 Mammonium acetate (pepsin 45 2.50, 5.00, and 10.00 ug/mL concentrations. These standards inhibitor) stock are added, for a final concentration of 0.5M. are used to generate a standard curve for quantification of All control and test samples (0,1, and 3 h) are incubated at 37° samples. When necessary, the standard curves are also used to C. for 5 min, while shaking. Fifty (50) uL of 0.1 mg/mL calculate the concentration of the corresponding digestion peptide and Insulin B chain, oxidized (Sigma-Aldrich, product. Ten (10) uL injections are made of each sample and 16383; positive control), stocks are added to the respective 50 standard. tubes. Samples are returned to the 37°C. shaking water bath. Carboxypeptidase A Identification of Proteolysis Product Reactions are quenched by the addition of 500 uL of 1 M To analyze carboxypeptidase Adigestion product, Samples ammonium acetate for a final concentration of 0.5M (except of 0.01 mg/mL peptide are prepared in the carboxypeptidase to the control samples, which already contained 0.5 Mammo A reaction buffer at a total volume of 500 uL in 2 mLeppen nium acetate). Samples are cooled on ice and stored at 4°C. 55 dorf tubes. Triplicate samples are prepared for the following until analysis. Upon analysis, samples are transferred to a 1 time points: 0, 15, 30, 60, 120, 180 and 240 min. The samples mL 96-well plate. Standards of peptide and Insulin B chain, are incubated at 37° C. for 5 min. Twenty (20) uL of a 0.01 oxidized, are prepared in 25 mM Tris-hydrochloric acid, 500 mg/mL carboxypeptidase A stock are added to each sample mM sodium chloride, pH 7.5 buffer at 0.625, 1.25, 2.50, 5.00, and returned to the 37° C. water bath. The reactions are and 10.00 ug/mL concentrations. These standards are used to 60 quenched with 5 LIL, of a 40 g/mL carboxypeptidase inhibi generate a standard curve for quantification of samples. Ten tor at the proper time points. Samples are Subsequently flash (10) uL injections are made of each sample and standard. frozen in liquid nitrogen, and stored at -80°C. until analysis. Aminopeptidase Upon analysis, Samples are thawed and transferred to a 1 mL 500 uL samples of 0.01 mg/mL peptide and chemically 96-well plate. Standards of peptide prepared in carboxypep synthesized wild type ST (positive control) are prepared in 65 tidase A reaction buffer at 0.625, 1.25, 2.50, 5.00, and 10.00 the aminopeptidase reaction buffer (5 mM Tris-HCl, 5 mM ug/mL concentrations. These standards are used to generate a MgCl, pH 7.5) in 2 mLeppendorf tubes. 5uIL of a 5 mg/mL standard curve for quantification of samples. When neces US 8,779,090 B2 33 34 sary, the standard curves are also used to calculate the con peptide and 5% activated carbon are administered simulta centration of the corresponding digestion product. Ten (10) neously (versus 7 minutes apart). In addition, the animals are LL injections are made of each sample and Standard. If the sacrificed 10 minutes after the administration of peptide and formation of a digestion product is evident, then a spectral test compound. The experiment can be performed in male and analysis used to determine the mass of the digestion product, female rats. and predict its possible identity. The gastrointestinal transit assay can also performed in Simulated Gastric Fluid (SGF) wild-type mice and mice lacking the guanylate cyclase C Samples of peptide are prepared in the simulated gastric receptor (GC-CKO: Mann etal 1997 Biochem and Biophysi fluid buffer (0.2% NaCl (w/v), 0.7% HCl (v/v), pH 1) to a total cal Research Communications 239:463). Wild type and volume of 500 uL in 2 mL eppendorf tubes. The reference 10 GC-C KO mice are fasted overnight and test peptide or control and test samples (0, 1 and 3 h) are incubated at 37°C. vehicle alone are orally administered 10 minutes prior to an for the time point indicated. The reference control sample is oral dose of a 10% Activated Carbon/10% Gum Arabic sus diluted 10-fold (1000 uL volume) in distilled water for a final pension. Animals are sacrificed 5 minutes after peptide or concentration of 10M and chilled on ice. At each time point, vehicle administration. samples are diluted 10-fold (1000 uL volume) in distilled 15 water for an expected concentration of 10 UV, and chilled on Example 8 ice, until analysis. Upon analysis, Samples are transferred to a 1 mL 96-well plate. Standards of peptide are prepared in Intestinal Secretion Assay in Suckling Mice (SuMi distilled water at 0.625, 1.25, 2.50, 5.00, and 10.00 uM con Assay) centrations. These standards are used to generate a standard curve for quantification of samples. Ten(10) uL injections are Peptides are tested for their ability to increase intestinal made of each sample and standard. secretion using a Suckling mouse model of intestinal Secre tion. In this model a test compound is administered to Suck Example 7 ling mice that are between 7 and 9 days old. After the mice are 25 sacrificed, the gastrointestinal tract from the stomach to the Rodent Intestinal Transit Assays cecum is dissected (“guts”). The remains (“carcass') as well as the guts are weighed and the ratio of guts to carcass weight In order to determine whether a peptide increases the rate is calculated. If the ratio is above 0.09, one can conclude that of gastrointestinal transit, the peptide and controls are tested the test compound increases intestinal secretion. Wild type using a murine gastrointestinal transit (GIT) assay (Moon et 30 ST peptide can be used as a control in this assay. al. Infection and Immunity 25:127, 1979). In this assay, char coal, which can be readily visualized in the gastrointestinal Example 9 tract, is administered to mice after the administration of a test compound. The distance traveled by the charcoal is measured Colonic Hyperalgesia Animal Models and expressed as a percentage of the total length of the colon. 35 Mice are fasted with free access to water for 12 to 16 hours Hypersensitivity to colorectal distension is common in before the treatment with peptide or control buffer. The pep patients with IBS and may be responsible for the major symp tides are orally administered at 1 g/kg-1 mg/kg of peptide in tom of pain. Both inflammatory and non-inflammatory ani buffer (20 mM Tris pH 7.5) 7 minutes before being given an mal models of visceral hyperalgesia to distension have been oral dose of 5% Activated Carbon (Aldrich 242276-250G). 40 developed to investigate the effect of compounds on visceral Control mice are administered buffer only before being given pain in IBS. a dose of Activated Carbon. After 15 minutes, the mice are I. Trinitrobenzenesulphonic Acid (TNBS)-Induced Rectal sacrificed and their intestines from the stomach to the cecum Allodynia in Two Rodent Models are dissected. The total length of the intestine as well as the TNBS Visceral Hypersensitivity Rat Model distance traveled from the stomach to the charcoal front is 45 Male Wistar rats (220-250 g) are premedicated with 0.5 measured for each animal and the results are expressed as the mg/kg of acepromazine injected intraperitoneally (IP) and percent of the total length of the intestine traveled by the anesthetized by intramuscular administration of 100 mg/kg of charcoal front. All results are reported as the average of 10 . Pairs of nichrome wire electrodes (60 cm in length miceitstandard deviation. A comparison of the distance trav and 80 um in diameter) are implanted in the striated muscle of eled by the charcoal between the mice treated with peptide 50 the abdomen, 2 cm laterally from the white line. The free ends versus the mice treated with vehicle alone is performed using of electrodes are exteriorized on the back of the neck and a Student's t test and a statistically significant difference is protected by a plastic tube attached to the skin. Electromyo considered for P-0.05. P-values are calculated using a two graphic (EMG) recordings are started 5 days after Surgery. sided T-Test assuming unequal variances. Controls include Electrical activity of abdominal striated muscle is recorded vehicle alone (e.g. Tris buffer) and ZelnormR). 55 with an electroencephalograph machine (Mini VIII, Alvar, An identical experiment can be performed to determine if Paris, France) using a short time constant (0.03 sec.) to peptides are effective in a chronic dosing treatment regimen. remove low-frequency signals (<3 HZ). Briefly, 8 week old CD1 female mice are dosed orally once a Ten days post Surgical implantation, trinitrobenzenesul day for 5 days with either peptide (0.06 mg/kg or 0.25 mg/kg phonic acid (TNBS) is administered to induce rectal inflam in 20 mM Tris pH 7.5) or vehicle alone (20 mM Tris pH 7.5). 60 mation. TNBS (80 mg kg in 0.3 ml 50% ethanol) is admin On the 5" day, a GIT assay is performed identical to that istered intrarectally through a silicone rubber catheter above except 200 ul of a 10% charcoal solution is adminis introduced at 3 cm from the anus under light diethyl-ether tered. anesthesia, as previously described (Morteau et al. 1994 Dig The gastrointestinal transit assay can also performed in Dis Sci 39:1239). Following TNBS administration, rats are male and female CD rats (Charles River; Wilmington, Mass.). 65 placed in plastic tunnels where they are severely limited in The assay is performed as described above for mice except an mobility for several days before colorectal distension (CRD). average of 5-8 animals are used for each test group and test Experimental compound is administered one hour before US 8,779,090 B2 35 36 CRD which is performed by insertion into the rectum, at 1 cm recording artifacts. The balloon used for distension is 4 cm of the anus, a 4 cm long balloon made from a latex condom long and made from a latex condom. It is fixed on a rigid (Gue et al., 1997 Neurogastroenterol. Motil. 9:271). The bal catheter taken from an embolectomy probe (Fogarty). CRD is loon is fixed on a rigid catheter taken from an embolectomy performed by insertion of the balloon in the rectum at 1 cm probe (Fogarty). The catheter attached balloon is fixed at the from the anus. The tube is fixed at the base of the tail. Isobaric base of the tail. The balloon, connected to a barostat, is distensions are performed from 0 to 60 mmHg, with each inflated progressively by steps of 15 mmHg, from 0 to 60 distension step lasting 5 minutes. The first distension is per mmHg, each step of inflation lasting 5 min. Evaluation of formed at a pressure of 15 mmHg and an increment of 15 rectal sensitivity, as measured by EMG, is performed before mmHg is added at each following step, until a maximal pres (1-2 days) and 3 days following rectal instillation of TNBS. 10 Sure of 60 mmHg is attained. Electromyographic recordings The number of spike bursts that corresponds to abdominal commence 5 days after Surgery. Electrical activity is recorded contractions is determined per 5 min periods. Statistical with an electroencephalograph (Mini VIII, Alvar, Paris, analysis of the number of abdominal contractions and evalu France) using a short time constant (0.03 sec.) to remove ation of the dose-effects relationships is performed by a one low-frequency signals (<3 Hz) and a paper speed of 3.6 way analysis of variance (ANOVA) followed by a post-hoc 15 cm/minute. Isobaric distensions of the colon are performed (Student or Dunnett tests) and regression analysis for ED50 if by connecting the balloon to a computerized barostat. appropriate. Colonic pressure and balloon Volume are continuously moni TNBS Visceral Hypersensitivity Model in Wild-Type tored on a potentiometric recorder (L6514, Linseis, Selb, (WT) Mice and Mice Lacking the Guanylate-Cyclase C Germany) with a paper speed of 1.0 cm/minute. The number Receptor (GC-C KO) of spike bursts, corresponding to abdominal contractions, is TNBS induced visceral hypersensitivity can be assessed in evaluated per 5-minute period. Colorectal volumes are deter WT and GC-C KO mice. Two groups (WT and GC-C KO) of mined as the maximal Volume obtained for each stage of male mice (22-25 g) are Surgically prepared for electromyo distension using the potentiometric recorder. Statistical graphic (EMG) recordings. Three electrodes are implanted in analysis of these two parameters is performed using a one the striated muscles of the abdomen for EMG recording of 25 way analysis of variance (ANOVA) followed by an unpaired abdominal contractions. Colorectal distension (CRD) is per two-tailed Student's t test using GraphPad Prism 4.0. p val formed with a balloon inflated by 10s steps of 0.02 ml from 0 ues<0.05 are considered significantly different. The values to 0.12 ml. Under basal conditions mice are submitted to are expressed as meaniSEM. control CRD (time 0) followed by oral administration of test III. Water Avoidance Stress-Induced Hyperalgesia Model peptide (0.01 and 0.3 g/kg) or vehicle only (distilled water, 30 The effect of peptides on basal visceral nociception can be 1 ml) at 3 hours. One hour post dosing the CRD procedure is tested using a model of water avoidance stress-induced vis repeated. Abdominal EMG contractile response to colorectal ceral hyperalgesia in adult male Wistar rats. The stress distension in basal conditions in both WT and GC-C KO mice involves confining rats to a platform Surrounded by water for (12-14 mice per group) is determined in the absence of a period of 1 hour and then measuring their visceromotor vehicle and test peptide, and the mean+/-standard error of the 35 response to colonic distension using electromyography mean (SEM) are determined. (EMG). For TNBS induced visceral hypersensitivity conditions, At least 7 days prior to stress measurements, animals are mice are submitted to control CRD (time 0) and TNBS (20 deeply anesthetized with pentobarbital sodium (45 mg/kg) mg/kg) is administered at 3 days. Three days post intracolonic and equipped with electrodes implanted into the external TNBS-induction animals are orally administered test peptide 40 oblique musculature, just Superior to the inguinal ligament. (0.01 and 0.3 ug/kg) or vehicle (distilled water, 1 ml) 1 hour Electrode leads are then tunneled subcutaneously and exter before CRD. The effect of test peptide (0.01 ug/kg) on nalized laterally for future access. Following Surgery, rats are abdominal response to colorectal distension after TNBS in housed in pairs and allowed to recover for at least 7 days. On WT and GC-C KO mice (12-14 per group) at a volume dis the day of the experiment, animals are lightly anesthetized tension of 0.8 ml is determined and the mean+/-standard 45 with halothane, and a lubricated latex balloon (6 cm) is error of the mean (SEM) is determined. inserted intra-anally into the descending colon. Animals are II. Partial Restraint Stress-Induced Hyperalgesia Model allowed to recover for 30 minutes, and colorectal distension Five groups of female Wistar rats (weighing 200-250 g (CRD) is initiated. The CRD procedure consists of graded each), are Surgically prepared for electromyography as intensities of phasic CRD (10, 20, 40, 60 mmHg: 20s dura described (Morteau et al. 1994 Dig Dis Sci 39:1239-48) and 50 tion; 4 min inter-stimulus interval). Visceromotor response can be used to evaluate the effects of a test peptide on col (VMR) to CRD is quantified by measuring EMG activity. orectal sensitivity and compliance after a 2 hour partial To determine the effect of a test peptide in a model of water restraint stress session. Partial restraint stress (PRS), a rela avoidance stress-induced visceral hyperalgesia, a baseline tively mild stress, is induced as previously described (Mor CRD is recorded and then the animals are subjected to 1 hour teau et al. 1994 Dig Dis Sci 39:1239-48). Female rats are 55 of water avoidance stress. For water avoidance stress, the test lightly anesthetized with and their shoulders, apparatus consists of a Plexiglas tank with a block affixed to upper forelimbs and thoracic trunkare wrapped in a confining the center of the floor. The tank is filled with fresh room harness of paper tape to restrict, but not prevent body move temperature water (25°C.) to within 1 cm of the top of the ments. Control sham-stress animals are anesthitized but not block. The animals are placed on the block for a period of 1 h. wrapped. Animals receive isobaric colorectal distensions 60 The sham water avoidance stress entails placing the rats on (CRD) directly prior to (control CRD) and 15 minutes after the same platform in a waterless container. two hours of partial restraint induced stress. Rats are treated Phenylbenzoquinone-Induced Writhing Model orally with test peptide (0.3, 3, 30 ug/kg) or vehicle only The PBQ-induced writhing model can be used to assess (distilled water 1 mL) one hour before the CRD procedure. pain control activity of the peptides and GC-C receptor ago For the CRD procedure, rats are acclimatized to restraint in 65 nists. This model is described by Siegmund et al. (1957 Proc. polypropylene tunnels (diameter: 7 cm; length: 20 cm) peri Soc. Exp. Bio. Med.95:729-731). Briefly, one hour after oral odically for several days before CRD in order to minimize dosing with a test compound, e.g., a peptide, morphine or US 8,779,090 B2 37 38 vehicle, 0.02% phenylbenzoquinone (PBQ) solution (12.5 vehicle (20 mM Tris) in a volume of 300 ul. 1 hour after mL/kg) is injected by intraperitoneal route into the mouse. dosing, intestinal transit rate is measured. Animals are again The number of stretches and writhings are recorded from the dosed with 300 ul of the peptide followed immediately by 500 5" to the 10" minute after PBQ injection, and can also be ul of a charcoal meal (10% charcoal, 10% gum arabic in counted between the 35' and 40' minute and between the water). To calculate the distance of the small intestine traveled 60" and 65' minute to provide a kinetic assessment. The by the charcoal front, after 20 minutes, the total length of the results are expressed as the number of stretches and writhings intestine as well as the distance traveled from the stomach to (meant-SEM) and the percentage of variation of the nocice the charcoal front are measured for each animal. ptive threshold calculated from the mean value of the vehicle treated group. The statistical significance of any differences 10 Example 13 between the treated groups and the control group is deter mined by a Dunnett's test using the residual variance after a Peptide Effect on coMP Levels and Secretion in one-way analysis of variance (P<0.05) using SigmaStat Soft Ligated Loops Rodent Models Wa. 15 The effect of peptide on coMP levels and secretion are Example 10 studied by injecting peptide directly into an isolated loop in either wild-type or GC-C KO mice. This done by surgically Measuring the Effect of Peptides on Bowel Habits ligating a loop in the Small intestine of the mouse. The meth odology for ligated loop formation is a similar to that Single doses of 30, 100,300, 1000 or 3000 ug of peptide are described in Londonetal. 1997 Am J Physiolp. G93-105. The given to healthy males and postmenopausal females. At each loop is roughly centered and is a length of 1-3 cm. The loops dose level (peptide or placebo (vehicle) is administered orally are injected with 100 ul of either peptide (5ug) or vehicle (20 in 5.0 mL 50 mM phosphate buffer (pH 6.0) plus 3x20 mL mM Tris, pH 7.5 or Krebs Ringer, 10 mM Glucose, HEPES water rinses and 175 mL water after at least a 10-hour fast. In buffer (KRGH)). Following a recovery time of 90 minutes the each dosing group, Subjects are randomized to receive either 25 loops are excised. Weights are recorded for each loop before placebo or peptide. Bowel habits (including Bristol Stool and after removal of the fluid contained therein. The length of Form Scale score (BSFS), stool frequency, and stool weight) each loop is also recorded. A weight to length ratio (W/L) for are evaluated for each collected bowel movement 48 hours each loop is calculated to determine the effects of peptide on prior to dose and up to approximately 48 hours postdose. The secretion. BSFS scale is as follows: (1) Separate hard lumps, like nuts; 30 To determine the effect of peptide on coMP activity, fluid (2) Sausage-shaped but lumpy, (3) Like a sausage or Snakebut from the loop is collected in ice-cold trichloracetic acid with cracks on its surface, (4) Like a sausage or Snake, Smooth (TCA) and stored at -80° C. for use in an assay to measure and soft, (5) Soft blobs with clear-cut edges, (6) Fluffy pieces cGMP levels in the fluid. Intestinal fluid samples are TCA with ragged edges, a mushy stool, and (7) Watery, no solid extracted, and cyclic GMP is measured by EIA according to pieces. 35 procedures outlined in the Cayman Chemical Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, Mich.) to determine Example 11 cyclic GMP levels in the intestinal fluid of the mouse in the presence of either peptide or vehicle. The effects of peptide on Examination of the Effect of Peptides on the cGMP levels and secretion in ligated loops in female CD rats Consistency and Timing of Bowel Movements in 40 can also be determined using protocols similar to those Humans after a Seven-Day Dosing Period described above. In the case of the rat, however four loops of intestine are Surgically ligated. The first three loops are dis Seven daily doses of 30, 100, 300, or 1000 ug of peptide are tributed equally in the small intestine and the fourth loop is given to healthy subjects. Peptide or placebo (vehicle) is located in colon. Loops are 1 to 3 centimeters, and are injected administered orally in 5.0 mL 50 mM phosphate buffer (pH 45 with 2004 of either peptide (5ug) or vehicle (Krebs Ringer, 6.0) plus 3x20 mL water rinses and 175 mL water after at least 10 mM glucose, HEPES buffer (KRGH)). a 10-hour fast. In each dosing group, Subjects are randomized to receive peptide or receive placebo. Daily mean BSFS Example 14 scores, mean stool weight and mean ease of passage for the different dosing groups during the seven days prior to and the 50 Peptide Effects on Opioid Induced Constipation seven days during dosing with peptide are collected. The Mean Ease of Passage Scale is as follows: (1) Manual disim The effect of peptide on opioid induced constipation is paction, (2) Enema needed, (3) Straining needed, (4) Normal, studied by dosing female rats (~160 g each) with 300 ul of the (5) Urgent without pain, (6) Urgent with pain, and (7) Incon opiate, morphine (2.5 mg/kg) via intra-peritoneal injection. tinent. 55 Thirty minutes postdosing, animals are treated with 300 ul of SEQID NO:3 or vehicle only. Ten minutes later, the animals Example 12 are orally dosed with 500 ul 10% charcoal, 10% gum arabic meal. After ten minutes, the animals are sacrificed and gas Peptide Effects in a Rat Model of Postoperative Ileus trointestinal transit is measured as in Example 3 above. 60 Female CD rats are used to test the effect of peptide on Example 15 delayed transit induced by abdominal Surgery and manual manipulation of the Small intestine. Groups of at least nine Mass Spectrometry Characterization of Disulfide rats undergo abdominal Surgery under isoflurane anesthesia. Bonds in Peptide Surgery consists of laparotomy and 5 minutes of gentle 65 manual intestinal massage. Following recovery from anesthe The position of disulfide bonds in a test peptide can be sia, rats are dosed orally with either 10 ug/kg peptide 3 or determined as follows. To identify the optimal conditions US 8,779,090 B2 39 40 required to partially reduce a test peptide, chemically synthe U.S. Pat. No. 4,904,584; U.S. Pat. No. 5,834,594; U.S. Pat. sized peptide is alkylated with iodoacetamide after TCEP No. 5,824,784 and U.S. Pat. No. 5,985,265). (tris(2-carboxyethyl) phosphine) treatment (0.1 to 10 mM for Administration of Peptides and GC-C Receptor Agonists 20 minutes at room temperature). After TCEP reduction, the For therapeutic and preventive treatment of disorders reaction is adjusted to pH 8.0 with Tris and iodoacetamide is described herein, the peptides and agonists described herein added to 50 mM. The reaction products are analyzed by can be administered orally, e.g., as a tablet or cachet contain LC-MS. Partially reduced peptide is then cyanylated, cleaved ing a predetermined amount of the active ingredient, pellet, with base and completely reduced to separate fragments. gel, paste, syrup, bolus, electuary, slurry, Sachet, capsule: After partial reduction, both cyanylation and cleavage of pep powder, lyophilized powder, granules; as a solution or a 10 Suspension in an aqueous liquid or a non-aqueous liquid; as an tide are performed either in a test tube or in an HPLC column. oil-in-water liquid emulsion ora water-in-oil liquid emulsion, A modified method of Wu and Watson ((2002) Methods Mol. via a liposomal formulation (see, e.g., EP 736299) or in some Biol. 194: 1-22) is used to determine the position of the other form. Orally administered compositions can include disulfide bonds. The steps are carried out manually, with binders, lubricants, inert diluents, lubricating, Surface active isolation of the alkylation products by Solid phase extraction 15 or dispersing agents, flavoring agents, and humectants. Orally (SPE), or in-line (automated), with reactions occurring in an administered formulations such as tablets may optionally be SPE column. Briefly, the manual procedure comprised the coated or scored and may be formulated so as to provide following. Chemically synthesized peptide is partially Sustained, delayed or controlled release of the active ingredi reduced with 1 mM tris(2-carboxyethyl) phosphine (TCEP) ent therein. The peptides and agonists can be co-administered at pH 3. The sulfhydryl groups of partially reduced peptide with other agents used to treat gastrointestinal disorders are cyanylated with 2.1 umoles of 1-cyano-4-dimethylamino including but not limited to the agents described herein. The pyridinium tetrafluoroborate (CDAP) for 15 minutes. The peptides and agonists can also be administered by rectal Sup reaction mixture is then diluted to 0.5 mL with 10 mMammo pository. For the treatment of disorders outside the gas nium acetate pH 5.8 and applied to an Amprep octadecyl C18 trointestinal tract Such as congestive heart failure and benign minicolumn (100 mg, GE HealthTech). The minicolumn is 25 prostatic hypertrophy, peptides and agonists are preferably washed with 1 mL of 10 mMammonium acetate pH 5.8 and administered parenterally or orally. peptides eluted with 0.6 mL methanol. After drying, the pep The peptides described hereincan be administered alone or tides are cleaved in 1 MNH4OH and fully reduced with 0.1 M in combination with other agents. For example, the peptides TCEP. After drying, the peptide fragments are reconstituted can be administered together with an analgesic peptide or in 0.1% formic acid and analyzed by LC-MS. Briefly, the 30 compound. The analgesic peptide or compound can be automated procedure comprised the following. Peptide is covalently attached to a peptide described herein or it can be loaded onto an Oasis HLB 2x15 mm column (Waters). Reac a separate agent that is administered together with or sequen tions are carried out by filling a 5 mL sample loop with 1.2 tially with a peptide described herein in a combination mMTCEP, 2.4 mMCDAP, 2 MNHOH or 6 mMTCEP and therapy. pushing each reagent through the column with 0.1% formic 35 Combination therapy can be achieved by administering acid in 5% methanol at a flow rate of 0.3 mL/min. The column two or more agents, e.g., a peptide described herein and an is then back-flushed and the cleaved peptides analyzed by analgesic peptide or compound, each of which is formulated LC-MS. and administered separately, or by administering two or more LC-MS analysis can be conduced using an Atlantis dC18 agents in a single formulation. Other combinations are also 2.1 x50 mm column (Waters) equilibrated in 98% buffer A 40 encompassed by combination therapy. For example, two (0.1% formic acid), 2% buffer B (0.1% formic acid: 85% agents can be formulated together and administered in con methanol, 15% CH3CN) at a flow rate of 0.3 mL/min. After a junction with a separate formulation containing a third agent. 4 min wash with the same buffers, peptides are eluted with a While the two or more agents in the combination therapy can linear gradient of 2% buffer B to 40% buffer B over 38 min be administered simultaneously, they need not be. For with a constant flow rate of 0.3 mL/min. Cleaved peptide 45 example, administration of a first agent (or combination of masses are determined using a Micromass Q-T of II instru agents) can precede administration of a second agent (or ment equipped with an electrospray ionization (ESI) source combination of agents) by minutes, hours, days, or weeks. operating in positive ion mode. The instrument is pro Thus, the two or more agents can be administered within grammed to scan in the mass range of m/z. 100 to 1000. minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 Molecular weight predictions and data analysis are carried 50 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 out with MassLynx version 4.0 software. Based on the days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks method of Wu and Watson (supra), a list of possible fragments of each other. In some cases even longer intervals are pos resulting from CN-induced cleavage of singly reduced and sible. While in many cases it is desirable that the two or more cyanylated species of peptide with all possible disulfide link agents used in a combination therapy be present in within the age combinations is generated. The list included the signature 55 patient’s body at the same time, this need not be so. fragments for each possible structure and is used to predict the Combination therapy can also include two or more admin disulfide bonding pattern of the test peptide. istrations of one or more of the agents used in the combina PEGylated Peptides tion. For example, if agent X and agent Y are used in a The in vivo half-life of peptides can be extended by con combination, one could administer them sequentially in any jugating the peptide to a water Soluble polymer, Such as 60 combination one or more times, e.g., in the order X-Y-X. polyethylene glycol (PEG) to create a PEGylated peptide. X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc. The polyethylene glycol molecules are usually connected to Combination therapy can also include the administration the peptide via a reactive group found on the peptide, e.g., an of two or more agents via different routes or locations. For amino group found within alysine or at the amino terminus of example, (a) one agent is administered orally and another the peptide. 65 agent is administered intravenously or (b) one agent is admin Various methods are known for attaching polyethylene istered orally and another is administered locally. In each glycol to a peptide (see, for example, U.S. Pat. No. 4,002.531; case, the agents can either simultaneously or sequentially. US 8,779,090 B2 41 42 Approximated dosages for some of the combination therapy potato or tapioca starch, other starches, pre-gelatinized agents described herein are found in the “BNF Recom starch, clays, other algins, other celluloses, gums (like gel mended Dose” column of tables on pages 11-17 of WO01/ lan), low-substituted hydroxypropyl cellulose, or mixtures 76632 (the data in the tables being attributed to the March thereof, 2000 British National Formulary) and can also be found in LUBRICANTS: calcium stearate, magnesium stearate, other standard formularies and other drug prescribing direc mineral oil, light mineral oil, glycerin, Sorbitol, mannitol, tories. For some drugs, the customary prescribed dose for an polyethylene glycol, otherglycols, Stearic acid, Sodium lauryl indication will vary somewhat from country to country. Sulfate, Sodium Stearyl fumarate, vegetable based fatty acids The agents, alone or in combination, can be combined with lubricant, talc, hydrogenated vegetable oil (e.g., peanut oil, any pharmaceutically acceptable carrier or medium. Thus, 10 cottonseed oil, Sunflower oil, Sesame oil, olive oil, corn oil they can be combined with materials that do not produce an and soybean oil), Stearate, ethyl oleate, ethyl laurate, adverse, allergic or otherwise unwanted reaction when agar, syloid silica gel (AEROSIL 200, W.R. Grace Co., Bal administered to a patient. The carriers or mediums used can timore, Md. USA), a coagulated aerosol of synthetic silica include Solvents, dispersants, coatings, absorption promoting (Deaussa Co., Plano, Tex. USA), a pyrogenic silicon dioxide agents, controlled release agents, and one or more inert 15 (CAB-O-SIL, Cabot Co., Boston, Mass. USA), or mixtures excipients (which include starches, polyols, granulating thereof, agents, microcrystalline cellulose (e.g. celphere, Celphere ANTI-CAKING AGENTS: calcium silicate, magnesium Beads(R), diluents, lubricants, binders, disintegrating agents, silicate, silicon dioxide, colloidal silicon dioxide, talc, or and the like), etc. If desired, tablet dosages of the disclosed mixtures thereof, compositions may be coated by Standard aqueous or non ANTIMICROBIAL AGENTS: benzalkonium chloride, aqueous techniques. benzethonium chloride, benzoic acid, benzyl , butyl Compositions of the present disclosure may also optionally paraben, cetylpyridinium chloride, cresol, chlorobutanol, include other therapeutic ingredients, anti-caking agents, pre dehydroacetic acid, ethylparaben, methylparaben, , servatives, Sweetening agents, colorants, flavors, desiccants, phenylethyl alcohol, phenoxyethanol, phenylmercuric plasticizers, dyes, glidants, anti-adherents, anti-static agents, 25 acetate, phenylmercuric nitrate, potassium Sorbate, propylpa Surfactants (wetting agents), anti-oxidants, film-coating raben, Sodium benzoate, Sodium dehydro acetate, sodium agents, and the like. Any Such optional ingredient must be propionate, Sorbic acid, thimersol, thymo, or mixtures compatible with the compound described herein to insure the thereof, and stability of the formulation. COATING AGENTS: sodium carboxymethyl cellulose, The composition may contain other additives as needed, 30 cellulose acetate phthalate, ethylcellulose, gelatin, pharma including for example lactose, glucose, fructose, galactose, ceutical glaze, hydroxypropyl cellulose, hydroxypropyl trehalose, sucrose, maltose, raffinose, maltitol, melezitose, methylcellulose (hypromellose), hydroxypropyl methyl cel stachyose, lactitol, palatinite, starch, Xylitol, mannitol, myo lulose phthalate, methylcellulose, polyethylene glycol, poly inositol, and the like, and hydrates thereof, and amino acids, vinyl acetate phthalate, shellac, Sucrose, titanium dioxide, for example alanine, glycine and betaine, and peptides and 35 carnauba wax, microcrystalline wax, gellan gum, maltodex proteins, for example albumen. trin, methacrylates, microcrystalline cellulose and carrag Examples of excipients for use as the pharmaceutically eenan or mixtures thereof. acceptable carriers and the pharmaceutically acceptable inert The formulation can also include other excipients and cat carriers and the aforementioned additional ingredients egories thereof including but not limited to L-histidine, Plu include, but are not limited to binders, fillers, disintegrants, 40 ronic R, Poloxamers (such as Lutrol(R) and Poloxamer 188), lubricants, anti-microbial agents, and coating agents such as: ascorbic acid, glutathione, permeability enhancers (e.g. lip BINDERS: corn starch, potato starch, other starches, gela ids. Sodium cholate, acylcarnitine, Salicylates, mixed bile tin, natural and synthetic gums such as acacia, Xanthan, salts, fatty acid micelles, chelators, fatty acid, Surfactants, Sodium alginate, alginic acid, other alginates, powdered medium chain glycerides), protease inhibitors (e.g. soybean tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl 45 trypsin inhibitor, organic acids), pH lowering agents and cellulose, cellulose acetate, carboxymethyl cellulose cal absorption enhancers effective to promote bioavailability (in cium, Sodium carboxymethyl cellulose), polyvinyl pyrroli cluding but not limited to those described in U.S. Pat. No. done (e.g., povidone, crospovidone, copovidone, etc), methyl 6,086,918 and U.S. Pat. No. 5,912,014), creams and lotions cellulose, Methocel, pre-gelatinized starch (e.g., STARCH (like maltodextrin and carrageenans); materials for chewable 1500R and STARCH 1500 LMR, sold by Colorcon, Ltd.), 50 tablets (like dextrose, fructose, lactose monohydrate, lactose hydroxypropyl methyl cellulose, microcrystalline cellulose and aspartame, lactose and cellulose, maltodextrin, maltose, (e.g. AVICELTM, such as, AVICEL-PH-101TM, -103TM and mannitol, microcrystalline cellulose and guar gum, Sorbitol -105TM, sold by FMC Corporation, Marcus Hook, Pa., USA), crystalline); parenterals (like mannitol and povidone); plas or mixtures thereof, ticizers (like dibutyl sebacate, plasticizers for coatings, poly FILLERS: talc, calcium carbonate (e.g., granules or pow 55 vinylacetate phthalate); powder lubricants (like glyceryl der), dibasic calcium phosphate, tribasic calcium phosphate, behenate); Soft gelatin capsules (like Sorbitol special solu calcium sulfate (e.g., granules or powder), microcrystalline tion); spheres for coating (like Sugar spheres); spheronization cellulose, powdered cellulose, dextrates, kaolin, mannitol, agents (like glyceryl behenate and microcrystalline cellu silicic acid, Sorbitol, starch, pre-gelatinized starch, dextrose, lose); Suspending/gelling agents (like carrageenan, gellan fructose, honey, lactose anhydrate, lactose monohydrate, lac 60 gum, mannitol, microcrystalline cellulose, povidone, sodium tose and aspartame, lactose and cellulose, lactose and micro starch glycolate, Xanthan gum); Sweeteners (like aspartame, crystalline cellulose, maltodextrin, maltose, mannitol, micro aspartame and lactose, dextrose, fructose, honey, maltodex crystalline cellulose & guar gum, molasses, sucrose, or trin, maltose, mannitol, molasses, Sorbitol crystalline, Sorbi mixtures thereof, tol special Solution, Sucrose); wet granulation agents (like DISINTEGRANTS: agar-agar, alginic acid, calcium car 65 calcium carbonate, lactose anhydrous, lactose monohydrate, bonate, microcrystalline cellulose, croScarmellose Sodium, maltodextrin, mannitol, microcrystalline cellulose, povi crospovidone, polacrilin potassium, Sodium starch glycolate, done, starch), caramel, carboxymethylcellulose Sodium, US 8,779,090 B2 43 44 cherry cream flavor and cherry flavor, citric acid anhydrous, tered orally, e.g., as a tablet or cachet containing a predeter citric acid, confectioner's sugar, D&C Red No. 33, D&C mined amount of the active ingredient, gel, pellet, paste, Yellow #10 Aluminum Lake, disodium edetate, ethyl alcohol syrup, bolus, electuary, slurry, capsule, powder, lyophilized 15%, FD&C Yellow No. 6 aluminum lake, FD&C Blue #1 powder, granules, Sachet, as a solution or a suspension in an Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 alu aqueous liquid or a non-aqueous liquid, as an oil-in-water minum lake, FD&C Green No. 3, FD&C Red No. 40, FD&C liquid emulsion or a water-in-oil liquid emulsion, via a micel Yellow No. 6 Aluminum Lake, FD&C Yellow No. 6, FD&C lar formulation (see, e.g. WO 97/1 1682) via a liposomal Yellow No. 10, glycerol palmitostearate, glyceryl monostear formulation (see, e.g., EP 736299, WO 99/59550 and WO ate, indigo carmine, lecithin, manitol, methyl and propyl 97/13500), via formulations described in WO 03/094886, via parabens, mono ammonium glycyrrhizinate, natural and arti 10 bilosome (bile-salt based vesicular system), via a dendrimer, ficial orange flavor, pharmaceutical glaze, poloxamer 188, or in Some other form. Orally administered compositions can Polydextrose, polysorbate 20, polysorbate 80, polyvidone, include binders, lubricants, inert diluents, lubricating, Surface pregelatinized corn starch, pregelatinized starch, red iron active or dispersing agents, flavoring agents, and humectants. oxide, Saccharin Sodium, Sodium carboxymethyl ether, Orally administered formulations such as tablets may option Sodium chloride, Sodium citrate, Sodium phosphate, Straw 15 ally be coated or scored and may be formulated so as to berry flavor, synthetic black iron oxide, synthetic red iron provide sustained, delayed or controlled release of the active oxide, titanium dioxide, and white wax. ingredient therein. The agents can also be administered trans Solid oral dosage forms may optionally be treated with dermally (i.e. via reservoir-type or matrix-type patches, coating systems (e.g. Opadry(R) fix film coating system, for microneedles, thermal poration, hypodermic needles, ionto example Opadry(R) blue (OY-LS-20921), Opadry(R) white phoresis, electroporation, ultrasound or other forms of Sono (YS-2-7063), Opadry(R) white (YS-1-7040), and black ink phoresis, jet injection, or a combination of any of the preced (S-1-8106). ing methods (Prausnitz et al. 2004, Nature Reviews Drug The agents either in their free form or as a salt can be Discovery 3:115-124)). The agents can be administered using combined with a polymer Such as polylactic-glycoloic acid high-velocity transdermal particle injection techniques using (PLGA), poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) 25 the hydrogel particle formulation described in U.S. (WO 01/12233), polyglycolic acid (U.S. Pat. No. 3,773.919), 20020061336. Additional particle formulations are described polylactic acid (U.S. Pat. No. 4,767,628), poly(M-caprolac in WO 00/45792, WO 00/53160, and WO 02/19989. An tone) and poly(alkylene oxide) (U.S. 2003.0068384) to create example of a transdermal formulation containing plaster and a Sustained release formulation. Such formulations can be the absorption promoter dimethylisosorbide can be found in used to implants that release a peptide or another agent over a 30 WO 89/04179. WO 96/11705 provides formulations suitable period of a few days, a few weeks or several months depend for transdermal administration. The agents can be adminis ing on the polymer, the particle size of the polymer, and the tered in the form a suppository or by other vaginal or rectal size of the implant (see, e.g., U.S. Pat. No. 6,620,422). Other means. The agents can be administered in a transmembrane Sustained release formulations and polymers for use in are formulation as described in WO 90/07923. The agents can be described in EP 0467389 A2, WO 93/24150, U.S. Pat. No. 35 administered non-invasively via the dehydrated particles 5,612,052, WO 97/40085, WO 03/075887, WO 01/01964A2, described in U.S. Pat. No. 6,485,706. The agent can be admin U.S. Pat. No. 5,922,356, WO 94/155587, WO 02/074247A2, istered in an enteric-coated drug formulation as described in WO 98/25642, U.S. Pat. No. 5,968,895, U.S. Pat. No. 6, 180, WO 02/49621. The agents can be administered intranassaly 608, U.S. 2003.0171296, U.S. 20020176841, U.S. Pat. No. using the formulation described in U.S. Pat. No. 5,179,079. 5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat. No. 5,134,122, 40 Formulations suitable for parenteral injection are described in U.S. Pat. No. 5,192,741, U.S. Pat. No. 5,192,741, U.S. Pat. WO 00/62759. The agents can be administered using the No. 4,668,506, U.S. Pat. No. 4,713,244, U.S. Pat. No. 5,445, casein formulation described in U.S. 20030206939 and WO 832 U.S. Pat. No. 4,931,279, U.S. Pat. No. 5,980,945, WO 00/06108. The agents can be administered using the particu 02/058672, WO 9726015, WO 97/04744, and late formulations described in U.S. 2002.0034536. US20020019446. In such sustained release formulations 45 The agents, alone or in combination with other Suitable microparticles (Delie and Blanco-Prieto 2005 Molecule components, can be administered by pulmonary route utiliz 10:65-80) of peptide are combined with microparticles of ing several techniques including but not limited to intratra polymer. One or more Sustained release implants can be cheal instillation (delivery of solution into the lungs by placed in the large intestine, the small intestine or both. U.S. Syringe), intratracheal delivery of liposomes, insufflation (ad Pat. No. 6,011,011 and WO 94/06452 describe a sustained 50 ministration of powder formulation by Syringe or any other release formulation providing either polyethylene glycols similar device into the lungs) and aerosol inhalation. Aerosols (i.e. PEG 300 and PEG 400) or triacetin. WO 03/053401 (e.g., jet or ultrasonic nebulizers, metered-dose inhalers describes a formulation which may both enhance bioavail (MDIs), and dry-powder inhalers (DPIs)) can also be used in ability and provide controlled release of the agent within the intranasal applications. Aerosol formulations are stable dis GI tract. Additional controlled release formulations are 55 persions or Suspensions of Solid material and liquid droplets described in U.S. Pat. No. 6,734,188, WO 02/38129, EP326 in a gaseous medium and can be placed into pressurized 151, U.S. Pat. No. 5,236,704, WO 02/30398, WO 98/13029; acceptable propellants, such as hydrofluoroalkanes (HFAS, U.S. 20030064105, U.S. 2003.0138488A1, U.S. i.e. HFA-134a and HFA-227, or a mixture thereof), dichlo 20030216307A1, U.S. Pat. No. 6,667,060, WO 01/49249, rodifluoromethane (or other chlorofluocarbon propellants WO 01/49311, WO 01/49249, WO 01/49311, and U.S. Pat. 60 Such as a mixture of Propellants 11, 12, and/or 114), propane, No. 5,877,224. nitrogen, and the like. Pulmonary formulations may include The agents can be administered, e.g., by intravenous injec permeation enhancers such as fatty acids, Saccharides, chelat tion, intramuscular injection, Subcutaneous injection, intrap ing agents, enzyme inhibitors (e.g., protease inhibitors), adju eritoneal injection, topical, Sublingual, intraarticular (in the vants (e.g., glycocholate, Surfactin, span 85, and nafamoStat), joints), intradermal, buccal, ophthalmic (including intraocu 65 preservatives (e.g., benzalkonium chloride or chlorobutanol), lar), intranasaly (including using a cannula), intraspinally, and ethanol (normally up to 5% but possibly up to 20%, by intrathecally, or by other routes. The agents can be adminis weight). Ethanol is commonly included in aerosol composi US 8,779,090 B2 45 46 tions as it can improve the function of the metering valve and elland et al., 1987: Quintanar-Guerrero et al., 1998; Douglas in Some cases also improve the stability of the dispersion. et al., 1987). To avoid side effects due to intracellular poly Pulmonary formulations may also include Surfactants which meric overloading, ultrafine particles (sized around 0.1 um) include but are not limited to bile salts and those described in can be designed using polymers able to be degraded in vivo U.S. Pat. No. 6,524,557 and references therein. The surfac (e.g. biodegradable polyalkyl-cyanoacrylate nanoparticles). tants described in U.S. Pat. No. 6,524,557, e.g., a C8-C16 Such particles are described in the prior art (Couvreur et al. fatty acid salt, a bile salt, a phospholipid, or alkyl saccaride 1980; 1988: Zur Muhlen et al., 1998; Zambaux et al. 1998: are advantageous in that some of them also reportedly Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684). enhance absorption of the peptide in the formulation. Also The agents described herein can be formulated with pH suitable in the disclosure are dry powder formulations com 10 sensitive materials which may include those described in prising a therapeutically effective amount of active com WOO4041195 (including the seal and enteric coating pound blended with an appropriate carrier and adapted for use described therein) and pH-sensitive coatings that achieve in connection with a dry-powder inhaler. Absorption enhanc delivery in the colon including those described in U.S. Pat. ers which can be added to dry powder formulations of the No. 4,910,021 and WO9001329. U.S. Pat. No. 4,910,021 present disclosure include those described in U.S. Pat. No. 15 describes using a pH-sensitive material to coat a capsule. 6,632,456. WO 02/080884 describes new methods for the WO9001329 describes using pH-sensitive coatings on beads Surface modification of powders. Aerosol formulations may containing acid, where the acid in the bead core prolongs include U.S. Pat. No. 5,230,884, U.S. Pat. No. 5,292,499, WO dissolution of the pH-sensitive coating. U.S. Pat. No. 5,175, 017/8694, WO 01/78696, U.S. 2003019437, U.S. 003 discloses a dual mechanism polymer mixture composed 20030165436, and WO 96/40089 (which includes vegetable of pH-sensitive enteric materials and film-forming plasticiz oil). Sustained release formulations suitable for inhalation are ers capable of conferring permeability to the enteric material, described in U.S. 20010036481A1, 20030232019A1, and for use in drug-delivery systems; a matrix pellet composed of U.S. 20040018243A1 as well as in WO 01/13891, WO a dual mechanism polymer mixture permeated with a drug 02/067902, WO 03/072080, and WO 03/079885. Pulmonary and sometimes covering a pharmaceutically neutral nucleus; formulations containing microparticles are described in WO 25 a membrane-coated pellet comprising a matrix pellet coated 03/015750, U.S. 20030008013, and WO 00/00176. Pulmo with a dual mechanism polymer mixture envelope of the same nary formulations containing stable glassy state powder are or different composition; and a pharmaceutical dosage form described in U.S. 20020141945 and U.S. Pat. No. 6,309,671. containing matrix pellets. The matrix pellet releases acid Other aerosol formulations are described in EP 1338272A1 soluble drugs by diffusion in acid pH and by disintegration at WO 90/09781, U.S. Pat. No. 5,348,730, U.S. Pat. No. 6,436, 30 pH levels of nominally about 5.0 or higher. The agents 367, WO 91/04011, and U.S. Pat. No. 6,294,153 and U.S. Pat. described herein may be formulated in the pH triggered tar No. 6,290,987 describes a liposomal based formulation that geted control release systems described in WOO4052339. can be administered via aerosol or other means. Powder for The agents described herein may be formulated according to mulations for inhalation are described in U.S. 2003.0053960 the methodology described in any of WO03105812 (extruded and WO 01/60341. The agents can be administered intrana 35 hyrdratable polymers); WO0243767 (enzyme cleavable sally as described in U.S. 20010038824. The agents can be membrane translocators); WO03007913 and WO03086297 incorporated into microemulsions, which generally are ther (mucoadhesive systems); WO02072075 (bilayer laminated modynamically stable, isotropically clear dispersions of two formulation comprising pH lowering agent and absorption immiscible liquids, such as oil and water, Stabilized by an enhancer); WOO4O64769 (amidated peptides); interfacial film of surfactant molecules (Encyclopedia of 40 WO05063156 (solid lipid suspension with pseudotropic and/ Pharmaceutical Technology (New York: Marcel Dekker, or thixotropic properties upon melting); WO03035029 and 1992), volume 9). For the preparation of microemulsions, WO03035041 (erodible, gastric retentive dosage forms): Surfactant (emulsifier), co-surfactant (co-emulsifier), an oil U.S. Pat. No. 5,007,790 and U.S. Pat. No. 5,972,389 (sus phase and a water phase are necessary. Suitable Surfactants tained release dosage forms); WOO4112711 (oral extended include any Surfactants that are useful in the preparation of 45 release compositions); WO05027878, WO02072033, and emulsions, e.g., emulsifiers that are typically used in the WO02072034 (delayed release compositions with natural or preparation of creams. The co-surfactant (or “co-emulsifer') synthetic gum); WO05030182 (controlled release formula is generally selected from the group of polyglycerol deriva tions with an ascending rate of release); WO05048998 (mi tives, glycerol derivatives and fatty . Preferred emul croencapsulation system); U.S. Pat. No. 5,952.314 (biopoly sifier/co-emulsifier combinations are generally although not 50 mer); U.S. Pat. No. 5,108,758 (glassy amylose matrix necessarily selected from the group consisting of glyceryl delivery); U.S. Pat. No. 5,840,860 (modified starch based monostearate and polyoxyethylene Stearate; polyethylene delivery). JP10324642 (delivery system comprising chitosan glycol and ethylene glycol palmitostearate; and caprilic and and gastric resistant material Such as wheat gliadin or Zein); capric triglycerides and oleoyl macrogolglycerides. The U.S. Pat. No. 5,866.619 and U.S. Pat. No. 6,368,629 (saccha water phase includes not only water but also, typically, buff 55 ride containing polymer); U.S. Pat. No. 6,531,152 (describes ers, glucose, propylene glycol, polyethylene glycols, prefer a drug delivery system containing a water soluble core (Ca ably lower molecular weight polyethylene glycols (e.g., PEG pectinate or other water-insoluble polymers) and outer coat 300 and PEG 400), and/or glycerol, and the like, while the oil which bursts (eg hydrophobic polymer-Eudragrit)); U.S. Pat. phase will generally comprise, for example, fatty acid esters, No. 6,234,464; U.S. Pat. No. 6,403,130 (coating with poly modified vegetable oils, silicone oils, mixtures of mono- di 60 mercontaining casein and high methoxy pectin; WOO174175 and triglycerides, mono- and di-esters of PEG (e.g., oleoyl (Maillard reaction product); WO05063206 (solubility macrogol glycerides), etc. increasing formulation); WOO4019872 (transferring fusion The agents described herein can be incorporated into phar proteins). The agents described herein may be formulated maceutically-acceptable nanoparticle, nanosphere, and nano using gastrointestinal retention system technology (GIRES; capsule formulations (Delie and Blanco-Prieto 2005 Mol 65 Merrion Pharmaceuticals). GIRES comprises a controlled ecule 10:65-80). Nanocapsules can generally entrap release dosage form inside an inflatable pouch, which is compounds in a stable and reproducible way (Henry-Mich placed in a drug capsule for oral administration. Upon disso US 8,779,090 B2 47 48 lution of the capsule, a gas-generating system inflates the terpolymers which are sensitive to pH and temperature which pouch in the stomach where it is retained for 16-24 hours, all are useful carriers for conducting bioactive agents through the the time releasing agents described herein. gastric juices of the stomach in a protected form. The terpoly The agents described herein can be formulated in an mers swell at the higher physiologic pH of the intestinal tract osmotic device including the ones disclosed in U.S. Pat. No. 5 causing release of the bioactive agents into the intestine. The 4,503,030, U.S. Pat. No. 5,609,590 and U.S. Pat. No. 5,358, terpolymers are linear and are made up of 35 to 99 wt % of a 502. U.S. Pat. No. 4,503,030 discloses an osmotic device for temperature sensitive component, which imparts to the ter dispensing a drug to certain pH regions of the gastrointestinal polymer LCST (lower critical solution temperature) proper tract. More particularly, the disclosure relates to an osmotic ties below body temperatures, 1 to 30 wt % of a pH sensitive device comprising a wall formed of a semi-permeable pH 10 component having a pKa in the range of from 2 to 8 which sensitive composition that Surrounds a compartment contain functions through ionization or deionization of carboxylic ing a drug, with a passageway through the wall connecting the acid groups to prevent the bioactive agent from being lost at exterior of the device with the compartment. The device low pH but allows bioactive agent release at physiological pH delivers the drug at a controlled rate in the region of the of about 7.4 and a hydrophobic component which stabilizes gastrointestinal tract having a pH of less than 3.5, and the 15 the LCST below body temperatures and compensates for device self-destructs and releases all its drug in the region of bioactive agent effects on the terpolymers. The terpolymers the gastrointestinal tract having a pH greater than 3.5, thereby provide for safe bioactive agent loading, a simple procedure providing total availability for drug absorption. U.S. Pat. Nos. for dosage form fabrication and the terpolymer functions as a 5,609.590 and 5.358.502 disclose an osmotic bursting device protective carrier in the acidic environment of the stomach for dispensing a beneficial agent to an aqueous environment. and also protects the bioactive agents from digestive enzymes The device comprises a beneficial agent and osmagent Sur until the bioactive agent is released in the intestinal tract. rounded at least in part by a semi-permeable membrane. The The agents described herein may be formulated in pH beneficial agent may also function as the osmagent. The semi sensitive polymers according to those described in U.S. Pat. permeable membrane is permeable to water and substantially No. 6,103,865. U.S. Pat. No. 6,103,865 discloses pH-sensi impermeable to the beneficial agent and osmagent. A trigger 25 tive polymers containing Sulfonamide groups, which can be means is attached to the semi-permeable membrane (e.g., changed in physical properties, such as Swellability and solu joins two capsule halves). The trigger means is activated by a bility, depending on pH and which can be applied for a drug pH of from 3 to 9 and triggers the eventual, but sudden, delivery system, bio-material, sensor, and the like, and a delivery of the beneficial agent. These devices enable the preparation method therefore. The pH-sensitive polymers are pH-triggered release of the beneficial agent core as a bolus by 30 prepared by introduction of Sulfonamide groups, various in osmotic bursting. pKa, to hydrophilic groups of polymers either through cou The agents described herein may be formulated based on pling to the hydrophilic groups of polymers, such as acryla the disclosure described in U.S. Pat. No. 5,316,774 which mide, N,N-dimethylacrylamide, acrylic acid, N-isopropy discloses a composition for the controlled release of an active lacrylamide and the like or copolymerization with other Substance comprising a polymeric particle matrix, where 35 polymerizable monomers. These pH-sensitive polymers may each particle defines a network of internal pores. The active have a structure of linear polymer, grafted copolymer, hydro substance is entrapped within the pore network together with gel or interpenetrating network polymer. a blocking agent having physical and chemical characteristics The agents described herein may be formulated according selected to modify the release rate of the active substance U.S. Pat. No. 5,656.292 which discloses a composition for pH from the internal pore network. In one embodiment, drugs 40 dependent or pH regulated controlled release of active ingre may be selectively delivered to the intestines using an enteric dients especially drugs. The composition consists of a com material as the blocking agent. The enteric material remains pactable mixture of the active ingredient and starch molecules intact in the stomach but degrades under the pH conditions of substituted with acetate and dicarboxylate residues. The pre the intestines. In another embodiment, the Sustained release ferred dicarboxylate acid is succinate. The average substitu formulation employs a blocking agent, which remains stable 45 tion degree of the acetate residue is at least 1 and 0.2-1.2 for under the expected conditions of the environment to which the dicarboxylate residue. The starch molecules can have the the active substance is to be released. The use of pH-sensitive acetate and dicarboxylate residues attached to the same starch materials alone to achieve site-specific delivery is difficult molecule backbone or attached to separate starch molecule because of leaking of the beneficial agent prior to the release backbones. The present disclosure also discloses methods for site or desired delivery time and it is difficult to achieve long 50 preparing said starch acetate dicarboxylates by transesterifi time lags before release of the active ingredient after exposure cation or mixing of starch acetates and starch dicarboxylates to high pH (because of rapid dissolution or degradation of the respectively. pH-sensitive materials). The agents described herein may be formulated according The agents may also be formulated in a hybrid system to the methods described in U.S. Pat. Nos. 5,554,147, 5,788, which combines pH-sensitive materials and osmotic delivery 55 687, and 6,306.422 which disclose a method for the con systems. These hybrid devices provide delayed initiation of trolled release of a biologically active agent wherein the agent Sustained-release of the beneficial agent. In one device a is released from a hydrophobic, pH-sensitive polymer matrix. pH-sensitive matrix or coating dissolves releasing osmotic The polymer matrix swells when the environment reaches pH devices that provide sustained release of the beneficial agent 8.5, releasing the active agent. A polymer of hydrophobic and see U.S. Pat. Nos. 4,578,075, 4,681,583, and 4,851,231. A 60 weakly acidic comonomers is disclosed for use in the con second device consists of a semipermeable coating made of a trolled release system. Also disclosed is a specific embodi polymer blend of an insoluble and a pH-sensitive material. As ment in which the controlled release system may be used. The the pH increases, the permeability of the coating increases, pH-sensitive polymer is coated onto a latex catheter used in increasing the rate of release of beneficial agent see U.S. Pat. ureteral catheterization. A ureteral catheter coated with a Nos. 4,096,238, 4,503,030, 4,522,625, and 4,587,117. 65 pH-sensitive polymer having an antibiotic or urease inhibitor The agents described herein may be formulated interpol trapped within its matrix will release the active agent when umers according to U.S. Pat. No. 5,484,610 which discloses exposed to high pH urine. US 8,779,090 B2 49 50 The agents described herein may be formulated in/with Ann. Hum. Genet. 37:219. For example, in EP 322,094, bioadhesive polymers according to U.S. Pat. No. 6,365,187. various shorter forms of HSA. Some of these fragments of Bioadhesive polymers in the form of, or as a coating on, HSA are disclosed, including HSA(1-373), HSA(1-388), microcapsules containing drugs or bioactive substances HSA(1-389), HSA(1-369), and HSA(1-419) and fragments which may serve for therapeutic, or diagnostic purposes in between 1-369 and 1-419. EP 399,666 discloses albumin diseases of the gastrointestinal tract, are described in U.S. Pat. fragments that include HSA(1-177) and HSA(1-200) and No. 6,365,187. The polymeric microspheres all have a bio fragments between HSA(1-177) and HSA(1-200). Methods adhesive force of at least 11 mN/cm (110 N/m2) Techniques related to albumin fusion proteins can be found in U.S. Pat. for the fabrication of bioadhesive microspheres, as well as a No. 7,056,701, U.S. Pat. No. 6,994,857, U.S. Pat. No. 6,946, method for measuring bioadhesive forces between micro 10 spheres and selected segments of the gastrointestinal tract in 134, U.S. Pat. No. 6,926,898, and U.S. Pat. No. 6,905,688 and vitro are also described. This quantitative method provides a the related priority documents and references cited therein. means to establish a correlation between the chemical nature, The agent can also be conjugated to polyethylene glycol the Surface morphology and the dimensions of drug-loaded (PEG) chains. Methods for pegylation and additional formu microspheres on one hand and bioadhesive forces on the 15 lations containing PEG-conjugates (i.e. PEG-based hydro other, allowing the screening of the most promising materials gels, PEG modified liposomes) can be found in Harris and from a relatively large group of natural and synthetic poly Chess, Nature Reviews Drug Discovery 2: 214-221 and the mers which, from theoretical consideration, should be used references therein. Peptides can also be modified with alkyl for making bioadhesive microspheres. Solutions of medica groups (e.g., C1-C20 straight or branched alkyl groups); fatty ment in buffered saline and similar vehicles are commonly acid radicals; and combinations of PEG, alkyl groups and employed to generate an aerosol in a nebulizer. Simple nebu fatty acid radicals (see U.S. Pat. No. 6,309,633; Soltero et al., lizers operate on Bernoulli’s principle and employ a stream of 2001 Innovations in Pharmaceutical Technology 106-110). air or oxygen to generate the spray particles. More complex The agent can be administered via a nanocochleate or cochle nebulizers employ ultrasound to create the spray particles. ate delivery vehicle (BioDelivery Sciences International). Both types are well known in the art and are described in 25 The agents can be delivered transmucosally (i.e. across a standard textbooks of pharmacy Such as Sprowls American mucosal Surface Such as the vagina, eye or nose) using for Pharmacy and Remington's The Science and Practice of mulations such as that described in U.S. Pat. No. 5,204,108. Pharmacy. Other devices for generating aerosols employ The agents can be formulated in microcapsules as described compressed gases, usually hydrofluorocarbons and chlorof in WO 88701165. The agent can be administered intra-orally luorocarbons, which are mixed with the medicament and any 30 using the formulations described in U.S. 20020055496, WO necessary excipients in a pressurized container, these devices 00/47203, and U.S. Pat. No. 6,495,120. The agent can be are likewise described in standard textbooks such as Sprowls delivered using nanoemulsion formulations described in WO and Remington. O1/91728A2. The agents can be a free acid or base, or a pharmacologi Controlled Release Formulations cally acceptable salt thereof. Solids can be dissolved or dis 35 In general, one can provide for controlled release of the persed immediately prior to administration or earlier. In some agents described herein through the use of a wide variety of circumstances the preparations include a preservative to pre polymeric carriers and controlled release systems including vent the growth of microorganisms. The pharmaceutical erodible and non-erodible matrices, osmotic control devices, forms suitable for injection can include sterile aqueous or various reservoir devices, enteric coatings and multiparticu organic Solutions or dispersions which include, e.g., water, an 40 late control devices. alcohol, an organic solvent, an oil or other solvent or dispers Matrix devices are a common device for controlling the ant (e.g., glycerol, propylene glycol, polyethylene glycol, and release of various agents. In such devices, the agents Vegetable oils). The formulations may contain antioxidants, described herein are generally present as a dispersion within buffers, bacteriostats, and solutes that render the formulation the polymer matrix, and are typically formed by the compres isotonic with the blood of the intended recipient, and aqueous 45 sion of a polymer/drug mixture or by dissolution or melting. and non-aqueous sterile Suspensions that can include Sus The dosage release properties of these devices may be depen pending agents, solubilizers, thickening agents, stabilizers, dent upon the solubility of the agent in the polymer matrix or, and preservatives. Pharmaceutical agents can be sterilized by in the case of porous matrices, the Solubility in the sink filter sterilization or by other suitable means. The agent can be solution within the pore network, and the tortuosity of the fused to immunoglobulins or albumin, albumin variants or 50 network. In one instance, when utilizing an erodible poly fragments thereof, or incorporated into a liposome to improve meric matrix, the matrix imbibes water and forms an aque half-life. Thus the peptides described herein may be fused ous-swollengel that entraps the agent. The matrix then gradu directly or via a peptide linker, water soluble polymer, or ally erodes, Swells, disintegrates or dissolves in the GI tract, prodrug linker to albumin or an analog, fragment, or deriva thereby controlling release of one or more of the agents tive thereof. Generally, the albumin proteins that are part of 55 described herein. In non-erodible devices, the agent is the fusion proteins of the present disclosure may be derived released by diffusion through an inert matrix. from albumin cloned from any species, including human. Agents described herein can be incorporated into an erod Human serum albumin (HSA) consists of a single non-gly ible or non-erodible polymeric matrix controlled release cosylated peptide chain of 585 amino acids with a formula device. By an erodible matrix is meant aqueous-erodible or molecular weight of 66,500. The amino acid sequence of 60 water-swellable or aqueous-Soluble in the sense of being human HSA is known See Meloun, et al. (1975) FEBS either erodible or swellable or dissolvable in pure water or Letters 58:136: Behrens, et al. (1975) Fed. Proc. 34:591; requiring the presence of an acid or base to ionize the poly Lawn, et al. (1981) Nucleic Acids Research 9:6102-6114; meric matrix Sufficiently to cause erosion or dissolution. Minghetti, et al. (1986) J. Biol. Chem. 261:6747, each of When contacted with the aqueous environment of use, the which are incorporated by reference herein. A variety of 65 erodible polymeric matrix imbibes water and forms an aque polymorphic variants as well as analogs and fragments of ous-swollen gel or matrix that entraps the agent described albumin have been described. See Weitkamp, et al., (1973) herein. The aqueous-swollen matrix gradually erodes, Swells, US 8,779,090 B2 51 52 disintegrates or dissolves in the environment of use, thereby lacrylate, (2-dimethylaminoethyl) methacrylate, and controlling the release of a compound described herein to the (trimethylaminoethyl) methacrylate chloride. environment of use. The erodible matrix polymer may contain a wide variety of The erodible polymeric matrix into which an agent the same types of additives and excipients known in the described herein can be incorporated may generally be pharmaceutical arts, including osmopolymers, osmagens, described as a set of excipients that are mixed with the agent solubility-enhancing or -retarding agents and excipients that following its formation that, when contacted with the aqueous promote stability or processing of the device. environment of use imbibes water and forms a water-swollen Alternatively, the agents of the present disclosure may be gel or matrix that entraps the drug form. Drug release may administered by or incorporated into a non-erodible matrix occur by a variety of mechanisms, for example, the matrix 10 device. In Such devices, an agent described herein is distrib may disintegrate or dissolve from around particles or granules uted in an inert matrix. The agent is released by diffusion of the agent or the agent may dissolve in the imbibed aqueous through the inert matrix. Examples of materials suitable for solution and diffuse from the tablet, beads or granules of the the inert matrix include insoluble plastics (e.g. methyl acry device. One ingredient of this water-swollen matrix is the late-methyl methacrylate copolymers, polyvinyl chloride, water-swellable, erodible, or soluble polymer, which may 15 polyethylene), hydrophilic polymers (e.g. ethyl cellulose, generally be described as an osmopolymer, hydrogel or cellulose acetate, crosslinked polyvinylpyrrolidone (also water-swellable polymer. Such polymers may be linear, known as crospovidone)), and fatty compounds (e.g. car branched, or crosslinked. The polymers may be homopoly nauba wax, microcrystalline wax, and triglycerides). Such mers or copolymers. In certain embodiments, they may be devices are described further in Remington: The Science and synthetic polymers derived from vinyl, acrylate, methacry Practice of Pharmacy, 20th edition (2000). late, urethane, ester and oxide monomers. In other embodi Matrix controlled release devices may be prepared by ments, they can be derivatives of naturally occurring poly blending an agent described herein and other excipients mers such as polysaccharides (e.g. chitin, chitosan, dextran together, and then forming the blend into a tablet, caplet, pill, and pullulan; gum agar, gum arabic, gum karaya, locust bean or other device formed by compressive forces. Such com gum, gum tragacanth, carrageenans, gum ghatti, guar gum, 25 pressed devices may beformed using any of a wide variety of Xanthan gum and Scleroglucan), starches (e.g. dextrin and presses used in the fabrication of pharmaceutical devices. maltodextrin), hydrophilic colloids (e.g. pectin), phosphati Examples include single-punch presses, rotary tablet presses, des (e.g. lecithin), alginates (e.g. ammonium alginate, and multilayer rotary tablet presses, all well known in the art. Sodium, potassium or calcium alginate, propylene glycol See for example, Remington: The Science and Practice of alginate), gelatin, collagen, and cellulosics. Cellulosics are 30 Pharmacy, 20th Edition, 2000. The compressed device may cellulose polymer that has been modified by reaction of at be of any shape, including round, oval, oblong, cylindrical, or least a portion of the hydroxyl groups on the saccharide repeat triangular. The upper and lower surfaces of the compressed units with a compound to form an ester-linked or an ether device may be flat, round, concave, or convex. linked substituent. For example, the cellulosic ethyl cellulose In certain embodiments, when formed by compression, the has an ether linked ethyl substituent attached to the saccharide 35 device has a strength of at least 5 Kiloponds (Kp)/cm (for repeat unit, while the cellulosic cellulose acetate has an ester example, at least 7 Kp/cm). Strength is the fracture force, linked acetate substituent. In certain embodiments, the cellu also known as the tablet hardness required to fracture a tablet losics for the erodible matrix comprises aqueous-soluble and formed from the materials, divided by the maximum cross aqueous-erodible cellulosics can include, for example, ethyl sectional area of the tablet normal to that force. The fracture cellulose (EC), methylethyl cellulose (MEC), carboxymethyl 40 force may be measured using a Schleuniger Tablet Hardness cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), Tester, Model 6D. The compression force required to achieve hydroxypropyl cellulose (HPC), cellulose acetate (CA), cel this strength will depend on the size of the tablet, but gener lulose propionate (CP), cellulose butyrate (CB), cellulose ally will be greater than about 5 kP/cm. Friability is a well acetate butyrate (CAB), CAP CAT, hydroxypropyl methyl know measure of a device's resistance to Surfaceabrasion that cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl 45 measures weight loss in percentage after Subjecting the methyl cellulose acetate trimellitate (HPMCAT), and ethyl device to a standardized agitation procedure. Friability values hydroxyethylcellulose (EHEC). In certain embodiments, the of from 0.8 to 1.0% are regarded as constituting the upper cellulosics comprises various grades of low viscosity (MW limit of acceptability. Devices having a strength of greater less than or equal to 50,000 daltons, for example, the Dow than 5 kP/cm generally are very robust, having a friability of MethocelTM series E5, E15LV, E50LV and K10OLY) and high 50 less than 0.5%. Other methods for forming matrix controlled viscosity (MW greater than 50,000 daltons, for example, release devices are well known in the pharmaceutical arts. See E4MCR, E1OMCR, K4M, K15M and K10OM and the for example, Remington: The Science and Practice of Phar MethocelTM K series) HPMC. Other commercially available macy, 20th Edition, 2000. types of HPMC include the Shin Etsu Metolose 90SH series. As noted above, the agents described herein may also be The choice of matrix material can have a large effect on the 55 incorporated into an osmotic control device. Such devices maximum drug concentration attained by the device as well generally include a core containing one or more agents as as the maintenance of a high drug concentration. The matrix described herein and a water permeable, non-dissolving and material can be a concentration-enhancing polymer, for non-eroding coating Surrounding the core which controls the example, as described in WO05/011634. influx of water into the core from an aqueous environment of Other materials useful as the erodible matrix material 60 use so as to cause drug release by extrusion of some or all of include, but are not limited to, pullulan, polyvinyl pyrroli the core to the environment of use. In certain embodiments, done, polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid the coating is polymeric, aqueous-permeable, and has at least esters, polyacrylamide, polyacrylic acid, copolymers of one delivery port. The core of the osmotic device optionally ethacrylic acid or methacrylic acid (EUDRAGITO, Rohm includes an osmotic agent which acts to imbibe water from America, Inc., Piscataway, N.J.) and other acrylic acid deriva 65 the Surrounding environment via Such a semi-permeable tives such as homopolymers and copolymers of butyl membrane. The osmotic agent contained in the core of this methacrylate, methylmethacrylate, ethylmethacrylate, ethy device may be an aqueous-swellable hydrophilic polymer or US 8,779,090 B2 53 54 it may be an osmogen, also known as an osmagent. Pressure palmitol), fatty acid esters (e.g. glyceryl (mono- and di-) is generated within the device which forces the agent(s) out of Stearates, triglycerides, glyceryl (palmitics tearic) ester, Sor the device via an orifice (of a size designed to minimize solute bitan esters (e.g. Sorbitan monostearate, saccharose diffusion while preventing the build-up of a hydrostatic pres monostearate, Saccharose monopalmitate, sodium Stearyl Sure head). fumarate), polyoxyethylene Sorbitan esters), Surfactants (e.g. Osmotic agents create a driving force for transport of water alkyl Sulfates (e.g. sodium lauryl Sulfate, magnesium lauryl from the environment of use into the core of the device. Sulfate), polymers (e.g. polyethylene glycols, polyoxyethyl Osmotic agents include but are not limited to water-swellable ene glycols, polyoxyethylene, polyoxypropylene ethers, hydrophilic polymers, and osmogens (or osmagens). Thus, including copolymers thereof), polytetrafluoroethylene), and the core may include water-swellable hydrophilic polymers, 10 inorganic materials (e.g. talc, calcium phosphate), cyclodex both ionic and nonionic, often referred to as osmopolymers trins, Sugars (e.g. lactose, Xylitol), Sodium starch glycolate). and hydrogels. The amount of water-swellable hydrophilic Nonlimiting examples of disintegrants are sodium starch gly polymers present in the core may range from about 5 to about colate (e.g., ExplotabM CLV. (microcrystalline cellulose 80 wt % (including for example, 10 to 50 wt %). Nonlimiting (e.g., AviceITM), microcrystalline silicified cellulose (e.g., examples of core materials include hydrophilic vinyl and 15 ProSolviM), croscarmellose sodium (e.g., Ac-Di-SolTM). acrylic polymers, polysaccharides such as calcium alginate, When the agent described herein is a solidamorphous disper polyethylene oxide (PEO), polyethylene glycol (PEG), sion formed by a solvent process, such additives may be polypropylene glycol (PPG), poly (2-hydroxyethyl meth added directly to the spray-drying solution when forming an acrylate), poly (acrylic) acid, poly (methacrylic) acid, poly agent described herein/concentration-enhancing polymer vinylpyrrolidone (PVP) and crosslinked PVP polyvinyl alco dispersion such that the additive is dissolved or suspended in hol (PVA). PVA/PVP copolymers and PVA/PVP copolymers the solution as a slurry, Alternatively, such additives may be with hydrophobic monomers such as methyl methacrylate, added following the spray-drying process to aid in forming vinyl acetate, and the like, hydrophilic polyurethanes con the final controlled release device. taining large PEO blocks, sodium croscarmellose, carrag A nonlimiting example of an osmotic device consists of eenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellu 25 one or more drug layers containing an agent described herein, lose (HPC), hydroxypropyl methyl cellulose (HPMC), Such as a Solid amorphous drug/polymer dispersion, and a carboxymethyl cellulose (CMC) and carboxyethyl cellulose sweller layer that comprises a water-swellable polymer, with (CEC), sodium alginate, polycarbophil, gelatin, Xanthan a coating Surrounding the drug layer and Sweller layer. Each gum, and sodium starch glycolat. Other materials include layer may contain other excipients such as tableting aids, hydrogels comprising interpenetrating networks of polymers 30 oSmagents, Surfactants, water-soluble polymers and water that may be formed by addition or by condensation polymer swellable polymers. ization, the components of which may comprise hydrophilic Such osmotic delivery devices may be fabricated in various and hydrophobic monomers such as those just mentioned. geometries including bilayer (wherein the core comprises a Water-swellable hydrophilic polymers include but are not drug layer and a Sweller layer adjacent to each other), trilayer limited to PEO, PEG, PVP, sodium croscarmellose, HPMC, 35 (wherein the core comprises a sweller layer sandwiched Sodium starch glycolate, polyacrylic acid and crosslinked between two drug layers) and concentric (wherein the core versions or mixtures thereof. comprises a central Sweller agent Surrounded by the drug The core may also include an osmogen (or osmagent). The layer). The coating of Such a tablet comprises a membrane amount of osmogen present in the core may range from about permeable to water but substantially impermeable to drug and 2 to about 70 wt % (including, for example, from 10 to 50 wt 40 excipients contained within. The coating contains one or %). Typical classes of Suitable osmogens are water-soluble more exit passageways or ports in communication with the organic acids, salts and Sugars that are capable of imbibing drug-containing layer(s) for delivering the drug agent. The water to thereby effect an osmotic pressure gradient across drug-containing layer(s) of the core contains the drug agent the barrier of the Surrounding coating. Typical useful (including optional osmagents and hydrophilic water-soluble osmogens include but are not limited to magnesium Sulfate, 45 polymers), while the sweller layer consists of an expandable magnesium chloride, calcium chloride, Sodium chloride, hydrogel, with or without additional osmotic agents. lithium chloride, potassium Sulfate, Sodium carbonate, When placed in an aqueous medium, the tablet imbibes Sodium sulfite, lithium sulfate, potassium chloride, sodium water through the membrane, causing the agent to form a Sulfate, mannitol. Xylitol, urea, Sorbitol, inositol, raffinose, dispensable aqueous agent, and causing the hydrogel layer to Sucrose, glucose, fructose, lactose, citric acid. Succinic acid, 50 expand and push against the drug-containing agent, forcing tartaric acid, and mixtures thereof. In certain embodiments, the agent out of the exit passageway. The agent can Swell, the osmogen is glucose, lactose, Sucrose, mannitol. Xylitol, aiding inforcing the drug out of the passageway. Drug can be Sodium chloride, including combinations thereof. delivered from this type of delivery system either dissolved or The core may include a wide variety of additives and dispersed in the agent that is expelled from the exit passage excipients that enhance the performance of the dosage form 55 way. or that promote stability, tableting or processing. Such addi The rate of drug delivery is controlled by such factors as the tives and excipients include tableting aids, Surfactants, water permeability and thickness of the coating, the osmotic pres soluble polymers, pH modifiers, fillers, binders, pigments, Sure of the drug-containing layer, the degree of hydrophilicity disintegrants, antioxidants, lubricants and flavorants. Non of the hydrogel layer, and the surface area of the device. Those limiting examples of additives and excipients include but are 60 skilled in the art will appreciate that increasing the thickness not limited to those described elsewhere herein as well as of the coating will reduce the release rate, while any of the microcrystalline cellulose, metallic salts of acids (e.g. alumi following will increase the release rate: increasing the per num Stearate, calcium Stearate, magnesium Stearate, sodium meability of the coating; increasing the hydrophilicity of the Stearate, Zinc stearate), pH control agents (e.g. buffers, hydrogel layer, increasing the osmotic pressure of the drug organic acids, organic acid salts, organic and inorganic 65 containing layer, or increasing the device's Surface area. bases), fatty acids, hydrocarbons and fatty alcohols (e.g. Other materials useful in forming the drug-containing Stearic acid, palmitic acid, liquid paraffin, Stearyl alcohol, and agent, in addition to the agent described herein itself, include US 8,779,090 B2 55 56 HPMC, PEO and PVP and other pharmaceutically acceptable tegrants known to those skilled in the art. Depending upon the carriers. In addition, osmagents such as Sugars or salts, particular formulation, Some disintegrants work better than including but not limited to Sucrose, lactose, Xylitol, manni others. Several disintegrants tend to form gels as they swell tol, or sodium chloride, may be added. Materials which are with water, thus hindering drug delivery from the device. useful for forming the hydrogel layer include sodium CMC, Non-gelling, non-Swelling disintegrants provide a more rapid PEO (e.g. polymers having an average molecular weight from dispersion of the drug particles within the core as water enters about 5,000,000 to about 7,500,000 daltons), poly (acrylic the core. In certain embodiments, non-gelling, non-Swelling acid), sodium (polyacrylate), sodium croscarmellose, sodium disintegrants are resins, for example, ion-exchange resins. In starch glycolat, PVP, crosslinked PVP, and other high one embodiment, the resin is AmberliteTM IRP 88 (available molecular weight hydrophilic materials. 10 from Rohm and Haas, Philadelphia, Pa.). When used, the In the case of a bilayer geometry, the delivery port(s) or exit disintegrant is present in amounts ranging from about passageway(s) may be located on the side of the tablet con 50-74% of the core agent. taining the drug agent or may be on both sides of the tablet or Water-soluble polymers are added to keep particles of the even on the edge of the tablet so as to connect both the drug agent suspended inside the device before they can be deliv layer and the sweller layer with the exterior of the device. The 15 ered through the passageway(s) (e.g., an orifice). High vis exit passageway(s) may be produced by mechanical means or cosity polymers are useful in preventing settling. However, by laser drilling, or by creating a difficult-to-coat region on the polymer in combination with the agent is extruded the tablet by use of special tooling during tablet compression through the passageway(s) under relatively low pressures. At or by other means. a given extrusion pressure, the extrusion rate typically slows The osmotic device can also be made with a homogeneous with increased viscosity. Certain polymers in combination core Surrounded by a semipermeable membrane coating, as in with particles of the agent described hereinform high viscos U.S. Pat. No. 3,845,770. The agent described herein can be ity solutions with water but are still capable of being extruded incorporated into a tablet core and a semipermeable mem from the tablets with a relatively low force. In contrast, poly brane coating can be applied via conventional tablet-coating mers having a low weight-average, molecular weight (