AU2007340369C1 - GLP-1 pharmaceutical compositions - Google Patents

Abstract

The present invention is directed to peptide analogues of glucagon-like peptide-1, the pharmaceutically-acceptable salts thereof, to methods of using such analogues to treat mammals and to pharmaceutical compositions useful therefore comprising said analogues.

Description

-1 GLP-1 PHARMACEUTIC L COMPOSITIONS 5 Background of t Invention The present invention relates to im ovements in compositions containing peptide analogues of glucagon-like peptide 1 and/or pharmaceutically-acceptable salts thereof, methods for preparing such compositions, pharmaceutical 10 compositions and methods of using such co positions to treat mammals. Glucagon-like peptide-1 (7-36) amide (GLP-1) is synthesized in the intestinal L-cells by tissue-specific post-translational processing of the glucagon precursor preproglucagon (Varndell, J.M., et al., J. Flistochem Cytochem, 1985:33:1080-6) and is released into the circulation systerfi in response to a meal. The plasma 15 concentration of GLP-1 rises from a fasting level of approximately 15 pmol/L to a peak postprandial level of 40 pmol/L. It hs been demonstrated that, for a given rise in plasma glucose concentration, the increase in plasma insulin is approximately threefold greater when glucose is administered orally compared with intravenously (Kreymann, B., et al., Lar et 1987:2, 1300-4). This alimentary 20 enhancement of insulin release, known as the incretin effect, is primarily humoral and GLP-1 is thought to be the most pot nt physiological incretin in humans, In addition to the insulinotropic effect, GLP-1 suppresses glucagon secretion, delays gastric emptying (Wettergren A., et al., [Dig Dis Sci 1993:38:665-73) and may enhance peripheral glucose disposal (E-'Alessio, D.A. et al., J. Clin Invest 25 1994:93:2293-6). In 1994, the therapeutic potential )f GLP-1 was suggested following the observation that a single subcutaneous s/c) dose of GLP-1 could completely normalize postprandial glucose levels i patients with non-insulin-dependent diabetes mellitus (NIDDM) (Gutniak, M.K., -t al., Diabetes Care 1994:17:1039-44). 30 This effect was thought to be mediated bo by increased insulin release and by a reduction in glucagon secretion. Furtherrr ore, an intravenous infusion of GLP-1 has been shown to delay postprandial gastric emptying in patients with NIDDM (Williams, B., et al., J. Clin Endo Metab 1996:81:327-32). Unlike sulphonylureas, -2 the insulinotropic action of GLP-1 is depen ent on plasma glucose concentration (Holz, G.G. 4 th, et al., Nature 1993:361:362-5). Thus, the loss of GLP-1-mediated insulin release at low plasma glucose concentration protects against severe hypoglycemia. This combination of acti)ns gives GLP-1 unique potential 5 therapeutic advantages over other agents c rrently used to treat NIDDM. Numerous studies have shown that then given to healthy subjects, GLP-1 potently influences glycemic levels as well qs insulin and glucagon concentrations (Orskov,'C, Diabetologia 35:701-711, 1992; Holst, J.J., et al., Potential of GLP-1 in diabetes management in Glucagon Ill, Han book of Experimental Pharmacology, 10 Lefevbre PJ, Ed. Berlin, Springer Verlag, 1996, p. 311-326), effects which are glucose dependent (Kreymann, B., et al., Lancet ii: 1300-1304, 1987; Weir, G.C., et al., Diabetes 38:338-342, 1989). More er, it is also effective in patients with diabetes (Gutniak, M., N. Engl J Med 226:1316-1322, 1992; Nathan, D.M., et al., Diabetes Care 15:270-276, 1992), norma izing blood glucose levels in type 2 15 diabetic subjects (Nauck, M.A., et al., piabetologia 36:741-744, 1993), and improving glycemic control in type 1 patie ts (Creutzfeldt, W.O., et al., Diabetes Care 19:580-586, 1996), demonstrating its ability to, inter alia, increase insulin sensitivity/reduce insulin resistance. G 1 and agonists thereof have been proposed for use in subjects at risk for developing non-insulin dependent diabetes 20 (see WO 00/07617) as well as for the tre tment of gestational diabetes mellitus (U.S. Patent Pub. No. 20040266670). In addition to the foregoing, there are a number of therapeutic uses in mammals, e.g., humans, for which GLIR-1 and agonists thereof have been suggested, including, without limitation: proving learning, enhancing neuro 25 protection, and/or alleviating a symptom <f a disease or disorder of the central nervous system, e.g., through modulation f neurogenesis, and e.g., Parkinson's Disease, Alzheimer's Disease, Huntingto 's Disease, ALS, stroke, ADD, and neuropsychiatric syndromes (U.S. Pat nt Pub. No.'s 20050009742 and 20020115605); converting liver stem/p ogenitor cells into functional cells 30 pancreatic (WO03/033697); preventing be a-cell deterioration (U.S. Patent Pub. No.'s 20040053819 and 20030220251) a id stimulation of beta-cell proliferation (U.S. Patent Pub. No. 20030224983); tr ating obesity (U.S. Patent Pub. No. 2967035 1 (GH Maer P81399AU l H -3 20040018975; W098/19698); suppressing appetite and inducing satiety (U.S. Patent Pub. No. 20030232754); treating irri able bowel syndrome (WO 99/64060); reducing the morbidity and/or mortality ass cited with myocardial infarction (US Patent Pub No. 20040162241, WO98f0831) and stroke (see WO 00/16797); 5 treating acute coronary syndrome chara terized by an absence of Q-wave myocardial infarction (U.S. Patent Pub. o. 20040002454); attenuating post surgical catabolic changes (US Patent No. 6,006,753); treating hibernating myocardium or diabetic cardiomyopathy (.S. Patent Pub. No. 20050096276); suppressing plasma blood levels of noiepinepherine (U.S. Patent Pub. No. 10 20050096276); increasing urinary sodium 4cretion, decreasing urinary potassium concentration (U.S. Patent Pub. No. 20050037958); treating conditions or disorders associated with toxic hypervolen ia, e.g., renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pul onary edema, and hypertension (U.S. Patent Pub. No. 20050037958); inducing an inotropic response and increasing 15 cardiac contractility (U.S. Patent Pub. No. 00500 37 9 58); treating polycystic ovary syndrome (U.S. Patent Pub. No.'s 20 0266678 & 20040029784); treating respiratory distress (U.S. Patent Pub. No. 0040235726); improving nutrition via a non-alimentary route, i.e., via intravenous, subcutaneous, intramuscular, peritoneal, or other injection or infusion U.S. Patent Pub. No. 20040209814); 20 treating nephropathy (U.S. Patent Pub. N4 20040209803); treating left ventricular systolic dysfunction, e.g., with abnormal[ left ventricular ejection fraction (U.S. Patent Pub. No. 20040097411); inhibiting antro-duodenal motility, e.g., for the treatment or prevention of gastrointestir al disorders such as diarrhea, post operative dumping syndrome and irritable owel syndrome, and as premedication 25 in endoscopic procedures (U.S. Patent Fub. No. 20030216292); treating critical illness polyneuropathy (CIPN) and systemic inflammatory response syndrome (SIRS) (U.S. Patent Pub. No. 20030199W45); modulating triglyceride levels and treating dyslipidemia (U.S. Patent Pub. Igo.'s 20030036504 and 20030143183); treating organ tissue injury caused by rep rfusion of blood flow following ischemia 30 (U.S. Patent Pub. No. 20020147131); tre ting coronary heart disease risk factor (CHDRF) syndrome (U.S. Patent Pub. No. 20020045636); and others.

-4 GLP-1 is, however, metabolically un able, having a plasma half-life (t1/) of only 1-2 min in vivo. Exogenously administered GLP-1 is also rapidly degraded (Deacon, C.F., et al., Diabetes 44:1126-1,, 31, 1995). This metabolic instability limits the therapeutic potential of native GL '-1. A number of attempts have been 5 taken to improve the therapeutic potential of GLP-1 and its analogues through improvements in formulation. For example, International patent publication no. WO 01/57084 describes a process for pi during crystals of GLP-1 analogues which are said to be useful in the prepare nation of pharmaceutical compositions, such as injectable drugs, comprising the cr stals and a pharmaceutical acceptable 10 carrier. Heterogeneous micro crystalline clusters of GLP-1(7-37)OH have been grown from saline solutions and examined fter crystal soaking treatment with zinc and/or m-cresol (Kim and Haren, Pharm Res. Vol. 12 No. 11 (1995)). Crude crystalline suspensions of GLP(7-36)NH: containing needle-like crystals and amorphous precipitation have been prepared from phosphate solutions containing 15 zinc or protamine (Pridal, et. al., internatioi al Journal of Pharmaceutics Vol. 136, pp. 53-59 (1996)). European patent publication no. EP 0619322A2 describes the preparation of micro-crystalline forms of GL P-1(7-37)OH by mixing solutions of the protein in pH 7-8.5 buffer with certain cci binations of salts and low molecular weight polyethylene glycols (PEG). U.S. Fatent No. 6,566,490 describes seeding 20 microcrystals of, inter alia, GLP-1 which ar* said to aid in the production of purified peptide products. U.S. Patent 6,555,521 (US '521) discloses GLP-1 crystals having a tetragonal flat rod or a plate-like khape which are said to have improved purity and to exhibit extended in vivo activity. US '521 teaches that such crystals are relatively uniform and remain in susp nsion for a longer period of time than 25 prior crystalline clusters and amorphous crystalline suspensions which were said to settle rapidly, aggregate or clump togefer, clog syringe needles and generally exacerbate unpredictable dosing. A biodegradable triblock copolynier of poly [(dl-lactide-co-glycolide)-#3 ethylene glycol-#-(-lactide-co-glycolide)] ihas been suggested for use in a 30 controlled release formulation of GLP-1. H ever like other polymeric systems, the manufacture of triblock copolymer invol s complex protocols and inconsistent particulate formation.

-5 Similarly, biodegradable polymers, e.1., poly(lactic-co-glycolic acid) (PLGA), have also been suggested for use in susta ned delivery formulations of peptides. However the use of such biodegradable p ymers has been disfavored in the art since these polymers generally have poor solubility in water and require water 5 immiscible organic solvents, e.g., methyl e chloride, and/or harsh preparation conditions during manufacture. Such orga ic solvents and/or harsh preparation conditions are considered to increase the riE k of inducing conformational change of the peptide or protein of interest, resultin in decreased structural integrity and compromised biological activity (Choi et E , Pharm. Research, Vol. 21, No. 5, 10 (2004).) Poloxamers have been likewise fated. (Id.) The GLP-1 compositions described in the foregoing references are less than ideal for preparing pharmaceutical fo mulations of GLP's since they tend to trap impurities and/or are otherwise diffi ut to reproducibly manufacture and administer. Also, GLP analogues are k own to induce nausea at elevated 15 concentrations, thus there is a need to provide a sustained drug effect with reduced initial plasma concentrations (R zel et al., Diabetologia, 38: 720-725 (1995); Gutniak et al., Diabetes Care, 17 9): 1039-1044 (1994); Deacon et al., Diabetes, 44: 1126-1131 (1995).) Hence there is a need for GLP-1 formulations which are more easily and reliably manufactured, that are more easily and 20 reproducibly administered to a patient, an that provide for reduced initial plasma concentrations in order to reduce or elimin te unwanted side-effects. Summary of tl e Invention The invention may be sumi arized in the following paragraphs as 25 well as the claims. Accordingly, th a invention provides a pharmaceutical composition comprising an analogue of GLP-1, wherein the GLP-1 analogue is [Aib 8 3 5 hGLP-1 (7-36) H 2 ,prepared with a pharmaceutically acceptable salt of said analogue o a mixture of the salt of said analogue and the analogue, thereby provi ng a molar ratio of analogue salt to 30 peptide analogue in said pharmaceutical composition, wherein the molar ratio can be adjusted to modulate .he solubility, the pH, and release effect on in vivo release profile of the pep de in said pharmaceutical composition, -6 said composition further comprising divalent metal and/or divalent metal salt, with a molar ratio of said GLP-1 analogue to said divalent metal and/or divalent metal salt in said pharmaceutical composition ranging from approximately 5.4:1 to approximately 1.5:1. 5 Preferably the pharmaceutical composition of the invention does not consist of a clear aqueous ZnCl 2 solution having pH 4 n which said [Aib"]lhGLP-1(7-36)NH2 is present at a concentration of 4 mgf i and said ZnC 2 is present at a concentration of 0.5 mg/ml. One embodiment of the invent on provides for a pharmaceutical 10 composition having an improved drug relIase profile, preferably with a reduced initial burst. In preferred features, t4 invention also provides for a pharmaceutical composition which precipit tes in vivo at physiological pH to form an in situ deposit for a sustained release dr pg profile. 15 In an embodiment the pharm ceutical composition comprises a pharmaceutically acceptable carrier or dil kent. Preferably said carrier or diluent comprises water. Preferably, the salt of the GLP-1 p ptide analogue in said pharmaceutical composition is selected from the list 0 pharmaceutically acceptable salts of 20 organic acids, such as those of acetic, Iltic, malic, ascorbic, succinic, benzoic, citric, methanesulphonic or toluenesulphortc acids, or pharmaceutically acceptable salts of inorganic acids, such as those f hydrochloric, hydrobromic, hydriodic, sulfuric or phosphoric acids. Pharmaceu ically acceptable salts of strong acids, such as hydrochloric acid, are particularly !referred. A strong acid is defined as an 25 acid having a pKA of less than 4.5. A ditional preferred peptide salts in said pharmaceutical composition are salts of o anic acids such as those of acetic acid or trifluoroacetic acid, lactic, malic, ascorbic, succinic, benzoic, or citric acid. The solubility, the pH, and the release profile of the pharmaceutical composition can be modulated by adjusti pg the molar ratio of GLP-1 analogue in 30 salt form to GLP-1 analogue not in salt form to extend the release profile and reduce the initial spike in GLP-1 analogue oncentration. | 67035 1 (GHMlters) P81399A -7 The. divalent metal can lower the w ter solubility of the composition and thereby extend the release profile while sim Itaneously reducing the initial burst or spike in plasma concentrations. Preferred d talent metals include zinc and copper. Salt forms of the divalent metals are particu rly preferred, including but not limited 5 to chloride and acetate salts of the divalen metals. CuAc 2 , CuC 2 , ZnAc 2 , and/or ZnCl 2 are most preferred. Preferably, the di alent metal and/or divalent metal salts in said pharmaceutical composition is pr sent in a concentration from about 0.0005mg/ml to about 50mg/m. Even mor preferably, the divalent metal and/or divalent metal salts in said pharmace tical composition is present in a 10 concentration from about 0.01 mg/mI to ab ut 0.50 mg/ml. More preferably, said pharmaceutical composition comprises a di uent, wherein said diluent comprises a pharmaceutically acceptable aqueous solu ion. The diluent may comprise sterile water. Ina further embodiment, said pharm iceutical composition further comprises 15 a divalent metal and/or divalent metal salt, wherein the molar ratio of said GLP-1 analogue to said divalent metal and/or di talent metal salt in said pharmaceutical composition ranges from approximately 6: to approximately 1:1. Preferably, said ratio ranges from approximately 5.5:1 to a proximately 1:1. More preferably, said ratio ranges from approximately 5.4:1 to approximately 1.5:1. Even more 20 preferably still, said ratio is approximately 5.4:1, 4.0:1, or 1.5:1. Most preferably, said ratio is approximately 1.5:1. What is ieant by approximately in this aspect of the invention is a ratio of 1.5:1 ± 10% Edach target value, thus expected ratios include ratios encompassing, e.g., 1.35-1. 5:0.85-1.15. Preferably, said pharmaceutical composition comprises an aqueous 25 mixture, suspension or solution, wherein said analogue of GLP-1, compound of formula (1), or salt thereof is present at , concentration of approximately 0.5% 30% (w/w). More preferably the concentr tion of said GLP-1 analogue and/or salt thereof in said aqueous mixture, suspense n or solution is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 30 18%, 19%, 20%, 21%, 22%, 23%, 24% 25%, 26%, 27%, 28%, 29%, or 30% (w/w). More preferably, the concentrating of said GLP-1 analogue and/or salt thereof in said aqueous solution is appr im 1%, 2%, 3%, 4%, 5%, 6%, 7%, I 29870AS5 1 CHMaterSl la) ~ fS ' ~ .~tc f~l -8 8%, 9%,, 10%, 11%, 14%, 15%, 16%, 190%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 29%, or 30% (wlw). More preferably still, the concentration of said analogue of GLP-1 analogue and/or salt thereof in s id aqueous solution is approximately 1%, 2%, 3%, 4%, 5%, 6%, 9%, 10%, 11%, 22%, 23%, 24%, 25%, or 26% (w/w). 5 Even more preferably still, the concentratior of said analogue of GLP-1 and/or salt thereof in said aqueous solution is approxin ately 1%, 2%, 3%, 4%, 5%, 6%, 10%, 22%, 23%, 24%, 25%, or 26% (wlw). Still more preferably, the concentration of said analogue of GLP-1 and/or salt tl ereof in said aqueous solution is approximately 1%, 2%, 5%, 10%, 23% or 2 % (w/w). By "approximately" is meant 10 the following: for concentrations of about 0 5% to about 4%, +0.5% of the target value is the desired range (for example, O 5% to 1.5% is approximately 1%); for target concentrations of about 5% and h her, 20% of the target value is the desired range (for example, 8% to 12% is al proximately 10%). Preferably, the concentration of {tibaas]hGLP-1(7-36)NH 2 , analogue of 15 GLP-1, or salt thereof in the pharmaceutical composition is about 1% (weight/volume) and the molar ratio of [Ai 8 35]hGLP-1(7-36)NH 2 to said divalent metal and/or divalent metal salt is about 1. :1. More preferably, the concentration of [Aib 8

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35 ]hGLP-1(7-36)NH 2 or salt thereof in said pharmaceutical composition is about 2% (weight/volume) and the molar r tio of [Aib 8

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5 ]hGLP-1(7-36)NH 2 or salt 20 thereof to said divalent metal and/or div lent metal salt is about 1.5:1. More preferably still, the concentration of [Aiba8,31 GLP-1 (7-36)NH 2 or salt thereof in said pharmaceutical composition is about 10% (weight/volume) and the molar ratio of [Aib 8

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35 ]hGLP-1(7-36)NH 2 or salt thereof said divalent metal and/or divalent metal salt is about 1.5:1. Most preferably, the concentration of [Aib 835 ]hGLP-1(7 25 36)NH 2 or salt thereof in said pharmaceutical composition is about 23% or about 25% (weight/volume) and the molar ratio' of [Aib 35 ]hGLP-1(7-36)NH 2 or salt thereof to said divalent metal and/or divaler t metal salt is about 1.5:1. In a preferred embodiment, the concentration of the analogue of GLP-1, [Aib" 35 ]hGLP-1(7-36)NH 2 , or salts thereof in the pharmaceutical composition is 30 about 5% (weight/volume) and the molar ratio of the peptide to the divalent metal and/or divalent metal salt is approxiately 5.4:1. More preferably, the concentration of [Aiba35]hGLP-1(7-36)NH1 or salt thereof in said composition is 2%7351 gl44GH)a PS flA -9- i about 5% (weight/volume) and said ratio is approximately 4.0:1. More preferably still, the concentration of [Aib 835 hGLP-'j(7-36)NH2 or salt thereof in said composition is about 10% (weight/volume) nd said ratio is approximately 5.4:1. Still further preferably, the concentration of [Aib 8 5]hGLP-1(7-36)NH2 or salt 5 thereof in said composition is about 10% (weight/volume) and said ratio is approximately 4.0:1. Preferably, said divalent metal and/o divalent metal salt is provided as zinc chloride or zinc acetate. More preferably, s id zinc acetate is provided as ZnAc2-2

H

2 0. 10 In an alternative embodiment, said d valent metal and/or divalent metal salt is provided as copper chloride or copper ac tate. In one embodiment, the pH of said F harmaceutical composition is adjusted upward using a base. More preferably, sai pH adjustment is made using NaOH. More preferably still, the pH of said pharrr aceutical composition is adjusted with 15 NaOH such that, when diluted to approxim tely % initial concentration using 0.9% NaCI, a pH value of approximately 5.0 - 5. is obtained using direct potentiometric determination. A preferred embodiment of the !invention features a pharmaceutical composition, wherein the composition is fcjmulated such that a peptide analogue 20 of GLP-1 or salt thereof, e.g., the compund according to formula (I) or salt thereof, is released within a subject in neeh thereof, e.g., a mammal, preferably a human, for an extended period of time. Prferably said release of said compound extends for at least one hour, more prefer bly at least 4, 6, 12, or 24 hours. More preferably still, said composition is formula ted such that the compound according 25 to formula (I) is released within a subject for at least 36, 48, 60, 72, 84, or 96 hours. More preferably still, said composition is formulated such that the compound according to formula (1) is leased within a subject for at least approximately 5, 6, 7, 8, 9, 10, 11, 12, 1 , or 14 days. More preferably still, said composition is formulated such that the compound according to formula (1) is 30 released'within a subject for at least approximately 2, 3 or 4 weeks. Even more preferably, said composition is formulate such that the compound according to forulte -10 formula (1) is released within a subject for, at least approximately 1, 1.5, 2, or 3 months, or longer. The modulation of the salt content Of the GLP-1 peptide analogue in said pharmaceutical composition can improveth solubility and the stability of the GLP 5 1 peptide analogue in the pharmaceutical imposition and furthermore provide an improvement on the in vivo release profile t decreasing the initial burst. The wording "modulation" means in ;his aspect of the invention adjustment of salt content by adjusting the molar ratic of the GLP-1 analogue in salt form to GLP-1 analogue not in salt form. 10 Even more preferably, the peptide salt in said pharmaceutical composition is a salt of hydrochloric or acetic acid, or hlorides or acetates of said peptide of formula (1). In said pharmaceutical compo ition the acetate or chlorides is present as final molar ratio of acetate or chloride to said compound of formula (1) in ranges from approximately of 0.5:1 to approximately 10:1. More preferably said ratio 15 ranges from approximately 0.8:1 to appro imately 9:1. Even more preferably said ratio is approximately 1:1 to approxima ly 6:1. Most preferably said ratio is approximately 3.0:1 in particular 3.2:1. In this aspect of the invention th molar ratio of acetate or chloride to peptide means the molar proportion of attate (CH 3 COO) or chloride (Cl) in the 20 pharmaceutical composition to the m ar proportion of the peptide in the pharmaceutical composition. In example for a molar ratio of 3:1 in the pharmaceutical composition, acetate is khree times the molar content of the peptide in proportion. This is a stoichiome ic ratio of a compound compared to the other. 25 The wording "approximately" mean in this aspect of the invention a ratio of 1.5:1 + 10% each target value, thus exp ted ratios include ratios encompassing, e.g., 1.35-1.65:0.85-1.15. In additional preferred aspects of the invention, the pharmaceutical composition pH is adjusted by modu ation of the acetate content of the 30 composition. Preferably, the pH ranged of said pharmaceutical composition is from pH 3 to pH 6. More preferably tid pH ranges of said pharmaceutical 2987035 ~ ~1 3 90QA' togi P35) 'L -11 composition is from pH 3.5 to 5.5. Mos preferably said pH ranges of said pharmaceutical composition is from pH 4.2 t4 pH 4.6. Preferably, to acidify the pharmaceical composition the acetate content may be increased by adding acetic acid. 5 In one embodiment, the pH of the sa pharmaceutical composition may be increased starting from a peptide salt of an analogue of GLP-1 having a low acetate or no acetate content by modulation f acetate content. In preferred embodiments, adjustme t of the pH in the final pharmaceutical composition by modulation of acetate or chloride content allows modulation of 10 parameters such as, the peptide concentration, the zinc concentration, the chemical stability, the physical stability an in vivo release profile by decreasing the initial burst. In an embodiment Zn or Cu content fixed, pH is controlled by modulating the acetate content. Increased content of estate shows an improvement on the 15 solubility and the physical stability and d reased content of acetate shows an increasing effect on the pH and decreasing effect on the Cmnx. In preferred embodiments, said ph maceutical composition comprises an aqueous mixture, suspension or solution. Aso. provided is a method of elicitin a GLP-1 agonist effect, said method 20 comprising contacting a receptor of the kLP-1(7-36)NH 2 ligand with a GLP-1 analogue or salt thereof, directly or indirectl In the foregoing method, said rec ptor of the GLP-1(7-36)NH 2 ligand is present in an animal subject, preferably a imate, more preferably a human being. Thus, in this embodiment the present inv tion provides a method of eliciting an 25 agonist effect from a GLP-1 receptor in a bject in need thereof which comprises administering to said subject a composition of the instant invention, wherein said composition comprises an effective a ount of a GLP-1 analogue or a pharmaceutically acceptable salt thereof. In a preferred aspect of the foreg ing method, said subject is a human 30 afflicted with, or at risk of developing, a isease or condition selected from the group consisting of Type I diabetes, Type I diabetes, gestational diabetes, obesity, -12- i excessive appetite, insufficient satiety, anj metabolic disorder. Preferably said disease is Type I diabetes or Type II diabetes. In another more preferred aspect of he foregoing method, said subject is a human afflicted with, or at risk of developi g, a disease selected from the group 5 consisting of Type I diabetes, Type II diab es, obesity, glucagonomas, secretory disorders of the airway, arthritis, osteopo sis, central nervous system disease, restenosis, neurodegenerative disease, r nal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, hypertension, and disorders wherein the reduction of food intake is desi ed, a disease or disorder of the central 10 nervous system, (e.g., through modulation of neurogenesis, and e.g., Parkinson's Disease, Alzheimer's Disease, Huntingtc 's Disease, ALS, stroke, ADD, and neuropsychiatric syndromes), irritable bow l syndrome, myocardial infarction (e.g., reducing the morbidity and/or mortality associated therewith), stroke, acute coronary syndrome (e.g., characterized t an absence of Q-wave) myocardial 15 infarction, post-surgical catabolic change , hibernating myocardium or diabetic cardiomyopathy, insufficient urinary sodiurr excretion, excessive urinary potassium concentration, conditions or disorders asTociated with toxic hypervolemia, (e.g., renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, and hypertension), polycystic vary syndrome, respiratory distress, 20 nephropathy, left ventricular systolic c function, (e.g., with abnormal left ventricular ejection fraction), gastroint stinal disorders such as diarrhea, postoperative dumping syndrome and irrit ble bowel syndrome, (i.e., via inhibition of antro-duodenal motility), critical illness polyneuropathy (CIPN), systemic inflammatory response syndrome (SIRS), yslipidemia, organ tissue injury caused 25 by reperfusion of blood flow following isc emia, and coronary heart disease risk factor (CHDRF) syndrome. In an additional aspect of the inver ion, the invention features a method of converting liver stem/progenitor cells into Functional pancreatic cells, of preventing beta-cell deterioration and of stimulatin beta-cell proliferation, of suppressing 30 plasma blood levels of norepinepherine, f inducing an inotropic response and of increasing cardiac contractility, of improv ng nutrition via a non-alimentary route, (e.g., via intravenous, subcutaneous, intra uscular, peritoneal, or other injection or 296705S I GHMaUrs) P81399AU -13-i infusion rout), of pre-treating a subject to u dergo an endoscopic procedures, and of modulating triglyceride levels, in a si bject in need thereof, said method comprising administering to said subject formulation of the present invention comprising an effective amount of a compo ind of formula (I) or a pharmaceutically 5 acceptable salt thereof. Preferably said s bject is a mammalian animal, more preferably a primate, more preferably still a uman being. Brief DescriDtic h of the Drawings Figure 1 depicts the plasma profiles median values) obtained after a single 10 subcutaneous (s.c.) administration to dogs f approximately 1mg of [Aib 8

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35 ]hGLP 1(7-36)NH 2 . In each case the peptide w4s administered as an aqueous zinc composition comprising approximately 1% (wt/vol) peptide and having a peptide:Zn molar ratio of approximately 1 5. Filled squares and open squares represent compositions in which the pH 1is adjusted with NaOH as described 15 herein; filled triangles represent a compostion in which the pH was not adjusted with NaOH; filled circles represent a compc ition in buffered with AcOH/AcO-. Figure 2 depicts the plasma profiles (median values) obtained after a single subcutaneous (s.c.) administration to dogs of approximately 15 mg of [Aib 8

'

35 ]hGLP-1(7-36)NH 2 . In each case khe peptide was administered as an 20 aqueous zinc composition comprising approximately 10% (wt/vol) peptide and having a peptide:Zn molar ratio of apprc ximately 1.5. Filled squares and open squares represent compositions in whict the pH is adjusted with NaOH as described herein; filled triangles represent'a composition in which the pH was not adjusted with NaOH; filled circles repr sent a composition in buffered with 25 AcOH/AcO-. Figure 3 depicts the plasma profileE (median values) obtained after a single subcutaneous (s.c.) administration to dogs of approximately 1 mg of Aib 835 ]hGLP 1(7-36)NH 2 . In each case the peptide wa, administered as an semisolid aqueous zinc composition as follows: solid circle: about 5% (wt/vol) peptide, peptide:Zn 30 molar ratio about 5.4:1, no pH adjustment; open circle: about 10% (wt/vol) peptide, peptide:Zn molar ratio about 5.4:1, no pl adjustment; open square: about 10% (wt/vol) peptide, peptide:Zn molar ratio a ut 5.4:1, pH adjusted with NaOH; solid -14- i square: about 10% (wt/vol) peptide, peptid(:Zn molar ratio about 4:1, pH adjusted with NaOH. i Figure 4 provides a schematic presentation of various devices useful in preparing certain formulations of the present invention. 5 Figure 5 depicts the plasma profiles median values) obtained after a single subcutaneous (s.c.) administration to dogs 1 1 f approximately 1 mg of [Aib 8 f5]hGLP 1(7-36)NH 2 . The peptide was administered s an aqueous zinc composition having a peptide concentration of about 2%, and a peptide:Zn molar ratio of about 1.5:1. Figure 6 depicts the plasma profiles median values) obtained after a single 10 subcutaneous (s.c.) administration to ogs of approximately 15 mg of [Aib 8

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3 ]hGLP-1(7-36)NH 2 . The peptide was administered as a semisolid zinc composition having a peptide concentration of about 25%, and a peptide:Zn molar ratio of about 4:1. Figure 7 depicts the plasma profiles (median values) obtained after a single 15 subcutaneous (s.c.) administration to [dogs of approximately 15 mg of [Aib 8 3]hGLP-1(7-36)NH 2 . The peptide %as administered as a semisolid zinc composition having a peptide concentratio of about 23%, and a peptide:Zn molar ratio of about 1.5:1. Figure 8 depicts the full time course plasma profiles (median values) 20 obtained after a single subcutaneous (s.c.) administration to rats of 0.3mg of (3 [L of 10% solution) of the GLP-1 analogue H 3 1 salt test formulations: (1) (Aib 8

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3 )hGLP-1(7-36)NH 2 Hd1 salt with CuCl 2 : the molar ratio of (Aib')hGLP-1(7-36)NH 2 /CuCl 2 is 1[5:1. The peptide concentration is 10% (30 mM) in water (w/w) with approxi nately pH5.5. 25 (2) (Aib 8

,

35 )hGLP-1(7-36)NH 2 H A salt with ZnCl 2 : the molar ratio of (Aib 835 )hGLP-1(7-36)NH 2 /ZnC 2 is t.5:1. The peptide concentration is 10% (30 mM) in water (w/w) with approxi jnately pH5.5. 30 Figure 9 depicts the full time c4 rse plasma profiles (median values) obtained after a single subcutaneous (s.c. administration to rats of 0.3mg of (3 pL of 10% solution) of the GLP-1 analogue acetate salt test formulation: (Aiba 35 )hGLP-1(7-36)NH 2 acetate salt with ZnCl 2 : the molar ratio of 35 (Aiba 35 )hGLP-1(7-36)NH 2 /ZnC 2 is .5:1. The peptide concentration is 10% (30 mM) in water (w/w) with approxi ately pH5.5. | rornas1 /G~alttM P139 -15 Figure 10 depicts the early time course plasma profiles (median values) obtained after a single subcutaneous (s.c.) a ministration to rats of 0.3mg of (3 pL of 10% solution) of the test formulations shoin in Figure 8. 5 Figure 11 depicts the early time co irse plasma profiles (median values) obtained after a single subcutaneous (s.c.) administration to rats of 0.3mg of (3 jL of 10% solution) of the test formulations sho n in Figure 9. Figure 12 depicts the estimated pe centage of (Aib 8

,

35 )hGLP-1(7-36)NH 2 remaining at the injection site of rats after a single subcutaneous (s.c.) 10 administration of 0.3mg of (3 ItL of 10% solution) of the three test formulations shown in Figure 8. Detailed Descriotion 15 A preferred GLP-1 peptide, to be util zed as a peptide salt of the invention, is denoted herein by the following format, e g., (Aib 835 )hGLP-1(7-36)NH 2 , with the substituted amino acids from the natural se uence placed between the first set of parentheses (e.g., Aib 8 ,3 5 denotes that Ail is substituted for Ala 8 and Gly 35 in hGLP-1). Aib is the abbreviation for a-amintisobutyric acid. The abbreviation GLP 20 1 means glucagon-like peptide-1; hGLP-1 ineans human glucagon-like peptide-1. The numbers between the second set of pa entheses refer to the number of amino acids present in the peptide (e.g., hGLP-1( 36) refers to amino acids 7 through 36 of the peptide sequence for human GLP- ). The sequence for hGLP-1(7-37) is listed in Mojsov, S., Int. J. Peptide Prot n Res,. 40, 1992, pp. 333-342. The 25 designation "NH 2 " in hGLP-1(7-36)NH 2 indicates that the C-terminus of the peptide is amidated. hGLP-1(7-36) means that the C-terminus is the free acid. In hGLP 1(7-38), residues in positions 37 and 38 re Gly and Arg, respectively, unless otherwise indicated. Particularly preferred GLP-1 peptide analogues used in this invention are in 30 the form of pharmaceutically acceptable sa s. Examples of such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, metha esulfonic, toluenesulfonic, or pamoic 2MS GMO)P 9 -16 acid), inorganic acids (e.g., hydrochloric a id, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-gly olic acids). A typical method of making a salt of a peptide of the present invention is well known in the art and can be 5 accomplished by standard methods of salt exchange. Accordingly, the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC, elutin with TFA containing buffer solutions) can be converted into another salt, such as an acetate salt by dissolving the peptide in a small amount of 0.25 N aceti acid aqueous solution. The resulting 10 solution is applied to a semi-prep HPLC column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammonium +etate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution for 0.E hrs. and (3) a linear gradient (20% to 100% of solution B over 30 min.) at a floi rate of 4 mI/min (solution A is 0.25N acetic acid aqueous solution; solution B is 0.25N acetic acid in acetonitrile/water, 15 80:20). The fractions containing the peltide are collected and lyophilized to dryness. As is well known to those skilled in he art, the known and potential uses of GLP-1 are varied and multitudinous (See, "odd, J.F., et al., Clinical Science, 1998, 95, pp. 325-329; and Todd, J.F. et al., Eu opean Journal of Clinical Investigation, 20 1997, 27, pp.533-536). Thus, the administ htion of the compounds of this invention for purposes of eliciting an agonist effect an have the same effects and uses as GLP-1 itself. These varied uses of GL P-1 may be summarized as follows, treatment of: Type I diabetes, Type 11 diabetes, obesity, glucagonomas, secretory disorders of the airway, metabolic disorde , arthritis, osteoporosis, central nervous 25 system diseases, restenosis, neurod generative diseases, renal failure, congestive heart failure, nephrotic syi drome, cirrhosis, pulmonary edema, hypertension, disorders wherein the reducion of food intake is desired, as well as the various other conditions and disord rs discussed herein. Accordingly, the present invention includes within its scope pharmaceutical compositions as defined 30 herein comprising, as an active ingredients compound of formula (1). The dosage of active ingredient in he formulations of this invention may be varied; however, it is necessary that the mount of the active ingredient be such -17 that a suitable dosage is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of a ministration, and on the duration of the treatment, and normally will be determined by the attending physician. In general, an effective dosage for the activities of this i vention is in the range of 1x10- 7 to 200 5 mg/kg/day, preferably 1x104 to 100 mg/kg day, which can be administered as a single dose or divided into multiple doses. The formulations of this invention a preferably administered parenterally, e.g., intramuscularly, intraperitoneally, intra enously, subcutaneously, and the like. Preparations according to this inven on for parenteral administration include 10 sterile aqueous or non-aqueous solution s, suspensions, gels, or emulsions, provided that the desired in vivo release profile is achieved. Examples of non aqueous solvents or vehicles are propylen glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin and injectable organic esters such as ethyl oleate. Such dosage forms may als( contain adjuvants such as preserving, 15 wetting, emulsifying, and dispersing age ts. They may be sterilized by, for example, filtration through a bacteria-retlining filter, by incorporating sterilizing agents into the compositions, by irradiati g the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile 20 injectable medium immediately before use{ Synthesis of Peptides Peptides useful for practicing thE present invention can be and were prepared by standard solid phase peptidE synthesis. See, e.g., Stewart, J.M., et 25 al., Solid Phase Synthesis (Pierce Chemic i Co., 2d ed. 1984). The following examples describe synthetic methods that can be and were used for making peptides with which the i stant invention may advantageously be practiced, which synthetic methods are wE l-known to those skilled in the art. Other methods are also known to those skilled iV the art. The examples are provided for 30 the purpose of illustration and are not rpeant to limit the scope of the present invention in any manner. 296735 1 GH~ers P$3_QaLQQ99W4-1I -18 Said peptides such as GLP-1 anal gue can be obtained with different synthesis known to those skilled in the art wt ich may comprise final precipitation of the peptide, freeze-drying process, vacuu n drying or other drying processes known in the art. Ion exchange chromatography, osmotic exchange of buffer and 5 difiltration could be suitable methods in this i ivention to purify or select the peptide in different salt form. Boc-ISAla-OH, Boc-D-Arg(Tos)-OH ar p Boc-D-Asp(OcHex) were purchased from Nova Biochem, San Diego, Californi4. Boc-Aun-OH was purchased from Bachem, King of Prussia, PA. Boc-Ava-C H and Boc-Ado-OH were purchased 10 from Chem-Impex International, Wood Da , IL. Boc-2Nal-OH was purchased from Synthetech, Inc. Albany, OR. The full names for other abbreviation. used herein are as follows: Boc for t butyloxycarbonyl, HF for hydrogen fluoride, jFm for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF for dimethylformamide, DCM 15 for dichloromethane, HBTU for 2-(11- Benzotriazol-I-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA for dpsopropylethylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for ;-chlorobenzyloxycarbony, 2BrZ for 2 bromobenzyloxycarbonyl, OcHex for O-cyclohexyl, Fmoc for 9 fluorenylmethoxycarbonyl, HOBt for N-hyfroxybenzotriazole; PAM resin for 4 20 hydroxymethylphenylacetamidomethy resin; Tris for Tris(hydroxymethyl)aminomethane; and Eis-Tris for Bis(2-hydroxyethyl)amino tris(hydroxymethyl)methane (i.e., 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl) 1,3-propanediol). The term "halo" or "halogpn" encompasses fluoro, chloro, bromo and iodo. 25 Unless defined otherwise, all technicI and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents and other references mentioned herein are incor orated by reference. 30 Exam 1 (Aiba, 35 )hGLP 1(7-36)NH 2 A detailed synthesis procedure f r (Aib 83 5)hGLP-1(7-36)NH 2 has been 1 2967035 1 GHMatters P13G9AU2Q 7 T c' -19 provided in International Patent Publication o. WO 00/34331 (PCT/EP99/09660), the contents of which are incorporated herein in their entirety. Briefly, the compound was synthesized on an Applied Biosystems (Foster City, CA) model 430A peptide synthesizer which was mod ied to do accelerated Boc-chemistry 5 solid phase peptide synthesis. See Schnol er, et al., Int. J. Peptide Protein Res., 40:180 (1992). 4-methylbenzhydrylamine ( BHA) resin (Peninsula, Belmont, CA) with the substitution of 0.91 mmol/g was u ed. The Boc amino acids (Bachem, CA, Torrance, CA; Nova Biochem., LaJolla, CA) were used with the following side chain protection: Boc-Ala-OH, Boc-Arg( ~os)-OH, Boc-Asp(OcHex)-OH, Boc 10 Tyr(2BrZ)-OH, Boc-His(DNP)-OH, Boc-Va OH, Boc-Leu-OH, Boc-Gly-OH, Boc Gin-OH, Boc-lie-OH, Boc-Lys(2CIZ)-OH, B c-Thr(Bzl)-OH, Boc-Ser(Bzl)-OH, Boc Phe-OH, Boc-Aib-OH, Boc-Glu(OcHex)-OH and Boc-Trp(Fm)-OH. The Boc groups were removed by treatment with 100% FA for 2 x 1 min. Boc amino acids (2.5 mmol) were pre-activated with HBTU (.0 mmol) and DIEA (1.0 ml) in 4 ml of 15 DMF and were coupled without prior neut alization of the peptide-resin TFA salt. Coupling times were 5 min. except for the Boc-Aib-OH residues and the following residues: Boc-Lys(2CIZ)-OH and Boc-HiskDNP)-OH wherein the coupling times were 2 hours. At the end of the assembly of the pe tide chain, the resin was treated with a 20 solution of 20% mercaptoethanol/10% DIE8 in DMF for 2 x 30 min. to remove the DNP group on the His side chain. The N-ttrminal Boc group was then removed by treatment with 100% TFA for 2 x 2 min. After neutralization of the peptide-resin with 10% DIEA in DMF (1 x 1 min), the fo myl group on the side chain of Trp was removed by treatment with a solution of 1 % ethanolamine/ 15% water/ 70% DMF 25 for 2 x 30 min. The peptide-resin was was ed with DMF and DCM and dried under reduced pressure. The final cleavage wa done by stirring the peptide-resin in 10 ml of HF containing i mi of anisole and dit iothreitol (24 mg) at 0*C for 75 min. HF was removed by a flow of nitrogen. The r sidue was washed with ether (6 x 10 ml) and extracted with 4N HOAc (6 x 10 ml). 30 The- peptide mixture in the aqueo s extract was purified on reverse-phase preparative high pressure liquid chromat graphy (HPLC) using a reverse phase VYDAC® C 18 column (Nest Group, Sout borough, MA). The column was eluted 1 29157M3 - (M attesI B39' 219' C-~Ic I -20 with a linear gradient (20% to 50% of solution B over 105 min.) at a flow rate of 10 ml/min (Solution A = water containing .1% TFA; Solution B = acetonitrile containing 0.1% of TFA). Fractions were collected and checked on analytical HPLC. Those containing pure product werg combined and lyophilized to dryness. 5 in one example of synthesis of this com ound, 135 mg of a white solid was obtained. Purity was 98.6% based on an ilytical HPLC analysis. Electro-spray mass spectrometer (MS(ES))S analysis gz ve the molecular weight at 3339.7 (in agreement with the calculated molecular wEight of 3339.7). 10 Examp 2 Formulation Pi cedures I 2.1 Materials, Stock Solutions, Calculations 15 A) Materials: ZnCl 2 , NaOH pellets, and hydrochloric acid, 35%, were obtained from Panreac Quimica, Barcelona, Spain. FI (sterile water for injection/irrigation) was obtained from B. Braun Medical, Barcelona, Spain. B) Stock solutions 20 (i) ZnCI 2 , pH=3: 1. With stirring, add 35%HCI -o WFI to achieve pH=3. 2. In a volumetric flask, tran fer a weighed amount of ZnC 2 . With stirring, add pH=3 HCI to achieve a final concentration of approximately 1-4 mg ZnC 2 /ml. 25 (ii) ZnCl, pH=2: 1. With stirring, add 35%HCI o WFI to achieve pH=2. 2. In a volumetric flask, tran fer a weighed amount of ZnCI 2 . With stirring, add pH=2 HCI to achieve a final concentration of approximately 4-12 mg ZnCl 2 /ml. 30 (iii) NaOH, 0.1 to 10 mg/mI: -21 1. In a volumetric flask, trans er a weighed amount of NaOH. With stirring, add WF1I to achieve a final con ntration of approximately 0.1-10 mg NaOH/ml (iv) Freeze-dried 20 mq aliquot (Aib 8 ' )HGLP-1(7-36)NH/vial: 5 1. Prepare a 0.04% (vlv) dilution of acetic acid and WFI. 2. In a volumetric flask, tran r a weighed amount of (Aiba, 3 6)HGLP 1(7-36)NH 2 (acetate salt). With stirring, a d sufficient 0.04% acetic acid to bring the final concentration to 20 mg (Aib 8 3 5 ) IGLP-1(7-36)NH 2 /ml. Following filter sterilization using 0.45 micron filters, 1 ml liquots of the solution were transferred 10 to lyophilization vials, freeze dried and the jried product stored at -22*C. (v) Freeze-dried 50 mq aliquot (Aib8,))HGLP-1(7-36)NH 2 /vial: 1. Prepare a 0.1% (v/v) dilution of acetic acid and WFI. 2. In a volumetric flask, transit r a weighed amount of (Aib 8 ,3 5

)HGLP

1(7-36)NH 2 (acetate salt). With stirring, ad sufficient 0.1% acetic acid to bring the 15 final concentration to 50 mg (Aiba, 35 ) [fLP-1(7-36)NH2/ml. Following filter sterilization, 1 ml aliquots of the solution a e transferred to lyophilization vials and freeze dried. C) Calculations 20 (i) To determine the total weight / vlume of excipient (E) for a composition: E = (A x 100) - (A/P) wherein: E = excipient in mg A = content of pure peptide (mg); 25 T = target concentration of the compsition; e.g., 2 if target is 2%; and P = concentration of pure peptide (rr g peptide/1 00 mg formulation) With respect to the total volume of Excipient, the assumption that 1 ml = 1 g is applied. (ii) To determine the volume/weigh (W) of ZnC 2 to add to each ml or g of 30 composition solution: I a) W=1 00% E for compositio s in which no pH adjustment is made; -22 b) W=80% E for liquid formulat ns in which the peptide is about 1%, or about 2% or up to about 10% and the pH i adjusted using a base; c) W=50% E for semi-solid or c formulations in which the peptide is about 1%, or about 2% or up to about 10% a Pd the pH is adjusted using a base; 5 d) W=66.66% E for semi-sold or gel formulations in which the peptide is about 25% and the pH is adjusted sing a base; e) W=90% E for formulations in which the peptide is reconstituted from a freeze-dried preparation and the pH i, adjusted using a base. (iii) To determine the volume/weight (jN) of NaOH to add to each ml or g of 10 composition solution: a) W=20% E for formulations in which the peptide is about 1%, or about 2% or up to about 10% and the pH is adjusted using a base; b) W=50% E for semi-solid or el formulations in which the peptide is about 1%, or about 2% or up to about 10% d the pH is adjusted using a base; 15 c) W=33.33% E for semi-so id or gel formulations in which the peptide is about 25% and the pH is adjusted using a base; d) W=10% E for formulations in which the peptide is reconstituted from a freeze-dried preparation and the pH is adjusted using a base. (iv). To determine the concentration Df ZnCl 2 (mg/mi or mg/g) to be used in 20 each composition: [ZnC1 2 ] = (136.29 x A)IW x 3339.76 x R) wherein: A = content of pure peptide (mg). R = molar ratio of peptide/Zn 25 R=1.5 for formulations in whic the peptide is about 1%, or about 2% or about 10% or up to about 23%; R=4.0 formulations in which th peptide is about 25%; and W = weight (g) or volume (ml) of Z 1 C1 2 solution to add to each g or ml of composition solution. 30 2.2 Preparation of compositions with 1-10% freeze-dried peptide and ZnCl, no pH adjustment I Mm 1GHMVU;VseA PR1 IAU2~l7I~~~~N -23 As used herein, a formulation compri 4 ng a percentage of peptide describes a formulation comprising a weight of peptide per total weight of the composition, e.g., 1% peptide, describes a formulation comprising 1g of peptide per 100g of total composition. Formulations comprisin about 1%, or about 2% up to about 5 10% peptide were prepared as follows. F Ieze-dried samples of (Aib 8 35

)HGLP

1(7-36)NH 2 prepared as described were thoroughly mixed with a ZnCl 2 stock solution pH 3 at 100% of the total excipient tiume and [peptide:Zn] = 1.5:1. A) 1% compositions are preparEd by mixing 20 mg freeze-dried (Aib 8

'

35 )HGLP-1(7-36)NH 2 (see 2.1 B (iv) ab ve) with 2 ml of ZnCl 2 solution (0.272 10 mg/ml; see 2.1 B (i) above) B) 2% compositions are prepared by mixing 20 mg freeze-dried (Aib 8 35 )HGLP-1(7-36)NH 2 (see 2.1 B (iv) at ve) with 1 ml of ZnC 2 solution (0.544 mg/ml; see 2.1 B (i) above) C) 10% compositions are prepa ed by mixing 50 mg freeze-dried 15 (Aiba 3 )HGLP-1(7-36)NH 2 (see 2.1 B (v) tbove) with 0.45 ml of ZnCl 2 solution (3.023 mg/ml; see 2.1 B (i) above) Freeze-dried peptides and solutions were allowed to equilibrate to room temperature. The designated volume of Z C12 solution was injected into the vial containing the freeze-dried peptide and hy' ration allowed to proceed for about 2 20 minutes for 1% or 2% peptide composition to about 60 minutes for 10% peptide composition, or until all freeze-dried peptide is completely hydrated and the solution is free of clumps of peptide. Following hydration, the reconstituted peptide is shaken for approximately 1 minute. The appropriate amount of dissolve peptide may be removed for dosing, 25 e.g., 100 ul of a 1% peptide solution prep red as per A above equates to a 1 mg dose, 50 ul of a 2% peptide solution prep red as per B above equates to a 1 mg dose, 150 ul of a 10% peptide solution pr pared as per C above equates to a 15 mg dose, etc. Using the teachings of the instant Opplication, one skilled in the art could 30 vary the amounts of peptide and ZnC 2 to chieve compositions other than the 1%, 2% and 10% compositions detailed below 7s well as desired dosages.

-24- 1 2.3 Preparation of compositions with 1-100/ freeze-dried peptide and ZnC 2 , with a pH adjustment Formulations comprising about 1%, r about 2% up to about 10% peptide were prepared as follows. Freeze-dried samples of (Aib 8

'

35 )HGLP-1(7-36)NH 2 5 prepared as described were thoroughly mi ed with a ZnC 2 stock solution pH 3 at 90% of the total excipient volume. The d sired pH of the solution is reached by the addition of diluted NaOH solution. A) 1% compositions are prepa rd by mixing 20 mg freeze-dried (Aib 8

.

35 )HGLP-1(7-36)NH 2 (see 2.1 B (iv) above) with 1.8 ml of ZnCI 2 solution (see 10 2.1 B (i) above) B) 2% compositions are prepaid by mixing 20 mg freeze-dried (Aib 8

'

35 )HGLP-1(7-36)NH 2 (see 2.1 B (iv) akove) with 0.9 ml of ZnCl 2 solution (see 2.1 B (i) above) C) 10% compositions are preped by mixing 50 mg freeze-dried 15 (Aiba" )HGLP-1(7-36)NH 2 (see 2.1 B (v) above)with 0.40 ml of ZnCl 2 solution (see 2.1 B (i) above) To the above solutions, add the ne essary volume (10% of total volume of excipient) of diluted NaOH solution to ac ieve the target concentration and pH. 20 For example, to each: 1% composition: Add 0.2 ml of NaOO solution of proper concentration 2% composition: Add 0.1 ml of NaO solution of proper concentration 1Q% composition: Add 0.05 ml of N OH solution of proper concentration Using the teachings of the instant application, one skilled in the art could 25 vary the amounts of peptide and ZnCl 2 to chieve compositions other than the 1 %, 2% and 10% compositions detailed below. 2.4 Preparation of liquid compositions vfith 1-10% peptide and ZnCI 2 , no pH adjustment 30 Liquid formulations comprising abIut 1%, or about 2% up to about 10% peptide were prepared as follows. Sa les of (Aib 8 35 )HGLP-1(7-36)NH 2 were weighed and mixed with a ZnCl 2 stocks solution pH 3 to achieve the target 20670S5 1 (GHWM)s PISS09A' 'i0f771 (C-L n sn -25 concentration of 1%, 2%, up to 10% peptid. Following mixing, the composition is filter sterilized and stored until use. 2.5 Preparation of liquid compositions ith 1-10% peptide and ZnCl2, pH 5 adjustment Liquid formulations comprising aboLt 1%, or about 2% up to about 10% peptide were prepared as follows. Sam pes of (Aib 8

,

35 )HGLP-1(7-36)NH 2 were weighed and thoroughly mixed with a ZnC stock solution pH 3 at 80% of the total excipient volume. The zinc solution may e either ZnCl 2 or ZnAc2-2H20. The 10 desired pH of the solution is reached by -he addition of diluted NaOH solution. Preparations C5 to C1 3 were prepared using this method. Uing the teachings of the instant application, one skilled in the art could vary the amounts of peptide and ZnCl 2 to a hieve compositions other than the 1%, 2% and 10% described herein. 15 2.6 Preparation of semi-solid/gel compositicfs with 25% peptide and ZnCI. no pH adjustment Semi-solid or gel formulations composing about 25% peptide were prepared as follows. Samples of (Aib835)HGLP-1(436)NH 2 were weighed and thoroughly 20 mixed with a ZnCl 2 stock solution pH 2 a4 66.66% of the total excipient volume. The zinc solution may be either ZnCl 2 or nAc2-2H20. Preparations C1 and C2 were prepared using this method. More specifically, the semi-solid or qel compositions were prepared using a "push-pull" mixing method: 25 a)' The desired amount of peptid was weighed into the barrel of a disposable syringe S1 previously fitted Ath a special two-way hand valve HV (1.D.=0.5 mm) and tubing was placed insid the syringe Luer hole; b) The syringe plunger was secured* ith a stainless steel rod SR; c) HV in SI was connected to a va um source and HV was opened. After 30 10 min HV was closed; d) The Zinc solution was accurateIV weighed into the barrel of a second disposable syringe S2; | I973 1(HatrsiS39 -26- l e) S2 was then connected to the free art of HV; f) HV was opened and the solvent w4 pulled by the vacuum into the barrel containing the peptide powder S1; g) HV was closed and the solvent sy inge S2 was removed, thus hydrating 5 the peptide powder in S1; h) SR was removed and the syringe p unger was slowly released; i) The syringe plunger is moved (push and pull), without opening HV, so that the powder mass is fully soaked by solvent; j) A two-way stainless connector SC (.D.=1.0 mm) was placed in syringe S2 10 with the tubing placed inside the syringe Lu r hole, and its plunger was pushed to the end; k) HV in S1 was opened to vent th4 vacuum and then HV was removed. The syringe plunger was moved so that ai in the syringe barrel was minimized; and 15 1) S1 and S2 were connected by SC 'nd the composition was kneaded from S1 to S2 through SC. Using the teachings of the instant publication, one skilled in the art could vary the amounts of peptide and ZnC1 2 tct achieve compositions other than the 25% described herein. 20 2.7 Preparation of semi-solid/gel compositicis with 25% peptide and ZnCI 2 pH adjustment Semi-solid or gel formulations compr sing about 25% peptide were prepared as follows. Samples of (Aib"35)HGLP-1(7D36)NH2 were weighed and thoroughly 25 mixed with a ZnCl 2 stock solution pH 2 at 66.66% of the total excipient volume. The zinc solution may be either ZnCl 2 o ZnAc22H20. The desired pH of the solution is reached by the addition of dilutEd NaOH solution. In this example, the total volume of liquid added to the powder must be divided between the zinc and the NaOH solutions. Therefore, the ct centration of the zinc solution was 30 adjusted so that the total volume of zinc solution needed was reduced to 50% of the total liquid volume added to the peptide powder (step d). The remaining 50% of pept9de7powde -27 the total liquid volume added to the peptide powder was added as NaOH solution as detailed below. Preparations C3 and C4 kere prepared using this method. The pH adjusted semi-solid or gel 'compositions were prepared using a "push-pull" mixing method: 5 a) The desired amount of peptid was weighed into the barrel of a disposable syringe SI previously fitted wh a special two-way hand valve HV (l.D.=0.5 mm) and tubing was placed insidE the syringe Luer hole; b) The syringe plunger was secured ith a stainless steel rod SR; c) HV in SI was connected to a vac um source and HV was opened. After 10 10 min HV was closed; d) The Zinc solution was accurately weighed into the barrel of a second disposable syringe S2; e) S2 was then connected to the free part of HV; f) HV was opened and the solvent wjs pulled by the vacuum into the barrel 15 containing'the peptide powder SI; g) HV was closed and the solvent s ringe S2 was removed, thus hydrating the peptide powder in SI; h) SR was removed and the syringe p lunger was slowly released; i) The syringe plunger is moved (push and pull), without opening HV, so that 20 the powder mass is fully soaked by solvent; j) A two-way stainless connector SC 1.D.=1.0 mm) was placed in syringe S2 with the tubing placed inside the syringe Uier hole, and its plunger was pushed to the end; k) HV in SI was opened to vent the vacuum and then HV was removed. 25 The syringe plunger was moved so that air in the syringe barrel was minimized; I) SI and S2 were connected by SC nd the composition was kneaded from S1 to S2 through SC; m) After homogenization, an aliquo of the mixed product was removed to determine the concentration of the peptide; 30 n) The remaining intermediate bulk product was accurately weighed and the amount of NaOH solution required to reach the desired pH was calculated; -28 o) The NaOH solution was accurai ly weighed into a third disposable syringe S3; and p) The syringe plungers were slowly compressed to minimize the air in the syringe chambers. Both syringes were connected by SC and the composition 5 was kneaded through SC. Using the teachings of the instant al plication, one skilled in the art could vary the amounts of peptide and ZnCl 2 to achieve compositions other than the 25% described herein. 10 Table 1 Ex. *Peptide **Peptide: Peptide No. % Solution Zn Ratio Dose 15 C1 10 ZnCl 2 0.846 mg/ml 5.4.1 1 mg C2 5 0.40 mg ZnCl 2 /ml 5.4:1 1 mg C3 10 50% ZnCl 2 1.69 mg/ml, 50% NaRH I mg/ml 5.4:1 1 mg C4 10 50% ZnCl 2 2.28 mg/ml, 50% NarH 1 mg/ml 4:1 1 mg C5 5 80% ZnCI 2 0.674 mg/ml, 20% N4OH 3.81 mg/ml 4:1 1 mg 20 C6 2 80% ZnC 2 0.26 mg/ml, 20% Na4H 2.15 mg/ml 5.4:1 1 mg C7 10 80% ZnCI 2 3.81 mg/ml, 20% NaH 4.47 mg/ml 1.5:1 1 mg C8 10 80% ZnAc 2 .2H 2 0 2.3 mg/ml, 20 o NaOH 6.1 mg/mI 4:1 1 mg C9 2 80% ZnCl 2 0.695 mg/ml, 20% N OH 1.75 mg/ml 1.5:1 1 mg C10 2 80% ZnAc 2 .2H 2 0 1.12 mg/ml, 2 )% NaOH 1.44 mg/ml 1.5:1 1 mg 25 C11 2 80% ZnC 2 0.695 mg/mI, 20% N, OH 1.75 mg/m 1.5:1 1 mg C12 1 80% ZnCl 2 0.384 mg/ml, 20% N OH 0.875 mg/ml 1.5:1 1 mg C13 10 80% ZnCl 2 3.85 mg/ml, 20% Na H 4.47 mg/ml 1.5:1 15 mg * Target value shown. Actual value w s within 5% of target in all cases ** Target value shown. Actual values were within 10% of target in all cases 30 3.0 Detdrmination of GLP-1 receptor affinity\ A compound useful to practice the present invention can be tested for its ability to bind to the GLP-1 receptor using tt e following procedure. Cell Culture: I 296M03 1 (GHMalerso) 3AAilM",i eie7 yn.

-29 RIN 5F rat insulinoma cells (ATCC # CRL-2058, American Type Culture Collection, Manassas, VA), expressing tI e GLP-1 receptor, were cultured in Dulbecco's modified Eagle's medium (DM FM) containing 10% fetal calf serum, and maintained at about 37 *C in a humidife atmosphere of 5% C0 2 /95% air. 5 Radioligand Binding: Membranes were prepared for radioligand binding studies by homogenization of the RIN cells in 20 rr I of ice-cold 50 mM Tris-HCI with a Brinkman Polytron (Westbury, NY) settingq 6, 15 sec). The homogenates were 10 washed twice by centrifugation (39,000 g / 10 min), and the final pellets were resuspended in 50 mM Tris-HCI, containin 2.5 mM MgCl 2 , 0.1 mg/ml bacitracin (Sigma Chemical, St. Louis, MO), and 0. % BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM ( 12 6l)GLP-1 7-36) (-2200 Ci/mmol, New England Nuclear, Boston, MA), with and without p.05 ml of unlabeled competing test 15 peptides. After a 100 min incubation (25 C), the bound ( 125 1)GLP-1(7-36) was separated from the free by rapid filter tion through GF/C filters (Brandel, Gaithersburg, MD), which had been previo sly soaked in 0.5% polyethyleneimine. The filters were then washed three times w th 5 ml aliquots of ice-cold 50 mM Tris HCI, and the bound radioactivity trapped n the filters was counted by gamma 20 spectrometry (Wallac LKB, Gaithersburg, IVD). Specific binding was defined as the total (1 2 -l)GLP-1(7-36) bound minus that bound in the presence of 1000 nM GLP1(7-36) (Bachem, Torrence, CA). 4. Determination of solubility vs pH 25 4.1. Determination of Compound Solubilit vs pH in Phosphate Buffered Saline (PBS) A compound that may advantageou Iy be used to practice the invention can be tested to determine its solubility in PBS t different pHs and temperatures using 30 the following procedure. A stock PBS buffered solution was made by dissolving one packet of pre mixed powder (SIGMA, Product No.: P-3 13) in one liter of de-ionized water to -30 yield 10 mM phosphate-buffered saline with 38 mM NaCl, 2.7 mM KCl, and a pH of 7.4. PBS buffers with different pH values ere made by adjusting the pH of this stock solution with phosphoric acid and/or sc ium hydroxide. Two mg samples of a compound to t e tested, e.g., 2 mg of the compound 5 of Example 1, were weighed into glass vi s. Into each vial was added a 50 ptl aliquot of PBS buffer at a certain pH. The s lution was vortexed, and if necessary sonicated, until clear. For each pH tested the total volume of buffer needed to dissolve 2 mg of the compound was recorde and the solubility was calculated. Peptide solutions that are clear at ro temperature (20-25* C) were placed 10 in a refrigerator (40 C) overnight and the so bility of the peptide at 40 C was then examined. 4.2. Determination of compound solubility v4 pH in saline A compound that may advantageous be used to practice the invention can 15 be tested to determine its solubility in saline at different pH values and temperatures using the following procedure.! A stock saline solution is prepared b4 dissolving 9 grams of NaCl in one liter of de-ionized water. Saline solutions with different pH values are made by adjusting the pH of this stock solution with I- Cl and/or NaOH. 20 Two mg samples of a compound to ie tested, e.g., 2 mg of a compound of example 1, are weighed into glass vials. In o each vial is added a 50 gI aliquot of saline solution at a certain pH. The vial is vortexed and, if necessary, sonicated until clear. For each tested pH the total vol me of saline needed to dissolve 2 mg of the compound is recorded and the solubi ty is calculated. 25 Solutions that are clear at room te nperature (20-254 C) are placed in a refrigerator (40 C) overnight and the solubilii at 40 C then examined. 4.3. Determination of compound solubility ih saline at pH 7.0 Compounds that may advantageous y be used to practice the invention can 30 be tested to determine their solubility at ro m temperature in saline having pH = 7 using the following procedure. 2967035 1 (GHMalters} PS1S99AU f Lo' -31 Saline solution is prepared by dissolv ng 9 grams of NaCI in one liter of de ionized water. A 2 mg sample of a compoiind to be tested, e.g., a compound of example 1, is weighed into a glass vial and I ml aliquots of saline are added, with vortexing and sonication, until clear. The to'al volume of saline used to dissolve 2 5 mg of peptide is recorded and the solubility t room temperature is calculated. 4.4. Determination of compound solubility in saline at various pH Compounds that may advantageous be used to practice the invention can be tested to determine their solubility at oom temperature in saline solutions 10 having various pH values using the following procedure. A stock saline solution is prepared by dissolving 9 grams of NaCI in one liter of de-ionized water. Saline solutions havir# various pH values are obtained by treating aliquots of this stock saline solution muith HCI and NaOH. A 2 mg sample of a compound to be tested, e.g., the compound of example 15 1, is weighed into a glass vial. Aliquots of 0 pl of a saline buffer at a certain pH are added. The solution is vortexed and soicated until clear. The total volume of buffer used to dissolve 2 mg of peptide is re orded and the solubility is calculated. 5. Determination of aqueous solubility of cc pound vs zinc concentration 20 A compound that may advantageou y be used to practice the invention can be tested to determine its solubility in pH water at different zinc concentrations using the following procedure. A stock zinc solution was prepared y dissolving ZnCl 2 in de-ionized water to a concentration of 100 mg/ml and adjus ing the pH to 2.7 using HCI. Solutions 25 having various ZnCI 2 concentrations ("Z| Test Solutions") were prepared by making appropriate dilutions of the stock sc ution. One mg of a compound to be te ted, e.g., 1 mg of the compound of Example 1, was dissolved in 250 lt of eah Zn Test Solution to yield a solution having 4 mg/ml of the compound. The pH Ef this solution was then adjusted using 30 0.2 N NaOH until white precipitat oer aq observed to form. The precipitation solution was centrifuged and the mother liquor analyzed using HPLC. The UV absorption area of test compound peak 4s measured and the concentration of -32 the test compound in the mother liquor +as determined via comparison to a calibration curve. As a representative example of a cc pound that may be used to practice the invention, the compound of Example 1 'as tested in the immediately foregoing 5 assay and the following results were obtained (aqueous, pH 7.0, room temperature): ! Table 2 ZnCl 2 concentration Solubility (fg/ml) (mg/ml) 10 0 5.788 80 0.0770 500 0.0579 1000 0.0487 1500 0.0668 15 2500 0.1131 6. Determination of isoelectric point (p1) usiq IEF gels Invitrogen's Novex IEF pH3-10 gels ere used to measure the pl of GLP-1 peptides, e.g., the compound of Example 1. Peptidyl compounds to be tested 20 were dissolved in water to a concentration c 0.5 mg/ml. For each such compound, 5 i±l of the resulting solution was mixed v ith 5 pl of Novex* Sample Buffer 2X (comprised of 20 mM Arginine free base 20 mM Lysine free base and 15% Glycerol) and the resulting 10 pl sample s lution was loaded onto the gel along with a protein standard sample. 25 Running buffers were also obtained from Invitrogen and the gel is run according to manufacture's instructions, generally as follows: 100 V constant for 1 hour, followed by 200 V constant for 1 h r, followed by 500 V constant for 30 minutes. The gel was then fixed in 12% TCA containing 3.5% sulfosalicylic acid for 30 30 minutes, and then stained for 2 hours wih Colloidal Coomassie Blue according to the instructions found on the Novex* olloidal Blue Kit thereafter, then de stained in water overnight. The gel was scanned and analyzed 'y the program Fragment Analysis 1.2. pi's of unknown peptides were calculal3d relative to the pi's of standard -33 compounds having pi values of: 10.7, 9.5, 83, 8.0, 7.8, 7.4, 6.9, 6.0, 5.3, 5.2, 4.5, 4.2, and 3.5. The measured pl of the compound of xampie 1 was 7.60. 5 7. In vivo assays in rat Compositions of the present inventi n can be tested to determine their ability to promote and enhanced effect in vi using the following assays. 7.1. Experimental procedure: 10 The'day prior to the experiment, aduli male Sprague-Dawley rats (Taconic, Germantown, NY) that weighed approxima ely 300-350g were implanted with a right atrial jugular cannula under chlorohy rate anesthetic. The rats were then fasted for 18 hours prior to the injection c the appropriate test composition or vehicle control at time 0. The rats continu d to be fasted throughout the entire 15 experiment. A 0.5 mg/ml ZnC 2 solution was prepared by dilution of a solution of 100 mg/mi ZnC1 2 in an HCI solution having pH 2.7 water. I mg of the compound of formula (1) ((Aib 5

'

35 )hGLPI(7-36)NH 2 ) was ?issolved in 250 pl of this solution to yield a clear solution having 4 mg/mI of the cmpound and 0.5 mg/mI Zn at pH 4. 20 At time zero the rats were injected *ibcutaneously (sc) either with (a) the immediately forgoing solution of (Aib8,")hGIjP-1(7-36)NH 2 ), or with vehicle control. In both cases the injection volume was very small (4-6 gL) and the dose of GLP-1 compound administered to the subject was r5 jig/kg. At the appropriate time after the sc injections a 500jI blood sample w s withdrawn via the intravenous (iv) 25 cannula and the rats were given an iv gluco e challenge to test for the presence of enhanced insulin secretion. The times of thE glucose challenge were 0.25, 1, 6, 12 and 24 hours post-compound injection. !After the initial blood sample was withdrawn glucose (1g/kg) was injected iv and flushed in with 500l heparinized saline (1OU/mi). Thereafter, 500p1l blood sar ples were withdrawn at 2.5, 5, 10 and 30 20 minutes post-glucose injection. Each of -hese was immediately followed by an iv injection of 500pl heparinized saline (10 /ml) through the cannula. The blood samples were centrifuged, plasma was collected from each sample and the 12967035 1 (GIMaOers PB1SOOAU29G60877 M~r- )P10 1 -34 samples were stored at -20 0 C until they iere assayed for insulin content. The amount of insulin in each sample was deten ined using a rat insulin enzyme-linked immunosorbant assay (ELISA) kit (Americi Laboratory Products Co., Windham, NH). 5 7.1.1. Results: A sustained insulin-enhancing activity was observed that was inducible by glucose injection over the full 24 hours of th experiment. 8. In vivo assays in doq There are a number of in vivo ass ys known in the art which enable the 10 skilled artisan to determine a composition's ability to promote extended release of active compound in vivo. 8.1. 1% Peptide Composi t ion: By way of example, an aqueous teE formulation was prepared comprising 1% (w/w) of the compound of formula (I) in buffered solution of ZnCl 2 (peptide:Zn 15 ratio = 1.5:1.0). A total of 6 male Beagle dogs, a es 42 - 78 months and 14 - 21 kg bodyweight were maintained with free access to water and once daily food (approx. 400 g of dry standard diet (SAFE 125). The dogs were fasted 18 hours before administration of test composition. i 20 The test composition was admini tered by subcutaneous route in the interscapular area by. The volume of administration (approx. 20 microliters per animal) was made by 0.3 ml Terumo syringes with 0.33-12 mm (BS=30M2913). A theoretical dose of approximately 0.2mg pe tide was thus achieved. Blood samples were taken periodic lly, at approx. time = 0, 8, 15, 30, 45 25 min, and 1, 2, 4, 8, and 12 hours, and 1, 2, 3, 4, 5, and 6 days after administration. The blood was rapidly chilled after sampl g until centrifugation, and the plasma decanted and rapidly frozen pending as ay. Determination of peptide plasma concentration was made after off line soli phase extraction, followed by on-line phase extraction coupled to LC-MS/MS,' and the data obtained managed by 30 Analyst v1.2 software. The composition demonstrated an extended release of the active peptide for at least 2 days. 29670D35 1 QHMangMts P1SOAU29EOI7Y1 21m)Ptff -35 8.2. 1% (Aib.3)hGLP1(7-36)NH 2 ) Solution: Using substantially the same in vi o assay procedure as described in section 8.1, above, the following compositions were examined for their ability to release the subject peptide over an exterided period of time. For each of the 5 following four compositions the concentration of peptide was about 1% (wt/wt), the ratio of peptide to zinc was about 1.5:1, an the dose of peptide administered was approximately 1 mg. Solution 8.2.A: (Aiba 35 )hGLP1(7-3 NH 2 in a solution containing (i) 90% ZnC 2 (0.298 mg/ml) and (ii) 10% NaOH (O. 5 mg/ml); 10 Solution 8.2.3: (Aib 8

'

35 )hGLPI(7-36) H 2 in a solution of ZnC 2 (0.286 mg/ml) Solution 8.2.C: Substantially similar to Solution 8.2.B, and buffered using AcOH/AcO Solution 8.2.D: Substantially similar tt Solution 8.2.A The compositions provided for an extended release of (Aib 835 )hGLP1(7 15 36)NH 2 , as depicted in Figure 1. 8.3. 1% (Aib 8 ')hGLP1(7-36)NH 2 ) Solution Using substantially the same in vro assay procedure as described in section 8.1, above, the following compo ition was examined for its ability to release the subject peptide over an exteried period of time. For the following 20 composition the concentration of peptide w s about 2% (wt/wt), the ratio of peptide to zinc was about 1.5:1, and the dose of pe tide administered was approximately 1 mg. Solution 8.3.: (Aib835)hGLP1(7-36JNH 2 in a solution containing (i) 80% ZnCl 2 (0.695 mg/ml) and (ii) 20% NaOH (1. 5 mg/ml); 25 The' composition provided for an Ixtended release of (Aib 8

'

5 )hGLP1(7 36)NH 2 , as depicted in Figure 5. 8.4. 10% Peptide Solutions: Using substantially the same in vkvo assay procedure as described in 30 section 8.1, above, the following composi ons were examined for their ability to release the subject peptide over an ext ded period of time. For each of the following four compositions the concentra on of peptide was about 10% (wt/wt), M705 I concentrajon -36 the ratio of peptide to zinc was about 1.5:1, and the dose of peptide administered was approximately 15 mg. Solution 8.4.A: (Aibaas)hGLP1(7-36 NH 2 in a solution containing (i) 90% ZnCl 2 (3.367 mg/ml) and (ii) 10% NaOH (5. mg/ml); 5 Solution 8.4.B: (Aib, 3 5)hGLP1(7-36)1 2 in a solution of ZnCl 2 (2.993 mg/ml) Solution 8.4.C: Substantially similar o Solution 8.4.B, and buffered using AcOH/AcO Solution 8.4.D: Substantially similar t( Solution 8.4.A The compositions provided for an xtended release of (Aiba 35 )hGLPI(7 10 36)NH 2 , as depicted in Figure 2. 8.5. Semisolid Compositions: Using substantially the same in v io assay procedure as described in section 8.1, above, the following semi-soli compositions were examined for their 15 ability to release the subject peptide o r an extended period of time. For composition 8.5.A., the concentration of ,he peptide was about 5%, while for compositions 8.5.B, 8.4.C, and 8.5.D., the c ncentration of peptide was about 10% (wt/wt). The ratio of peptide to zinc for compositions 8.5.A, 8.5.B, and 8.5.C was about 5.4:1, while for composition 8.5.D e ratio was about 4.0:1. For all four 20 compositions the dose of peptide administered was approximately 1 mg. Composition 8.5.A: (Aib 8

,

3 5)hGLP1 7-36)NH 2 in a semisolid composition containing ZnC2 (0.40 mg/ml) in WFL. Composition 8.5.6: Substantially sirrlilar to Composition 8.5.A., wherein the ZnCL2 concentration has been adjusted u ward to maintain a peptide:Zn ratio of 25 about 5.4:1. Composition 8.5.C: (Aib"- 35 )hGLP1( -36)NH 2 in a semisolid containing (i) 50% ZnC 2 (1.69 mg/ml) and (ii) 50% NaO (1mg/ml). Composition 8.5.D: (Aib 8

,

35 )hGLPI( -36)NH 2 in a semisolid containing (i) 50% ZnCl 2 (2.28 mg/ml) and (ii) 50% NaO (1mg/ml). 30 The compositions provided for an xtended release of (Aiba, 35 )hGLP1(7 36)NH 2 , as depicted in Figure 3.

-37 8.6. Semisolid Compositions: Using substantially the same in vivo assay procedure as described in section 8.1, above, the following semi-sol composition was examined for its ability to release the subject peptide ov an extended period of time. This 5 composition was formulated using a 5.22 mg/ml ZnCl 2 solution, at pH = 2.0. Sufficient peptide was provided to result in 25% peptide semisolid composition having a peptide to zinc ratio of about 4:1. T e pH of the composition was adjusted as provided herein using 10 mg/ml NaOH. ]fhe dose of peptide administered was approximately 15 mg. 10 Composition 8.6 provided for an e tended release of (Aib' 35 )hGLP1(7 36)NH 2 , as depicted in Figure 6. 8.7. Semisolid Compositions: Using substantially the same in vh o assay procedure as described in section 8.1, above, the following semi-sol composition was examined for its 15 ability to5 release the subject peptide ovar an extended period of time. This composition was formulated using a 8.5 1mg/ml ZnCl 2 solution, at pH = 2.0. Sufficient peptide was provided to result in [a 23% peptide semisolid composition having a peptide to zinc ratio of about 1. :1. The composition was formulated according to the process detailed in sect n 2.6, above. The dose of peptide 20 administered was approximately 15 mg (co responding to about 65 microliters of the composition). Composition 8.6 provided for an tended release of (Aib"' 3 )hGLP1(7 36)NH 2 , as depicted in Figure 7. Further assays with various permutalons of the disclosed formulation have 25 likewise been subject to in vivo assay and h ve confirmed that compositions of the present invention provide a useful drug d ivery platform for the compound of formula (1). Using the teachings of the in ant application, one skilled in the art could vaiythe amounts of peptide, ZnCl 2 d pH to prepare compositions of the present invention as described herein. 30 Example 9 -38- 1' 1. PK profile modulation by Acetate intent in 10% peptide solutions. This example discloses a pharmaokinetic study of (Alb 8 .3 5 )hGLP1(7 5 36)NH 2 in male beagle dogs following by ingle subcutaneous administration of two extemporaneous compositions contain ng 10% (Aiba 35 )hGLP1(7-36)NH 2 and zinc chloride [(Aibaas)hGLP1(7-36)NH2:Zn=1.5:1 at dose level of 15mg/dog. The method to conduct the in vivo Essay is the same as disclosed under paragraph 8.1. 10 This example illustrates PK profile odulation by acetate content in the pharmaceutical composition and thus the in luence of the ratio [acetate/peptide] in the pharmaceutical composition on the pH. The pH modulation is controlled by th way of modulation of acetate content a decreasing content of acetate shows an in reasing effect on the pH. 15 A variation of acetate also shows an effect on the Cmax.. In general a decreasing content of acetate decreases thE Cmax value. An increased content of acetate sh $ws an improvement on the solubility and the physical stability. According to the formulation chosen the improvement by the modulation of 20 the ratio acetate/peptide on solubility 6r stability, is compensated by the modulation of the ratio peptide/Zn for instaiice on the Cmax. This can be seen as a system with three possible variables to adju t stability, solubility, the pH or C max. In this example the abbreviation SD eans standard deviation. AUC means the Artemisinin areas under the plasma con entration-time curve. 25 The meaning of the abbreviation M T is mean residence time (MRT) is a parameter for estimating the rate of bioava ability to compare MRT with tmax wich is the time of peak drug concentration. M t was calculated using data from zero time through the last sampling time. In Table 3 are gathered the results df the 10% peptide composition batches 30 having different [Acetate/Peptidel ratios and subcutaneous administration in beagle dogs. The peak drug in plasma co centration values, the (Cmax) was 8.10 ng/ml (SD=1.80 ng/ml) for an [Acetate/Pep tide] molar ratio of the [3.7:1], whereas 2967035 1 -39- il the batch having a lower ratio [3.2:1] provided a Cmax value of 5.65 ng/ml (SD=2.61 ng/ml). 5 table 3 Formulation 10% 5mg 10% 15mg Ratio peptide/Zn 1.t:1 1.5:1 Parameter Units MEAN S.D. MEAN S.D. (n=5)L (n=4) Dose pg-kg- 1 857.7 131.0 694.8 46.5 tmax d 0.208 0.167 0.111 0.068 Cmax ng-mr' 8.10 1.80 5.65 2.61 ti2 app d 3.32 0.66 6.77 2.04 AUCt ng-mt-d 53.5 14.3 38.2 9.2 AUC ng-mf'-d 55.4 15.7 41.6 8.9 AUCextrap. % 2.99 1.83 8.44 5.00 MRTi d 9.31 2.25 7.48 1.39 MRT d 9.96 2.60 9.85 2.54 [Acetate:Peptide] 31:1 3.2:1 10. GLP-1 peptide salt/divalent metal formula tions 10 10.1. Methods (Aibaa 3 )hGLP-1(7-36)NH 2 1 mg/mL water and PBS solutions were prepared and the pH was adjusted to 7.0. 10 mg/mL qtock solutions of CaCl 2 , CuC1 2 , MgC 2 , and ZnC1 2 in water were prepared. The pH of CaCl 2 , MgCl 2 , and ZnC1 2 solutions 15 was adjusted to 7.0. The pH of CuCl2 solujon could not be basified because Cu precipitated out. Therefore, CuCl2 solution of pH 4.4 was used. 4 pL metal ion water or PBS solution were added to 200 p±L (Aib 8

,

35 )hGLP 1(7-36)NH 2 1 mg/mL solution to make a final metal ion concentration of 200 pig/mL. The resulting solution was mixed and checked for precipitation. If 20 precipitation formed, the suspension was centrifuged. The (Aib 8

.

35 )hGLP-1 (7 36)NH 2 concentration in the supernatant wa, determined by HPLC.

-40 10.2. Results 5 Table 4. Solubility of (Aib, 35 )hGLP-1(7-36)l H2 in the presence of divalent metal ions Water solution, mg/mL PBS solution, mg/mL CaCl 2 >1 (pH 7.1) >1 (pH 6.8) CuC1 2 0.058 (pH 7.1) 0.039 (pH 6.8) MgC 2 >1 (pH 7.2) >1 (pH 6.9) ZnC1 2 0.108 (pH 6.9) 0.056 (pH 6.8) 10 10.3. Pharmacokinetic studies of (Aiba,35)h LP-1(7-36)NH?/divalent metal PH 5.5 clear solution formulations Three different formulations of (Aibaf)hGLP-1(7-36)NH2 were prepared by using the following procedures: 15 (1) (Aib' 35 )hGLP-1(7-36)NH2 HCI sa with CuCI 2 (2) (Aib' 3 )hGLP-1(7-36)NH 2 HCI sa with ZnCl 2 (3) (Aib 8

,

35 )hGLP-1 (7-36)NH 2 acetatej salt with ZnC 2 20 A TFA salt of a GLP-1 analogue (the FA salt results from the purification of the peptide by using preparative HPLC eluting with TFA containing buffer solutions) can be converted into another sa , such as an acetate salt by dissolving the peptide in a small amount of 0.25 N acefic acid aqueous solution. The resulting 25 solution is applied to a semi-prep HPLC column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammonium a etate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution for 0.5 hrs. and (3) a linear gradient (20% to 100% of solution B over 30 min.) at a flov rate of 4 ml/min (solution A is 0.25N acetic acid aqueous solution; solution B is 0.25N acetic acid in acetonitrile/water, 30 80:20). The fractions containing the pe ide are collected and lyophilized to dryness.

-41 (Aib'35)hGLP-1(7-36)NH 2 HCI salt was prepared by a lyophilization procedure. 20 mg (Aiba')hGLP-1(7-36)NH2 1 acetate was dissolved in 4 mL 20 mM HCI aqueous solution and incubated at roor temperature for 10 min. The sample 5 was frozen and lyophilized overnight. Lyo hilization was performed for another two times and the chloride content of the final product was determined. The determined chloride content was 5.38%. (1) (Aib, 3 5 )hGLP-1(7-36)NH 2 HCl salt with uCI2: 10 (Aib 8

,

3 5 )hGLP-1(7-36)NH 2 HCI 5.3 mg (pepti e content is 95%) was dissolved in 50 pL 20 mM CuC1 2 aqueous solution. The pH was adjusted with approximately 2 [tL 1 N NaOH to about 5.5. The molar ratio c (Aib 83 5 )hGLP-1(7-36)NH 2 /CuCl 2 was 1.5:1. The peptide concentration was 10% (30 mM) in water (w/w) with a pH of approximately 5.5. 15 (2) (Aib 8

,

35 )hGLP-1(7-36)NH 2 HCI salt with ZCa: (Aib 8 35 )hGLP-1(7-36)NH 2 HCI 5.3 mg (pepti e content is 95%) was dissolved in 50 pL 20 mM ZnCl 2 aqueous solution. The pH was adjusted with approximately 2 iL of I N NaOH to about 5.5. The molar ratio f (Aib' 35 )hGLP-1(7-36)NH 2 /ZnCI2 was 20 1.5:1. The peptide concentration was 10%[(30 mM) in water (w/w) with a pH of approximately 5.5. (3) (Aib, 35 )hGLP-1 (7-36)NH 2 acetate salt wi h ZnCI,: (Aibs 35 )hGLP-1(7-36)NH 2 acetate 5.5 mg ( eptide content is 92%) was dissolved 25 in 50 iL 20 mM ZnCl 2 aqueous solution. The resulting solution was Iyophilized overnight and redissolved in 50 pL water. T e pH was adjusted with approximately 1 tiL of 1 N NaOH to about 5.5. The molar tio of (Aiba 35 )hGLP-1(7-36)NH 2 /ZnCI 2 was 1.5:1. The peptide concentration was 0% (30 mM) in water (w/w) with a pH of approximately 5.5. 30 -42- I 10.4. Dosing and blood sample collection Rats were dosed at 0.3 mg/rat (3 pL 'of 10% solution) subcutaneously with these three formulations of (Aib 8 l 35 )hGL -1(7-36)NH 2 . Blood samples were collected at 5, 10, 15, 30 min, 1, 2, 4, 8 hou s, and 1, 2, 3, 4, 7, 10 days. Plasma 5 was collected from the blood by centrifugati n and stored at -804C. The tissue at the injection site was also collected, homog pnized in 5x methanol, and stored at 80"C. Two rats were used for the 5, 10, 1, 30 min, and 1, 2, 4, 8 hours data points. One rat was used for 1, 2, 3, 4, 7, 10 ays data points. 10 10.5. LC-MS/MS sample preparation Plasma (200 pL) was acidified with 1J pL formic acid and precipitated with 600 ptL acetonitrile. The supernatant kas collected by centrifugation and concentrated to dryness under vacuum. T ie residues were dissolved in 150 L 15 30% acetonitrile in water and centrifuged. Sc pL of the supernatant was injected for LC-MS/MS analysis. Tissue methanol extract (10 pL) wt diluted to 1 mL 30% acetonitrile in water and 50 pL was injected for LC-MS/MS analysis. 20 10.6. LC-MS/MS analysis LC-MS/MS analysis was done with an APl4000 mass spectrometer system equipped With a Turbo lonspray probe. The MRM mode of molecular ion detection was used with the ion pair of 668. and 136.1. HPLC separation was performed with a Luna C8(2) 2x30 mm 3[ column run 25 from 10% B to 90% B in 10 minutes at a fl w rate of 0.30 mL/minute. Buffer A is 1% formic acid in water and buffer B is 1% f rmic acid in acetonitrile. LOQ was 0.5 ng/mL. 10.7. Results and summary 30 The plasma concentrations of the peptide were calculated with its standard calibration plot. 0.06 mg/mL (Aib835)hGt P-1(7-36)NH 2 (0.3 mg/rat in 5 mL -43- Ii methanol extract) was used as the 100% t< calculate the percentages left at the injection sites. Table 5. (Aib 8 35)hGLP-1(7-36)NH2 plasma concentrations 5 H lasma Plasma Plasma concentration con entration concentration (nq/mL) of n /mL) of np/mL) of (Aib 3 )hGLP-1(7- (Aib h )hGLP-1(7- (Aib 3 )hGLP-1 (7 36)NH 2 HCI and CuC1 2 36)N 2 HCI and 36)NH 2 acetate and Time h dose Zr 12 dose ZnCl 2 dose 0.083 4.76 5.)6±3.85 25.9±14.57 0.17 3.18 13.)4±12.81 16.35±5.02 0.25 3.44 13 65±8.14 32.2 0.5 7.95±5.3 13 86±11.8 19.5±3.68 1 11.8 1 24±10.61 11.5 2 11.4±1.27 12.9±0.35 8.64 4 5.9±5.2 6. 9±4.62 5.48 8 0.9±0.37 0.72 6.41 24 1.35 1.08 0.94 48 0.68 111.21 72 0.66 0.47 0.77 96 0.15 1.35 0.33 168 0.17 0.74 0.82 240 0.35 0.6 1.09 A full time course plot of the pha macokinetics profile of the HCI salt formulations of (Aib 8

.

3 )hGLP-1(7-36)NH 2 iS shown in Figure 8. An early time 10 course plot of the pharmacokinetics prcile of the HCI salt formulations of (Aib 35 )hGLP-1(7-36)NH 2 is shown in Fig re 9. A full time course plot of the pharmacokinetics profile of the acetate ?alt formulation of (Aib, 35 )hGLP-1(7 36)NH 2 is shown in Figure 10. An early tine course plot of the pharmacokinetics profile of the acetate salt formulation of Aiba, 35 )hGLP-1(7-36)NH 2 is shown in 15 Figure 11. 20 |I973a 1nHatrs 839 Table 6. Estimated percentages of (Aib,35)h9LP-1 (7-36)NH 2 left at the injection sites Estimated Es- mated Estimated percentage (%) left percent ge (%) left percentage (%) left at injection site of at inje ion site of at injection site of (Aib )hGLP-1(7- (Aib,3- hGLP-1(7- (Aib*,' 5 )hGLP-1(7 Time 36)NH 2 HCI and 36)N 2 HCI and 36)NH2 acetate and days CuCl 2 dose ZnG12 dose ZnCl 2 dose 1 1.58 '0.59 6.96 2 24.88 6.94 9.97 3 12 1.87 11.6 4 0.14 ).04 0.23 7 0.47 b.06 0.03 10 0.01 D.02 0.01 Tissue accumulation profile of (Aib 3 3 hGLP-1 (7-36)NH 2 at the injection site is further shown in Figure 12. Table 7. PK parameters 10 Plasma Plasma Plasma concentration concentration concentration ng/mL) of ny/mL) of (nw/mL) of (Aib '5 )hGLP-1(7- (Aib )hGLP-1(7- (Aib ' ,3)hGLP-1(7 36)NH 2 HCI and 361NH 2 HCI and 36)NH 2 acetate CuC1 2 dose ZnC2 dose and ZnCl 2 dose Tmax, h 1 0.5 0.25 Cmax, ng/ml 11.8 13.8 32.2 AUC ng-hrlml 204 514 394 The results indicate that salt forms of LP-1 analogues, particularly in combination with a divalent metal salts, pro de for acceptable sustained release formulations with reduced initial plasma con entrations, which may reduce or 15 eliminate unwanted side-effects. The data indicate that strong acid sal s, for example, HCI salts of the GLP-1 analogue, show a further reduction in initial lasma concentrations. Without being bound to this theory, it is believed that the s perior reduction in initial plasma 20 concentrations of the HCI salts of GLP-1 an logues relate to the neutralization 298703S 1 (GHMfM I P81SQQ9AU2OO~l c~sc- [ -45 process in vivo. In compositions (1) and (2) above at pH 5.5, 100% of the acid is in the chloride form and there is no free acic Accordingly, after the subcutaneous injection the body fluid is able to neutralize t e solution more quickly thereby precipitating the solution more rapidly. TheE e decrease in neutralization time leads 5 to a smaller, less pronounced, initial plasma concentration or spike. The publications cited above are incc porated herein by reference. Additional embodiments of the present inver tion will be apparent from the foregoing disclosure and are intended to be ncompassed by the invention as 10 described fully herein and defined in the fol( ing claims. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" ar variations such as "comprises" or 15 "comprising" is used in an inclusive sense, i. . to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prio art publication is referred to herein, 20 such reference does not constitute an admi sion that the publication forms a part of the common general knowledge in the art in Australia or any other country. 25 n&7OaS. (GHMsftmrs Pal SQGAU

Claims (15)

1. A pharmaceutical composition comprising an analogue of GLP-1, wherein the GLP-1 analogue is [Aibs 35 ]hGLP-1(7-36)NH 2 ,prepared with a pharmaceutically acceptable salt of said analogue or a mixture of the salt of said analogue and the analogue, thereby providing a molar ratio of analogue salt to peptide analogue in said pharmaceutical composition, wherein the molar ratio can be adjusted to modulate the solubility, the pH, and release effect on in vivo release profile of the peptide in said pharmaceutical composition, said composition further comprising a divalent metal and/or divalent metal salt, with a molar ratio of said GLP-1 analogue to said divalent metal and/or divalent metal salt in said pharmaceutical composition ranging from approximately 5.4:1 to approximately 1.5:1; and wherein the analogue salt in said pharmaceutical composition is a salt of hydrochloric or acetic acid, or chlorides or acetates of said GLP-1 peptide analogue and the acetate or chloride is present as final molar ratio of acetate or chloride to said peptide analogue in the pharmaceutical composition ranging from approximately of 0.5:1 to approximately 10:1.

2. The pharmaceutical composition according to Claim 1, wherein said selected divalent metal is zinc or copper.

3. The pharmaceutical composition according to Claim 1, wherein said composition contains divalent metal salts selected from the group consisting of CuAc 2 , CuCl 2 , ZnAc 2 , and/or ZnCl 2 .

4. The pharmaceutical composition according to Claim 1 or 3, wherein said salt of [Aib 8 ' 5 ]hGLP-1(7-36)NH 2 is a pharmaceutically acceptable salt of an organic acid.

5. The pharmaceutical composition according to Claim 4, wherein said organic acid is selected from the group consisting of acetic, trifluoroacetic, lactic, malic, ascorbic, succinic, benzoic, citric, methanesulphonic and toluenesulphonic acids. 2097151 1 (CHMtte) P81390 AU 47

6. The pharmaceutical composition according to Claim 4, wherein said acid is acetic acid.

7. The pharmaceutical composition according to Claim 1 or 3, wherein said salt of [Aib 8 ' 3 ]hGLP-1(7-36)NH 2 is a pharmaceutically acceptable salt of an inorganic acid.

8. The pharmaceutical composition according to Claim 7, wherein said inorganic acid is selected from the group consisting of hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acids.

9. The pharmaceutical composition according to Claim 7, wherein said acid is hydrochloric acid,

10. A pharmaceutical composition according to Claim 1, wherein the molar ratio ranges of said [Aib 835 ]hGLP-1(7-36)NH2 salt to GLP-1 peptide analogue is approximately 0.5:1 to approximately 10:1.

11. The pharmaceutical composition according to claim 1, wherein said pharmaceutically acceptable salt is [Aiba-aslhGLP-1(7-36)NH2-HCI-Zn.

12. The pharmaceutical composition according to claim 1, wherein said pharmaceutically acceptable salt is [Alb 8 . 35 ]hGLP-1(7-36)NH2-acetate-Zn.

13. The pharmaceutical composition according to claim 1, wherein said pharmaceutically acceptable salt is [Aib , 3 s JhGLP-1 (7-36)NH 2 -HCI-copper.

14. The pharmaceutical composition according to any of the preceding claims, wherein the final molar ratio of acetate or chloride to said GLP-1 peptide analogue ranges from approximately of 0.8:1 to approximately 9:1. 2%S7151.1 (GHMnters) P61319AU 48

15. The pharmaceutical composition according to any of the preceding claims, wherein the final molar ratio of acetate or chloride to said GLP-1 peptide analogue ranges from approximately of 1:1 to approximately 6:1 2997151_1 (GHMattsM Pa1390.AU