USOO8933O3OB2
(12) United States Patent (10) Patent No.: US 8,933,030 B2 Fretzen et al. (45) Date of Patent: Jan. 13, 2015
(54) TREATMENTS FOR GASTROINTESTINAL 5,654,278 A 8, 1997 Sorensen DSORDERS 5,904,935 A 5/1999 Eckenhoff et al. 6,068,850 A 5, 2000 Stevenson et al. 6,124,261 A 9, 2000 Stevenson et al. (75) Inventors: Angelika Fretzen, Somerville, MA 6,541,606 B2 4/2003 Margolin et al. (US); Hong Zhao, Lexington, MA (US); 6,734,162 B2 5/2004 Van Antwerp et al. Marco Kessler, Danvers, MA (US) 6,828.303 B2 12/2004 Kim et al. 6,979,437 B2 12/2005 Bartus et al. (73) Assignee: Ironww.od Pharmaceuticals, Inc., 6,995,200 B2 2/2006 Krohnke. Cambridge, MA (US 7,056,942 B2 6/2006 Hildesheim et al. ambridge, MA (US) 7,141,254 B2 11/2006 Bhaskaran et al. 7,304,036 B2 12/2007 Currie et al. (*) Notice: Subject to any disclaimer, the term of this 7.351,798 B2 4/2008 Margolin et al. patent is extended or adjusted under 35 7,371,727 B2 5/2008 Currie et al. U.S.C. 154(b) by 0 days. 7,494,979 B2 2/2009 Currie et al. 7,704,947 B2 4/2010 Currie et al. 7,745.409 B2 6, 2010 Currie et al. (21) Appl. No.: 13/579,685 7,767,644 B2 8/2010 Schumann et al. 7,772,188 B2 8, 2010 Currie et al. (22) PCT Filed: Feb. 17, 2011 7.910,546 B2 3/2011 Currie et al. 8,080,526 B2 12/2011 Currie et al. (86). PCT No.: PCT/US2011/025274 8, 101,579 B2 1/2012 Currie et al. 8, 110,553 B2 2/2012 Currie et al. S371 (c)(1), 2003,0003563 A1 1/2003 Vinkemeier et al. (2), (4) Date: Mar. 29, 2013 2003, OO691.82 A1 4/2003 Rinella 2003, OO73628 A1 4/2003 Shailubhai et al. (87) PCT Pub. No.: WO2011/103311 2003. O104996 A1 6/2003 Li et al. PCT Pub. Date: Aug. 25, 2011 (Continued)Continued FOREIGN PATENT DOCUMENTS (65) Prior Publication Data US 2013/0190238 A1 Jul 25, 2013 E. 208,383 4.38 US 2014/OO73571 A9 Mar. 13, 2014 (Continued) Related U.S. Application Data OTHER PUBLICATIONS (60) Provisional application No. 61/305,465, filed on Feb. Heck etal “Modification and Inhibition of Vancomycin Group Anti 17, 2010. biotics by Formaldehyde and Acetaldehyde' Chemistry a European s Journal 7:910-916. Published Feb. 16, 2001.* (51) Int. Cl Moss, Alex “Peptides Notes' Downloaded from
(56) References Cited Capasso et al., “Deamidation via Cyclic Imide of Asparaginyl Peptides: Dependence on Salts, Buffers and Organic Solvents.” U.S. PATENT DOCUMENTS Peptide Research, 4(4): 234-238, 1991. Carpenter et al., “Rational Design of Stable Lyophilized Protein 2003/0175230 A1 9, 2003 Dubief 2004/0265242 A1 12/2004 Bartus et al. Formulations: Some Practical Advice.' Pharmaceutical Research, 2004/0266989 A1 12/2004 Currie et al. 14(8): 969-975, 1997. 2005.0020811 A1 1/2005 Currie et al. Carson et al., “Effect of Linaclotide on Quality of Life in Adults With 2005, 0080009 A1 4/2005 Metzner et al. Chronic Constipation: Results From 2 Randomized, Double-Blind, 2007/O122354 A1 5, 2007 Hastedt et al. Placebo-Controlled Phase III Trials.” Gastroenterology, 139(1):E19. 2007. O1544.06 A1 7/2007 Moon et al. 2010. 2007/0202165 A1 8, 2007 Heuer et al. Chourasia, M.K. and Jain, S.K., “Pharmaceutical approaches to 2009, OO62207 A1* 3/2009 Currie et al...... 514f14 2009/O110729 A1 4/2009 Giovannone et al. colon targeted drug delivery systems.” J Pharm Pharmaceut Sci 2009,0253634 A1 10/2009 Currie et al. (www.ualberta.ca/-csps), 6(1): 33-66, 2003. 2009/0305993 A1 12/2009 Currie Cleland et al., “The Development of Stable Protein Formulations: A 2010.0048489 A1* 2/2010 Fretzen et al...... 514f14 Close Look at Protein Aggregation, Deamidation, and Oxidation.” 2010, 0221329 A1 9, 2010 Shailubhai et al. Critical Reviews in Therapeutic Drug Carrier Systems, 10(4): 307 2012,0009225 A1 1/2012 Fretzen et al. 377, 1993. 2012/0039949 A1 2/2012 Fretzen et al. 2012fO213846 A1 8, 2012 Fretzen et al. Constantino, H.R. et al., “Moisture-Induced Aggregation of 2013/OO.124.54 A1* 1/2013 Mo et al...... 514, 21.1 Lyophilized Insulin'. Pharmaceutical Research, 11(1): 21-29, 1994. 2013, O190239 A1 7, 2013 FRETZEN et al. Cook et al., "Chronic constipation: Overview and Challenges.” 2013/0273.169 A1 10/2013 Fretzen et al. Neurogastroenterology and Motility, 21 (supp2): 1-8, 2009. Dulbecco et al., “Plaque formation and isolation of pure liner with FOREIGN PATENT DOCUMENTS poliomyelitis viruses.” Journal of Experimental Medicine, 99(2): 167-182, 1954. WO WO9012029 10, 1990 Elan Pharmaceuticals, Inc. (2004). Prialt (Ziconotide) injection, solu WO WO9104743 4f1991 tion, Product Label). San Diego, CA 92121, USA. WO WO9703692 2, 1997 Fix, J.A., “Oral Controlled Release Technology for Peptides: Status WO WO9704796 2, 1997 WO WO98OO152 1, 1998 and Future Prospects.” Pharmaceutical Research, 13(12): 1760 WO WO98OO157 1, 1998 1764, 1996. WO WOOOO488O 2, 2000 Forest, "Phase III randomized double-blind placebo-controlled trial WO WOOO32172 6, 2000 of linaclotide administered to patients with chronic constipation.” WO WOO226248 4/2002 2008. Retrieved from
(56) References Cited teins in Lyophilized Dosage Forms.” Journal of Parenteral Science & Technology, 44(6): 300-301, 1990. OTHER PUBLICATIONS Reporter'S Guide to Irritable Bowel Syndrome, retrieved from <
CIWNI-ISKOWu00I U.S. Patent Jan. 13, 2015 Sheet 2 of 4 US 8,933,030 B2
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L se S. S S se U.S. Patent Jan. 13, 2015 Sheet 3 of 4 US 8,933,030 B2
-o-Linaciotide -o- Cys-IMD
50
2 5
O -12 -11 -10 -9 -8 7 6 5 4 log (Peptide Concentration, M)
FIGURE 3 U.S. Patent Jan. 13, 2015 Sheet 4 of 4 US 8,933,030 B2
Fi{RE :
- 2 - - - - -8 - 6 -5 - as eitie Cescentratists vi US 8,933,030 B2 1. 2 TREATMENTS FOR GASTRONTESTINAL tion, a sensation of incomplete evacuation, and fewer than DISORDERS three bowel movements per week. The discomfort and bloat ing of CC significantly affects patients’ quality of life by PRIORITY CLAIM impairing their ability to work and participate in typical daily activities. This application is a national phase application of PCT/ Half of CC patients are not satisfied with currently avail US2011/025274, filed on Feb. 17, 2011, which claims prior able treatments for CC. Thus, there remains a need for new ity to U.S. Provisional Patent Application Ser. No. 61/305, compounds and methods of treating CC. 465, filed Feb. 17, 2010. The entire contents of the U.S. Pat. Nos. 7,304,036 and 7,371,727 disclose peptides aforementioned applications are incorporated herein by ref 10 that act as agonists of the guanylate cyclase C (GC-C) recep CCC. tor for the treatment of gastrointestinal disorders. One par ticular peptide disclosed is linaclotide, which consists of the FIELD OF THE INVENTION following amino acid sequence: CyS CyS Glu Tyr CyS Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:1). These This invention relates to peptides, compositions and meth 15 patents also disclose methods for preparing linaclotide and ods for treating gastrointestinal disorders. related peptides. Linaclotide has the amino acid structure of: SEQUENCE LISTING This application incorporates by reference in its entirety H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. the Sequence Listing entitled “IW087 seq. ST25.txt” (527 bytes), which was created Feb. 17, 2011 and filed electroni cally herewith. H-H BACKGROUND 25 Linaclotide is orally administered and currently in clinical Gastrointestinal disorders (GI) include irritable bowel syn trials for treatment of irritable bowel syndrome with consti drome (IBS) which is a common chronic disorder of the pation (IBS-c) and chronic constipation (CC), has numerous intestine that affects 20 to 60 million individuals in the US effects on GI physiology including: (1) reduced visceral pain, alone (Lehman Brothers, Global Healthcare-Irritable bowel 30 (2) reduced bloating, and (3) increased GI transit, which can syndrome industry update, September 1999). IBS is the most lead to increased stool frequency and improved stool consis common disorder diagnosed by gastroenterologists and tency. Orally administered linaclotide acts locally by activat accounts for 12% of visits to primary care physicians (Cam ing GC-C at the luminal surface; there are no detectable levels illeri 2001, Gastroenterology 120:652-668). In the US, the of linaclotide seen systemically after oral administration at economic impact of IBS is estimated at S25 billion annually, 35 therapeutic dose levels. Thus, the results from clinical trials of through direct costs of health care use and indirect costs of linaclotide, as well as preclinical studies that have been done absenteeism from work (Talley 1995, Gastroenterology 109: with linaclotide and related peptides, Suggest that GC-C pep 1736-1741). Patients with IBS have three times more absen tide agonists may be used therapeutically. teeism from work and report a reduced quality of life. There The contents of the U.S. Pat. Nos. 7,304,036 and 7,371,727 is a tremendous unmet medical need for patients Suffering for 40 are incorporated herein by reference in their entirety. IBS since few prescription options exist to treat IBS. The present invention feature peptides which may be modi Patients with IBS suffer from abdominal pain and a dis fied at their amine groups into imidazolidinone derivatives turbed bowel pattern. Three subgroups of IBS patients have and/or modified at their carboxyl groups into alkyl esters that been defined based on the predominant bowel habit: consti are capable of activating and/or binding the guanylate pation-predominant irritable bowel syndrome (c-IBS), diar 45 cyclase-C (GC-C) receptors at different affinities. GC-C is a rhea-predominant irritable bowel syndrome (d-IBS) or alter key regulatorin mammals of intestinal function, although low nating between the two irritable bowel syndromes (a-IBS). levels of GC-C have been detected in other tissues. GC-C Estimates of individuals who suffer from c-IBS range from responds to the endogenous hormones, guanylin and urogua 20-50% of the IBS patients with 30% frequently cited. In nylin, and to enteric bacterial peptides from the heat stable contrast to the other two subgroups that have a similar gender 50 enterotoxin family (ST peptide). When agonists bind to ratio, c-IBS is more common in women (ratio of 3:1) (Talley GC-C, there is an elevation of the second messenger, cyclic et al. 1995, Am J Epidemiol 142:76-83). GMP (c-GMP), and an increase in chloride and bicarbonate The definition and diagnostic criteria for IBS have been secretion, resulting in an increase in intestinal fluid secretion. formalized in the “Rome Criteria' (Drossman et al. 1999, Gut In some examples of the present invention, the peptides 45: Suppl II: 1-81), which are well accepted in clinical prac 55 described herein may produce increased elevation of c-GMP tice. Recently, there has been increasing evidence for a role of levels and provide a therapeutic option for treating gas inflammation in etiology of IBS. Reports indicate that subsets trointestinal disorders. of IBS patients have Small but significant increases in colonic inflammatory and mast cells, increased inducible nitric oxide SUMMARY (NO) and synthase (iNOS) and altered expression of inflam 60 matory cytokines (reviewed by Talley 2000, Medscape Cov The present invention features peptides, compositions, and erage of DDW week). related methods for treating gastrointestinal disorders and Gastrointestinal disorders can also include constipation conditions, including but not limited to, irritable bowel syn wherein as many as 34 million Americans suffer from Symp drome (IBS) gastrointestinal motility disorders, constipation, toms associated with chronic constipation (CC) and 8.5 mil 65 functional gastrointestinal disorders, gastroesophageal reflux lion patients have sought treatment. Patients with CC often disease (GERD), duodenogastric reflux, Crohn's disease, experience hard and lumpy stools, straining during defeca ulcerative colitis, inflammatory bowel disease, functional US 8,933,030 B2 3 4 heartburn, dyspepsia, Visceral pain, gastroparesis, chronic comprises the amino acid sequence CyS CyS Glu Tyr CyS Cys intestinal pseudo-obstruction (or colonic pseudo-obstruc ASn Pro Ala Cys Thr Gly Cys Tyr (SEQID NO:1), wherein at tion), and other conditions and disorders described herein least one carboxyl group of the peptide is an alkyl ester having using peptides and compositions that activate the guanylate cyclase C (GC-C) receptor. the formula ( COOR) in which R is a C- alkyl. One aspect of the present invention provides a peptide or a In one embodiment, the peptide comprises the amino acid pharmaceutically acceptable salt thereof, wherein the peptide structure of:
R OH
CH Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-R', | 8 ||-s-s-— | |
S-S
or a pharmaceutically acceptable salt thereof, wherein R is H 25 or a C- alkyl, and at least one R' is C alkyl. A second aspect of the present invention provides a peptide that comprises the amino acid structure of
30
35 CH CH Cys-Cys-NH- i -Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr, 40 H. O. L- S-S- — S-S S-S
45 or a pharmaceutically acceptable salt thereof, wherein R is a C. alkyl; or a peptide that comprises the amino acid structure of:
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-R-
65 or a pharmaceutically acceptable salt thereof, wherein R is C- alkyl. US 8,933,030 B2 5 6 A third aspect of the present invention provides a peptide or Description of Exemplary Peptides: pharmaceutically acceptable salt thereof, wherein the peptide In various embodiments, a peptide may be modified comprises the amino acid structure of wherein at least one carboxyl group of the amino acid residue
S-S
"Nu Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr.—s-s—
A fourth aspect of the present invention provides a phar- 15 of the peptide is modified to an alkyl ester. This modification maceutical composition comprising a peptide of the present may be produced, for example, by treating a carboxylic acid invention. with an alcohol in the presence of a dehydrating agent A fifth aspect of the present invention provides a method wherein the dehydrating agent can include but is not limited for treating a gastrointestinal disorder, which includes admin- to a strong acid Such as Sulfuric acid. Other methods of istering a pharmaceutical composition according to the 20 producing alkyl esters from carboxyl groups are readily present invention. known in those skilled in the arts and are incorporated herein. The details of one or more embodiments of the invention As used herein, a carboxyl group has the formula: are set forth in the accompanying description. (-COOH). As used herein, the term “alkyl, refers to a saturated linear BRIEF DESCRIPTION OF THE FIGURE 2 or branched-chain monovalent hydrocarbon radical. As used herein, a group is terminal or terminus when the FIG. 1 shows the dose response of exemplary peptides of group is present at the end of the amino acid sequence. the present invention in a T84 cell c-GMP assay. As used herein, an amine group on a peptide has the for FIG. 2 demonstrates an example of an analysis of exem plary peptides by RP-HPLC, wherein “Cys-IMD” refers to 30 mula: the linaclotide imidazolidinone derivative modified on its N-terminal amine group. FIG.3 shows specific binding of linaclotide and Cys-IMD to cell-surface GC-C receptors on T84 cells in a competitive HN R2, radioligand binding assay. 35 FIG. 4 shows the dose response of exemplary peptides of the present invention in a T84 cell c-GMP assay. The figures are provided by way of example and are not wherein R is the rest of the peptide. intended to limit the scope of the present invention. As used herein, an imine group on a peptide has the for 40 mula: DETAILED DESCRIPTION Guanylate cyclase C (GC-C) is a transmembrane receptor that is located on the apical surface of epithelial cells in the HC=N R2, stomach and intestine. The receptor has an extracellular 45 ligand-binding domain, a single transmembrane region and a C-terminal guanylyl cyclase domain. When a ligand binds to the extracellular domain of GC-C, the intracellular catalytic wherein R is the rest of the peptide. domain catalyzes the production of c(GMP from GTP. In vivo, In some embodiments, the carboxylic acid of the side chain this increase in intracellular cc MP initiates a cascade of 50 of a glutamate amino acid in a peptide sequence is modified events that leads to increased secretion of chloride and bicar- into an alkyl ester. bonate into the intestinal lumen, increased luminal pH, In further embodiments, the carboxylic acid on the side decreased luminal Sodium absorption, increased fluid secre- chain of a glutamate amino acid a peptide sequence is modi tion, and acceleration of intestinal transit. cQMP is secreted fied into an ethyl ester. bidirectionally from the epithelium into the mucosa and 55 In other embodiments, the C-terminus carboxylic acid of a lumen. The peptides and compositions of the present inven- tyrosine amino acid in a peptide sequence is modified into an tion bind to the intestinal GC-C receptor which is a regulator alkyl ester. of fluid and electrolyte balance in the intestine. In further embodiments, the C-terminus carboxylic acid of In some circumstances it can be desirable to treat patients a tyrosine amino acid of a peptide sequence is modified into with a variant or modified peptide that binds to and activates 60 an ethyl ester. intestinal GC-C receptors, but is less active or more active In several embodiments, the present invention provides a than the non-variant form of a peptide. Reduced activity can peptide or a pharmaceutically acceptable salt thereof, arise from reduced affinity for the receptor or a reduced wherein the peptide comprises the amino acid sequence Cys ability to activate the receptor once bound or reduced stability Cys Glu Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ of the peptide. Increased activity can arise from increased 65 ID NO:1), wherein at least one carboxyl group of the peptide affinity for the receptor or an increased ability to activate the is an alkyl ester having the formula ( COOR) in which R is receptor once bound or increased stability of the peptide. a C- alkyl. US 8,933,030 B2 7 8 In several embodiments, the peptide comprises an amino In some embodiments, the peptide or pharmaceutically acid structure of: acceptable salt thereof comprises an amino acid structure of
R OH o hi, t CH Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-R', | 8 ||-s-s-
S-S
2O or a pharmaceutically acceptable salt thereof, wherein R is H or a C- alkyl, and at least one R' is C alkyl. CH2CH2CH In some embodiments, the peptide or pharmaceutically h acceptable salt thereof comprises a peptide having an amino 25 acid structure of: o= ". R CH 30 Cys-Cys-NH- i -Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. O H. O. L- S-S- — CH S-S- 35 S-S
Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyrt C H.| O.|| L- S-S- — S-S- 40 S-S In some embodiments, the peptide or pharmaceutically acceptable salt thereof comprises an amino acid structure of wherein R is a C- alkyl (“Glu-alkyl ester'). In other embodiments, R is a C alkyl. 45 CH In further embodiments, R is methyl, ethyl, or propyl. h In some embodiments, the peptide or pharmaceutically o= acceptable salt thereof comprises an amino acid structure of (“Glu-ethyl ester): 50 t ". fict pre-Ni---is press-to-air-tigliety. 55 H. O. L- S-S- — OC S-S- S-S fl. CH 60 Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. | || -s-s- In some embodiments, the C-terminal tyrosine of the Glu S S-S alkyl ester or pharmaceutically acceptable salt is absent. S 65 In some embodiments, the peptide or pharmaceutically acceptable salt thereof comprises an amino acid structure of US 8,933,030 B2 10
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-R
H. O
wherein R is C alkyl. In other embodiments, R is a C alkyl. In further embodiments, R is methyl, ethyl, or propyl. In some embodiments, the peptide or pharmaceutically acceptable salt thereof comprises an amino acid structure of (“Tyra-ethyl ester):
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CH3.
H. O
In some embodiments, the peptide or pharmaceutically acceptable salt thereof comprises an amino acid structure of
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CHCH
H. O
65 In some embodiments, the peptide or pharmaceutically acceptable salt thereof comprises an amino acid structure of US 8,933,030 B2 11 12
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH
H. O
Without wishing to be bound by any theory, a peptide may 20 be modified wherein at least one amine group of the amino acid residues of the peptide is modified into an imine. This HC=N R2, modification may be produced, for example, by treating an amine group with a carbonyl, such as an aldehyde or ketone, in the presence of acid catalyst. Other methods of producing 25 imines from amine groups are readily known to those skilled wherein R is the rest of the peptide. in the arts and are incorporated herein. In further embodiments, the peptide or pharmaceutically In Some embodiments, the imine modification may be pro acceptable salt thereof comprises an amino acid structure of duced by a formaldehyde mediated reaction in the presence of 3 acid catalyst. O
In further embodiments, the imine carbon may be cross HCFCys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. linked to another amine group of the peptide. In other embodiments, a peptide may be modified into an imine at the C-amine group of the N-terminal amino acid, 35 wherein the imine carbon is cross-linked with an amine group of the second amino acid residue of the peptide forming a five membered ring. In other embodiments, a peptide comprising the amino acid 40 In several embodiments, the peptide or pharmaceutically sequence Cys CysGlu Tyr Cys Cys Asn Pro Ala Cys Thr Gly acceptable salt thereof comprises an amino acid structure of Cys Tyr (SEQID NO:1) may be modified with the addition of methylene at the O.-amine group of the N-terminal Cys which is cross-linked to the amine group of Cys to form an imida Zolidinone 5 membered ring at the N-terminus of the peptide 45 (“Cys-IMD).
In one aspect, the invention provides novel GC-C peptide urgesS-S are alsTrobesets. agonists useful for the treatment of gastrointestinal disorders. In several embodiments, the present invention provides a 50 peptide or a pharmaceutically acceptable salt thereof, In some embodiments, the C-terminal tyrosine of the Cys wherein the peptide comprises the amino acid sequence Cys IMD peptide or pharmaceutically acceptable salt thereof is Cys Glu Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ absent. In some embodiments, the Cys-IMD peptide orphar ID NO:1), wherein at least one amine group of the peptide is maceutically acceptable salt thereof further comprises one or an imine having the formula 55 more peptide modifications, wherein at least one carboxyl group of the peptide is an alkyl ester having the formula ( COOR) in which R is a C- alkyl. In several embodiments, the present invention provides a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:1), wherein at least one carboxyl group of the peptide wherein R is the rest of the peptide. is an alkyl ester having the formula ( COOR) in which R is In some embodiments, the peptide or a pharmaceutically 65 a C- alkyl. acceptable salt comprises a peptide wherein the N-terminal In several embodiments, the peptide consists of an amino amine group of the peptide is an imine having the formula acid structure of: US 8,933,030 B2 13 14
R OH
O
OEC CH CH CH Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-R', | || H. O H. O
S-S
S-S or a pharmaceutically acceptable salt thereof, wherein R is H or a C- alkyl, and at least one R' is C alkyl. In some embodiments, the peptide or pharmaceutically acceptable salt thereof consists of an amino acid structure of
R
O
OEC CH CH Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr | || H. O
S-S
SHS
wherein R is a C- alkyl. In further embodiments, the O C-terminal tyrosine is absent. In other embodiments, R is a C alkyl. In further embodiments, R is methyl, ethyl, or propyl. In some embodiments, the peptide or pharmaceutically acceptable salt thereof consists of a peptide having an amino acid structure of:
CHCH O
OEC CH CH Cys-Cys-NH- o i -Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr.
H. O
S-S
SHS
In some embodiments, the peptide or pharmaceutically 65 acceptable salt thereof consists of a peptide having an amino acid structure of: US 8,933,030 B2 15 16
CHCH2CH3 O
OC CH CH Cys-Cys-NH- o i -Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. H O
S-S
S-S
In some embodiments, the peptide or pharmaceutically 2 O acceptable salt thereof consists of a peptide having an amino acid structure of:
CH3 O
OC CH CH Cys-Cys-NH- o i -Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr.
H. O
S-S
S-S
40 In some embodiments, the peptide or pharmaceutically acceptable salt thereof consists of a peptide having an amino acid structure of:
OH
CH
Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-R
H O
wherein R is C alkyl. In other embodiments, R is a C alkyl. In further embodiments, R is methyl, ethyl, or propyl. 65 In some embodiments, the peptide or pharmaceutically acceptable salt thereof consists of an amino acid structure of US 8,933,030 B2 17 18
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CH3.
H. O
In some embodiments, the peptide or pharmaceutically acceptable salt thereof consists of an amino acid structure of
OH
CH2 Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CH2CH3. H-s-s- - |-- In some embodiments, the peptide or pharmaceutically acceptable salt thereof consists of an amino acid structure of
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH3. H-s-s- s-l |-- In several embodiments, the present invention provides a 55 In some embodiments, the peptide or a pharmaceutically peptide or a pharmaceutically acceptable salt thereof, acceptable salt consists of a peptide wherein the N-terminal wherein the peptide consists of the amino acid sequence Cys amine group of the peptide is an imine having the formula Cys Glu Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:1), wherein at least one amine group of the peptide is an imine having the formula 60 HC=N R2,
65 wherein R is the rest of the peptide. In further embodiments, the peptide or pharmaceutically wherein R is the rest of the peptide. acceptable salt thereof consists of an amino acid structure of US 8,933,030 B2 19 20 Drosophila Sf9 cell systems), yeast cell systems (e.g., S. HC=Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. cerevisiae, S. Saccharomyces) or filamentous fungal expres sion systems, or animal cell expression systems (e.g., mam s-s- malian cell expression systems). Peptides or precursor pep 5 tides of the invention may also be chemically synthesized. If the peptide or variant peptide is to be produced recom binantly, e.g., E. coli, the nucleic acid molecule encoding the peptide may also encode a leader sequence that permits the In some embodiments, the peptide or pharmaceutically secretion of the mature peptide from the cell. Thus, the acceptable salt thereof consists of an amino acid structure of 10 sequence encoding the peptide can include the pre sequence and the pro sequence of for example, a naturally-occurring bacterial ST peptide. The secreted, mature peptide can be S-S purified from the culture medium. O The sequence encoding a peptide described herein can be O 15 inserted into a vector capable of delivering and maintaining HN\- N S-S — the nucleic acid molecule in a bacterial cell. The DNA mol Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. ecule may be inserted into an autonomously replicating vec S-S — tor (suitable vectors include, for example, pGEM37 and pcDNA3, and derivatives thereof). The vector nucleic acid may be a bacterial or bacteriophage DNA such as bacterioph In some embodiments, the C-terminal tyrosine of the Cys age lambda or M13 and derivatives thereof. Construction of a IMD peptide or pharmaceutically acceptable salt thereof is vector containing a nucleic acid described herein can be fol absent. In some embodiments, the Cys-IMD peptide orphar lowed by transformation of a host cell such as a bacterium. maceutically acceptable salt thereof further comprises one or Suitable bacterial hosts include but are not limited to, E. coli, more peptide modifications, e.g., wherein at least one car 25 B. subtilis, Pseudomonas and Salmonella. The genetic con boxyl group of the peptide is an alkyl ester having the formula struct also includes, in addition to the encoding nucleic acid ( COOR), wherein R is a C- alkyl. molecule, elements that allow expression, Such as a promoter In some embodiments, the peptide or pharmaceutically and regulatory sequences. The expression vectors may con acceptable salt thereof activates the guanylate cyclase C tain transcriptional control sequences that control transcrip receptor. 30 tional initiation, Such as promoter, enhancer, operator, and In other embodiments, the peptide or pharmaceutically repressor sequences. A variety of transcriptional control acceptable salt thereof comprises 30 or fewer amino acids. sequences are well known to those in the art. The expression In further embodiments, the peptide or pharmaceutically vector can also include a translation regulatory sequence acceptable salt thereof comprises 20 or fewer amino acids. (e.g., an untranslated 5' sequence, an untranslated 3' In other embodiments, the peptide or pharmaceutically 35 sequence, or an internal ribosome entry site). The vector can acceptable salt thereof comprises a peptide wherein fewer be capable of autonomous replication or it can integrate into than five amino acids precede the first Cys residue of the host DNA to ensure stability during peptide production. amino acid sequence. The protein coding sequence that includes a peptide In some embodiments, the peptide or pharmaceutically described herein can also be fused to a nucleic acid encoding acceptable salt thereof is isolated. 40 a peptide affinity tag, e.g., glutathione S-transferase (GST), In other embodiments, the peptide or pharmaceutically maltose E binding protein, protein A, FLAG tag, hexa-histi acceptable salt thereof is purified. dine, myc tag or the influenza HA tag, in order to facilitate In some embodiments, a pharmaceutically acceptable salt purification. The affinity tag or reporter fusion joins the read of the peptide is provided. In some instances, the pharmaceu ing frame of the peptide of interest to the reading frame of the tically acceptable salt is a chloride salt. 45 gene encoding the affinity tag such that a translational fusion Variant or Modified Peptides is generated. Expression of the fusion gene results in transla In various embodiments, the peptide includes two Cys that tion of a single peptide that includes both the peptide of form one disulfide bond, the peptide includes four Cys that interest and the affinity tag. In some instances where affinity form two disulfide bonds, or the peptide includes six Cys that tags are utilized, DNA sequence encoding a protease recog form three disulfide bonds. 50 nition site will be fused between the reading frames for the In some peptides one or both members of one or both pairs affinity tag and the peptide of interest. of Cys residues which normally form a disulfide bond can be Genetic constructs and methods suitable for production of replaced by homocysteine, penicillamine, 3-mercaptoproline immature and mature forms of the peptides and variants (Kolodzie et al. 1996 Int J Pept Protein Res 48:274); B.B described herein in protein expression systems other than dimethylcysteine (Hunt et al. 1993 Int J Pept Protein Res 55 bacteria, and well knownto those skilled in the art, can also be 42:249) or diaminopropionic acid (Smith et al. 1978 J Med used to produce peptides in a biological system. Chem 21:117) to form alternative internal cross-links at the In other embodiments, peptides containing amino acids not positions of the normal disulfide bonds. In other embodi normally incorporated by the translation machinery and ments, the disulfide bonds may be replaced by hydrocarbon described above (e.g.—B-carboxylated Asp, Y-carboxylated crosslinking (Schafmeister et al. 2000 JAm ChemSoc. 122: 60 Glu, Asu, Aad and Apm) may be recombinantly produced by 5891, Patgiri et al. 2008 Acc Chem Res 41:1289, Henchey et tRNA modification methods. Methods for modifying tRNA al. 2008 Curr Opin Chem Biol 12:692). including, but not limited to, modifying the anti-codon, the Production of Peptides amino acid attachment site, and/or the accepter stem to allow In one embodiment, peptides or precursor peptides of the incorporation of unnatural and/or arbitrary amino acids are invention can be produced recombinantly in any known pro 65 known in the art (Biochem. Biophys. Res. Comm. (2008) tein expression system, including, without limitation, bacte 372: 480-485; Chem. Biol. (2009) 16:323-36; Nat. Methods ria (e.g., E. coli or Bacillus subtilis), insect cell systems (e.g., (2007) 4:239-44; Nat. Rev. Mol. Cell Biol. (2006) 7:775-82; US 8,933,030 B2 21 22 Methods (2005) 36:227-238; Methods (2005) 36:270-278: and Annu. Rev. Biochem. (2004) 73:147-176: Nuc. Acids Res. iii. a peptide or a pharmaceutically acceptable salt thereof, (2004) 32:6200-6211; Proc. Natl. Acad. Sci. USA (2003) 100:6353-6357; Royal Soc. Chem. (2004) 33:422-430). wherein the peptide comprises the amino acid sequence Cys In some embodiments, peptides may be chemically pro Cys Glu Tyr Cys Cys ASn Pro Ala Cys Thr Gly Cys Tyr (SEQ duced. Peptides can be synthesized by a number of different ID NO:1), wherein at least one carboxyl group of the peptide methods including Solution and solid phase synthesis using is an alkyl ester having the formula ( COOR) in which R is traditional BOC or FMOC protection. For example, the pep a C- alkyl. tide can be synthesized on 2-Chlorotrity1 or Wang resin using consecutive amino acid couplings. The following protecting In other embodiments, the pharmaceutical composition groups can be used: Fluorenylmethyloxycarbonyl or tert-bu 10 comprises linaclotide and a peptide or a pharmaceutically tyloxycarbonyl (alpha-amino groups, N-terminus); trity1 or acceptable salt thereof, wherein the peptide comprises an tert-butyl (thiol groups of Cys); tert-butyl (Y-carboxyl of amino acid structure of glutamic acid and the hydroxyl group of threonine, if present); and trityl (3-amid function of the asparagine side chain and the phenolic group of tyrosine, if present). Cou 15 pling can be effected with DIC and HOBt in the presence of a tertiary amine, and the peptide can be deprotected and cleaved from the Solid Support in using cocktail K (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%, 1.2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide 1.5% w/w). After removal of trifluoroacetic acid and other volatiles Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr; the peptide can be precipitated using an organic solvent. Disulfide bonds between Cys residues can be formed using dimethyl sulfoxide (Tam et al. (1991) J. Am. Chem. Soc. 113:6657-62) or using an air oxidation strategy. The resulting 25 peptide can be purified by reverse-phase chromatography and lyophilized. These peptides can be made, isolated or used either inform and the peptide or pharmaceutically acceptable salt thereof of the base or as pharmaceutically acceptable salts thereof. comprises less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, Examples of salts include, without limitation, acetate, chlo 30 80%, or 90% by weight compared to the weight of linaclotide. ride, Sulfate and phosphate salts of the peptide. Compositions of Peptides and GC-C Receptor Agonists In further embodiments, the pharmaceutical composition In another aspect, pharmaceutical compositions are pro comprises linaclotide and a peptide or a pharmaceutically vided wherein the peptides, alone or in combination, can be acceptable salt thereof, wherein the peptide comprises an combined with any pharmaceutically acceptable carrier or 35 amino acid structure of medium. In several embodiments, the pharmaceutical composition comprises a peptide or pharmaceutically acceptable salt thereofas described herein. The pharmaceutical composition may comprise two or more peptides or pharmaceutically 40 acceptable salts thereof described herein. In some embodiments, the pharmaceutical composition comprises two or more peptides selected from: i. a peptide or a pharmaceutically acceptable salt thereof, Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr; wherein the peptide comprises the amino acid structure of: 45
and the peptide orpharmaceutically acceptable Salt thereof comprises less than 9%, 8%, 7%, 6%. 5%, 4%,3%,2%, or 1% 50 by weight compared to the weight of linaclotide. Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr; In some embodiments, the imidazolidinone derivative of linaclotide comprises less than about 15% by weight of the composition, less than about 10% by weight of the composi 55 tion, less than about 7% by weight of the composition or less ii. a peptide or a pharmaceutically acceptable salt thereof, than about 5% by weight of the composition. In other exem wherein the peptide comprises an amino acid structure of: plary embodiments, the imidazolidinone derivative of lina clotide comprises from about 0.01% to about 15% by weight of the composition, about 0.05% to about 10% by weight of H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH: 60 the composition, about 0.05% to about 7% by weight of the composition or about 0.05% to about 5% by weight of the composition. In other embodiments, the pharmaceutical composition 65 comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid structure of US 8,933,030 B2 23 24
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CH3: H-s-s- s-l |-- and the peptide or pharmaceutically acceptable salt thereof comprises less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weightoflinaclotide. In further embodiments, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid structure of
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CH: H-s-s-
and the peptide or pharmaceutically acceptable salt thereof 45 comprises less than 9%, 8%, 7%, 6%. 5%, 4%,3%,2%, or 1% CHCH by weight compared to the weight of linaclotide.
In some embodiments, the Tyra-ethyl ester peptide com 50 prises less than about 15% by weight of the composition, less SH: than about 10% by weight of the composition, less than about Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr; 7% by weight of the composition or less than about 5% by weight of the composition. In other exemplary embodiments, 55 the Tyra-ethyl ester comprises from about 0.01% to about 15% by weight of the composition, about 0.05% to about 10% by weight of the composition, about 0.05% to about 7% by weight of the composition or about 0.05% to about 5% by weight of the composition. 60 and the peptide orpharmaceutically acceptable Salt thereof comprises less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide. In other embodiments, the pharmaceutical composition In further embodiments, the pharmaceutical composition comprising linaclotide and a peptide or a pharmaceutically 65 comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an acceptable salt thereof, wherein the peptide comprises an amino acid structure of amino acid structure of US 8,933,030 B2 25 26 dispersants, coatings, absorption promoting agents, con trolled release agents, and one or more inert excipients (which include starches, polyols, granulating agents, microcrystal line cellulose (e.g. celphere, Celphere Beads(R), diluents, lubricants, binders, disintegrating agents, and the like), etc. If desired, tablet dosages of the disclosed compositions may be SH: coated by standard aqueous or nonaqueous techniques. Cys-Cys-NH-C-C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr; Examples of excipients for use as the pharmaceutically acceptable carriers and the pharmaceutically acceptable inert 10 carriers and the aforementioned additional ingredients include, but are not limited to binders, fillers, disintegrants, lubricants, anti-microbial agents, and coating agents. L As used herein, the term “binder refers to any pharmaceu tically acceptable binder that may be used in the practice of and the peptide or pharmaceutically acceptable salt thereof 15 the invention. Examples of pharmaceutically acceptable comprises less than 9%, 8%, 7%, 6%. 5%, 4%,3%,2%, or 1% binders include, without limitation, a starch (e.g., corn starch, by weight compared to the weight of linaclotide. potato starch and pre-gelatinized Starch (e.g., STARCH In some embodiments, the Glus-ethyl ester peptide com 1500R and STARCH 1500LM(R), sold by Colorcon, Ltd.)and prises less than about 15% by weight of the composition, less other starches), maltodextrin, gelatin, natural and synthetic than about 10% by weight of the composition, less than about gums such as acacia, powdered tragacanth, guar gum, cellu 7% by weight of the composition or less than about 5% by lose and its derivatives (e.g., methylcellulose, hydroxyethyl weight of the composition. In other exemplary embodiments, cellulose, hydroxyethyl methylcellulose, hydroxypropyl cel the Glu-ethyl ester comprises from about 0.01% to about lulose and hydroxypropyl methylcellulose (hypromellose), 15% by weight of the composition, about 0.05% to about 10% ethyl cellulose, cellulose acetate, carboxymethyl cellulose by weight of the composition, about 0.05% to about 7% by 25 calcium, Sodium carboxymethyl cellulose, carboxymethyl weight of the composition or about 0.05% to about 5% by cellulose, microcrystalline cellulose (e.g. AVICELTM, such weight of the composition. as, AVICEL-PH-101TM, -103TM and -105TM, sold by FMC In some embodiments, the pharmaceutical composition Corporation, Marcus Hook, Pa., USA)), polyvinyl alcohol, comprises a peptide or pharmaceutically acceptable salt, polyvinyl pyrrolidone (e.g., polyvinyl pyrrolidone K30), and wherein the peptide consists of the amino acid structure of: 30 mixtures thereof. As used herein, the term “filler” refers to any pharmaceu tically acceptable filler that may be used in the practice of the S-S invention. Examples of pharmaceutically acceptable fillers O include, without limitation, talc, calcium carbonate (e.g., 35 granules or powder), dibasic calcium phosphate, tribasic cal HN\- N O s-s— cium phosphate, calcium Sulfate (e.g., granules or powder), Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. microcrystalline cellulose (e.g., Avicel PH101 or Celphere S-S — CP-305), powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch (e.g., Starch 1500), pre-gelati 40 nized starch, lactose, glucose, fructose, galactose, trehalose, In other embodiments, the pharmaceutical composition Sucrose, maltose, isomalt, raffinose, maltitol, melezitose, consists essentially of a peptide or pharmaceutically accept stachyose, lactitol, palatinite, Xylitol, myoinositol, and mix able salt thereof, wherein the peptide comprises the amino tures thereof. acid structure of: Examples of pharmaceutically acceptable fillers that may 45 be particularly used for coating the peptides include, without limitation, talc, microcrystalline cellulose (e.g., Avicel S-S PH101 or Celphere CP-305), powdered cellulose, dextrates, O S-S kaolin, mannitol, silicic acid, Sorbitol, starch, pre-gelatinized O starch, lactose, glucose, fructose, galactose, trehalose, HN N 50 Sucrose, maltose, isomalt, dibasic calcium phosphate, raffi ne-1 Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. nose, maltitol, melezitose, stachyose, lactitol, palatinite, Xyli s-s- tol, mannitol, myoinositol, and mixtures thereof. As used herein, the term “additives' refers to any pharma The term “consisting essentially of, and variants thereof, ceutically acceptable additive. Pharmaceutically acceptable when used to refer to the composition, are used hereinto mean 55 additives include, without limitation, disintegrants, dispers that the composition includes a sole active peptide and other ing additives, lubricants, glidants, antioxidants, coating addi desired pharmaceutically inactive additives, excipients, and/ tives, diluents, Surfactants, flavoring additives, humectants, or components (e.g., polymers, sterically hindered primary absorption promoting additives, controlled release additives, amines, cations, filling agents, binders, carriers, excipients, anti-caking additives, anti-microbial agents (e.g., preserva diluents, disintegrating additives, lubricants, solvents, dis 60 tives), colorants, desiccants, plasticizers and dyes. As used persants, coating additives, absorption promoting additives, herein, an “excipient' is any pharmaceutically acceptable controlled release additives, anti-caking additives, anti-mi additive, filler, binder or agent. crobial additives, preservatives, Sweetening additives, colo Compositions of the present invention may also optionally rants, flavors, desiccants, plasticizers, dyes, or the like), and include other therapeutic ingredients, anti-caking agents, pre no other active pharmaceutical ingredient(s). 65 servatives, Sweetening agents, colorants, flavors, desiccants, The peptides described herein can be combined with any plasticizers, dyes, glidants, anti-adherents, anti-static agents, pharmaceutically tolerable carrier or medium, e.g. solvents, Surfactants (wetting agents), anti-oxidants, film-coating US 8,933,030 B2 27 28 agents, and the like. Any Such optional ingredient must be Aquacoat Ethylcellulose Aquaeous Dispersion, 15% w/w, compatible with the compound described herein to insure the FMC Biopolymer, ECD-30), Eudragit (e.g. Eudragit E PO stability of the formulation. The composition may contain PE-EL, Roehm Pharma Polymers) and Opadry (e.g. Opadry other additives as needed, including for example lactose, AMB dispersion, 20% w/w, Colorcon). glucose, fructose, galactose, trehalose, Sucrose, maltose, In certain embodiments, suitable additives for the peptides raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, composition include one or more of Sucrose, talc, magnesium starch, Xylitol, mannitol, myoinositol, and the like, and stearate, crospovidone or BHA. hydrates thereof, and amino acids, for example alanine, gly The compositions of the present invention can also include cine and betaine, and peptides and proteins, for example other excipients, agents, and categories thereof including but albumen. 10 not limited to L-histidine, Pluronic R, Poloxamers (such as The compositions can include, for example, various addi Lutrol.R and Poloxamer 188), ascorbic acid, glutathione, per tional Solvents, dispersants, coatings, absorption promoting meability enhancers (e.g. lipids, Sodium cholate, acylcar additives, controlled release additives, and one or more inert nitine, Salicylates, mixed bile salts, fatty acid micelles, chela additives (which include, for example, starches, polyols, tors, fatty acid, Surfactants, medium chain glycerides), granulating additives, microcrystalline cellulose, diluents, 15 protease inhibitors (e.g. soybean trypsin inhibitor, organic lubricants, binders, disintegrating additives, and the like), etc. acids), pH lowering agents and absorption enhancers effec If desired, tablet dosages of the disclosed compositions may tive to promote bioavailability (including but not limited to be coated by standard aqueous or non-aqueous techniques. those described in U.S. Pat. No. 6,086,918 and U.S. Pat. No. Compositions can also include, for example, anti-caking 5.912.014), materials for chewable tablets (like dextrose, additives, preservatives, Sweetening additives, colorants, fla fructose, lactose monohydrate, lactose and aspartame, lactose Vors, desiccants, plasticizers, dyes, and the like. and cellulose, maltodextrin, maltose, mannitol, microcrystal Suitable disintegrants include, for example, agar-agar, cal line cellulose and guar gum, Sorbitol crystalline); parenterals cium carbonate, microcrystalline cellulose, croScarmellose (like mannitol and povidone); plasticizers (like dibutyl seba Sodium, crospovidone, povidone, polacrilin potassium, cate, plasticizers for coatings, polyvinylacetate phthalate); Sodium starch glycolate, potato or tapioca starch, other 25 powder lubricants (like glyceryl behenate); Soft gelatin cap starches, pre-gelatinized starch, clays, otheralgins, other cel Sules (like Sorbitol special Solution); spheres for coating (like luloses, gums, and mixtures thereof. Sugar spheres); spheronization agents (like glyceryl behenate Suitable lubricants include, for example, calcium Stearate, and microcrystalline cellulose); Suspending/gelling agents magnesium Stearate, mineral oil, light mineral oil, glycerin, (like carrageenan, gellan gum, mannitol, microcrystalline Sorbitol, mannitol, polyethylene glycol, other glycols, Stearic 30 cellulose, povidone, Sodium starch glycolate, Xanthan gum); acid, sodium lauryl Sulfate, talc, hydrogenated vegetable oil Sweeteners (like aspartame, aspartame and lactose, dextrose, (e.g., peanut oil, cottonseed oil, Sunflower oil, sesame oil, fructose, honey, maltodextrin, maltose, mannitol, molasses, olive oil, corn oil and Soybean oil), Zinc Stearate, ethyl oleate, Sorbitol crystalline, Sorbitol special Solution, Sucrose); wet ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R. granulation agents (like calcium carbonate, lactose anhy Grace Co., Baltimore, Md. USA), a coagulated aerosol of 35 drous, lactose monohydrate, maltodextrin, mannitol, micro synthetic silica (Evonik Degussa Co., Plano, Tex. USA), a crystalline cellulose, povidone, starch), caramel, carboxym pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, ethylcellulose sodium, cherry cream flavor and cherry flavor, Mass. USA), and mixtures thereof. citric acid anhydrous, citric acid, confectioner's Sugar, D&C Suitable glidants include, for example, leucine, colloidal Red No. 33, D&C Yellow #10 Aluminum Lake, disodium silicon dioxide, magnesium trisilicate, powdered cellulose, 40 edetate, ethyl alcohol 15%, FD&C Yellow No. 6 aluminum starch, talc, and tribasic calcium phosphate. lake, FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, Suitable anti-caking additives include, for example, cal FD&C blue no. 2 aluminum lake, FD&C Green No. 3, FD&C cium silicate, magnesium silicate, silicon dioxide, colloidal Red No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C silicon dioxide, talc, and mixtures thereof. Yellow No. 6, FD&C Yellow No. 10, glycerol palmitostear Suitable anti-microbial additives that may be used, e.g., as 45 ate, glyceryl monostearate, indigo carmine, lecithin, manitol, a preservative for the peptides compositions, include, for methyl and propyl parabens, mono ammonium glycyrrhiz example, benzalkonium chloride, benzethonium chloride, inate, natural and artificial orange flavor, pharmaceutical benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium glaze, poloxamer 188, Polydextrose, polysorbate 20, polysor chloride, cresol, chlorobutanol, dehydroacetic acid, ethylpa bate 80, poly Vidone, pregelatinized corn Starch, pregelati raben, methylparaben, phenol, phenylethyl alcohol, phe 50 nized starch, red iron oxide, saccharin Sodium, Sodium car noxyethanol, phenylmercuric acetate, phenylmercuric boxymethyl ether, sodium chloride, sodium citrate, Sodium nitrate, potassium Sorbate, propylparaben, Sodium benzoate, phosphate, Strawberry flavor, synthetic black iron oxide, Syn Sodium dehydroacetate, Sodium propionate, Sorbic acid, thetic red iron oxide, titanium dioxide, and white wax. thimersol, thymo, and mixtures thereof. In some embodiments, there is provided a pharmaceutical Suitable antioxidants include, for example, BHA (buty 55 composition comprising a peptide described herein and one lated hydroxyanisole), BHT (butylated hydroxytoluene), or more agents selected from Mg, Ca", Zn, Mn", K", Vitamin E, propyl gallate, ascorbic acid and salts or esters Na" or Al", a combination thereof, and/or a sterically hin thereof, tocopherol and esters thereof, alpha-lipoic acid and dered primary amine. In further embodiments, the agent is beta-carotene. Mg", Ca" or Zn" or a combination thereof. In some Suitable coating additives include, for example, sodium 60 embodiments, the cation is provided, without limitation, as carboxymethyl cellulose, cellulose acetate phthalate, ethyl magnesium acetate, magnesium chloride, magnesium phos cellulose, gelatin, pharmaceutical glaze, hydroxypropyl cel phate, magnesium Sulfate, calcium acetate, calcium chloride, lulose, hydroxypropyl methylcellulose, hydroxypropyl calcium phosphate, calcium Sulfate, Zinc acetate, Zinc chlo methyl cellulose phthalate, methylcellulose, polyethylene ride, Zinc phosphate, Zinc sulfate, manganese acetate, man glycol, polyvinyl acetate phthalate, shellac, Sucrose, titanium 65 ganese chloride, manganese phosphate, manganese Sulfate, dioxide, carnauba wax, microcrystalline wax, and mixtures potassium acetate, potassium chloride, potassium phosphate, thereof. Suitable protective coatings include Aquacoat (e.g. potassium sulfate, sodium acetate, sodium chloride, sodium US 8,933,030 B2 29 30 phosphate, sodium sulfate, aluminum acetate, aluminum In a further embodiment, the pharmaceutical composition chloride, aluminum phosphate or aluminum Sulfate. In fur further comprises a pharmaceutically acceptable binder or ther embodiments, the cation is provided as magnesium chlo additive, and/or a pharmaceutically acceptable glidant, lubri ride, calcium chloride, calcium phosphate, calcium Sulfate, cant or additive that acts as both a glidant and lubricant and/or Zinc acetate, manganese chloride, potassium chloride, an antioxidant. sodium chloride or aluminum chloride. In other embodi Suitable pharmaceutical compositions in accordance with ments, the cation is provided as calcium chloride, magnesium the invention will generally include an amount of the active chloride or Zinc acetate. compound(s) with an acceptable pharmaceutical diluent or In another embodiment, the agent is a sterically hindered excipient, such as a sterile aqueous solution, to give a range of 10 final concentrations, depending on the intended use. The primary amine. In a further embodiment, the sterically hin techniques of preparation are generally well known in the art, dered primary amine is an amino acid. In yet a further as exemplified by Remington's Pharmaceutical Sciences embodiment, the amino acid is a naturally-occurring amino (18th Edition, Mack Publishing Company, 1995). acid. In a still further embodiment, the naturally-occurring The compositions described herein may be administered amino acid is selected from the group consisting of histidine, 15 systemically or locally, e.g.: orally (e.g. using capsules, pow phenylalanine, alanine, glutamic acid, aspartic acid, ders, Solutions, Suspensions, tablets, Sublingual tablets and glutamine, leucine, methionine, asparagine, tyrosine, threo the like), by inhalation (e.g. with an aerosol, gas, inhaler, nine, isoleucine, tryptophan, glycine and Valine; yet further, nebulizer or the like), to the ear (e.g. using ear drops), topi the naturally-occurring amino acid is leucine, isoleucine, ala cally (e.g. using creams, gels, liniments, lotions, ointments, nine or methionine. In a still further embodiment, the natu pastes, transdermal patches, etc), ophthalmically (e.g. with rally-occurring amino acid is leucine. In another embodi eye drops, ophthalmic gels, ophthalmic ointments), rectally ment, the sterically hindered primary amine is a non-naturally (e.g. using enemas or Suppositories), nasally, buccally, vagi occurring amino acid (e.g., 1-aminocyclohexane carboxylic nally (e.g. using douches, intrauterine devices, vaginal Sup acid). In a further embodiment, the sterically hindered pri mary amine is cyclohexylamine, 2-methylbutylamine or chi positories, vaginal rings or tablets, etc), via an implanted 25 reservoir or the like, or parenterally depending on the severity tosan. In another embodiment, one or more sterically hin and type of the disease being treated. The term “parenteral as dered primary amines may be used in a composition. used herein includes, but is not limited to, Subcutaneous, In some cases, the sterically hindered primary amine has intravenous, intramuscular, intra-articular, intra-synovial, the formula: intrasternal, intrathecal, intrahepatic, intralesional and intrac 30 ranial injection or infusion techniques. Preferably, the com positions are administered orally, intraperitoneally or intra venously. For treatment of gastrointestinal disorders, the peptides described herein are preferably administered orally, e.g., as a 35 tablet, capsule, Sachet containing a predetermined amount of the active ingredient pellet, gel, paste, syrup, bolus, electuary, wherein R, R and R are independently selected from: H, slurry, powder, lyophilized powder, granules, as a Solution or C(O)OH, C1-C6 alkyl, C1-C6 alkylether, C1-C6 alkylthioet a suspension in an aqueous liquid or a non-aqueous liquid; as her, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylamide an oil-in-water liquid emulsion or a water-in-oil liquid emul and alkylaryl, wherein any group can be singly or multiply 40 sion, via a liposomal formulation (see, e.g., EP 736299) or in Substituted with: halogen or amino, and provided that no Some other form. Orally administered compositions can more than two of R, RandR are H. In another embodiment, include binders, lubricants, inert diluents, lubricating, Surface no more than one of R, R and R is H. active or dispersing agents, flavoring agents, and humectants. In other embodiments, there is provided a pharmaceutical Orally administered formulations such as tablets may option composition comprising a pharmaceutically acceptable car 45 ally be coated or scored and may be formulated so as to rier, peptide, a cation selected from Mg, Ca", Zn", Mn", provide sustained, delayed or controlled release of the active K", Na" or Al", or a mixture thereof, and a sterically hin ingredient therein. dered primary amine. In one embodiment, the cation is Mg", The peptides can be co-administered with other agents Ca" or Zn" or a mixture thereof. In a further embodiment, used to treat gastrointestinal disorders including but not lim the pharmaceutical composition further comprises a pharma 50 ited to the agents described herein. ceutically acceptable binder and/or a pharmaceutically In another aspect, Suitable pharmaceutical compositions acceptable glidant, lubricant or additive that acts as both a may comprise one or more other therapeutic agents. Such glidant and lubricant and/or an antioxidant. In some embodi therapeutic agents include, without limitation, analgesic ments, the pharmaceutical composition is applied to a carrier. agents; anti-secretory agents, including proton pump inhibi In some embodiments, the carrier is a filler. 55 tors, acid pump antagonists, H2 receptor antagonists; PDE5 In some cases the molar ratio of cation:sterically hindered inhibitors; GABA-Bantagonists; bile acid sequestrants; pro primary amine: peptide in the aqueous solution applied to the kinetic and promotility agents; antidepressants; antibiotics; carrier is 5-100:5-50:1. In some cases, the molar ratio of antiemetics; and mucosal-protecting agents. cation:sterically hindered primary amine may be equal to or Methods of Treatment greater than 2:1 (e.g., between 5:1 and 2:1). Thus, in some 60 In various embodiments, the peptides and compositions cases the molar ratio of cation:sterically hindered primary described herein are useful for the treatment of patient gas amine: peptide applied to the carrier is 100:50:1, 100:30:1. trointestinal disorder. 80:40:1, 80:30:1, 80:20:1, 60:30:1, 60:20:1, 50:30:1, 50:20: In some embodiments, the gastrointestinal disorder is 1, 40:20:1, 20:20:1, 10:10:1, 10.5:1 or 5:10:1. When binder, selected from the group consisting of irritable bowel syn e.g., methylcellulose, is present in the GC-C agonist peptide 65 drome (IBS), constipation, a functional gastrointestinal dis solution applied to the carrier it can be present at 0.5%-2.5% order, gastroesophageal reflux disease, functional heartburn, by weight (e.g., 0.7%-1.7% or 0.7%-1% or 1.5% or 0.7%). dyspepsia, visceral pain, gastroparesis, chronic intestinal US 8,933,030 B2 31 32 pseudo-obstruction, colonic pseudo-obstruction, Crohn's In another aspect, the invention features a method of disease, ulcerative colitis, and inflammatory bowel disease. increasing the level of cyclic guanosine 3'-monophosphate In a further embodiment, the gastrointestinal disorder is (cGMP) in a biological sample, tissue (e.g., the intestinal constipation. The constipation can be chronic constipation, mucosa), or cell (e.g., a cell bearing GC-A receptor), or whole idiopathic constipation, due to post-operative ileus, or caused 5 organism by contacting the sample, tissue, or organism to a by opiate use. Clinically accepted criteria that define consti peptides described herein. pation include the frequency of bowel movements, the con The peptide GC-C receptor agonists described herein can sistency of feces and the ease of bowel movement. One com be administered in combination with other agents. For mon definition of constipation is less than three bowel example, the peptides can be administered with an analgesic movements per week. Other definitions include abnormally 10 peptide or compound. The analgesic peptide or compound hard stools or defecation that requires excessive straining (Schiller 2001, Aliment Pharmacol Ther 15:749-763). Con can be covalently attached to a peptide described herein or it stipation may be idiopathic (functional constipation or slow can be a separate agent that is administered together with or transit constipation) or secondary to other causes including sequentially with a peptide described herein in a combination neurologic, metabolic or endocrine disorders. These disor 15 therapy. The peptides described herein may also be adminis ders include diabetes mellitus, hypothyroidism, hyperthy tered in combination with other agents used to treat GI dis roidism, hypocalcaemia, Multiple Sclerosis, Parkinson's dis orders including antidepressants, promotility or prokinetic ease, spinal cord lesions, Neurofibromatosis, autonomic agents, antiemetics, antibiotics, proton pump inhibitors, acid neuropathy, Chagas disease, Hirschsprung's disease and Cys blockers (e.g., histamine 112 receptor antagonists), acid tic fibrosis. Constipation may also be the result of Surgery pump antagonists, PDE5 inhibitors, GABA-B agonists, bile (postoperative ileus) or due to the use of drugs such as anal acid sequestrants, and mucosal protecting agents. gesics (like opioids), antihypertensives, anticonvulsants, In some embodiments, useful analgesic agents that may be antidepressants, antispasmodics and antipsychotics. used with the peptides described herein include Ca channel In other embodiments, the gastrointestinal disorder is irri blockers (e.g., Ziconotide), 5HT receptor antagonists (e.g., table bowel syndrome (IBS). The irritable bowel syndrome 25 5HT3, 5HT4 and 5HT1 receptor antagonists), 5HT4agonists can be constipation-predominant irritable bowel syndrome (e.g., tegaserod Zelnorm.R., mosapride, Zacopride, (c-IBS), diarrhea-predominant irritable bowel syndrome cisapride, renzapride, prucalopride Resolor R, benzimida (d-IBS) or alternating between the two irritable bowel syn Zolone derivatives such as BIMU 1 and BIMU 8, and lirex dromes (a-IBS). apride), 5HT1 agonists (e.g., Sumatriptan and buspirone), In other embodiments, the gastrointestinal disorder is dys 30 opioid receptor agonists (e.g., loperamide, fedotozine, pepsia. enkephalin pentapeptide, morphine, diphenyloxylate, frake In other embodiments, the gastrointestinal disorder is gas famide, trimebutine and fentanyl), CCK receptor agonists troparesis. The gastroparesis can be selected from idiopathic, (e.g., loxiglumide and dexloxiglumide), NK1 receptor diabetic or post-Surgical gastroparesis. antagonists (e.g., aprepitant, Vofopiitant, eZlopitant, R-673 In still other embodiments, the gastrointestinal disorder is 35 chronic intestinal pseudo obstruction. (Hoffmann-LaRoche Ltd), SR-48968 and SR-14033, (Sanofi In other embodiments, the gastrointestinal disorder is Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo Crohn's disease. Smith Kline) and TAK-637 (Takeda/Abbot)), NK2 receptor In some embodiments, the gastrointestinal disorder is antagonists (e.g., nepadutant, saredutant, GW5975.99 (Glaxo ulcerative colitis. 40 Smith Kline), SR-144190 (Sanofi-Synthelabo) and In some embodiments, the gastrointestinal disorder is UK-290795 (Pfizer Inc)), NK3 receptor antagonists (e.g., inflammatory bowel disease. osanetant (SR-142801; Sanofi-Synthelabo), SR-241586 and In another embodiment, the gastrointestinal disorder is talnetant), norepinephrine-serotonin reuptake inhibitors visceral pain. In a further embodiment, the present invention (NSRI) (e.g., milnacipran), mixed and selective dopamine features a method for decreasing gastrointestinal pain or vis 45 receptor antagonists (e.g. —metoclopramide, itopride, dom ceral pain in a patient, the method comprising, administering peridone), Vanilloid and cannabanoid receptor agonists, sia to the patient a pharmaceutical composition comprising of lorphin and sialorphin-related peptides. Analgesics agents in peptide described herein. The peptide agonists described the various classes are described in the literature. herein can be used alone or in combination therapy for the In some embodiments, one or more other therapeutic treatment, prevention or reduction of visceral pain associated 50 agents may be used in combination with the peptides with a gastrointestinal disorder or pain associated with described herein. Such agents include antidepressants, pro another disorder. motility or prokinetic agents, antiemetics, antibiotics, proton In another embodiment, the invention features a method for pump inhibitors, acid blockers (e.g., histamine H2 receptor treating inflammation, including inflammation of the gas antagonists), acid pump antagonists, PDE5 inhibitors, trointestinal tract, e.g., inflammation associated with a gas 55 GABA-B agonists, bile acid sequestrants, and mucosal pro trointestinal disorder or infection or some other disorder, the tecting agents. method comprising administering to a patient a pharmaceu Examples of antidepressants include, without limitation, tical composition comprising a purified peptide described tricyclic antidepressants such as amitriptyline (ElavilR), herein. desipramine (Norpramin(R), imipramine (TofranilR), amox In another embodiment, the invention features a method for 60 apine (AsendinR), nortriptyline; the selective serotonin treating a gastrointestinal disorder comprising administering reuptake inhibitors (SSRIs) such as paroxetine (Paxil.R.), an agonist of the intestinal guanylate cyclase (GC-C) receptor fluoxetine (ProzacR), sertraline (Zoloft(R), and citralopram either orally, by rectal Suppository, or parenterally. (CelexaR); and others such as doxepin (Sinequan R) and tra In still another embodiment, the invention features a Zodone (DesyrelR). method for treating a gastrointestinal disorder comprising 65 Examples of promotility and prokinetic agents include, administering an agonist of the intestinal guanylate cyclase without limitation, itopride, octreotide, bethanechol, meto (GC-C) receptor. clopramide (ReglanR), domperidone (Motilium(R), erythro US 8,933,030 B2 33 34 mycin (and derivatives thereof) and cisapride (Propulsider). day, with food after an overnight fast (e.g. with breakfast), at An example of antiemetics includes, without limitation, bedtime after a low fat snack. In one particular embodiment, prochlorperazine. the dosage unit is administered prior to or Subsequent to food Examples of antibiotics that may be used include those that consumption (e.g., a meal). In a further embodiment, the may be used to treat Heliobacter pylori infections, such as dosage unit is administered approximately 15 minutes to 1 amoxicillin, tetracycline, metronidazole, or clarithromycin. hour prior to food consumption. In various embodiments, the Other antibiotics such as erythromycin and derivatives dosage unit is administered once a day, twice a day, three thereof may also be used in combination with the peptides times a day, four times a day, five times a day or six times a described herein. day. In certain embodiments the dosage unit and daily dose Examples of proton pump inhibitors include, without limi 10 tation, omeprazole (PrilosecR), esomeprazole (Nexium(R), are equivalent. lansoprazole (PrevacidR), pantoprazole (Protonix(R) and The precise amount of each of the two or more active rabeprazole (Aciphex(R). Examples of H2 receptor blockers ingredients in a dosage unit will depend on the desired dosage include, without limitation, including cimetidine, ranitidine, of each component. Thus, it can be useful to create a dosage famotidine and nizatidine. Examples of acid pump antago 15 unit that will, when administered according to a particular nists include, without limitation, revaprazan, CS-526 (J. dosage schedule (e.g., a dosage schedule specifying a certain Pharmacol. Exp. Ther. (2007) 323:308-317), PF-03716556 number of units and a particular timing for administration), (J. Pharmacol. Exp. Ther. (2009)328(2):671-9), andYH1885 deliver the same dosage of each component as would be (Drug Metab. Dispos. (2001) 29(1):54-9). administered if the patient was being treated with only a Examples of PDE5 inhibitors include, without limitation, single component. In other circumstances, it might be desir avanafil, lodenafil, mirodenafil, sildenafil citrate, tadalafil, able to create a dosage unit that will deliver a dosage of one or Vardenafil and udenafil. GABA-B agonists include, without more components that is less than that which would be admin limitation, baclofen and XP 19986 (CAS Registry No. istered if the patient was being treated only with a single 847353-30-4). Examples of bile acid sequestrants include, component. Finally, it might be desirable to create a dosage without limitation, GT102-279, cholestyramine, coleseve 25 unit that will deliver a dosage of one or more components that lam, colesevelam hydrochloride, urSodeoxycholic acid, is greater than that which would be administered if the patient colestipol, colestilan, sevelamer, polydiallylamine cross was being treated only with a single component. linked with epichlorohydrin, dialkylaminoalkyl derivatives The pharmaceutical composition can include additional of a cross-linked dextran, and N-(cycloalkyl)alkylamines. ingredients including but not limited to the excipients Examples of mucosal protecting agents include, without limi 30 described herein. In certain embodiments, one or more thera tation, Sucralfate (Carafate), teprenone, polaprezinc, cetrax peutic agents of the dosage unit may exist in an extended or ate and bismuth subsalicyclate. control release formulation and additional therapeutic agents Combination therapy can be achieved by administering may not exist in extended release formulation. For example, a two or more agents, e.g., a peptide described herein and peptide or agonist described herein may exist in a controlled another therapeutic peptide or compound, each of which is 35 release formulation or extended release formulation in the formulated and administered separately, or by administering same dosage unit with another agent that may or may not be two or more agents in a single formulation. Other combina in eithera controlled release or extended release formulation. tions are also encompassed by combination therapy. For Thus, in certain embodiments, it may be desirable to provide example, two agents can be formulated together and admin for the immediate release of one or more of the agents istered in conjunction with a separate formulation containing 40 described herein, and the controlled release of one or more a third agent. While the two or more agents in the combination other agents. therapy can be administered simultaneously, they need not be. The present invention has been described with reference to For example, administration of a first agent (or combination certain exemplary embodiments thereof. However, it will be of agents) can precede administration of a second agent (or readily apparent to those skilled in the art that it is possible to combination of agents) by minutes, hours, days, or weeks. 45 embody the invention in specific forms other than those of the Thus, the two or more agents can be administered within exemplary embodiments described above. This may be done minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 without departing from the spirit of the invention. The exem hours of each other or within 1,2,3,4,5,6,7,8,9, 10, 12, 14 plary embodiments are merely illustrative and should not be days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks considered restrictive in any way. The scope of the invention of each other. In some cases even longer intervals are pos 50 is defined by the appended claims and their equivalents, rather sible. While in many cases it is desirable that the two or more than by the preceding description. agents used in a combination therapy be present in within the patient’s body at the same time, this need not be so. EXAMPLES Dosage The dose range for adult humans may be generally from 5 55 The GC-Cagonist peptides orpharmaceutically acceptable ug to 100 mg/day orally of the peptides described herein. salts thereofas described herein were prepared by solid phase Tablets, capsules, or other forms of presentation provided in chemical synthesis and natural folding (air oxidation) by discrete units may conveniently contain an amount of com American Peptide Company (Sunnyvale, Calif.). In some pound described herein which is effective at such dosage or as cases, the peptides were modified after synthesis as described a multiple of the same, for instance, units containing 25ug to 60 herein. 2 mg or around 100 ug to 1 mg. The precise amount of The Cys-IMD peptide was synthesized by mixing 4.6 g. compound prescribed to a patient will be the responsibility of (3.0 mmol) of linaclotide in 200 ml of EtOH. Formaldehyde the attendant physician. However, the dose employed will at 37% (1.12 ml/5 eq) was added to this mixture. The reaction depend on a number of factors, including the age and sex of mixture was incubated in a water bath (45° C.) for overnight. the patient, the precise disorder being treated, and its severity. 65 The following day the solvent was removed by rota-evapora In various embodiments, the dosage unit is administered tion. The peptide was further purified through reverse-phase with food at anytime of the day, without food at anytime of the chromatography. US 8,933,030 B2 35 36 The Glu-ethyl ester peptide was synthesized on a 20 mmol Supernatants from the previous spin were removed and Fmoc-Tyr(tBu)-Wang resin. Protecting groups used for placed into a fresh96 well HPLC plate. amino acids are: t-Butyl group for Tyr and Thr, Trt group for cGMP concentrations were determined from each sample ASn and Cys. The peptide chain was assembled on the resin using the LC/MS conditions (Table 1 below) and calculated by repetitive removal of the Fmoc protecting group and cou standard curve. ECso values were calculated from concentra pling of protected amino acid. DIC and HOBt were used as tion-response curves generated with GraphPad Prism Soft coupling reagents and NMM was used as the base for this Wa. reaction. 20% piperidine in DMF was used as de-Fmoc-re agent. After removal of last Fmoc protecting group, resin was TABLE 1 treated with cocktail K for 3 hours to cleave the peptide from 10 resin and removal of the side chain protecting groups. The LCMS conditions eluted peptide was precipitated in cold ether and dried. The MS: Thermo Quantum dried peptide was dissolved in a mixture of TFA/TIS/water Ion Mode: ESIt (95:3:2v/v) in a ratio of 1 to 10 (g/v). This mixture was stirred Scan Type: MRM 15 at room temperature for 1 hour. The isolated peptide was also Dwell Collision Retention precipitated in cold ether, collected by filtration and dried Time Energy Tube Time under high vacuum. Compound: Transition (msec) (V) Lens (min) The Tyra-ethyl ester peptide was synthesized by a frag ment condensation method. Fragment A (Boc-Cys(Trt)-Cys cGMP 346 - 152 100 28 139 1.O (Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro HPLC: Agilent Technologies 1200 Series with CTC Ala-Cys(Trt)-Thr(tBu)-Gly-OH) was prepared on 15 mmol Analytics HTS PAL CTC resin using Fmoc chemistry. This peptide chain was also Column: Thermo Hypersil Gold 2.1 x 50 mm 5 micron particle size assembled on the resin by repetitive removal of the Fmoc Flow Rate: 400 uL/min protecting group and coupling of protected amino acid. DIC Column RT and HOBt were used as coupling reagents and NMM was 25 Temperature: used as the base. 20% piperidine in DMF was used as de AutoSampler 6° C. Fmoc-reagent. After removal of last Fmoc protecting group, Temperature: Injection 20 uL Boc was coupled to protect the N-terminal amino group. The Volume: peptide resin was washed, dried, and treated with 1% TFA/ Mobile Phases: A = 98:2 Water:Acetonitrile + 0.1% Formic Acid DCM to cleave peptide from resin. Fragment B (Cys(Trt)- 30 B = 2:98 Water:Acetonitrile + 0.1% Formic Acid Tyr-OEt) was prepared from coupling of Fmoc-Cys(Trt)-OH and Tyr-OEt. HC1. The Fmoc group was removed by treating Time (min) % A % B this di-peptide with 20% piperidine in DMF. Gradient: O 100 O The Tyra-ethyl ester peptide was finally synthesized by O.3 3O 70 coupling the two fragments in DMF. HBTU/HOBt/NMM 35 2.OO 3O 70 was used as the coupling reagent for this reaction. The pro 2.01 100 O tecting groups were removed by treating the peptide with 4 100 O cocktail K for 2 hours. This peptide was precipitated in cold ether and dried. The dried peptide was dissolved in a mixture of TFA/TIS/water (95:3:2v/v) in a ratio of 1 to 10 (g/v). This 40 Example 2 mixture was stirred at room temperature for 1 hour. The isolated peptide was again precipitated in coldether, collected Relative Binding Affinity of Exemplary Peptides to by filtration and dried under high vacuum. the GC-C Receptor of T84 Cells Example 1 45 The relative binding affinities of linaclotide and Cys-IMD to the guanylate cyclase-C receptor (GC-C) were determined cGMP Accumulation in T84 Cells for Analysis of using a competitive-binding assay in which the peptides com GC-C Activity peted with a known GC-C agonist, porcine-derived heat stable enterotoxin (pSTa), for binding sites on cell-surface For the coMP assay, 4.5x10 cells/mL of T84 cells were 50 GC-C receptors on human colonic epithelial (T84) cells. The grown overnight in 24 well tissue culture plates. On next day, pSTa was radiolabeled with 'I to enable measurement of its the T84 cells were washed twice with 1 mL of DMEM (pH 7). receptor binding. The competitive-binding assay was per After the second wash, the cells were incubated with 450 LL of 1 mM isobutylmethylxanthine (IBMX) in pH7 buffer for formed by adding various concentrations of each peptide (0.1 10 minutes at 37°C. to inhibit any phosphodiesterase activity. 55 to 3,000 nM) to 0.20 mL reaction mixtures containing Dul The peptides were then diluted in DMEM buffer (pH 7) to a becco's modified Eagle's medium (DMEM), 0.5% bovine 10x concentration. The peptide solution of 50 uL was diluted serum albumin (BSA), 2.0x10T84 cells, and 170 pM'I- to a final volume of 500 uL with the T84 cells, bringing each pSTa (200,000 cpm). The data were used to construct com peptide concentration to lx. The peptides were tested in dupli petitive radioligand-binding curves and determine the rela cate at 100 nM. 60 tive binding affinities of linaclotide and Cys-IMD, as There was no peptide control used to determine endog measured by their ICso and K values. enous levels of c(GMP Peptides were incubated for 30 min Both linaclotide and Cys-IMD competitively inhibited the utes at 37° C. After 30 minutes, the supernatants were specific binding of 'I-pSTa to cell-surface GC-C recep removed and the cells were lysed with 0.1M HC1. The cells tors on T84 cells. Their relative binding affinities, as mea were lysed for 30 minutes on ice. After 30 minutes, lysates 65 sured by their inhibition constants (K), were as follows: were pipetted off and placed into a 96 well HPLC plate and Linaclotide K-3.9+1.6 nM and Cys-IMD K=1.4+0.5 nM spun at 10,000xG for 10 minutes to remove any cell debris. (FIG. 3). US 8,933,030 B2 37 38 Example 3 15-45% in 60 minutes of buffer Bat flow rate of 100 mL/min. The pooled fractions with purity around 95% were loaded cGMP Response in T84 Cells Induced by Exemplary onto C18 column. After equilibrating the column with TEAP Peptides buffer and AA buffer, the peptide was purified and eluted out with HAC buffer with a linear gradient of 15-75% of buffer B Linaclotide and Cys-IMD were tested for guanylate in 60 minutes. Pooled fractions with purified peptide were cyclase-C (GC-C) agonist activity in T84 cells as follows. In lyophilized to dryness. An example of an analysis of lina each well of a 96-well plate, approximately 200,000 T84 clotide and Cys-IMD product by RP-HPLC is shown in FIG. cells/well was first incubated with 1 mM 3-isobutyl-1-meth 2. ylxanthine (IBMX) in 0.18 mL of Dulbecco's modified 10 Glus-Ethyl Ester Peptide Eagle's medium (DMEM) for 10 minutes at 37° C. Each The Glu-ethyl ester peptide (6.0 g) was dissolved in 12 L peptide was diluted to final concentrations ranging from 0.1 of 0.05Mammonium bicarbonate in water, and the oxidation to 10,000 nM, and 0.02 mL of each dilution was added in process was monitored by Ellman's test, MS and analytical duplicate to a 96-well plate containing the T84 cells, for a HPLC. The oxidation process took approximately 48 hours final volume of 0.20 mL per well. The peptide reactions were 15 for completion. incubated for 30 min at 37° C. Following the incubation, the The above solution was filtered and loaded onto a 2-inch Supernatants were removed and discarded and the cells were C18 column, and purified by using 0.05Mammonium acetate lysed with cold 0.1 M hydrochloric acid (HCl) for 30 min on buffer with a linear gradient of 10-40% of buffer B in 60 ice. The cell debris was removed by centrifugation and the minutes at flow rate of 100 mL/min. concentration of guanosine 3',5'-cyclic monophosphate (cy The pooled fractions with purity of 95% were lyophilized clic GMP) in each lysate was determined using liquid chro to dryness. After the peptides were dried, the peptide was matography with tandem mass spectrometry. The data were re-dissolved in acetonitrile-water and acidified to pH around used to construct dose-response curves and calculate half 4-5 by addition of acetic acid and re-lyophilized to dryness. maximal effective concentration (ECso) values for each test Tyr-Ethyl Ester Peptide article. 25 The Tyra-ethyl ester peptide was purified by dissolving Linaclotide and Cys-IMD showed GC-C agonist activity 2.5 g of the isolated peptide in 5 L of 0.05M ammonium in T84 cells, as measured by the increase in intracellular bicarbonate in water, and the oxidation process was moni cGMP (FIG. 4). The ECs values for linaclotide and Cys tored by Ellman's test, MS and analytical HPLC. This oxida IMD were 315+105 nM and 172+32 nM, respectively. tion process took approximately 16 hours for completion. 30 The above solution was filtered and loaded onto a 2-inch Example 4 Polymer column, and purified by using 0.05M ammonium bicarbonate buffer with a linear gradient of 15-45% of buffer Measurement of Content and Purity of Exemplary B in 60 minutes at flow rate of 100 mL/min. The pooled Peptides fractions with the peptide were lyophilized to dryness. After 35 the peptide was dried, the peptide was re-dissolved in aceto Content and purity of the peptides of the present invention nitrile-water and acidified to pH 4-5 by addition of acetic acid may be determined by reverse phase gradient liquid chroma and re-lyophilized to dryness. tography using an Agilent Series 1100 LC System with The contents of the purified peptides were measured by Chemistation Rev A.09.03 software or equivalent. A YMC determining the peptide concentration in the prepared sample ProTMC18 column (dimensions: 3.0x150 mm, 3.5um, 120 A; 40 against a similarly prepared external peptide standard. Waters Corp., Milford, Mass.) or equivalent is used and is maintained at 40°C. Mobile phase A (MPA) consists of water Other Embodiments with 0.1% trifluoroacetic acid while mobile phase B (MPB) consists of 95% acetonitrile:5% water with 0.1% trifluoro All publications and patents referred to in this disclosure acetic acid. Elution of the peptides is accomplished with a 45 are incorporated herein by reference to the same extent as if gradient from 0% to 47% MPB in 28 minutes followed by a each individual publication or patent application were spe ramp to 100% MPB in 4 minutes with a 5 minute hold at 100% cifically and individually indicated to be incorporated by MPB to wash the column. Re-equilibration of the column is reference. Should the meaning of the terms in any of the performed by returning to 0% MPB in 1 minute followed by patents or publications incorporated by reference conflict a 10 minute hold at 100% MPA. The flow rate is 0.6 mL/min 50 with the meaning of the terms used in this disclosure, the and detection is accomplished by UV at 220 nm. meaning of the terms in this disclosure are intended to be Samples for analysis are prepared by addition of the con controlling. Furthermore, the foregoing discussion discloses tents of capsules of exemplary peptides to 0.1 NHCl to obtain and describes merely exemplary embodiments of the present a target concentration of 20 ug peptide/mL. 100 uL of this invention. One skilled in the art will readily recognize from Solution is injected onto the column. 55 Such discussion and from the accompanying drawings and Cys-IMD Peptide claims, that various changes, modifications and variations can The Cys-IMD peptide was purified using a 2-inch Waters be made therein without departing from the spirit and scope of C18 column with 0.1% TFA buffer with a linear gradient of the invention as defined in the following claims.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS : 1
<21 Os SEQ ID NO 1 &211s LENGTH: 14 212s. TYPE: PRT US 8,933,030 B2 39 40 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetically generated peptide
<4 OOs, SEQUENCE: 1 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O
What is claimed is: 3. The peptide of claim 1, wherein the peptide activates the 1. A peptide or a pharmaceutically acceptable salt thereof, guanylate cyclase C receptor. wherein the peptide comprises the amino acid structure of 15
S-S O 4. A pharmaceutical composition comprising a peptide or O HN S-S pharmaceutically acceptable salt thereof according to claim \l-N – — 1. Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. 2O s-s— 2. The peptide of claim 1, wherein the peptide consists of the amino acid structure of 25 5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition further comprises linaclotide. S-S O O HN -S-S 30 MN Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr.– — S-S — 6. The pharmaceutical composition of claim 4, wherein the pharmaceutical compositions further comprises a peptide comprising an amino acid structure selected from:
OH
CH Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-NH-C-C-O-CH2CH3He O CHCH O
OEC CH CH Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-s-s- - US 8,933,030 B2 41 42 7. The pharmaceutical composition of claim 4, wherein the an antioxidant, wherein said antioxidant is BHA, vitamin E pharmaceutical composition comprises less than 5% by or propyl gallate; or weight of the peptide or pharmaceutically acceptable salt a pharmaceutically acceptable filler, wherein the pharma ceutically acceptable filler is cellulose, microfine cellu thereof relative to the total weight of linaclotide. lose, microcrystalline cellulose, isomalt, mannitol or 8. The pharmaceutical composition of claim 7, wherein the dibasic calcium phosphate. pharmaceutical composition comprises less than 4% by 15. A method for treating a gastrointestinal disorder com weight of the peptide or pharmaceutically acceptable salt prising administering the pharmaceutical composition thereof relative to the total weight of linaclotide. according to claim 7, wherein the gastrointestinal disorder is 9. The pharmaceutical composition of claim 7, further selected from the group consisting of irritable bowel syn comprising one or more agents selected from (i) a cation 10 drome (IBS); constipation-predominant irritable bowel syn selected from Mg, Ca", Zn", Mn, K", Na or Al", or (ii) drome (c-IBS); constipation; chronic constipation, idiopathic a sterically hindered primary amine. constipation: constipation due to post-operative ileus; consti 10. The pharmaceutical composition of claim 9, wherein pation caused by opiate use; and visceral pain. said agent is Mg, Ca", Zn", Mn", K", Na" or Al" is 16. The pharmaceutical composition of claim 9 compris provided as magnesium acetate, magnesium chloride, mag 15 ing: nesium phosphate, magnesium Sulfate, calcium acetate, cal iCa?"; cium chloride, calcium phosphate, calcium Sulfate, Zinc ii leucine; acetate, Zinc chloride, Zinc phosphate, Zinc sulfate, manga iiilinaclotide; and nese acetate, manganese chloride, manganese phosphate, iv one or more peptides orpharmaceutically acceptable salt manganese Sulfate, potassium acetate, potassium chloride, thereof, wherein the peptide comprises an amino acid potassium phosphate, potassium Sulfate, Sodium acetate, structure selected from: Sodium chloride, Sodium phosphate, Sodium sulfate, alumi num acetate, aluminum chloride, aluminum phosphate or S-S aluminum sulfate. 25 11. The pharmaceutical composition of claim 9, wherein O said agent is a sterically hindered primary amine which is an HN O S-S amino acid selected from: \l-N- 1. Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr;– — a naturally-occurring amino acid, wherein the naturally S-S — occurring amino acid is histidine, phenylalanine, ala 30 nine, glutamic acid, aspartic acid, glutamine, leucine, fict methionine, asparagine, tyrosine, threonine, isoleucine, O tryptophan or valine; or a non-naturally occurring amino acid or an amino acid o= derivative, wherein the non-naturally occurring amino 35 acid is 1-aminocyclohexane carboxylic acid, lanthanine ". or theanine. it. 12. The pharmaceutical composition of claim 9, wherein Cys-Cys-NH- -C-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr; the sterically hindered primary amine has the formula: 40 H S-S- —
R
O Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys NH 'N' 45 NH2 s-s- wherein R, R and R are independently selected from: H, S-S C(O)OH, C1-C6 alkyl, C1-C6 alkylether, C1-C6 alkylthioet OH her, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylamide 50 and alkylaryl, wherein any group can be singly or multiply Substituted with: halogen or amino, and provided that no more than one of R, R2 and R is H. 13. The pharmaceutical composition of claim 12, wherein said pharmaceutical composition further comprises Mg", 55 Ca?", Zn, Mn, K, Na' or Al". CH 14. The pharmaceutical composition of claim 9, further -C-C-O-CHCH. comprising one or more of a pharmaceutically acceptable binder or additive, wherein H. O the pharmaceutically acceptable binder or additive is 60 selected from polyvinyl alcohol, polyvinylpyrrolidone 17. The pharmaceutical composition of claim 14, wherein (poVidone), a starch, maltodextrin or a cellulose ether, the pharmaceutically acceptable binder is hydroxypropyl wherein the cellulose ether is selected from: methylcel methylcellulose and the pharmaceutically acceptable filler is lulose, ethylcellulose, carboxymethylcellulose, microcrystalline cellulose. hydroxyethyl cellulose, hydroxyethyl methylcellulose, 65 18. The pharmaceutical composition of claim 7, wherein hydroxypropyl cellulose and hydroxypropyl methylcel the pharmaceutical composition further comprises a peptide lulose; comprising an amino acid structure selected from:
UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,933,030 B2 Page 1 of 1 APPLICATIONNO. : 13/579685 DATED : January 13, 2015 INVENTOR(S) : Angelika Fretzen et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
In the Claims,
Claim 6, column 40, line 32 and claim 7, column 41, line 1, for the claim reference numeral '4', each occurrence, should read --5--.
Signed and Sealed this Fourth Day of August, 2015 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office