TW201023877A - 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

.201023877 VI. Description of the Invention: [Technical Field] The present invention relates to improving a peptide analog containing glucagon-like peptide-1 and/or a pharmaceutically acceptable salt thereof The composition, the method of preparing the composition, the pharmaceutical composition, and a method of treating a mammal using the composition. [Prior Art] The glucagon-like hyperglycemic hormone-like-1 (7-3 6) guanamine (GLP-1) is a specialized tissue of the glucagon in the gut of the intestinal L- fine sputum via the glucagon hormone precursor. Post-translational processing and synthesis (Varndell, JM·, et al·, J. Histochem

Cytochem, 1 9 8 5:3 3:1 0 8 0-, 6), is released into the circulatory system in response to a meal. The plasma concentration of GLP-1 increased from a fasting degree of about 15 pmol/L to a maximum postprandial level of 40 pmol/L. It has been shown that for known plasma glucose % 'increased sugar concentrations, plasma glucose is increased nearly three-fold compared to intravenous glucose (Kreymann, B., et al., Lancet 1987: 2, 1 300- 4). This digestive enhancement of insulin release is known to be an incretin action, primarily humoral and is considered to be the most potent physiological enteroprotein in humans. In addition to insulin secretion, GLP-1 inhibits pancreatic hyperglycemic hormone secretion, delays gastric emptying (Wettergren A., et al., Dig Dis Sci 1993: 38: 665-73) and improves peripheral glucose clearance (D) 'Alessio, D_A. et al., J. Clin Invest 1 994:93:2293 -6). After i 994 observed that subcutaneous (s/c) single dose of GLP-1 completely normalizes the postprandial glucose level in patients with non-insulin dependent diabetes mellitus (NIDDM), the therapeutic potential of GLP-1 was proposed (Gutniak, Μ· Κ·, et al., Diabetes Care 1994: 17: 1039-44). This effect is thought to be mediated by increased insulin 201023877 release and decreased pancreatic hyperglycemic hormone secretion. Furthermore, intravenous infusion of GLP-1 has been shown to delay postprandial gastric emptying in NIDDM patients (Williams, B., et al., J. Clin Endo Metab 1996: 81: 327-32). Unlike diuretic hormones, the insulin-promoting effect of GLP-1 is plasma glucose concentration-dependent (Holz, G.G_ 4th, et al., Nature 1 993:3 6 1:3 62-5). Therefore, GLP-1-mediated insulin release is lost when plasma glucose concentration is low to avoid severe hypoglycemia. This combination of effects confers a unique potential therapeutic benefit to GLP-1 remaining in other agents currently used to treat NID D Μ. ❹ There have been many studies showing that GLP-1 effectively affects blood glucose levels and insulin and glucagon concentrations when administered to healthy subjects (Orskov, C, Diabetologia 35:70 1 -7 1 1 , 1 992; Holst, JJ, et a 1., Potential of GLP-1 in diabetes management in Glucagon III, Handbook of Experimental Pharmacology, Lefevbre PJ, Ed. Experimental Berlin, Springer Verlag, 1 996, p. 311-326), The role is glucose dependent (Kreymann, B., et al., Lancet ii: 1 300-1304, 1 9 8 7; Weir, G. C., et al., Diabetes ® 38:338-342, 1989) . Furthermore, it is also effective in diabetic patients (Gutniak, Μ., N. Engl J Med 226:1 3 1 6- 1 322,1 992; Nathan, D. M., et al., Diabetes Care 1 5: 270-276, 1 992), normalizing blood glucose levels in patients with type 2 diabetes (Nauck, MA, et al., Diabetologia 36: 741-744, 1993), and improving glycemic regulation in type 1 patients (Creutzfeldt, WO) ·, et al., Diabetes Care 1 9:5 80-586, 1 9 96), in particular, its ability to increase insulin sensitivity/inhibition of insulin resistance. GLP-1 and its agonists have been proposed for use in subjects developing non-insulin dependent diabetes (see WO 00/076 17) and for 201023877 treatment of gestational diabetes (US Patent Publication No. 20040266670). In addition to the foregoing, there are a number of therapeutic uses for mammals, for example, it has been suggested that GLP-1 and its agonists may include, but are not limited to, improving learning, enhancing neuroprotection and/or alleviating symptoms of central nervous system diseases or disorders, For example, through neuronal neonatal regulation and, for example, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, ALS, Stroke, ADD, and Neuropsychiatric Syndromes) (U.S. Patent Application Publication No. 20050009742 and No. No. No. No. No. No. No. No. No. No. No. No. No. Publication No 2003022025 No. 1) and stimulating β-cell proliferation (U.S. Patent Publication No. 20030224983); treating obesity (US Patent Publication No. 2004001 8975; 'W098/1 9698); suppressing appetite and inducing satiety (US Patent Publication No. 20030232754) No.); treatment of irritable bowel syndrome (WO 99/6 4060); reduction associated with myocardial infarction Morbidity and/or mortality (U.S. Patent Publication No. 20040 1 62241, WO 9 8/0853 1 ) and stroke (see WO 00/1 6797); treatment of acute coronary heart disease characterized by a lack of Q-wave myocardial infarction (U.S. Patent Publication No. 20040002454); mitigating catabolic changes after surgery (U.S. Patent No. 6,006,753); treatment of dormant myocardial or diabetic cardiomyopathy (U.S. Patent Publication No. 20050096276); inhibiting the amount of ortho-adrenalin in plasma blood (U.S. Patent Publication No. 20050096276); increasing urine sodium excretion and reducing urine potassium concentration (U.S. Patent Publication No. 2005003 795 8); treating conditions or disorders associated with excessive toxic body fluids, such as renal failure, congestive heart failure , renal syndrome, cirrhosis, 201023877 pulmonary edema, and hypertension (U.S. Patent Publication No. 20050037958); induces muscle contraction and increases cardiac contractility (U.S. Patent Publication No. 200 5 003 795 8); treatment of polycystic ovarian syndrome (U.S. Patent Publication Nos. 20040266678 and 20040029784); Treatment of Respiratory Distress (US Patent Publication No. 2004023 5726); improving nutrition through non-nutrition pathways, ie by intravenous, subcutaneous, intramuscular, peritoneal, or other injection or infusion (US Patent Publication No. 200402098 14); treatment of renal lesions (US Patent Publication No. 20040209 8 03); treatment of left ventricular systolic heart failure, such as sputum with abnormal left ventricular ejection rate (US Patent Publication No. 2004009741 No. 1); inhibition of gastric treasure duodenal movement, for example for the treatment or prevention of gastrointestinal disorders such as diarrhea, Post-operative pour syndrome and irritable bowel syndrome and pre-dosing as an endoscopic procedure (US Patent Publication No. 20030216292); treatment of severe disease polyneuropathy (CIPN) with 'and systemic inflammatory response syndrome (SIRS) (U.S. Patent Publication No. 20030199445); modulating the amount of triglyceride and treating dyslipidemia (U.S. Patent Publication Nos. 20030336504 and 20030143183); Treatment® Organ Tissue Injury Caused by Post-Ischemic Reperfusion Blood Flow (US Patent Disclosure) No. 20010 1 47 1 3 No. 1); treatment of coronary heart disease risk factor (CHDRF) Waiting group (U.S. Patent Publication No. 20020045636); and others. However, GLP-1 is metabolically unstable, and the plasma half-life (t1/2) in vivo (in Wv〇) is only 1-2 minutes. Exogenously administered GLP-1 was also rapidly degraded (Deacon, C.F., et al., Diabetes 44: 1126-1131, 1995). This metabolic instability limits the therapeutic potential of native GLP-1. Many attempts have been made to improve the therapeutic potential of GLP-1 and its analogs by improving the formulation. For example, International Patent Publication No. WO 0 1/57084 describes a process for the production of GLP-1 analog crystals 201023877 which can be used to prepare pharmaceutical compositions containing crystalline and pharmaceutically acceptable carriers, such as injectables. A heterogeneous microcrystal cluster of GLP-1 (7-37) OH has been grown from a saline solution and examined after crystallization by zinc and/or m-cresol soaking (Kim and Haren, Pharma. Res. Vol. 12No. 11 (1995)). Preparation of a crude crystal suspension of GLP(7-3 6)NH2 containing similar needle crystals from a phosphate solution containing zinc or protamine and an amorphous precipitate (Pridal, et. al., International Journal of Pharmaceutics) Vol. 136, pp. O 53-59 (1 996)). European Patent Publication No. EP 061 9322 A2 describes the preparation of the GLP-1 (7-37) OH microcrystalline form via a mixed protein in a pH 7-8.5 buffer with a specific combination of salts and a low molecular weight polyethylene glycol (PEG) solution. . No. U.S. Patent No. 6,555,521 (U.S. Patent No. 5,521), which is incorporated herein by reference in its entirety, is incorporated herein by reference. US '521 teaches that such crystals are fairly uniform and present in comparison to previous crystal group® and amorphous crystal suspensions (which are believed to precipitate rapidly, aggregate together or clump together, block the needle and often exacerbate unpredictable doses) In the suspension for a longer period of time. Biodegradable poly[(dl-lactic acid-polyglycolic acid)-β-ethylene glycol·Ρ-(-lactic acid-polyglycolic acid)] triblock copolymer has been proposed for controlled release of GLP-1 Formulation. However, like other polymer systems, the manufacture of triblock copolymers involves complex procedures and inconsistent particulate formation. Likewise, biodegradable copolymers such as poly(lactic-glycolic acid) (PLGA) have also been proposed for the sustained delivery of peptide formulations. Conventional 201023877, however, does not favor the use of such biodegradable copolymers because these copolymers generally have poor solubility in water and require water-immiscible organic solvents (e.g., dichloromethane) and/or stringent manufacturing conditions during manufacture. Such organic solvents and/or stringent preparation conditions are believed to increase the risk of inducing a change in the peptide or protein of interest, resulting in reduced structural integrity and compromised biological activity (Choi et al., Pharm. Research, Vol.). 21, No. 5, (2004).) The poloxamers also have the same drawbacks. The GLP-1 compositions described in the above references are less desirable for the preparation of GLP medical formulations because they tend to trap impurities and/or are difficult to reproducibly manufacture and provide. It is also known that GLP analogs cause nausea when the concentration is increased, so there is a need to provide sustained pharmaceutical efficacy with reduced initial plasma concentration (Ritzel et al., Diabetologia, 3 8: 72 0-725 (1 995);

Gutniak et al., Diabetes Care, 1 7(9): 1 039- 1 044 (1 994);

Deacon et al., Diabetes, 44: 1126-1131 (1995).). There is therefore a need for a glp-1 formulation that is easier and more reliable to manufacture' to more easily and reproducibly administer patients and to provide reduced initial plasma concentrations to reduce or eliminate undesirable side effects. SUMMARY OF THE INVENTION The present invention is described in the following paragraphs and claims. Accordingly, the present invention provides a pharmaceutical composition comprising a GLP-ι analog. Particularly preferred is a GLP-1 analogue according to formula (I) or a pharmaceutically acceptable salt thereof: (Aib8 > 35) hGLP-1 (7-36) NH2 (I) wherein the formulation of the composition provides excellent manufacture, Investment, drug power 201023877 and pharmacodynamics, as well as reducing negative side effects. Preferably, the pharmaceutical composition of the present invention is not composed of a transparent aqueous ZnCl2 solution of pH 4, wherein the [Aib8,35]hGLP-1 (7-36)NH2 is present at a concentration of 4 mg/ml and the ZnCl2 is 0.5 mg. The concentration of /ml is present. A preferred embodiment of the present invention provides a pharmaceutical composition having a drug release improving pro file, preferably having a reduced initial burst. The present invention also provides a pharmaceutical composition comprising a compound of the formula (I) and having an extended action period. Ο In a preferred feature, the present invention also provides a pharmaceutical composition which precipitates in situ to precipitate in situ at physiological pH for sustained release of the drug. . A further embodiment of the invention provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. Preferably, the carrier or diluent contains water. In a preferred feature, the invention provides a pharmaceutical composition comprising a compound prepared as a salt of a peptide or a mixture of a peptide and a salt thereof, or a GLP-1 ® peptide analog. Preferably, the salt of the GLP-1 peptide analog in the pharmaceutical composition is a pharmaceutically acceptable salt selected from the group consisting of organic acids such as acetic acid, lactic acid, malic acid, ascorbic acid, succinic acid, benzoic acid, citric acid, Methanesulfonic acid or toluenesulfonic acid; or a pharmaceutically acceptable salt of a mineral acid such as hydrochloric acid 'hydrobromic acid, hydroiodic acid, sulfuric acid or phosphoric acid. Pharmaceutically acceptable salts of strong acids, such as hydrochloric acid, are particularly preferred. A strong acid is defined as an acid having a ρ ΚΑ less than 4.5. Further preferred salts in the pharmaceutical composition are salts of organic acids such as acetic acid or trifluoroacetic acid, lactic acid, malic acid, ascorbic acid, succinic acid, benzoic acid 201023877 or salts of citric acid. In a preferred embodiment, the solubility and pH of the pharmaceutical composition can be extended by adjusting the GLP-1 analog to the salt form to the molar ratio of the GLP-1 to the salt form. The initial spike in the concentration of GLP-1. In a preferred embodiment, the pharmaceutical composition further contains divalent to reduce the water solubility of the composition and thereby prolong the release condition and an initial burst or spike in plasma concentration. Preferred divalent metals include beryllium copper. Salt forms of divalent metals are particularly preferred, including but not limited to divalent metals and acetates. Preferably, CuAc2, CuCl2, ZnAc2 and/or ZnCl2 are divalent metals and/or divalent metal salts present in the pharmaceutical composition at a concentration of from about 0.5 mg/ml to about 50 mg/m. Even more preferably, the metal and/or divalent metal salt is present in the pharmaceutical composition at a concentration of from < 0.01 mg/ml to about 0.50 mg/ml. More preferably, the pharmaceutical composition is a diluent, wherein the diluent contains a pharmaceutically acceptable aqueous solution. Rarely contains sterile water. In a further embodiment, the pharmaceutical composition further comprises a di- and/or divalent metal salt, wherein the GLP-1 class has a molar ratio to the divalent metal and/or divalent metal salt in the pharmaceutical composition. The range is from about 6 to about 1:1. Preferably, the ratio ranges from about 5.5:1 to about 1:1. The ratio ranges from about 5.4:1 to about 1.5:1. Even more preferably it is about 5.4:1, 4.0:1 or 1.5:1. Preferably, the ratio is about 1. The term "about" in this context means that the ratio is 1.5: 1 ± 10% per 値, so it is preferred that the ratio includes, for example, 1.35 - 1.65: 0.85-1.15, preferably The pharmaceutical composition contains an aqueous mixture, a suspension, and a release similar to a metal to reduce zinc and chlorine. In the case of a divalent valence from an extract containing a valence of gold: 1 to better ratio 5: 卜 target ratio. Or a solution of -10-201023877, wherein the analog of GLP-1, the compound of formula (I) or a salt thereof is present at a concentration of from about 0.5% to about 30% (w/w). More preferably, the concentration of the GLP-1 analogue and/or its salt in the aqueous mixture, suspension or solution is about 1%, 2%, 3%, 4%, 5%, 6%, 7% '8% , 9% ' 10%, 11%, 12%, 13%, 14% ' 15%, 16% ' 17%, 1 8 % ' 1 9 % ' 2 0 % > 21%' 22% ' 23%' 2 4%, 25% > 26%, 27%, 28%, 29% or 30% (w/w). More preferably, the concentration of the GLP-1 analogue and/or its salt in the aqueous solution is about 1%, 2%, 3% '4%, 5%, 6% '7%, 8%, 9% '10% , 11%, 14%, φ 15% ' 16%' 1 9 %, 2 0 %, 2 1 %, 2 2 %, 2 3 %, 2 4 %, 2 5 %, 2 6 %, 29% or 3 0% (w/w). Still more preferably, the concentration of the GLP-1 analogue and/or its salt analog in the aqueous solution is about 1%, 2%, 3%, 4%, 5%, 6%, 9% '10%' 11 % ' 22% ' 23%, 24%, 2 5 % or 26% (w/w) » and even more preferably the concentration of the analog of GLP-1 and/or its salt in the aqueous solution is about 1%, 2%, 3%, 4%, 5%, 6%, 10%, 2 2%, 23%, 24%, 25% or 26% (w/w). Still more preferably, the concentration of the analog of GLP-1 and/or its salt in the aqueous solution is about 1%, 2%, 5%, 10%, 23% or 25% (w/w). "About" means the following meaning: for a concentration of about 0.5% to about 4%, the target 値± 〇. 5 % is the desired range (for example, 〇·5 % to 1.5% is about 1%); for the target concentration 5% and higher, 20% of the target 是 is the desired range (for example, 8% to 12% is about 10%). Preferably, [Aib8,35]hGLP-1(7-36)NH2, an analog of GLP-1, or a salt thereof, is at a concentration of about 1% (weight/volume) of the pharmaceutical composition and [Aib8'35]hGLP The molar ratio of -1(7-36)NH2 to the divalent metal and/or divalent metal salt is about 1.5:1. More preferably, [Aib8,35]hGLP-1 (7-36)NH2 or a salt thereof is present in the pharmaceutical composition at a concentration of about 2% (weight/volume) and -11 · 201023877 [Aib8'35]hGLP-l ( 7-3 6) The molar ratio of NH2 or its salt to the divalent metal and/or divalent gold cerium salt is about 1.5:1. More preferably, [Aib8'35]hGLP-1 (7-3 6)NH2 or a salt thereof is present in the pharmaceutical composition at a concentration of about 10% (w/v) and [Aib8,35]hGLP-1 (7- 36) The molar ratio of NH2 or its salt to the divalent metal and/or divalent metal salt is about K5:; Most preferably [Aib8'35]hGLP-l(7-3 6)NH2 or a salt thereof at a concentration of the pharmaceutical composition of about 23% or about 25% (weight/volume) and [Aib8'35]hGLP-l (7-3 6) The molar ratio of NH2 or its salt to the divalent metal and/or divalent europium metal salt is about 1.5:1. In a preferred embodiment, the analog of GLP-1, [Aib8'35]hGLP-1(7-3 6)NH2, or a salt thereof, is at a concentration of about 5% (weight/volume) of the pharmaceutical composition and The molar ratio of the peptide to the divalent metal and/or divalent metal salt is about 5.4:1. More preferably, [Aib8,35]hGLP-1 (7-36)NH2' or a salt thereof is present in the composition at a concentration of about 5% (weight/volume) and the ratio is about 4.0:1. Still more preferably [Aib8'35]hGLP-l(7-36)NH2 or a salt thereof is present in the composition at a concentration of about 10% (weight/volume) and the ratio is about 5.4:1. Still further preferably [eight 丨 1) 8'35] 11 〇 1 ^ -1 (the concentration of 7-36 mouth 112 or its salt at the composition is about 10% (weight/volume) and the ratio is about 4.0 Preferably, the divalent metal and/or divalent metal salt provided is zinc chloride or zinc acetate. More preferably, the zinc acetate provided is ZnAc; r2 H20. In another embodiment, The divalent metal and/or divalent metal salt provided is copper chloride or copper acetate. In one embodiment, the pH of the pharmaceutical composition is adjusted upward using hydrazine. More preferably, the pH adjustment is carried out using NaOH. More preferably, the pH of the pharmaceutical composition is adjusted by NaOH-12-201023877, so that when diluted with 0.9% NaCl to an initial concentration of about */2, a direct potential measurement is used to obtain a pH of about 5.0-5.5. A preferred embodiment is characterized by a pharmaceutical composition wherein the composition is formulated such that the peptide analog of GLP-1 or a salt thereof, for example, a compound according to formula (I) or a salt thereof, is released from the need thereof The tester (eg, a mammal, preferably a human) is extended for a period of time. Preferably, the release of the compound is extended by at least 1 More preferably, it is at least 4, 6, 12 or 24 hours. Further preferably, the composition is formulated such that the compound according to formula (I) is released to the subject for at least 36, 48, 60, 72, 84 or More preferably, the composition is formulated such that the compound according to formula (I) is released to the subject for at least about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. More preferably, the composition is formulated such that the compound according to formula (I) is released to the subject for at least about 2, 3 or 4 weeks. Even more preferably the composition is formulated such that the compound according to formula (I) is released The subject is at least about 1, 1.5, 2 or 3 months or longer. © In one aspect of the invention, modulating the salt content of the GLP-1 peptide analog in the pharmaceutical composition improves GLP -1 solubility and stability of the peptide analog in the pharmaceutical composition, and further improving the release of the in vivo by reducing the initial burst. In this aspect of the invention, the term "modulating" means via adjusting the salt form Modification of the molar ratio of the GLP-1 analogue to the molar ratio of the non-salt form of the GLP-1 analogue. More preferably, the peptide salt is a salt of hydrochloric acid or acetic acid in the pharmaceutical composition, or a chloride or acetate of the peptide of the formula (1). In the pharmaceutical composition-13- '201023877, acetic acid The salt or chloride is present in the final molar ratio of the compound of formula (1) from acetate or chloride in a range from about 0.5:1 to about 10:1. More preferably, the ratio ranges from about 〇8:1 to Approximately 9: 1. Even more preferably the ratio is from about 1:1 to about 6: 1. Preferably, the ratio is about 3.0:1, especially 3.2: 1. In this aspect of the invention, acetate or The molar ratio of chloride to peptide means the molar ratio of acetate (CH3COO_) or chloride (Cl_) to the pharmaceutical composition to the molar ratio of the peptide to the pharmaceutical composition. For example, a 3:1 molar ratio in a pharmaceutical composition means that the molar amount of acetate is 3 Φ times that of the peptide. This is the stoichiometric ratio of the compound compared to the others. The phrase "about" in this aspect of the invention means a ratio of 1.5:1 to ±10% per target 値, so the ratio 预计 includes a ratio of, for example, 1.35 - 1.65: 0.85-1.15. In an additional preferred aspect of the invention, the pH of the pharmaceutical composition is adjusted by adjusting the amount of acetic acid of the composition. Preferably, the pharmaceutical composition has a pH ranging from pH 3 to pH 6. More preferably, the pH of the pharmaceutical composition ranges from pH 3.5 to 5.5. The pH range most preferably for the pharmaceutical composition is from pH 4.2 to pH 4.6. Preferably, the medicinal composition is acidified, and the amount of acetic acid can be increased by adding acetic acid. In one embodiment, the pH increase of the pharmaceutical composition can be initiated from a peptide salt of a GLP-1 analog having a low or no acetic acid amount by adjusting the amount of acetic acid. In a preferred embodiment, the pH adjustment of the final pharmaceutical composition is adjusted by adjusting the amount of acetic acid or chloride to enable adjustment parameters such as peptide concentration, zinc concentration, chemical stability, physical stability, and living organisms via reduced initial bursts - Release conditions within 14-201023877. In one aspect of the invention, the Zn or Cu content is fixed and the pH is controlled by adjusting the amount of acetic acid. Increasing the amount of acetic acid showed an improvement in solubility and physical stability, and a decrease in the amount of acetic acid showed an effect on pH increase and a decrease in Cmax. In a preferred embodiment, the pharmaceutical composition contains an aqueous mixture, suspension or solution. The present invention also provides a method of causing a GLP-1 agonist comprising directing a receptor GLP-1 (7-36) NH2 receptor with a GLP-1 analogue or a salt thereof Or indirect contact. In the above method, the receptor of the ligand GLP-1(7-36)NH2 is present in an animal subject, preferably a primate, more preferably a human. Accordingly, in this particular embodiment the invention provides a method of eliciting an agonist effect from a 'GLP-1 receptor in a subject in need thereof, comprising administering to the subject a composition of the invention, wherein the composition The article contains an effective amount of a GLP-1 analog or a pharmaceutically acceptable salt thereof. © In a preferred aspect of the above method, the subject is suffering from or at risk of developing from type I diabetes, type II diabetes, gestational diabetes, obesity, excessive appetite, insufficient satiety, and metabolic disorders Humans with the disease or symptoms of the group. Preferably, the disease is Type I diabetes or Type II diabetes. In another preferred aspect of the above method, the subject is suffering from or at risk of developing from a type I diabetes, type II diabetes, obesity, glucagonomas, respiratory secretion disorders Inflammation, osteoporosis, central nervous system diseases, vascular restenosis, neurodegenerative diseases-15-201023877 Disease, renal failure, congestive heart failure, renal syndrome, cirrhosis, pulmonary edema, high blood pressure disease, desire to reduce Deregulation of food intake, central nervous system diseases or disorders (eg, through regulation of neuronal regeneration, and, for example, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease) , ALS, stroke, ADD, and neuropsychiatric syndromes, irritable bowel syndrome, myocardial infarction (eg, reducing morbidity and/or mortality associated with this), stroke, acute coronary heart disease (eg, Characterized by a lack of Q-waves) myocardial infarction, postoperative catabolic changes, dormant myocardium or diabetic cardiomyopathy Insufficient urinary sodium excretion, excessive potassium in urine, conditions or disorders associated with excessive toxic body fluids (eg renal failure, congestive heart failure, renal syndrome, cirrhosis, pulmonary edema, and hypertension), polycystic ovarian syndrome , respiratory distress, nephropathy, left ventricular systolic heart failure (eg, abnormal left ventricular ejection rate), gastrointestinal disorders such as diarrhea, post-operative dumping syndrome, and irritable bowel syndrome (ie, by suppressing stomach sales of duodenal movement) , severe disease neuropathy (CIPN), systemic inflammatory response syndrome® (SIRS), dyslipidemia, organ damage caused by post-ischemic reperfusion, and coronary heart disease risk factor (CHDRF) syndrome group The disease of humans. In another aspect of the invention, the invention features a subject in need thereof to switch liver stem cells/precursor cells into functional pancreatic cells, prevent P-cell deterioration and stimulate β-cell proliferation, and inhibit plasma blood. Pre-adrenergic dose, induces myocardial contractile response and increases cardiac contractility, improves nutrition through non-nutrient pathways (eg, through intravenous, subcutaneous, intramuscular, peritoneal, or other injection or infusion routes), pre-treating subjects for The endoscopic procedure is performed by the method of -16-201023877 and adjusting the amount of triglyceride, the method comprising administering to the subject a formulation of the invention comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Preferably, the subject is a mammal, more preferably a primate, and more preferably a human. [Embodiment] The preferred GLP-1 peptide used as a peptide salt of the present invention is represented by the following form in the text. For example, (Aib8, 35) hGLP-1 (7-36) NH2, the native sequence placed in the first set of parentheses has a substituted amino acid (eg, Aib8'35 indicates that Ala8 in hGLP-1 is replaced by ® Aib And Gly35). Aib is an abbreviation for α-amine isobutyric acid. The abbreviation GLP-1 means the pancreatic glucagon hormone peptide-1; hGLP-1 means human leukemia hormone glycopeptide-1. The numbers in the second set of parentheses refer to the amino acid number present in the peptide (e.g., hGLP-1 (7-36) refers to the amino acid 7 to 36 of the peptide sequence of human GLP-1). The sequence of hGLP-1 (7-3 7) is listed in Mojsov, S., Int. J. Peptide Protein Res,. 40, 1992, pp. 333-342. The designation "NH2" in hGLP-1 (7-36)NH2 represents the guanidine amination of the C-terminus of the peptide. hGLP-1 (7-3 6) means that the C-terminus is a free acid. Unless otherwise indicated, residues 37 and 38 in ® hGLP-1 (7-38) are Gly and Arg, respectively. A particularly preferred GLP-1 peptide analog for use in the present invention is in the form of a pharmaceutically acceptable salt. Examples of such salts include, but are not limited to, organic acids (eg, acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, methanesulfonic acid, toluenesulfonic acid or double Pamoic acid), inorganic acids (such as hydrochloric acid, sulfuric acid or phosphoric acid) and copolymeric acids (such as tannic acid, carboxymethyl cellulose, polylactic acid, polyglycolic acid, or polylactic acid-glycolic acid) Copolymer) formed by. Typical methods for making the peptide salts of the present invention are well known in the art and can be accomplished by standard methods of salt exchange. Thus, the present invention is a TFA salt of the form -17-201023877 (this TFA salt is produced from the use of preparative HPLC, purification of the peptide eluted with a TFA-containing buffer solution) can be converted to another salt, for example via dissolution The peptide is converted to acetate in a small amount of 0.25 N aqueous acetic acid. The resulting solution was applied to a semi-preparative HPLC column (Zorbax, 300 SB, C-8). The column was eluted with (1) 0.1 N ammonium acetate aqueous solution for 0.5 hour, (2) 0.25 N aqueous acetic acid solution for 0.5 hour, and (3) at a flow rate of 4 ml/min (solution A was 0.25 N aqueous acetic acid; solution B) A linear gradient of 0.25 N acetic acid in acetonitrile/water, 80:20) (20% to 100% solution B for 30 minutes). The sputum contains the layer of the peptide and is lyophilized to dryness. As is well known to those skilled in the art, the known and possible uses of GLP-1 are diverse and numerous (see Todd, JF, et al., Clinical Science, 1998, 95, ρρ·325-329; and Todd, JF). Et al., European Journal of Clinical Investigation, 1997, 27, pp. 533-536). Therefore, the administration of the compound of the present invention for the purpose of causing agonist action can have the same effect and use as GLP-1 itself. These different GLP-1 uses can be summarized as follows: treatment of type 1 diabetes, type 2 diabetes, obesity, glucagonomas, respiratory secretion disorders, metabolic disorders, arthritis, osteoporosis, central nervous system Disease, vascular restenosis, neurodegenerative diseases, renal failure, congestive heart failure, renal syndrome, cirrhosis, pulmonary edema, high blood pressure, dysregulation to reduce food intake, and various other conditions and disorders discussed in the text . Thus, the invention includes within its scope a pharmaceutical composition as defined herein containing a compound of formula (I) as the active ingredient. The dosage of the active ingredient in the formulations of the invention may be varied' but the amount of active ingredient must be an appropriate amount to obtain. The dosage chosen will vary depending on the desired therapeutic effect, route of administration, and duration of treatment' and is usually determined by the attending physician -18 - 201023877. Generally, an effective dose of the activity of the present invention is in the range of 1 x 1 (T7 to 200 mg/kg/day, preferably 1 x 1 (Γ4 to 1 mg/kg/day, which may be administered in a single dose or in multiple doses). Formulations of the invention are preferably administered parenterally, for example intramuscularly, intraperitoneally, intravenously, subcutaneously, etc. The preparations according to the invention for parenteral administration comprise sterile aqueous or nonaqueous solutions, Suspensions, gels or emulsions, but can achieve the desired release in vivo. Examples of non-aqueous solvents or vehicles are poly-propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil). ), animal glues and injectable organic esters such as ethyl oleate. These dosage forms may also contain adjuvants such as preservatives, humectants, emulsifiers and dispersing agents which can be filtered and added, for example, through a filter that blocks bacteria. The bactericide is sterilized in the composition, the radiation illuminating composition or the heating composition. They can also be prepared as a sterile solid composition which can be dissolved in sterile water or other sterile injectable medium which can be dissolved immediately before use. Synthesis of the peptides useful in the practice of the present invention can be prepared via standard solid phase peptide synthesis. See, for example, Stewart, JM, et al., Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984). Descriptions of synthetic methods that can be used to make peptides that are advantageous for practicing the present invention are well known to those skilled in the art. Other methods are also known to those skilled in the art. Examples are provided by way of illustration only and not The scope of the invention is therefore limited in any respect. The peptide, such as a GLP-1 analog, can be obtained by various synthetic methods known to those skilled in the art, which can include final precipitation of the peptide, freeze-drying-19- 201023877 Process, vacuum drying or other drying processes known in the art. Ion exchange chromatography, osmotic exchange of buffers, and difiltration should be suitable methods for purifying or screening peptides of different salt forms in the present invention.

Boc-/Ala-OH, Boc-D-Arg(Tos)-OH and Boc-D-Asp (OcHex) were purchased from Nova Biochem, San Diego, California. Boc-Aun-OH was purchased from Bachem, King of Prussia, PA. Boc-Ava-OH and Boc-Ado-OH were purchased from Chem-Impex International, Wood Dale, IL. Boc-2Nal-OH was purchased from Synthetech, Inc. Albany, OR. The full names of other abbreviations used in the text are as follows: Boc is a third butoxycarbonyl group, HF is hydrogen fluoride, Fm is a formazan group, Xan is xanthyl 'Bzl is a benzyl group, and Tos is toluene sulfonate. Base, DNP is 2,4-dinitrophenyl, DMF is dimethylformamide, DCM is dichloromethane, and HBTU is 2-(1Η-benzotriazol-1-yl)-1,1. 3,3-Tetramethylurea hexafluorophosphate, 'DIEA is diisopropylethylamine, h〇Ac is acetic acid, TFA is trifluoroacetic acid, 2C1Z is 2-chlorobenzyloxycarbonyl, 2BrZ is 2· Bromobenzyloxycarbonyl, OcHex is 〇-cyclohexyl, Fmoc is 9-fluorenylmethoxycarbonyl, HOBt is N-hydroxy® benzotriazole; PAM resin is 4-hydroxymethylphenylacetamidomethyl Resin; Tris is tris(hydroxymethyl)aminomethane; and Bis_Tris is bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (ie 2-di(2-hydroxyethyl)amino group -2-(hydroxymethyl)-1,3-propanediol). The term "halo" or "halogen" includes fluoro, chloro, bromo and iodo. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents and other references mentioned herein are also incorporated herein by reference. Example 1 -20- .201023877 (Aib8'35) hGLP-1 (7-36) NH2 Detailed synthesis steps for (Aib8'35) hGLP-1 (7-36) NH2 have been provided in International Patent Publication No. WO 00 /343 3 No. 1 (PCT/EP99/09660) 'The contents of which are incorporated herein. Briefly, compounds synthesized by the Applied Biosystems (Foster City, CA) Model 43A peptide synthesizer were modified to facilitate Boc-chemical solid phase peptide synthesis. See Schnolzer, et al., Int. J. Peptide Protein Res., 40:180 (1992). A 4-methyldiphenylmethylamine (MBHA) resin (Peninsula, Belmont, CA) containing 0.91 mmol/g was used. Use Boc amino acid with the following side-chain protection (Bachem, CA, Torrance, CA; Nova Biochem., LaJolla, CA): Boc-Ala-OH ' Boc-Arg(Tos)-OH ' Boc-Asp (OcHex )-OH ' Boc-Tyr(2BrZ)-OH, Boc-His(DNP)-OH ' Boc-Val-OH, Boc-Leu-OH, Boc-Gly-OH, Boc-Gln-OH, Boc-Ile- OH , Boc-Lys(2ClZ)-OH > Boc-Thr(Bzl)-OH, Boc-Ser(Bzl)-OH, Boc-Phe-OH, Boc-Aib-OH, Boc-Glu(OcHex)-OH And Boc-Trp(Fm)-OH. The Boc base was removed twice by 100% TFA for 1 minute. Boc amino acid (2.5 mmol) was pre-activated with 4 ml of DMF in HBTU (2.0 mmol) and DIEA (1.0 ml), and the peptide-resin TFA salt was not neutralized first. The coupling time was 5 minutes, except for the Boc-Aib-OH residue and the following residues, the coupling time of Boc-Lys(2ClZ)-OH and B〇c-His(DNP)-OH was 2 hours. At the end of the time, the resin was treated with 20% mercaptoethanol/10% DIEA in DMF for 30 minutes to remove the DNP group on the His side chain. The N-terminal Boc group was then removed 2 times with 100% TFA for 2 minutes. After neutralizing the peptide/resin with 10% DIEA in DMF (1 minute), the solution of 15% ethanolamine/15% water/70% DMF was applied twice for 30 minutes to remove the formazan group on the Trp side chain. The peptide-resin was washed with DMF and DCM and dried under reduced pressure -21 - 201023877. The final cleavage was carried out by stirring the peptide-resin at 10 °C in 10 ml of HF containing 1 ml of anisole and dithiothreitol (24 mg) for 75 minutes. The HF is removed via a large amount of nitrogen. The residue was washed with diethyl ether (6×10 ml) and extracted with 4N HOAc (6 X 10 ml). The peptide mixture in the aqueous extract was purified by reverse phase preparative high pressure liquid chromatography (HP LC) using a reverse phase VYDAC® Cis column (Nest Group, Southborough, ΜΑ). A linear gradient (20% to 50% solution B over 10 5 minutes) at a flow rate of 10 ml/min (solution A = water with 0.1% TFA; solution B = acetonitrile with 0.1% TFA) Strip the column. The layers were collected and examined by analytical HPLC. The combined product contains the purified product and is lyophilized to dryness. In one of the synthetic examples of this compound, 135 mg of a white solid was obtained. According to analytical HPLC analysis, the purity was 98.6%. Electrospray mass spectrometry (MS(ES)) S analysis gave a molecular weight of 3 3 3 9.7 (compared to the calculated molecular weight of 3 3 3 9.7). Example 2

Formulation Procedure Step I 2^1 Materials, stock solutions, calculations © A) Materials: ZnCl2, NaOH particles and hydrochloric acid 35% were obtained from Panreac Quimica, Barcelona, Spain. WFI (sterile water for injection/rinsing) was obtained from B. Braun Medical, Barcelona, Spain. B) Reserve roll (nZnCl,, τ > Η = 3 : 1 . Add 35% HCl to WFI with stirring to achieve ρ Η = 3. 2. Transfer one of the weighed ZnCl2 to the volumetric flask. Add PH = 3 HC1 to reach a final concentration of approximately 1-4 mg ZnC h/ml. -22- 201023877 nnZnCl,. dH = 2 : 1. Add 35% HCl to WFI with stirring to reach pH = 2. Transfer one of the weighed ZnCl2 to the measuring flask and add PH = 2 HC1 with stirring to reach a final concentration of approximately 4-12 mg ZnCl2/ml. (iii) NaOH, 0.1 stroke 1 〇 mg/ml: 1. Transfer has been weighed One of the quantitative NaOH was added to the measuring flask. WFI1 was added with stirring to reach a final concentration of approximately 〇.ll〇mg NaOH/ml. (iv) Freeze-dried 20 ms tiller (Aib8'35, HGLP-l (7-36) NH , / small bottles: 1. Prepare 0.04% (v / ν) dilution of acetic acid and WFI. 2. Transfer one of the weighed (Aib8'35) HGLP-l (7-36) NH2 (acetate In a measuring flask, add enough 0.04% acetic acid to a final concentration of 20 mg (Aib8'35) HGLP-1 (7-36) NH2/ml with stirring. After filtering the bacteria with a 〇·45 μιη filter, 1 ml Transfer the solution to a lyophilized vial, freeze-dry and The dried product is stored at -22 ° C. (v) lyophilized 50 mg aliquot (Aib8, 35) HGLP-lf7-36) NH, / ❹ bottle: 1. Prepare 0.1% of acetic acid and WFI (v /·ν) Dilution 2. Transfer one of the weighed (Aib8'35) HGLP-1 (7-36) NH2 (acetate) in a measuring flask. Add enough 〇.1% acetic acid to the final concentration with stirring. 50 mg (Aib8'35)HGLP-l(7-36)NH2/ml. After filter sterilization, transfer 1 ml of the solution to the lyophilized vial and freeze-dry. C) Calculate (i) Determine the composition Total weight/volume of excipient (E): E = (AX 100/T) - (A/P) -23- 201023877 where: E = amount of excipient in mg A = purified peptide (mg); T = target concentration of the composition ·, for example 2 is the target of 2%: and P = concentration of the purified peptide (peptide mg / formulation 100 mg) About the total volume of the excipient 'assuming 1 ml = 1 g application. (ii) Determine the volume/weight (W) of ZnCh to be added to the composition solution per mi or g: 〇a) W = 100% E in the composition 'without pH adjustment; b) W = 8 0% E In a liquid formulation, wherein the scent is about 1%, or about 2% or to about 10%, and the pH is adjusted using a base; c) W = 50% E in a semi-solid or gel formulation, wherein the peptide Is about 1%, or about 2% or to about 10%, and adjusts the pH with a base; 'd)W = 66.66% E in a semi-solid or gel formulation, wherein the peptide is about 25% and used Adjust pH; e) W = 90% E in the formulation 'where the composition is reconstituted from the freeze-dried preparation, and the pH is adjusted using a base. (iii) Determine the volume/weight (W) of NaOH to add to the composition solution per mi or g: a) W = 2 0% E in the formulation, wherein the peptide is about 1%, or about 2% or Approximately 10%, and adjust the pH with a base; b) W = 50% E in a semi-solid or gel formulation in which the winning form is about 1%, or about 2% or to about 1%, and the pH is adjusted using a base. c) W = 33.33% E in a semi-solid or gel formulation in which the peptide is about 25% and the pH is adjusted using a base; -24-201023877 d) W=10% E in the formulation, from freeze-drying The preparation reconstitutes the peptide and the pH is adjusted using a base. (iv) Determine the concentration of ZnCl2 (mg/ml or mg/g) for each composition: [ZnCl2] = (1 36.29 x A) / (W x 3 3 3 9.7 6 x R) where: A = purification The content of the peptide (mg); R = the peptide/Zri molar ratio R = 1.5, wherein the peptide is about 1%, or about 2% or about 1% φ or to about 2 3 %; R = 4.0 formulation wherein the peptide is about 25%; and W = ZnCl2 solution weight (g) or volume (ml) added to each g or ml of the composition solution. 2.2 Containing 1-10% freeze-dried fat peptide and ZnCh, composition without adjustment of 1?11 Preparation The use of a formulation containing a percentage peptide is described as a formulation containing a weight peptide in each composition. The total weight, for example, 1% peptide is described as a formulation containing 1 g of peptide in every 100 g of total composition. Formulations containing from about 1%, or from about 2% to about 10% peptide are prepared as follows. The lyophilized sample of (Aib8'35)HGLP-1 (7-3 6)NH2 prepared as described was mixed with ZnCl2 stock solution (pH 3) at 100% total excipient volume and [peptide: Zn] = 1.5: 1 mix well. A) 1% of the composition was prepared by mixing 20 mg of freeze-dried (Aib8'35) HGLP-1 (7-36) NH2 (see 2.1 B(iv) above) with 2 ml of ZnCl2 solution (0.272 mg/ml; See 2.1 B (i) above. B) The 2% composition was prepared by mixing 20 mg freeze-dried -25 - 201023877 (Aib8,35) HGLP-1 (7-36) NH2 (see 2.1 B (iv) above) With 1 ml of ZnCl2 solution (0.544 mg/ml; see 2.1 B (i) above) C) 10% of the composition was prepared by mixing 50 mg of freeze-dried (Aib8, 35) HGLP-1 (7-36) NH2 ( Refer to 2.1 B (v) above and 0.45 ml of ZnCl 2 solution (3.023 mg/ml; see 2.1 B (i) above) to equilibrate the freeze-dried peptide and solution to room temperature. A specified volume of ZnCl2 solution is injected into a vial containing the freeze-dried peptide and the 1% or 2% peptide composition water contract is allowed for 2 minutes, or even 10% of the composition water contract for 60 minutes, or until all frozen The dried succulent is completely hydrated and the solution is free of peptide peptides. After hydration, shake the reconstituted peptide for about 1 minute. An appropriate amount of dissolved peptide can be removed for administration. For example, loo Ul is prepared according to the above-mentioned A. The 1% peptide solution is equivalent to the 1 mg dose, and 50 ul of the 2% peptide solution prepared according to the above B is equivalent to the 1 mg dose, 150 ul. The 10% peptide solution prepared according to the above C' is equivalent to the 15 mg dose and the like. Using the teachings of the present application, those skilled in the art will be able to vary the amount of peptide and ZnCl2 to obtain compositions other than the 1%, 2%, and 10% compositions described below and the desired amount of ruthenium. 2.3 Preparation of Composition Containing 1-10% Freeze-Dried Fat Peptide and ZiiCl7, County pH Adjustment A formulation containing about 1%, or about 2% to about 10% peptide was prepared as follows. The lyophilized sample prepared as described (Aib8'35)HGLP-1 (7-36)NH2 was thoroughly mixed with a ZnCl2 stock solution (pH 3) at 90% of the total excipient volume. The desired pH of the solution is achieved by the addition of a diluted NaOH solution. A) 1% of the composition was prepared by mixing 20 mg of freeze-dried (Aib8'35) HGLP-1 (7-36) NH2 (see 2.1 B (i v) above) with 1.8 ml of -26-201023877

ZnCl2 solution (see 2.1 B (i) above) B) 2% of the composition was prepared by mixing 20 mg of freeze-dried (Aibs, 35) HGLP-1 (7-36) NH2 (see 2.1 B(iv) above) 0·9 ml of ZnCh solution (see 2.1 B (i) above) C) 10% of the composition was prepared by mixing 50 mg of freeze-dried (Aib8, 35) HGLP-1 (7-36) NH2 (see 2.1 B above) (v)) Add the necessary volume of diluted NaOH solution (10% of the total φ volume of the excipient) to the above solution with 0.40 ml of ZnCl 2 solution (see 2.1 B(1) above) to reach the target concentration and pH. For example: for each: 1% composition: add 0.2 ml of the right concentration of NaOH solution 2% composition: add 0.1 ml of the appropriate concentration of NaOH solution I) 10% composition: add 0.05 ml of the appropriate concentration of NaOH solution using this application In the teachings of the case, those skilled in the art can change the amount of the peptide and 'ZnCl2 to obtain a composition other than the 1%, 2%, and 10% compositions detailed below. 2.4 Preparation of liquid compositions containing 1-10% peptide and ZnCb, pH-free adjustment. Liquid formulations containing from about 1%, or from about 2% to about 10%, of the peptide are prepared as follows. The (Aib8'35)HGLP-1 (7-36) NH2 sample was weighed and mixed with a ZnCl2 stock solution (pH 3) to achieve a target concentration of 1%, 2%, to 10% peptide. After mixing, the composition is sterilized by a filter and stored for use. 2.5 Preparation of a liquid composition containing 1-10% fat peptide and ZnCb, pH adjustment A liquid formulation containing about 1%, or about 2% to about 10%, of the peptide is prepared as follows. The (Aib8'35)HGLP-1 (7-36) NH2 sample was weighed and thoroughly mixed with a ZnCl2 stock solution (pH 3) at 80% of the total excipient volume. The zinc solution can be ZnCl2 or ZnAc2*2H20. The pH of the desired solution -27-201023877 is achieved by adding a diluted NaOH solution. Preparation of Preparations C5 to C13 using this method. Using the teachings of the present application, those skilled in the art will be able to vary the amount of peptide and ZnCl2 to obtain compositions other than 1%, 2%, and 10% as described herein. 2.6 Preparation of a half-factor/gel composition containing 25% peptide and ZnCl without pH adjustment A semi-solid or gel formulation containing about 25% peptide was prepared as follows. Weigh (Bagua 1 > 8'35) 1101^-1 (7-36) 1^2 sample and mix well with 211 (:12 stock solution (?112) with 66.66% total excipient volume. Zinc solution can be Is ZnCl2 or 〇ΖηΑ (ί2·2Η20. Preparation of preparations C1 and C2 using this method) More specifically, a semi-solid or gel composition is prepared using a “push-pull” mixing method: a) weighing the desired peptide In the 'tube of the disposable syringe S1 with a special two-way hand valve HV (ID = 0.5 mm), and put the tube into the Luer hole of the syringe; b) with a stainless steel rod SR Close the syringe plug; OHV in S1, connect to the vacuum source and turn on the HV for 10 minutes, then turn off the HV; d) Accurately weigh the zinc solution in the barrel of the second disposable syringe S2; e) Then connect the S2 To the free portion of the HV; f) open the HV and draw the solvent via vacuum to the cartridge S j containing the peptide powder; g) turn off the HV and remove the solvent syringe S2, thereby hydrating the peptide powder in S1; h) Remove the SR and slowly loosen the syringe plug; 〇 push the syringe plug (push and pull) but not open the HV, so that the powder mass is completely -28- 201023877 as solvent soak; j) The stainless connector SC (ID = l.〇mm) is placed in the syringe S2 with the tube inserted into the Luer hole of the syringe, and the stopper is pushed to the extreme end; k) the HV in the S1 is opened to discharge the vacuum, and then moved In addition to HV. Pushing the syringe plug to minimize air in the syringe; and l) connecting S1 and S2 with SC and kneading the composition from S1 to S2 through SC. Using the teachings of this application, those skilled in the art can change the peptide and

The amount of ZnCl2 was such that a composition other than 25% as described herein was obtained. 2.7 2.7 Preparation of a semi-solid/gel composition containing 25% peptide and ZnCl2, pH adjustment A semi-solid or gel formulation containing about 25% peptide is prepared as follows. Weigh ((8丨1> 8'35) 1101^-1(7-36)^^2 sample and mix well with 211(:丨2 stock solution (?112) with 66.66% total excipient volume. Zinc solution It may be ZnCl2 or . ' ZnAc 2 « 2 H 20. The desired solution pH is achieved by adding a diluted (mg) NaOH solution. In this embodiment, the total volume of liquid added to the powder must be distributed between the zinc and NaOH solutions. Adjust the zinc solution concentration so that the total volume of the required zinc solution is reduced to 50% of the total liquid volume added to the peptide powder (step d). The remaining 50% total liquid volume added to the peptide powder is detailed below. Add to NaOH solution. Prepare preparations C3 and C4 using this method. Prepare pH adjusted semi-solid or gel composition using "push-pull" mixing method: a) Weigh the desired amount of peptide beforehand with special two To the barrel of the disposable syringe S1 of the two-way hand valve HV (ID = 0.5 mm), and place the tube into the Luer hole of the syringe; b) close the syringe plug with the stainless steel rod SR; -29 - 201023877 Closed; Powder ❿ is dissolved into the needle plug; the desired connection ZnCl Ο ΗV in S 1 , connected to Vacuum the source and turn on the HV. 1 〇 After HV; d) accurately weigh the zinc solution in the barrel of the second disposable syringe S2; e) then connect S2 to the free part of the HV; Ο open the HV and draw the solvent via vacuum to the peptide containing the peptide Cartridge S1 g) Turn off the HV and remove the solvent syringe S2, thereby hydrating the peptide in S1 th) Remove the SR and slowly loosen the syringe plug; i) Push the syringe plug (push and pull) but not open the HV So that the powder agglomerate is completely soaked; j) Place the two-way stainless connector SC (ID = 1.0 mm) in the syringe S2 with the tube barrel Luer hole, and push the stopper to the extreme end; k) Open The HV in S1 is to evacuate the vacuum and then remove the HV. Pushing the syringe to minimize the air in the syringe; and l) connecting S1 and S2 with SC and kneading the composition from S1 to S2 through SC. m) After homogenization, 'removal of a mixture of mixed products to determine the peptide concentration η) accurately weigh the remaining intermediate bulk product and calculate the amount of NaOH solution to achieve the desired pH; 〇) accurately weigh the NaO Η solution The third disposable syringe S3; and Ρ) slowly compresses the syringe plug to minimize air in the syringe chamber. The composition was kneaded by SC two syringes' and by SC. Using the teachings of this application, those skilled in the art will be able to vary the amount of peptide and 2 to obtain a composition other than 25% as described herein. -30- 201023877 Table 1 Example No. of this peptide solution Zn ratio ** peptide: wins % dose C1 10 Z11CI2 0.846 mg / ml 5.4: 1 1 mg C2 5 0.40 mg ZnCb / ml 5.4: 1 1 mg C3 10 50% ZnCl2 1.69 mg/ml, 50% NaOH 1 mg/ml 5.4:1 1 mg C4 10 50% Z11CI2 2.28 mg/ml5 50% NaOH 1 mg/ml 4:1 1 mg C5 5 80% ZnCl2 0.674 mg/ml , 20% NaOH 3.81 mg/ml 4:1 1 mg C6 2 80% ZnCl2 0.26 mg/ml, 20% NaOH 2.15 mg/ml 5.4:1 1 mg C7 10 80% ZnCl2 3.81 mg/ml, 20% NaOH 4.47 mg /ml 1.5:1 1 mg C8 10 80% Z11AC2.2H2O 2.3 mg/ml, 20% NaOH 6.1 mg/ml 4:1 1 mg C9 2 80% Z11CI2 0.695 mg/ml, 20% NaOH 1.75 mg/ml 1.5: 1 1 mg C10 2 80% ZnAc2.2H20 1.12 mg/ml, 20% NaOH 1.44 mg/ml 1.5:1 1 mg C11 2 80% ZnCl2 0.695 mg/ml, 20% NaOH 1.75 mg/ml 1.5:1 1 mg C12 1 80% ZnCl2 0.384 mg/ml, 20% NaOH 0.875 mg/ml 1.5:1 1 mg C13 10 80% ZnCl2 3.85 mg/ml, 20% NaOH 4.47 mg/ml 1.5:1 15 mg Target 値 is displayed. The actual case of all cases is less than 5% of the target

**Show target 値. The actual range of all cases is within 10% of the target. • Determination of 3.0 GL·P _ 1 receptor affinity • Compounds useful in the practice of the present invention can be tested for their ability to bind to the GLP-1 receptor using the following procedure. Cell Culture = Φ RIN 5F rat insulinoma cells (ATCC-#CRL-2058 > American Types Center, Manassas, VA) expressing GLP-1 receptor were cultured in DMEM containing 10% fetal bovine serum (Dulbecco's) Modified Eagle's medium) and maintained at a temperature of about 37 ° C in a humidified atmosphere of 5% C02 / 95% air. Radiation ligand binding = RIN cells were homogenized in 20 ml ice-cold 50 mM Tris-HC1 and homogenized in Brinkman Polytron (Westbury, NY) (set 6, 15 seconds) to prepare for radioligand binding studies. membrane. The homogenate was washed twice by centrifugation (39,000 g/10 -31 - 201023877 minutes) and the final pellet was resuspended in 2.5 mM MgCl2, 0.1 mg/ml bacitracin (Sigma Chemical, St. Louis) , MO) and 0.1% BSA 50 mM Tris-HCl» For analysis, the aliquot (0.4 ml) was incubated at 0. 05 nM (125I) GLP-l (7-36) (~2200)

Ci/mmol, New England Nuclear, Boston, ΜΑ), with and without 0.05 ml unlabeled competitive test peptide. After incubation (25 °C) for 1 minute, the (125I)GLP-1 (7I) was combined by rapid filtration through a GF/C filter (Brandel, Gaithersburg, MD) previously immersed in 0.5% polyethyleneimine. -36) Separate from ❹ free. The filter was then washed 3 times with 5 ml of ice-cold 50 mM Tris-HCl, and the bound radioactivity captured on the filter was counted by a gamma ray spectrometer (Wall ac LKB, Gaithersburg, MD). The specific combination is defined as the total (125I)GLP-1 (7-36) binding minus the binding in the presence of 1000 nM GLP1 (7-36) (Bachem, Torrence, CA). 4. Dissolution of pH to pH 4.1. Compound Solubility Determination of pH in phosphate I buffer (PBS) may be beneficial for the practice of the compounds of the invention, which can be tested to determine at different pH and temperature using the following procedure Its solubility in PBS. A pack of premixed powder (SIGMA, product number: P-3813) was dissolved in 1 liter of deionized water to produce a 10 mM phosphate buffer containing 138 mM NaCM, 2.7 mM KCl and pH 7.4 to make a stock PBS buffer solution. The pH of this stock solution was adjusted with phosphoric acid and/or sodium hydroxide to prepare a PBS buffer having various pH値. A 2 mg sample of the compound will be tested, for example, 2 mg of the compound of Example 1 in a glass vial. Add 50 μL of the specific pH PBS buffer to each vial. Rotate this solution and, if necessary, oscillate with ultrasound to clear -32-201023877. Record the total buffer volume required to dissolve 2 mg of compound per test pH and calculate the solubility. The clear peptide solution was placed in a cold room (4 ° C) overnight at room temperature (20-25 ° C), and then the solubility of the peptide was tested at 4 ° C. 4.2. Dissolution of Compounds to pH Measurements of Salts It may be beneficial to practice the compounds of the present invention which can be tested to determine their solubility in the salt liquor at various pH and temperature. 9 g of NaCl was dissolved in 1 liter of deionized water to prepare a stock salt solution. φ The pH of the stock solution is adjusted with HC1 and/or NaOH to produce a salt solution having various pH enthalpy. A 2 mg sample of the compound will be tested, for example, 2 mg of the compound of Example 1 in a glass vial. A 50 μί dispense of a specific pH salt solution was added to each vial. Rotate the vial and converge to clear as needed. ' Record the total volume of salt solution required to dissolve 2 mg of compound per test pH and calculate the solubility. Place the clear solution at room temperature (20-25 °C) in a freezer (4 °C) for a night, then check the solubility at 4 °C. 4.3. Solubility Measurement of Compounds in a pH 7.0 Salt Solution It may be beneficial to practice the compounds of the present invention, which can be tested to determine its solubility at room temperature in a saline solution having a pH of 7 using the following procedure. 9 g of NaCl was dissolved in 1 liter of deionized water to prepare a salt solution. A 2 mg sample of the compound will be tested, for example, by weighing the compound of Example 1 in a glass vial and adding 1 ml of the dispensed salt, rotating and ultrasonically oscillating to clear. The total volume of salt required to dissolve the 2 mg peptide was recorded and the solubility at room temperature was calculated. -33- 201023877 4.4. Solubility Measurement of Compounds at Salts of Various pH It may be beneficial to practice the compounds of the present invention, which can be tested to determine their solubility in the salt solution at room temperature using the following procedure. 9 g of NaCl was dissolved in 1 liter of deionized water to prepare a stock salt solution. The pH of the stock salt solution was treated with HC1 and NaOH to obtain a salt solution having various pH hydrazines. A 2 mg sample of the compound will be tested, e.g., the compound of Example 1 is weighed into a glass vial. Add 50 μί of the specific pH of the salt buffer.旋转 Rotate and ultrasonically oscillate the solution to clarity. The total buffer volume required to dissolve the 2 mg peptide was recorded and the solubility was calculated. 5. Measurement of Water Solubility of Compounds to Zinc Concentration Macro» may be beneficial for use in the practice of the compounds of the invention, which may be tested to determine their solubility in water at pH 7 at different zinc concentrations. 'Dissolve ZnCl2 in deionized water to prepare a stock zinc solution at a concentration of 100 ing/ml and adjust the pH to 2.7 using HC1. The stock solution was appropriately diluted to prepare a solution having various ZnCl2 concentrations ("Zn test solution"). ® 1 mg of the compound will be tested, for example, by dissolving 1 mg of the compound of Example 1 in 25 μL of each Zn test solution to produce a solution having the compound 4 mg/ml. The pH of the solution was then adjusted using 0.2 N NaOH until a white precipitate formed. This precipitation solution was centrifuged and the mother liquor was analyzed using HP LC. The UV absorption region spike of the test compound is measured, and the concentration of the test compound in the mother liquor is determined via a comparison calibration curve. As an example of a representative compound which can be used in the practice of the present invention, the compound of Test Example 1 was analyzed as described above and the following results were obtained (aqueous solution, p Η 7 · 0, room temperature): -34 - 201023877 Table 2

ZnCl2 concentration (pg/ml) Solubility (mg/ml) 0 5.788 80 0.0770 500 0.05 79 1000 0.0487 1500 0.0668 2500 0.1131 0 6. Use the IFE gel and other lightning spots (dH lacrimal sinus using Invitrogen's Novex IEFpH3-10 condensate The gel measures the pi of the GLP-1 peptide (such as the compound of Example 1.) The peptide compound to be tested is dissolved in water to a concentration of 0.5 mg/ml. For each of these compounds, a solution of '5 pL is mixed. With 5pL of Novex® Sample Buffer 2X (containing 20 - 11^ arginine (no base) '2〇1111^ lysine (no base) and 15% glycerol), and the resulting 1 〇pL sample solution and protein The standard sample was loaded into the gel. The running buffer was also obtained from Invitrogen, and φ was geled according to the manufacturer's instructions, usually as follows: Fix 100 v for 1 hour, then fix 200 V for 1 hour, then fix 500 V for 30 minutes. The gel was fixed to 12% TCA containing 3,5% sulfonylic acid for 30 minutes and then stained with Colloidal Coomassie Blue for 2 hours according to the instructions of the Novex® Colloidal Blue kit> Decolorization. Scanning The gel was analyzed by Fragment Analysis Program 1.2. Pis with unknown wins were calculated relative to standard compounds with pi 値: 10.7, 9_5, 8.3, 8.0, 7.8, 7.4, 6.9, 6.0, 5.3, 5.2, 4.5, 4.2, and 3.5. -35- 201023877 The pi measured by the compound of Example 1 was 7.60. 7. In vivo analysis of rats The following analysis can be used to test the composition of the present invention to determine its in vivo

The ability to promote and enhance the efficacy within I. 7.1. Experimental Procedure: One day prior to the experiment, approximately 300-3 50 g of mature male Sprague-Dawley rats (Taconic, Germantown, NY) were weighed and implanted into the right atrial neck cannula under chlorinated anesthesia. The rats were then fasted for 18 hours prior to injection of time 0 适当 appropriate test composition or vehicle control group. The rats continued to fast for the entire experiment. Dilute 10〇11^/1111211(:12 solution in 11 with 112.7 7# (1 solution to prepare 0.5 mg/ml ZnCl2 solution. 1 mg of compound of formula (I) ((Aib8'35) hGLPl ( 7-36) NH2) is dissolved in 250 μM of this solution to produce a clear solution with a pH 4 compound of 4 mg/ml and 0.5 mg/ml 。η. At time ,, the rats are injected subcutaneously (sc) (a) ( Aib8, 35) hGLP-1 (7-36) NH2) in the aforementioned solution or vehicle control group. In both cases, the injection volume is very small (4-6 μΙ〇, and the GLP-1 compound administered to the subject) The dose was 75 pg/kg. After subcutaneous injection, 500 μl blood samples were taken through the intravenous (iv) cannula at appropriate time, and glucose challenge was given intravenously to test for enhanced insulin secretion. The time of glucose provocation was after compound injection. 2 5, 1, 6, 1 2 and 24 hours. After taking the initial blood sample, intravenous glucose (lg/kg) was injected with 500 μl of heparinized saline (10 μL/ml). After that, after glucose injection 500 μl of blood sample was taken at 2.5, 5, 10, and 20 minutes. Each of these was immediately injected intravenously with 500 μl of heparin through the cannula. Brine (l〇U/ml). Centrifuge the blood sample, -36- 201023877 Collect plasma from each sample and store the sample at -20 °C until the insulin content is analyzed. Use rat insulin enzyme binding immunosorbent assay (ELISA) The amount of insulin in each sample was determined by a kit (American Laboratory Products Co., Windham, NH). 7.1.1. Results: Continuously enhanced insulin activity induced by glucose injection was observed over all 24 hours of the experiment. There are a number of in vivo assays available in vivo to enable the skilled artisan to determine the ability of the composition to promote prolonged release of the active compound in vivo. 8.1. 1% fat peptide fines: prepared by example An aqueous test formulation containing 1% (w/w) of the compound of formula (I) in a ZnCl2 buffer solution (peptide: Zn ratio = 1.5: 1.0). 'Feeding a total of 6 42-78 months old and weighing 14-21 The kg of the male beagle's free access to water and fed once a week (about 400 g dry standard foraging (SAFE 12 5). The dog was fasted for 18 hours before the test composition was administered. ® was administered via the subcutaneous route in the interscapular region Test composition A 0.3 ml Terumo syringe (BS = 30M2913) of 0.33-12 mm was used to make a volume (about 20 μM per animal). This gave a theoretical dose of about 0.2 mg of peptide. Blood samples are taken periodically, approximately at the time of administration = 0, 8, 15, 5, 45 minutes, and 1, 2, 4, 8 and 12 hours, and 1, 2, 3, 4, 5 and 6 day. The blood was rapidly cooled after sampling until centrifugation' and then the hemorrhage was gently poured and rapidly frozen during the analysis. Determination of peptide plasma concentration after offline solid phase extraction followed by LC-MS/MS via on-line phase extraction, -37-.201023877

The data obtained by the Analyst v 1.2 software. The composition was shown to prolong the release of the active peptide for at least 2 days. 8.2. l% (~Aib8'35) hGLPn7-36) NHd solution: The following compositions were tested for their ability to release the peptide of the subject for an extended period of time using an in vivo assay procedure substantially as described in Section 8.1 above. For each of the following four compositions, the concentration of the peptide is about 1% (wt/wt), the ratio of the peptide to zinc is about 1.5:1, and the dose of the peptide is about 1 mg. φ solution 8.2.A : (Aib8'35)hGLPl(7-36)NH2 contains (i)90%

a solution of ZnCl2 (0.29 8 mg/ml) and (ii) 10% NaOH (0.975 mg/ml); solution 8.2.B: (Aib8,35) hGLP1(7-36) NH2 in ZnCl2 (0.286 mg/ml) Solution 8.2.C: Substantially similar to solution 8.2.B, and using AcOH/AcO· • buffer; solution 8.2.D: substantially similar to solution solution 8.2.A. An extended release (Aib8'35) hGLP1(7-36)NH2 composition is provided, Φ as depicted in Figure 1. 8.3. 1% fAib8, 35MiGLP1(7-36)NH7) Solution: The following composition was tested for its ability to release the peptide of the subject for an extended period of time using an in vivo assay procedure substantially as described in Section 8.1 above. For the following compositions, the concentration of the peptide was about 2% (wt/wt), the ratio of the triumph to the zinc was about 1.5:1, and the dose of the peptide was about 1 mg. Solution 8.3. : (Aib 8 3 5 )hGLPl(7-36)NH2 in a solution containing (i) 80% ZnCl2 (0.695 mg/ml) and (ii) 20% NaOH (1.75 mg/ml); Release (Aib8'35) hGLP1(7-36) NH2 composition '-38- 201023877 as described in Figure 5. 8.4. 10% fat peptide solution: The following composition was tested for its ability to release the peptide of the subject for an extended period of time using an in vivo assay procedure substantially as described in Section 8.1 above. For each of the following four compositions, the concentration of the winning form was about 10% (wt/wt), the ratio of the peptide to zinc was about 1.5:1, and the dose of the peptide was about 15 mg. Solution 8.4.A: (Aib8'35) hGLP1(7-36)NH2 in a solution containing (i) 90% ❹ ZnCl2 (3.3 67 mg/ml) and (ii) 10% NaOH (5.01 mg/ml); 8.4.B: (Aib8'35) hGLP1(7-36) NH2 dissolved in ZnCl2 $ (2.993 mg/ml); solution 8.4.C: substantially similar to solution 8.4.B, using AcOH/AcO_ buffer;

' Solution 8.4.D: Substantially similar to the composition of extended release (Aib8,35) hGLP1(7-36)NH2 provided by solution 8.4.A, as depicted in Figure 2. ❹ 8.5. Semi-solid composition: The following semi-solid compositions were tested for their ability to release the peptide of the subject for an extended period of time using an in vivo assay procedure substantially as described in Section 8.1 above. For composition 8.5.A, the concentration of the peptide was about 5%, and the concentration of the peptides of the compositions 8·5·Β, 8.4.C and 8.5.D. was about 10% (wt/wt). As for the composition 8.5. Α, 8.5. Β and 8.5. C, the ratio of the peptide to zinc was about 5.4:1, and the ratio of the composition 8.5.D was about 4.0:1. For all four components, the dose of peptide administered was approximately 1 mg. Composition 8.5.A : (Aib8'35)hGLPl(7-36)NH2 containing -39- 201023877

Semi-solid composition of ZnCl2 (0.40 mg/ml) in WFI. Composition 8.5.B: Substantially similar composition 8·5·Α·, where the ZnCLz concentration has been adjusted upward to maintain the peptide: Zn ratio is about 5.4:1. Composition 8.5.C: (Aib8'35) hGLP1(7-36)NH2 is a semisolid containing (1) 50% ZnCl2 (1.69 mg/ml) and (ii) 50% NaOH (1 mg/ml). Composition 8.5.D: (Aib8'35) hGLP1(7-36)NH2 is a semisolid containing (i) 50% ZnCl2 (2.2 8 mg/ml) and (ii) 50% NaOH (1 mg/ml). The composition of the extended release (Aib8'35) hGLP1(7-36)NH2 is provided, as described in Figure 3. 8.6. Semi-body fines: The following semi-solid compositions were tested for their ability to release the subject peptide over an extended period of time using the same in vivo assay step as described in Section 8.1 above. This group of compounds was formulated using a 5.22 mg/ml ZnCl2 solution at pH = 2.0. Sufficient peptides are provided to produce a 25% peptide semisolid composition having a peptide to zinc ratio of about 4:1. The composition pH was adjusted using 1 〇 mg/ml NaOH as provided herein. The dose of peptide administered was approximately 15 mg. G provides a composition of extended release (Aib8'35) hGLP1(7-36)NH2 8.6, as depicted in Figure 6. 8.7. Semi-factor composition: The ability of the following semi-solid composition to release the subject over a prolonged period of time is tested using an in vivo assay step substantially identical to that described in Section 8.1 above. This composition was formulated using 8.5 mg/ml ZnCl2 solution with ρΗ = 2.0. Sufficient peptides are provided to produce a 23% peptide semisolid composition having a peptide to zinc ratio of about 1.5:1. The composition was formulated as detailed in Section 2.6 above. The dose of peptide administered was approximately 15 mg (equivalent to a composition of approximately 65 μM). -40- 201023877 Provides extended release (Aib8,35) hGLP1(7-36) NH2 composition 8.6, as depicted in Figure 7. Further analysis of the various transformations of the disclosed formulations has also been performed in vivo and it has been confirmed that the compositions of the present invention provide a pharmaceutical delivery platform useful for the compounds of formula (I). Using the techniques of this application, those skilled in the art will be able to modify the peptide, ZnCl2 amount and pH to prepare a composition of the invention as described herein. Example 9 Φ 1. PK in an amount of acetic acid in a 10% peptide solution. This example discloses that a single subcutaneous administration of 10% (Aib8'35) hGLP1(7-36)NH2 is carried out at a 15 mg/dog dose level. Two of zinc chloride [(Aib8'35)hGLPl(7-36)NH2:Zn=1.5:1] immediately after preparation of the composition (Aib8'35) hGLPl(7-36)NH2 in the drug power of the male beagle Learning 硏'. The method of performing in vivo analysis is the same as disclosed in Section 8.1. This example illustrates the adjustment of the PK ❹ condition of the pharmaceutical composition via the amount of acetic acid, and thereby the effect of the ratio [acetic acid/peptide) on the pH in the pharmaceutical composition. The pH adjustment was controlled by adjusting the amount of acetic acid, and the decrease in the amount of acetic acid showed an effect on pH increase. The change in acetic acid also showed an effect on Cmax. Usually the reduction in the amount of acetic acid reduces C m a X 値. An increase in the amount of acetic acid shows an improvement in solubility and physical stability. The improvement in solubility or stability via adjustment of the acetate/peptide ratio is offset by adjusting the peptide/Zn ratio (e.g., to Cmax), depending on the formulation selected. -41- 201023877 This can be considered as a variable with three adjustable stability, solubility, pH or Cmax for a system. In this embodiment, the abbreviation SD means the standard deviation. AUC means the area under the blood concentration-time curve of the artemisinin. The abbreviation MRT means mean residence time (MRT), a parameter used to estimate the rate of bioavailability compared to MRT with a drug concentration spike time tmax. The MRTt is calculated using the data obtained from the time 最后 to the last sampling time. φ Table 3 contains the results of different [acetic acid / peptide) ratio of 10% peptide composition batch and beagle subcutaneous administration. For [3.7:1] [acetic acid/victoric] molar ratio 'plasma concentration 药剂 medium drug spike Kmad sound 810 1^/1111 (3〇 = 1.80 11§/1111), with a lower ratio [3.2:1] The batch provides a Cmax 5.6 of 5.65 ng/ml (SD = 2.61 ng/ml). Table 3 调 Formulation 10% 15mg 10% 15mg peptide/Zn ratio 1.5:1__!·5:1. Parameter unit average (n=5) SD average (n=4) SD dose Mgkg1 857.7 131.0 694.8 46.5 tmax Imax d 0.208 0.167 0.111 0.068 ngmT1 8.10 1.80 5.65 2.61 tl/2jqip d 3.32 0.66 6.77 2.04 AUQ ngmr]d 53.5 14.3 38.2 9.2 AUC ngmr1 d 55.4 15.7 41.6 8.9 AUCextr^j. % 2.99 1.83 8.44 5.00 MRTt d 9.31 2.25 7.48 1.39 MRT d 9.96 2.60 9.85 2.54 [Acetic acid: wins] 3.7:1 3.2:1 10. GLP-1 fat salt / divalent metal formulation -42- 201023877 1 0.1 . Method preparation (Aib8'35) hGLP-l (7-36 NH2 1 mg/mL water and PBS solution and adjust the pH to 7.0» Prepare a stock solution of CaCl2, CuCl2, MgCl2 and ZnCl2 in water at 10 mg/mL. The pH of the CaCl2, MgCl2 and ZnCl2 solutions was adjusted to 7.0. Since the sinking source Cu, the pH of the CuCl2 solution cannot be alkalized. Therefore, a CuCl2 solution of pH 4.4 was used.

Add 4 μΙ> metal ion water or PBS solution to 200 pL (Aib8, 35) hGLP-1 (7-36) NH2 1 mg/mL solution to make 200 pg/mL 最终 final metal ion concentration. The resulting solution was mixed and the precipitate was checked. If a precipitate is formed, the suspension is centrifuged. Determination by HPLC (Aib8, 35) hGLP-1 (7-36) NH2 concentration 1 0 · 2 . Results Table 4: (Aib8, 35) hGLP-1 (7-3 6) NH2 in the presence of divalent metal ions Solubility aqueous solution, mg/mL PBS solution, mg/mL CaCl2 >1 (pH 7.1) >1 (pH 6.8) CuCl2 0.058 (pH 7.1) 0.039 (pH 6.8) MgCl2 >1 (pH 7.2) >1 (pH 6.9) ZnCl2 0.108 (pH 6.9) 0.056 (pH 6.8) 10.3. (Aib8'35) hGLP-1 (7-36) NH, / bismuth ruthenium pH 5.5 The pharmacokinetics of the clear solution formulation Three different (Aib8'35) hGLP-1 (7-36) NH2 formulations were prepared by the following procedure: (1) (Aib8'35) hGLP-1 (7-36) NH2 hydrochloride and CuCl2 (2) (Aib8 '35) hGLP-1 (7-36) NH2 hydrochloride and ZnCl2 -43- 201023877 (3) (Aib*'35) hGLP-1 (7-36) NH2 acetate and TFA of ZnCl2 GLP-1 analogue The salt (purified peptide using preparative HPLC 'TFA salt eluted with TFA containing buffer solution) can be converted to another salt, for example, the peptide is dissolved in a small amount of 0.25 N aqueous acetic acid to obtain an acetate. The resulting solution was applied to a semi-preparative HPLC column (Zorbax '300 38, (:-8). With (1) 0.11 ^ ammonium acetate aqueous solution for 0.5 hour, (2) 0.25 / acetic acid aqueous solution for 0.5 hour and (3) linear gradient (20% to 100% solution B over 30 minutes) eluted at a flow rate of 4 ml/min (solution A was 0.25 N aqueous acetic acid; φ solution B was 0.25 N acetic acid in acetonitrile/water, 80:20). The peptide was layered and lyophilized to dryness. (Aib8, 35) hGLP-1 (7-36) NH2 guanidine hydrochloride salt was prepared via a lyophilization step. 20 mg (Aib8'35) hGLP-1 (7-36) NH2 The acetate was dissolved in 4 mL of 20 mM HCl solution and incubated for 10 minutes at room temperature. The sample was frozen and lyophilized 'overnight. Freeze-dried twice and the chloride content of the final product was determined. The chloride content was determined to be 5.3 8 %. mrAib8,35, hGLP-l(7-36)NH, hydrochloride and CuCU: 溶解(Aib8'35)hGLP-l(7-36)NH2 HC1 5.3 mg (yield peptide content 9 5%) dissolved In 50 μL of 20 mM CuCl 2 aqueous solution, adjust the pH to about 5.5 with about 2 μL of 1N NaOH. (Aib8'35) hGLP-1 (7-36) NH2/CuCl2 has a molar ratio of 1.5: 1. The peptide concentration is 10% ( 30 mM) in water (w/w) With a pH of about 5.5. (2) (Άίΐ) 8,35,1ιΟΕΡ-Π7-36)ΝΗ, hydrochloric acid to ZnCl?: (Aib8'35)hGLP-l(7-36)NH2 HC1 5.3 mg (peptide) The content was 95%) dissolved in 50 pL of 20 mM ZnCl 2 aqueous solution. Adjust the pH to approximately 5.5 with approximately 2 pL of 1 N NaOH. (Aib8'35) hGLP-1 (7-36) NH2/ZnCl2 -44 - 201023877 has a molar ratio of 1.5:1. The peptide concentration is 10% (30 mM) and water (w/w), with a pH of about 5.5 » L3_) (Aib8'35, hGLP-U7-36) NH, acetate and ZnCl,: (Aib8'35) hGLP -l (7-36) NH2 acetate 5.5 mg (92% peptide) was dissolved in 50 pL of 20 mM ZnCl2 aqueous solution. The resulting solution was lyophilized overnight and redissolved in 50 μί water. Adjust the pH to approximately 5.5 with approximately 1 μM of 1 N NaOH. (Aib 8, 35) The molar ratio of hGLP-1 (7-36) NH2/ZnCl2 was 1.5: the concentration of the peptide was 10% (30 mM) in water (w/w) with a pH of about 5.5. ❹ 1〇.4. Dose and blood sample collection The three (Aib8'35) hGLP-1 (7-36) NH2 formulations were administered subcutaneously in 0.3 mg/rat (3 kL 10% solution). Blood samples were collected at 5, 10, 15, 30 minutes, 1, 2, 4, 8 hours and 1, 2, 3, 4, 7, and 10 days. Plasma was collected from the blood by centrifugation and stored at -80 °C. The tissue at the injection site was also collected, homogenized in 5x methanol, and stored at -80 °C. Two rats were used for 5, 10, 15, 30 minutes and 1, 2, 4, and 8 hour data points. One rat was used for 1, 2, 3, 4, 7, and 10 day data points. ❿ 1 0.5. LC-MS/MS sample preparation Acidified plasma with ΙΟμί carboxylic acid (200 μΙ〇 and precipitated with 600 pL of acetonitrile. The supernatant was collected by centrifugation and concentrated to dryness under vacuum. The residue was dissolved in 150 μί of 3 0 % acetonitrile in water and centrifuged. Inject 50 μί of the supernatant for LC-MS/MS analysis. Dilute tissue methanol extract (1 μμΜ to 1 mL 30% acetonitrile in water and inject 50 μΙ^ supernatant for LC- MS/MS analysis. 1 0.6. LC-MS/MS analysis was performed on a 400014000 mass spectrometer system equipped with a Turbo Ionspray probe. LC-MS/MS analysis was performed at -45-201023877. MRM mode using molecular ion detection with 668.9 and 136.1 Ion pairing. HPLC separation was performed using a Luna C8(2) 2x30 mm 3μ column at a flow rate of 〇3〇mL/min from 10% B to 90% B for 10 minutes. Buffer A was 1% formic acid in water and buffered. Liquid B was 1% formic acid in acetonitrile. LOQ was 0.5 ng/mL. 1 0.7. Results and summary The plasma concentration of the peptide was calculated using its standard calibration plot. Using 〇.〇6 mg/mL (Aib8'35) hGLP- l (7-36) NH2 (0.3 mg/rat in 5 mL methanol extract) as 100% to calculate the percentage remaining at the injection site. Table 5: (A Ib8'35) hGLP-1 (7-36) NH2 plasma concentration time administration (Aib8, 35) hGLP-1 (7-36) NH2 HC1 and CuCl2 blood concentration (ng/mL) Administration (Aib8'35) hGLP-1(7-36) Plasma concentration of NH2 HC1 and ZnCl2 (ng/mL) Administration (AibM5) hGLP-1 (7-36) Plasma concentration of NH2 acetate and ZnCl2 (ng/mL) 0.083 4.76 5·06 ±3·85 25.9±14.57 0.17 3.18 13·04±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 12.4±10.61 11.5 2 11.4±1.27 12.9±0_35 8.64 4 5.9±5.2 6.39±4.62 5.48 8 0.9±0.37 0.72 6.41 24 1.35 1.08 0.94 48 0.68 1.21 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 (Aib8'35)hGLP-l(7-3 6) The overall time course of the pharmacokinetic profile of the NH2 hydrochloride formulation is illustrated in Figure 8. -46-201023877 (Aib8'35) The early time course of the pharmacokinetic profile of the hGLP-1 (7-3 6) NH2 hydrochloride formulation is illustrated in Figure 9. The overall time course of the pharmacokinetic profile of the (Aib8'35) hGLP-1 (7-36) NH2 acetate formulation is illustrated in Figure 10. The early time course of the pharmacokinetics of the (Aib8'35) hGLP-1 (7-36) NH2 acetate formulation is illustrated in Figure 11. Table 6: (Aib8'35) hGLP-1 (7-3 6) NH2 remained at the injection site for the estimated percentage of time (Aib8'35) hGLP-1 (7-3 6) NH2 HC1 and CuC12 administration remained at the injection site Percentage of evaluation (%) (Aib8, 35) hGLP-1 (7-36) NH2 HC1 and ZnCl2 administration percentage at the injection site (%) (Aib8, 35) hGLP-1 (7-36) NH2 acetate And ZnCl2 administration remains at the injection site _ percentage (%) 1 1.58 10.59 6.96 2 24.88 26.94 9.97 3 12 21.87 11.6 4 0.14 0.04 0.23 7 0.47 0.06 0.03 10 0.01 0.02 0.01 (Aib8'35) hGLP-l (7-3 6 The tissue accumulation of NH2 at the injection site is further shown in Fig. 12. Table 7: PK parameters for administration and administration (Aib8, 35) hGLP-1 (7-36) (Aib8 > 35) hGLP-1 (7-36) (Aib8'35) hGLP-1 (7-36) NH2HC1 and CuCl2 NH2 acetate and ZnCl2 NH2 acetate and ZnCl2 plasma concentration (ng/mL) Blood (ng/mL) blood concentration (ng/mL) Tmax · h 1 0.5 0.25 cmax » ng/ml 11.8 13.8 32.2 AUC ng- Hr/ml 204 514 394 The results indicate that the salt form of the GLP-1 analog, particularly the combined divalent metal salt, provides an acceptable sustained release formulation that reduces the initial plasma concentration, which reduces or eliminates unwanted side effects. . This data indicates that strong acid salts, such as the HC1 salt of the GLP-1 analog, show a further reduction in the initial plasma concentration. Without being bound by this theory, it is believed that the large reduction in the initial plasma concentration of the HC1 salt of the GLP-1 analogue is related to the neutralization process in vivo. The above compositions (1) and (2) having a pH of 5.5, the 100% acid is in the form of a chloride and has no free acid. Therefore, the body fluid can neutralize the solution more quickly after subcutaneous injection, thereby precipitating the solution faster. These reductions in neutralization time result in smaller, less defined initial plasma concentrations or spikes. The above cited publications are incorporated herein by reference. Additional specific examples of the present invention will be apparent from the above disclosure, and are intended to be included in the scope of the invention as described herein and are defined in the following claims. Plasma conditions (median 値) obtained after subcutaneous (SC) administration of approximately 1 mg of [Aib8,35]hGLP-1 (7-36)NH2. In each case, the peptide was administered as an aqueous zinc composition containing about 1% (wt/vol) peptide and having a peptide: Zn molar ratio of about 1.5. Solid squares and open squares indicate that the pH of the composition is adjusted with NaOH as described herein; solid triangles indicate that the pH of the composition is not adjusted with NaOH; solid circles indicate that the composition is buffered with AcOH/AcO-. ® Figure 2 depicts the plasma status (median 値) obtained after administration of approximately 15 mg of [Aib8'35]hGLP-1 (7-36) NH2 subcutaneously (s.c.) to a dog. In each case, the peptide was administered as an aqueous zinc composition containing about 10% (wt/vol) peptide and having a peptide: Zn molar ratio of about 1.5. Solid squares and open squares indicate that the pH of the composition is adjusted with NaOH as described herein; solid triangles indicate that the pH of the composition is not adjusted with NaOH; solid circles indicate that the composition is buffered with AcOH/AcO-. Figure 3 depicts the plasma status (median -48-201023877 値) obtained after administration of approximately 1 mg of [Aib8'35]hGLP-1 (7-36) NH2 subcutaneously (S.C.) in dogs. In each case, the win was administered as a semi-solid aqueous zinc composition containing: solid circles: about 5% (wt/vol) peptide, peptide: Zn molar ratio about 5.4: 1, none pH adjustment: open circle: about l〇% (wt/vol) peptide, peptide: Zn molar ratio about 5.4:1, pH adjustment; hollow square: about 10% (wt/vol) peptide, peptide: The Zn molar ratio is about 5.4:1, the pH is adjusted by NaOH; the solid square: about 10% (wt/vol) peptide, the peptide: Zri molar ratio is about 4:1, and the pH is adjusted by NaOH. Figure 4 provides a graphical representation of various devices that can be used to prepare certain formulations of the present invention. Φ Figure 5 depicts the plasma status (median 値) obtained after administration of approximately 1 mg of [Aib8'35]hGLP-1 (7-36) NH2 subcutaneously (s.c.) to a dog. The peptide is administered as 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 status (median 値) obtained after administration of approximately 15 mg of [Aib8'35]hGLP-1 (7-36) NH2 subcutaneously (s.c.) to a dog. The peptide is administered as a semi-solid zinc composition having a peptide concentration of about 25% and a peptide:Zn molar ratio of about 4:1. © Figure 7 depicts the plasma status (median 値) obtained after administration of approximately 15 mg of [Aib8,35]hGLP-1 (7-36)NH2 to a single dose of dog subcutaneously (s.c.). The peptide is administered as a semi-solid zinc composition having a winning concentration of about 23% and a peptide:Zn molar ratio of about 1.5:1. Figure 8 depicts plasma conditions of the entire time course obtained after subcutaneous (sc) administration of the following 3 mg of GLP-1 analogue hydrochloride test formulation (10 pL of 10% solution) to a single dose of rats (middle) The number of digits 値): (l) (Aib8, 35) hGLP-l (7-36) NH2 hydrochloride and CuCl2: (Aib8, 35) hGLP-l (7-36) NH2 / CuCl2 molar ratio of 1.5 :1. The peptide -49-201023877 has a concentration of 10% (30 mM) in water (w/w) and is approximately pH 5.5. (2) (Aib8'35) hGLP-1 (7-36) NH2 hydrochloride and ZnCl2: (Aib8'35) hGLP-1 (7-3 6) NH2/ZnCl2 have a molar ratio of 1.5:1. The concentration of the peptide is 1% (3〇1111^) in water (%/rated) and approximate? 115.5. Figure 9 depicts the plasma condition of the entire time course obtained after subcutaneous (sc) administration of the following 3 mg of the GLP-1 analogue acetate test formulation (3 μί 10 〇 / 〇 solution) to a single dose of rats ( Median 値): (Aib8'35) hGLP-l (7-36) ΝΗ2 acetate and ZnCl2: φ (Aib8'35) hGLP-l(7-36)NH2/ZnCl2 has a molar ratio of 1.5:1 . The peptide concentration is 10% (30〇11^) in water (%/^) and approximate? 115.5. Figure 10 depicts plasma sputum (median 値) obtained after a single dose of subcutaneous (sc) administration of 0.3 mg of the test formulation (10 pL of 10% solution) shown in Figure 8 ). Figure 11 depicts the plasma status (median 値) of the initial time course obtained after subcutaneous (sc) administration of a single dose of 3 mg of the test formulation (3 μί of 10% solution) shown in Figure 9. . ❹ Figure 12 depicts the administration of a single dose of a subcutaneous (sc) to the sputum of 3 mg of the test formulation (3 μί of 10% solution) shown in Figure 8, which is estimated to continue to exist at the injection site (Aib8, 35) hGLP-1 (7-36) NH2 percentage. [Main component symbol description] HV palm control valve S 1 disposable syringe S2 second disposable cylinder SC two-way stainless steel connector SR stainless steel rod -50-

Claims (1)

201023877 VII. Patent application scope: 1. A pharmaceutical composition comprising a GLP-1 analogue prepared by using a pharmaceutically acceptable salt of a GLP-1 analogue or a mixture of a salt of the analog and the analog, thereby providing a similar The molar ratio of the salt to the peptide analog in the pharmaceutical composition, wherein the molar ratio can be adjusted to adjust the solubility, pH, and release to affect the release of the peptide in the living body of the pharmaceutical composition. 2. The pharmaceutical composition of claim 1, wherein the GLP-1 analog is [Aib8'35]hGLP-1 (7-36)NH2. 0. The pharmaceutical composition of claim 2, further comprising a divalent metal and/or a divalent metal salt. 4. The pharmaceutical composition of claim 3, wherein the divalent metal is zinc or copper. The pharmaceutical composition of claim 3, wherein the composition contains a divalent metal salt selected from the group consisting of CuAc2, CuCl2, ZnAc2, and/or ZnCh. 6. The pharmaceutical composition according to claim 2, wherein the salt of φ [Aib8'35]hGLP-1 (7-36)NH2 is a pharmaceutically acceptable salt of an organic acid. 7. The pharmaceutical composition according to claim 6 wherein the organic acid is selected from the group consisting of acetic acid, trifluoroacetic acid, lactic acid, malic acid, ascorbic acid, succinic acid, benzoic acid, citric acid, methanesulfonic acid and toluenesulfonic acid. Group of groups. 8. The pharmaceutical composition of claim 7, wherein the acid is acetic acid. 9. The pharmaceutical composition according to claim 2 or 5, wherein the salt of [Aib8'35]hGLP_l(7-3 6)NH2 is a pharmaceutically acceptable salt of a mineral acid. 10. The pharmaceutical composition according to claim 9 wherein the inorganic acid is -51-201023877 selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid. A pharmaceutical composition of 10, wherein the acid is hydrochloric acid. 12. The pharmaceutical composition of claim 3, wherein the molar ratio of the [Aib8'35]hGLP-1 (7-36)NH2 salt to the GLP-1 peptide analog is about 0.5:1 to About 10:1. 1 3 · The pharmaceutical composition of claim 3, wherein in the pharmaceutical group φ, the [八丨1) 8'35]11〇1^-1(7-36)^^2 salt pair The molar ratio of the divalent metal and/or divalent metal salt ranges from about 6:1 to about 1:1. 14. The pharmaceutical composition of claim 13, wherein the [Aib8'35]hGLP-1 (7-3 6)NH2 salt is a divalent metal and/or a divalent 'metal salt The molar ratio ranges from about 5.4:1 to about 1.5:1. 15. The pharmaceutical composition of claim 14 wherein the pharmaceutically acceptable salt is [8 to 8, 35] 11 〇 1 ^ -1 (7-36 chuan 112.11 (: 1.211. 16. The pharmaceutical composition of Item 14, wherein the pharmaceutically acceptable salt is [Aib 8 3 5 ]hGLP-l(7-36)NH2 ·acetate·Zn. 17. The pharmaceutical composition as claimed in claim 14 And the pharmaceutically acceptable salt is [8 to 8, 35]] ^ 1 ^ -1 (7-36) 1 ^ 2.11 (: 1. copper. - 52-