KR20090096739A - 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

JP-1 pharmaceutical composition {GLP-1 PHARMACEUTICAL COMPOSITIONS}

The present invention relates to a method for improving a composition comprising a peptide analog of glucagon-based peptide-1 and / or a pharmaceutically acceptable salt thereof, a method for preparing the composition, a pharmaceutical composition, and a method for using the composition for treating a mammal. It is about.

Glucagon-based peptide-1 (7-36) amides (GLP-1) are synthesized in intestinal L-cells by tissue-specific post-translational processing of preproglucagon, a precursor of glucagon (Varndell, JM, et al., J. Histochem Cytochem, 1985: 33: 1080-6), secreted into the circulation in response to a meal. Plasma concentrations of GLP-1 increase from fasting levels of approximately 15 pmol / l to 40 pmol / l postprandial. In blood glucose elevation, it has been demonstrated that plasma insulin increases approximately threefold when administered orally compared to intravenous glucose (Kreymann, B., et al., Lancet 1987: 2, 1300-4). ). Increasing insulin secretion by food, known as the incretin effect, is primarily humoral, and GLP-1 is considered to be the most potent physiological incretin in the human body. In addition to the insulin effect, GLP-1 can inhibit glucagon secretion, delay fasting (Wettergren A, et al., Dig Dis Sci 1993: 38: 665-73), and increase glucose utilization in peripheral blood vessels. (D'Alessio, DA et al., J. Clin Invest 1994: 93: 2293-6).

In 1994, treatment of GLP-1 was found, as one subcutaneous (s / c) dose of GLP-1 was found to be able to completely normalize postprandial glucose levels in patients with insulin independent diabetes (NIDDM). Scientific availability has been proposed (Gutniak, MK, et al., Diabetes Care 1994: 17: 1039-44). This effect was believed to be mediated by both increased insulin secretion and decreased glucagon secretion. In addition, intravenous infusion of GLP-1 has been shown to delay postprandial fasting in NIDDM patients (Williams, B., et al., J. Clin Endo Metab 1996: 81: 327-32). Unlike sulfonylureas, the insulinogenic action of GLP-1 is dependent on blood glucose levels (Holz, GG 4 ^th , et al., Nature 1993: 361: 362-5). Thus, reduced GLP-1 mediated insulin secretion at low blood sugar levels prevents severe hypoglycemia. Taken together, these actions yield a unique and potent therapeutic effect of GLP-1 that outperforms other agents currently used for NIDDM therapy.

Several studies have shown that GLP-1 has a strong effect on blood glucose levels as well as insulin and glucagon levels in healthy individuals (Orskov, C, Diabetologia 35: 701-711, 1992; Holst, JJ, et al., Potential of GLP -1 in diabetes management in Glucagon III, Handbook of Experimental Pharmacology, Lefevbre PJ, Ed. Berlin, Springer Verlag, 1996, p. 311-326), these effects were found to be glucose dependent (Kreymann, B., et al., Lancet ii; 1300-1304, 1987; Weir, GC, et al., Diabetes 38: 338-342, 1989) . In addition, GLP-1 is also effective in diabetic patients (Gutniak, M., N. Engl J Med 226: 1316-1322, 1992; Nathan, DM, et al., Diabetes Care 15: 270-276, 1992), More specifically, it normalizes blood glucose levels in type 2 diabetics (Nauck, MA, et al., Diagbetologia 36: 741-744, 1993) and improves blood sugar control in type 1 diabetics (Creutzfeldt, WO, et al., Diabetes Care 19: 580-586, 1996), among others, demonstrated the ability of GLP-1 to increase insulin sensitivity / reduce insulin resistance. GLP-1 and its analogs have been proposed for use in individuals at risk of developing insulin independent diabetes (WO 00/07617) and as a therapeutic for gestational diabetes (US Patent Publication 20040266670).

In addition, GLP-1 and its analogues have numerous therapeutic uses for mammals, such as humans, such as central nervous system diseases or disorders such as Parkinson's disease, such as through improved learning, enhanced neuroprotection and / or modulating neurogenesis, for example. , Symptomatic relief of Alzheimer's disease, Huntington's disease, ALS, seizures, ADD and neuropsychological syndromes (US Patent Publications 20050009742 and 20020115605); Conversion of hepatic stem / progenitor cells into functional pancreatic cells (WO03 / 033697); Preventing degeneration of beta cells (US Patent Publications 20040053819 and 20030220251) and stimulating proliferation of beta cells (US Patent Publication 20030224983); Treatment of obesity (US Patent Publication 20040018975; WO98 / 19698); Suppressing appetite and causing bloating (US Patent Publication 20030232754); Treatment of irritable bowel syndrome (WO 99/64060); Reduction in morbidity and / or mortality due to myocardial infarction (US Patent Publication No. 20040162241, WO98 / 08531) and seizures (WO 00/16797); The treatment of acute coronary syndromes characterized by the absence of Q-wave myocardial infarction (US Patent Publication No. 20040002454); Attenuating catabolic changes after surgery (US Pat. No. 6,006,753); Treatment of hibernating myocardium or diabetic cardiomyopathy (US Patent Publication 20050096276); Inhibiting blood levels of norepinephrine (US Patent Publication 20050096276); Increased sodium excretion through urine, decreased urine potassium concentration (US Patent Publication 20050037958); Treatment of conditions or disorders associated with deleterious blood overload, such as renal failure, congestive heart disease, nephrotic syndrome, sclerosis, pulmonary edema and hypertension (US Patent Publication 20050037958); Inducing muscle contractile response and increasing cardiac contractility (US Patent Publication 20050037958); Polycystic ovary syndrome treatment (US Patent Publications 20040266678 &20040029784); Respiratory distress treatment (US Patent Publication 20040235726); Improved nutritional supply through non-digestive pathways, ie intravenous, subcutaneous, intramuscular, peritoneal, or other injections or infusions (US Patent Publication 20040209814); Treating kidney disease (US Patent Publication 20040209803); Left ventricular contractile dysfunction treatment, eg, with abnormal left ventricular bleeding rates (US Patent Publication 20040097411); Use as preliminary anesthetics in pyloric-duodenal motility inhibition, and in endoscopic surgery for the treatment or prevention of gastrointestinal disorders such as diarrhea, post-operative rapid dumping syndrome and irritable bowel syndrome (US Patent Publication 20030216292); Treatment of critical illness polyneuropathy (CIPN) and systemic inflammatory response syndrome (SIRS) in critical diseases (US Patent Publication 20030199445); Triglyceride level control and treatment of lipid metabolism abnormalities (US Patent Publications 20030036504 and 20030143183); Treatment of organ damage due to blood perfusion following ischemia (US Patent Publication No. 20020147131); Treatment of coronary heart disease risk factor (CHDRF) syndrome (US Patent Publication No. 20020045636) and the like, but is not limited thereto.

However, GLP-1 is the only 1-2 minutes is only in vivo plasma half-life (t _1/2) unstable metabolically. Externally administered GLP-1 is also rapidly degraded (Deacon, CF, et al., Diabetes 44: 1126-1131, 1995). This metabolic instability limits the therapeutic potential of native GLP-1. There have been numerous attempts to improve the therapeutic potential of GLP-1 and its analogs by improving the formulation form. For example, in WO 01/57084, a process for preparing crystals of GLP-1 analogs known to be available for the preparation of pharmaceutical compositions, such as injectable drugs, comprising crystals and a pharmaceutically acceptable carrier. Is mentioned. Non-uniform microcrystalline clusters of GLP-1 (7-37) OH were grown from saline, and the crystals were examined after zinc and / or m-cresol immersion treatment (Kim and Haren, Pharma. Res. Vol. 12 No. 11 (1995)). Crude suspensions of GLP (7-36) NH ₂ containing needle-like crystals and amorphous precipitates were prepared from phosphate solutions containing zinc or protamine (Pridal, et. Al., International Journal of Pharmaceutics Vol. 136, pp. 53-59 (1996)). EP 0619322A2 discloses a micro- of GLP-1 (7-37) OH comprising mixing a specific combination of salt and low molecular weight polyethylene glycol (PEG) with a protein solution in a pH 7-8.5 buffer. A method for producing a crystalline form is disclosed. US Pat. No. 6,566,490 discloses a microcrystalline seeding method of GLP-1 that aids in the preparation of purified peptide products. 6,555,521 (US '521) discloses GLP-1 crystals in the form of square flat bars or plates that have improved purity and show increased activity in vivo. In US '521, the above-mentioned crystals are relatively homogeneous, and also have a problem in that they are precipitated rapidly and aggregated together or agglomerated together to prevent syringe needles from being able to carry out generally prescribed dosages. It is described as maintaining suspension for a longer period of time.

Poly [(dl-lactide-co-glycolide)-[beta] -ethylene glycol- [beta]-(-lactide-co-glycolide), a biodegradable triblock copolymer, is used in controlled release formulations of GLP-1. It is suggested to do so. However, as with other polymer systems, the protocol of the process for producing triblock copolymers is complex and difficult to produce uniform particulates.

Similarly, biodegradable polymers such as poly (lactic acid-co-glycolic acid) (PLGA) have also been proposed for use in sustained release delivery formulations of peptides. However, such biodegradable polymers are generally reluctant in the art because they are poorly water soluble and require water-immiscible organic solvents such as methylene chloride and / or stringent manufacturing conditions during the manufacturing process. Such organic solvents and / or stringent manufacturing conditions are believed to increase the risk of inducing structural changes in the subject peptide or protein, thereby reducing structural integrity and detrimental to biological activity (Choi et al., Pharm. Research, Vol. 21, No. 5, (2004). Poloxamers have the same problem (Id.).

The GLP-1 compositions described above contain impurities and / or tend to be difficult to reproduce and administer, so that the GLP-1 compositions are not ideal for preparing pharmaceutical formulations of GLP. In addition, GLP analogs are known to induce nausea at increased concentrations, requiring a sustained release drug effect with reduced initial plasma concentrations (Ritzel 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)). Thus, there is a need for a GLP-1 formulation that is easier and more reliable to manufacture, simpler and more reproducible to patients, and can provide lower initial plasma concentrations to reduce or eliminate unwanted side effects. .

Summary of the Invention

The invention can be summarized by the following description and claims. That is, the present invention provides a pharmaceutical composition comprising a GLP-1 analog. Particularly preferred are GLP-1 analogs according to formula (I) or pharmaceutically acceptable salts thereof, wherein the formulations of the compositions provide good preparation, administration, pharmacokinetics and pharmacodynamic properties as well as attenuation of negative side effects.

^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 (I)

Preferably, the pharmaceutical compositions of the present invention with 4 mg / ㎖ of ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 and a transparent aqueous solution of ZnCl ₂ pH4 0.5 mg / ㎖ of ZnCl _2, which is present It is not configured.

One preferred embodiment of the present invention provides a pharmaceutical composition with improved drug release profile, preferably attenuated initial burst.

The present invention also provides a pharmaceutical composition comprising a compound of formula (I) with an extended duration of action.

In a preferred aspect, the present invention also provides pharmaceutical compositions that precipitate at in vivo physiological pH to form in situ deposits of long-term release drug profiles.

Another example 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 is water.

In a preferred aspect, the present invention provides a pharmaceutical composition comprising a compound or GLP-1 peptide analog, formulated with a salt of a GLP-1 peptide or a mixture of said peptide with a salt thereof.

Preferably, the GLP-1 peptide analog in the pharmaceutical composition is a pharmaceutically acceptable salt of an organic acid such as acetic acid, lactic acid, malic acid, ascorbic acid, succinic acid, benzoic acid, citric acid, methanesulfonic acid or toluenesulfonic acid, or hydrochloric acid, It is selected from the list of pharmaceutically acceptable salts of inorganic acids such as hydrobromic acid, hydroiodic acid, sulfuric acid or phosphoric acid. Particular preference is given to pharmaceutically acceptable salts of strong acids such as hydrochloric acid. Strong acids are defined as acids with a pKA of less than 4.5. Further preferred peptide salts in the pharmaceutical composition are salts of organic acids such as acetic acid, trifluoroacetic acid, lactic acid, malic acid, ascorbic acid, succinic acid, benzoic acid or citric acid.

In a preferred embodiment, the solubility, pH, and release profile of the pharmaceutical composition is such that the GLP-1 analog in salt form: GLP, not in salt form, to extend the release profile and reduce the initial spike of GLP-1 analog concentration. By adjusting the molar ratio of the -1 analog, it can be adjusted.

In a preferred embodiment, the pharmaceutical composition further comprises a divalent metal to lower the water solubility of the composition, thereby extending the release profile while simultaneously reducing the initial ejection or spike at the plasma concentration. Preferred divalent metals include zinc and copper. Particularly preferred salt forms of divalent metals include, but are not limited to, chloride and acetate salts of divalent metals. Most preferred are CuAc ₂ , CuCl ₂ , ZnAc ₂ and / or ZnCl ₂ . Preferably, the divalent metal and / or divalent metal salt in the pharmaceutical composition is present at a concentration of about 0.0005 mg / ml to about 50 mg / ml. Even more preferably, the divalent metal and / or divalent metal salt in the pharmaceutical composition is present at a concentration of 0.01 mg / ml to about 0.50 mg / ml. More preferably, the pharmaceutical composition comprises a diluent, the diluent comprising a pharmaceutically acceptable aqueous solution. Diluents can include sterile water.

In another embodiment, the pharmaceutical composition comprises a divalent metal and / or a divalent metal salt, wherein the molar ratio of the GLP-1 analog: divalent metal and / or divalent metal salt is from about 6: 1 to about 1: 1. Additionally included. Preferably the ratio is about 5.5: 1 to about 1: 1. More preferably, the ratio is about 5.4: 1 to about 1.5: 1. Even more preferably, the ratio is about 5.4: 1, 4.0: 1, or 1.5: 1. Most preferably, the ratio is about 1.5: 1. Since in this aspect of the invention the drug is 1.5: 1 ± 10% at each target value, the expected ratio is therefore in the range covering, for example, 1.35-1.65: 0.85-1.15.

Preferably, the pharmaceutical composition comprises an aqueous mixture, suspension or solution, wherein the GLP-1 analog, or salt thereof, of the compound of formula (I) is present at a concentration of about 0.5% -30% (w / w). More preferably, the concentration of the GLP-1 analog and / or salt thereof 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%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% (w / w). More preferably, the concentration of GLP-1 analogs and / or salts thereof in the aqueous solution is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10 %, 11%, 14%, 15%, 16%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 29%, or 30% (w / w) . Even more preferably, the concentration of GLP-1 analog and / or salt thereof in the aqueous solution is about 1%, 2%, 3%, 4%, 5%, 6%, 9%, 10%, 11%, 22%, 23%, 24%, 25% or 26% (w / w). Even more preferably, the concentration of GLP-1 analog and / or salt thereof in the aqueous solution is about 1%, 2%, 3%, 4%, 5%, 6%, 10%, 22%, 23%, 24%, 25% or 26% (w / w). Also more preferably, the concentration of GLP-1 analog and / or salt thereof in the aqueous solution is about 1%, 2%, 5%, 10%, 23% or 25% (w / w). “About” means: at a concentration of about 0.5% to about 4%, ± 0.5% is the preferred range for the target value (eg, 0.5% to 1.5% is about 1%); For about 5% and higher target concentrations, 20% of the target value is the preferred range (eg 8% to 12% is about 10%).

Preferably, in a pharmaceutical composition ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2, GLP-1 analogs or a salt concentration is about 1% (w / ^{v), [Aib 8, 35]} hGLP The molar ratio of -1 (7-36) NH ₂ : divalent metal and / or divalent metal salt is about 1.5: 1. More preferably, the pharmaceutical composition within the ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 or a salt concentration is about 2% (w / ^{v), [Aib 8,35] hGLP-} 1 (7 -36) NH ₂ or a salt thereof: molar ratio of divalent metal and / or divalent metal salt is about 1.5: 1. Also more preferably, the pharmaceutical composition within the ^{[Aib 8,35] hGLP-1 (} 7-36) NH 2 or a salt concentration is about 10% (w / ^{v), [Aib 8, 35]} hGLP-1 ( 7-36) NH ₂ or a salt thereof: The molar ratio of divalent metal and / or divalent metal salt is about 1.5: 1. And most preferably, the pharmaceutical composition within the ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 or a salt thereof is in a concentration of about 23% or about 25% (w / ^{v), [Aib 8, 35]} hGLP The molar ratio of -1 (7-36) NH ₂ or a salt: divalent metal and / or divalent metal salt is about 1.5: 1.

In a preferred embodiment, pharmaceutical compositions within the GLP-1 ^{analog, [Aib 8, 35] hGLP} -1 (7-36) NH 2 or a salt concentration is from about 5% (w / v), peptide: divalent metal and / Or the molar ratio of divalent metal salt is about 5.4: 1. Decided more preferably, the composition within the ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 or a salt concentration is from about 5% (w / v) wherein the molar ratio is about 4.0: 1. Decided more preferably, the composition within the ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 or a salt concentration is about 10% (w / v) wherein the molar ratio is about 5.4: 1. Decided and even more preferably, the composition within the ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 or a salt concentration is about 10% (w / v) wherein the molar ratio is about 4.0: 1.

Preferably, the divalent metal and / or divalent metal salt is provided with zinc chloride or zinc acetate. More preferably, the zinc acetate is provided by ZnAc ₂ .2H ₂ O.

In another example, the divalent metal and / or divalent metal salt is provided with copper chloride or copper acetate.

In one embodiment, the pH of the pharmaceutical composition is up-regulated using a base. More preferably, the pH adjustment is performed using NaOH. More preferably, the pH of the pharmaceutical composition is adjusted with NaOH so that the pH is about 5.0-5.5 in direct potentiometer measurements when diluted to about 1/2 the initial concentration with 0.9% NaCl.

Preferred embodiments of the invention are pharmaceuticals, wherein the peptide analogues or salts thereof of GLP-1, a compound according to formula (I) or a salt thereof, are formulated for long-term release in a subject in need thereof, such as a mammal, preferably a human. Composition. Preferably, the release of the compound is continued for at least 1 hour, more preferably for at least 4, 6, 12 or 24 hours. Also more preferably, the composition is formulated such that the compound according to formula (I) is released in the subject for at least 36, 48, 60, 72, 84 or 96 hours. Also more preferably, the composition is formulated such that the compound according to formula (I) is released into 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 in 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 for at least about 1, 1.5, 2 or 3 months, or longer.

In one aspect of the invention, the salt content of the GLP-1 peptide analog in the pharmaceutical composition is adjusted to improve the solubility and safety of the GLP-1 peptide in the pharmaceutical composition, and also to reduce the initial ejection to provide an improvement in in vivo release profile. do.

The term "modulation" means adjusting the salt content by adjusting the molar ratio of GLP-1 analogs in salt form to GLP-1 analogs that are not in salt form.

Even more preferably, the peptide salt in the pharmaceutical composition is a hydrochloride, acetate salt, chloride or acetate of the peptide of formula (I). In the pharmaceutical composition, the acetate or chloride is acetate or chloride: the final molar ratio of the compound of formula (I) is from about 0.5: 1 to about 10: 1. More preferably, the ratio is about 0.8: 1 to about 9: 1. Even more preferably, the ratio is about 1: 1 to about 6: 1. Most preferably, the ratio is about 3.0: 1, in particular 3.2: 1.

In this aspect of the invention, the molar ratio of acetate or chloride to peptide refers to the molar ratio of acetate (CH ₃ COO ⁻ ) or chloride (Cl ⁻ ) in the pharmaceutical composition to the molar ratio of peptide in the pharmaceutical composition. In the example for molar ratio 3: 1 in the pharmaceutical composition, acetate is 3 times the molar content of the peptide. This is the stoichiometric ratio of the compound to the other compound.

In the ratio 1.5: 1 of the present invention, the term "about" is + 10% of each target value, so the expected ratio is in a range including, for example, 1.35-1.65: 0.85-1.15.

In a further preferred aspect of the invention, the pH of the pharmaceutical composition is adjusted by adjusting the acetate content of the composition. Preferably, the pH range of the pharmaceutical composition is pH 3-pH 6. More preferably, the pH range of the pharmaceutical composition is pH 3.5-5.5. Most preferably, the pH range of the pharmaceutical composition is pH 4.2-pH 4.6.

Preferably, in order to acidify the pharmaceutical composition, the acetate content may be increased by adding acetic acid.

In one embodiment, the pH of the pharmaceutical composition can be increased starting with the peptide salt of the GLP-1 analogue with or without acetate content by adjusting the acetate content.

In a preferred embodiment, the pH adjustment of the final pharmaceutical composition by controlling the content of acetate or chloride can control parameters such as peptide concentration, zinc concentration, chemical stability, physical stability and in vivo release profile with reduced initial ejection.

In one aspect of the invention, the Zn or Cu content is fixed and the pH is controlled by acetate content control. Increasing the acetate content showed an improvement in solubility and physical stability, and decreasing the acetate content showed an increase in action on pH and a decrease in action on C _max .

In a preferred embodiment, the pharmaceutical composition comprises an aqueous mixture, suspension or solution.

The present invention also provides a method for abolishing the agonist effect of GLP-1 comprising directly or indirectly contacting a receptor of GLP-1 (7-36) NH ₂ ligand with a GLP-1 analog or salt thereof.

In this method, the receptor of the GLP-1 (7-36) NH ₂ ligand is present in an animal subject, preferably in primates, more preferably in humans. Thus, in this example, the present invention comprises administering to a subject a composition of the invention comprising an effective amount of a GLP-1 analog or a pharmaceutically acceptable salt thereof, in a subject in need thereof. Provided are methods for canceling agonist effects from receptors.

In a preferred aspect of the method, the subject is a human or at risk of developing a disease or condition selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, excessive appetite, insufficient satiety, metabolic disorders. Preferably, the disease is type 1 diabetes or type 2 diabetes.

In another preferred embodiment of the method, the subject has type 1 diabetes, type 2 diabetes, obesity, glucagonomas, airway secretion disorders, arthritis, osteoporosis, central nervous system disease, restenosis, neurodegenerative disease, Renal failure, congestive heart failure, nephrotic syndrome, liver cirrhosis, pulmonary edema, diseases in which high blood pressure and reduced food intake are desirable, central nervous system diseases or disorders (e.g., through neuronal control, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, seizures , ADD and neuropsychiatric syndrome), irritable bowel syndrome, myocardial infarction (e.g. reduced morbidity and / or mortality), seizures, acute coronary syndrome (e.g. characterized by absence of Q-wave), myocardial infarction, surgery Changes in postcatabolism, hibernating myocardium or diabetic cardiomyopathy, poor sodium excretion through urine, excessive potassium concentration in urine, harmful conditions or disorders associated with excessive blood volume (e.g., Insufficiency, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema and hypertension), polycystic ovary syndrome, dyspnea, kidney disease, left ventricular systolic failure (e.g. abnormal left ventricular bleeding rate), diarrhea, postoperative dumping syndrome and irritable bowel syndrome (i.e. , Gastrointestinal disorders such as pyloric-duodenal motility), critical illness polyneuropathy (CIPN), systemic inflammatory response syndrome (SIRS), lipid metabolism abnormality, and blood flow due to ischemia Long-term tissue damage due to reperfusion and coronary heart disease risk factor (CHDRF) syndrome is a human at risk of developing a disease selected from the group consisting of syndrome.

In another aspect of the present invention, the present invention provides a method for converting liver stem / progenitor cells into functional pancreatic cells in a subject in need, a method for preventing beta cell degeneration, and a method for stimulating proliferation of beta cells. To inhibit blood levels of norepinephrine, to induce muscle contractile responses, to increase cardiac contractility, through non-digestive pathways (eg, intravenous, subcutaneous, intramuscular, peritoneal or other infusion or injection routes) To improve the nutritional supply, to pretreat the subject to perform an endoscopic procedure, and to adjust the triglyceride concentration, the method comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable Administering to the subject a formulation of the invention comprising a possible salt. Preferably, the subject is a mammalian animal, more preferably a primate, more preferably a human.

Detailed description of the invention

Preferred GLP-1 peptide used as a peptide salt of the invention, the following format, e.g., ^{(Aib 8, 35) hGLP-} 1 (7-36) is represented by NH _2, the native sequence as set forth in the first bracket; substituted amino acid ^(e, Aib ^8, at ^35, is also substituting Aib Ala Gly ⁸ and ³⁵ in hGLP-1). Aib is an abbreviation for alpha-aminoisobutyric acid. The abbreviation GLP-1 means glucagon-based peptide-1, and hGLP-1 means human glucagon-based peptide-1. The number in the second parenthesis is the number of amino acids present in the peptide (eg hGLP-1 (7-36) means amino acids 7 to 36 in the peptide sequence of human GLP-1). hGLP-1 (7-37) sequences are described in Mojsov, S., Int. J. Peptide Protein Res ,. 40, 1992, pp. 333-342. “NH ₂ ” in hGLP-1 (7-36) NH ₂ means that the C-terminus of the peptide is amidated. hGLP-1 (7-36) means that the C-terminus is the free acid. Residues 37 and 38 in hGLP-1 (7-36) are Gly and Arg, respectively, unless otherwise noted.

Particularly preferred GLP-1 peptide analogs used in the present invention are in the form of pharmaceutically acceptable salts. Examples of such salts include organic acids (e.g. acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, methanesulfonic acid, toluenesulfonic acid or pamoic acid), inorganic acids (e.g. hydrochloric acid, sulfuric acid or phosphoric acid). And salts formed with polymeric acids (eg, tannic acid, carboxymethyl cellulose, polylactic acid, polyglycolic acid or copolymers of polylactic acid-glycolic acid), but are not limited thereto. Typical methods for preparing salts of the peptides according to the invention are well known in the art and can be achieved by standard salt substitution methods. Thus, the TFA salt of the peptide according to the invention (prepared TFA salt by using preparative HPLC and eluting with TFA-containing buffer) to dissolve the peptide in a small amount of 0.25 N acetic acid aqueous solution such as acetate salt Can be converted to salts. The solution prepared is subjected to a semi-preparative HPLC column (Zorbax, 300 SB, C-8). The column is flow rate 4 ml / min, (1) 0.1N ammonium acetate solution for 0.5 hour, (2) 0.25N acetate solution for 0.5 hour, and (3) linear concentration gradient (20% solution B for 30 minutes). At 100%) (solution A is 0.25N acetic acid aqueous solution; solution B is acetonitrile / water, 0.25N acetic acid in 80:20). Fractions containing peptides are combined, lyophilized and dried.

As is well known to those skilled in the art, the known and potential uses of GLP-1 are diverse and diverse (Todd, JF, et al., Clinical Science, 1998, 95, pp. 325-329; and Todd, JF et al., European Journal of Clinical Investigation, 1997, 27, pp. 533-536). Thus, administration of a compound of the invention for the purpose of erasing agonist effects may have the same effects and uses as GLP-1 itself. The various uses of GLP-1 can be summarized for the following treatments: Type 1 diabetes, Type 2 diabetes, obesity, glucagonosis, airway secretion disorders, metabolic disorders, arthritis, osteoporosis, central nervous system disease, restenosis , Neurodegenerative diseases, renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, hypertension, reduced food intake, and other conditions and disorders disclosed herein. Accordingly, the present invention includes the pharmaceutical composition of the present invention comprising a compound of formula (I) as an active ingredient.

The dosage of the active ingredient in the formulations of the invention may vary, but the amount of active ingredient is necessarily the amount by which a suitable dosage is obtained. The dosage chosen will depend on the desired therapeutic effect, route of administration and duration of treatment and will usually be determined by the attending physician. In general, the effective amount for the activity of the invention is 1 x 10 ^-7 to 200 mg / kg / day, preferably 1 x 10 ^-4 to 100 mg / kg / day, which is divided into single or multiple doses. Can be administered.

The formulations of the present invention are preferably administered parenterally, such as intramuscularly, intraperitoneally, intravenously, subcutaneously and the like.

Parenteral dosage formulations according to the present invention include sterile aqueous or non-aqueous solutions, suspensions, gels or emulsions that provide the desired in vivo release profile. Examples of non-aqueous solvents or vehicles include vegetable oils such as propylene glycol, polyethylene glycol, olive oil and corn oil, injectable organic esters such as gelatin and ethyl oleate. Such dosage forms may also include adjuvant such as preservatives, wetting agents, emulsifiers, dispersants. It can be sterilized, for example, by filtration through a bacterial retention filter, by mixing the sterilant into the composition, by irradiating the composition with radiation, or by heat treating the composition. It may also be prepared in the form of a sterile solid composition which can be dissolved in sterile water or other sterile medium for injection immediately prior to use.

Synthesis of Peptides

Peptides usable in the practice of the present invention can be prepared by standard solid phase peptide synthesis. See, eg, Stewart, JM, et al., Solid See Phase Synthesis (Pierce Chemical Co., 2d ed. 1984).

The following embodiments represent synthetic methods well known to those skilled in the art, which may be used to prepare peptides which may advantageously practice the invention. In addition, other methods are known to those skilled in the art. The embodiments are intended to be illustrative only and do not limit the scope of the invention in any way.

Peptides, such as GLP-1 analogs, can be obtained by various synthesis known in the art, which may include final precipitation of the peptide, freeze-drying process, vacuum drying or other drying processes known in the art. Ion exchange chromatography, osmotic exchange and difiltration of buffers may be suitable methods of the present invention for purifying and selecting peptides in various salt forms.

Boc-βAla-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-lmpex International, Wood Dale, IL. Boc-2Nal-OH is manufactured by Synthetech, Inc. It was purchased from Albany, OR.

The full names of other abbreviations used herein are as follows: Boc means t-butyloxycarbonyl, HF means hydrogen fluoride, Fm means formyl, Xan means xantyl, Bzl means benzyl, Tos means tosyl, DNP means 2,4-dinitrophenyl, DMF means dimethylformamide, DCM means dichloromethane, HBTU means 2- (1H -Benzotriazol-1-yl) -1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA means diisopropylethylamine, HOAc means acetic acid, TFA Trifluoroacetic acid, 2ClZ means 2-chlorobenzyloxycarbonyl, 2BrZ means 2-bromobenzyloxycarbonyl, OcHex means O-cyclohexyl, Fmoc 9-flu Orenylmethoxycarbonyl, HOBt means N-hydroxybenzotriazole, PAM number And is 4-hydroxy-phenyl-acetamido methyl resin means; Tris means tris (hydroxymethyl) aminomethane; Bis-Tris is bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane (ie 2-bis (2-hydroxyethyl) amino-2- (hydroxymethyl) -1,3-propanediol ). The term "halo" or "halogen" includes fluoro, chloro, bromo and iodine.

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, all publications, patent applications, patents, and other references cited herein are hereby incorporated by reference.

Figure 1 is a ^{[Aib 8, 35] hGLP-} 1 (7-36) 1 times the dog an NH ₂ at about 1 mg subcutaneous (sc) administration of the plasma profiles obtained after a median. In each case, the peptide contained about 1% (wt / vol) peptide and was administered as an aqueous zinc composition with a molar ratio of peptide: Zn of about 1.5. And and □ indicate compositions adjusted to pH with NaOH as disclosed herein, and ▲ are compositions not adjusted to pH with NaOH; Is a composition buffered with AcOH / AcO-.

Figure 2 is ^{[Aib 8, 35] hGLP-} 1 (7-36) 1 times the dog a NH ₂ to about 15 mg subcutaneously (sc) administered the plasma profiles obtained after the (median). In each case, the peptide contained about 10% (wt / vol) peptide and was administered as an aqueous zinc composition with a molar ratio of peptide: Zn of about 1.5. And and □ indicate compositions adjusted to pH with NaOH as disclosed herein, and ▲ are compositions not adjusted to pH with NaOH; Is a composition buffered with AcOH / AcO-.

Figure 3 is ^{[Aib 8, 35] hGLP-} 1 (7-36) 1 times the dog an NH ₂ at about 1 mg subcutaneous (sc) administration of the plasma profiles obtained after a median. In each case, the peptide was administered as a semi-solid aqueous zinc composition as follows: the pH was not adjusted to about 5% (wt / vol) peptide, peptide: Zn molar ratio of about 5.4: 1; O is about 10% (wt / vol) peptide, no peptide: Zn molar ratio of about 5.4: 1; □ is pH adjusted with NaOH of about 10% (wt / vol) peptide, peptide: Zn molar ratio of about 5.4: 1; Is about 10% (wt / vol) peptide, peptide: Zn molar ratio of about 4: 1, pH adjusted with NaOH.

4 schematically depicts the various instruments available for making certain formulations of the present invention.

5 is ^{[Aib 8, 35] hGLP-} 1 (7-36) 1 times the dog an NH ₂ at about 1 mg subcutaneous (sc) administration of the plasma profiles obtained after a median. The peptide was administered as an aqueous zinc composition containing the peptide at a concentration of about 2% (wt / vol) and having a molar ratio of peptide: Zn of about 1.5: 1.

6 is ^{[Aib 8, 35] hGLP-} 1 (7-36) 1 times the dog a NH ₂ to about 15 mg subcutaneously (sc) administered the plasma profiles obtained after the (median). The peptide was administered as a semi-solid zinc composition, comprising the peptide at a concentration of about 25% (wt / vol) and having a molar ratio of peptide: Zn of about 4: 1.

Figure 7 is a ^{[Aib 8, 35] hGLP-} 1 (7-36) 1 times subcutaneously to dogs for NH ₂ to about 15 mg (sc) administered after the plasma profile obtained (median). The peptide was administered as a semisolid zinc composition, comprising the peptide at a concentration of about 23% (wt / vol) and having a molar ratio of peptide: Zn of about 1.5: 1.

FIG. 8 is the plasma profile (median) over the entire time course obtained after subcutaneous (s.c.) administration of 0.3 mg (3 μl of 10% solution) of the GLP-1 analog HCl salt test formulation.

(1) CuCl ₂ and ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl ^{salt: (Aib 8, 35) hGLP} -1 (7-36) , the molar ratio of NH ₂ / CuCl ₂ 1.5: 1 Peptide concentration is 10% (30 mM) in water (w / w), about pH5.5.

₍₂₎ ZnCl 2 and ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl ^{salt: (Aib 8, 35) hGLP} -1 (7-36) , the molar ratio of NH ₂ / ZnCl ₂ is from 1.5: 1 Peptide concentration is 10% (30 mM) in water (w / w), about pH5.5.

FIG. 9 is the plasma profile (median) over the entire time course obtained after subcutaneous (s.c.) administration of 0.3 mg (3 μl of 10% solution) of the GLP-1 analog acetate salt test formulation.

And ZnCl ₂ ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 acetate ^{salt: (Aib 8, 35) hGLP} -1 (7-36) NH 2 / ZnCl ₂ molar ratio of 1.5: 1 Im. Peptide concentration is 10% (30 mM) in water (w / w), about pH5.5.

FIG. 10 is the initial plasma profile (median) obtained after subcutaneous (s.c.) administration of 0.3 mg (3 μl of 10% solution) of the test formulation shown in FIG. 8.

FIG. 11 is the initial plasma profile (median) obtained after subcutaneous (s.c.) administration of 0.3 mg (3 μl of 10% solution) of the test formulation shown in FIG. 9.

12 is that (Aib ^{8, 35)} remaining on the three test formulations of the rats to 0.3 mg (10% solution of 3 ㎕) 1 times subcutaneously (sc) administered after rats injection site shown in Figure 8 hGLP-1 Expected percentage of (7-36) NH ₂ .

Example  One

^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2

^{[Aib 8, 35] hGLP-} 1 (7-36) NH ₂ in the specific synthetic method is disclosed in International Patent Publication No. WO 00/34331 (PCT / EP99 / 09660 ), in its entirety by reference herein, Included. Briefly, the compounds were synthesized on a model 430A peptide synthesizer from Applied Biosystems (Foster City, CA), modified to accelerate Boc-chemical solid phase peptide synthesis. Int., Schnolzer et al. See J. Peptide Protein Res., 90: 180 (1992). 0.91 mmol / g of substituted 4-methylbenzhydrylamine (MBHA) resin (Peninsula, Beltmont, Calif.) Was used. Boc amino acids include 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) Amino acids with side chain protecting groups such as -OH, Boc-Phe-OH, Boc-Aib-OH, Boc-Glu (OcHex) -OH and Boc-Trp (Fm) -OH (Bachem, CA, Torrance, CA; Nova Biochem , LaJolla, CA). The Boc group was removed by treatment with 100% TFA for 2 × 1 min. Boc amino acid (2.5 mmol) was preactivated using HBTU (2.0 mmol) and DIEA (1.0 mL) in 4 mL of DMF and coupled without prior neutralization of the peptide-resin TFA salt. The coupling time of the Boc-Aib-OH residue and the following residues, Boc-Lys (2ClZ) -OH and Boc-His (DNP) -OH, was 2 hours, and the coupling time for the remaining residues was 5 minutes.

In the final step of peptide chain assembly, the resin was treated with a 20% mercaptoethanol / 10% DIEA solution in DMF for 2 x 30 minutes to remove DNP groups on His side chain. Then, 100% TFA was treated for 2 x 2 minutes to remove the N-terminal Boc group. Formyl group on Trp side chain by neutralizing peptide-resin using 10% DIEA in 10% DMF (1 × 1 min) and then treated with 15% ethanolamine / 15% water / 70% DMF solution for 2 × 30 min. Was removed. The peptide-resin was washed with DMF and DCM and dried under reduced pressure. Peptide resin was stirred at 0 ° C. in 10 ml HF containing 1 ml of anisole and dithiothreitol (24 mg) for 75 minutes to effect final cleavage. Nitrogen was introduced to remove HF. The residue was washed with ether (6 × 10 mL) and the residue was extracted with 4N HOAc (6 × 10 mL).

The peptide mixture in the aqueous extract was purified on preparative reverse phase high pressure liquid chromatography (HPLC) using a reverse phase VYDAC ^® C ₁₈ column (Nest Group, Southborough, Mass.). The column was eluted at a flow rate of 10 ml / min using a linear concentration gradient (20%-> 50% of B solution for 105 minutes) (A solution = water with 0.1% TFA; B solution = 0.1% TFA Acetonitrile). Fractions were collected and confirmed on analytical HPLC. Pure products were collected and lyophilized. As a synthesis example of the compound, 135 mg of a white solid were obtained. It was 98.6% pure based on analytical HPLC analysis. The molecular weight by electrospray mass spectrometer (MS (ES)) S analysis was 3339.7 (which corresponds to the calculated molecular weight of 3339.7 g).

Example 2

Formulation Process I

2.1 materials, stock  Solution, calculation

A) material

ZnCl ₂ , NaOH pellets and hydrochloric acid 35% were purchased from Panreac Quimica, Barcelona, Spain. WFI (sterile w ater f or i injection / irrigation) was purchased from B. Braun Medical, Barcelona, Spain.

B) stock  solution

(i) ZnCl ₂ , pH = 3:

1. While stirring, add 35% HCl to WFI to adjust pH = 3.

2. Transfer the weighed ZnCl ₂ to the volumetric flask. PH = 3 HCl was added with stirring to bring the final concentration to about 1-4 mg ZnCl ₂ / ml.

( ii ) ZnCl ₂ , pH = 2:

1. While stirring, add 35% HCl to WFI to adjust pH = 2.

2. Transfer the weighed ZnCl ₂ to the volumetric flask. Add pH = 2 HCl with stirring to bring the final concentration to about 4-12 mg ZnCl ₂ / ml.

( iii ) NaOH , 0.1-10 mg / Ml:

1. Transfer the weighed NaOH to the volumetric flask. WFII is added to a final concentration of about 0.1-10 mg NaOH / mL while stirring.

( iv ) frozen- Dried  20 mg of Aliquot [ Aib ^{8 , 35} HGLP-1 (7-36) NH ₂ Of Vial :

1. Prepare 0.04% (v / v) acetate dilution and WFI.

2. Place the flask on a capacitance measurement, weighing ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 ( acetate salt). Final concentration of ^{20 mg [Aib 8, 35]} HGLP-1 (7-36) 0.04% acetic acid is added with stirring so that the NH ₂ / ㎖. After sterilization by filtration using a 0.45 μm filter, 1 ml of the solution was placed in a lyophilization vial and freeze-dried, and the dry product was stored at -22 ° C.

(v) freeze-dried 50 mg of Aliquot [ Aib ^{8 , 35} HGLP-1 (7-36) NH ₂ Of Vial :

1. Prepare 0.1% (v / v) acetic acid dilution and WFI.

2. Place the flask on a capacitance measurement, weighing ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 ( acetate salt). Final concentration of 0.1% acetic acid with stirring ^{50 mg [Aib 8, 35]} HGLP-1 (7-36) is added to the NH ₂ / ㎖. After sterilization by filtration, 1 ml of the solution is placed in a lyophilization vial and freeze-dried.

C) calculation

(i) The total weight / volume of excipient (E) in the composition is determined by the formula:

E = (A x 100 / T)-(A / P)

In the above,

E = mg excipient

A = amount of pure peptide (mg);

T = target concentration of the composition; Yes, 2 if goal is 2%; And

P = concentration of pure peptide (mg peptide / 100 mg formulation)

In the total volume of excipients, assume 1 ml = 1 g.

(ii) to determine the volume / weight (W) of ZnCl ₂ added to each ml or g of the composition solution:

a) W = 100% E for compositions without pH adjustment;

b) W = 80% E when the peptide is about 1%, about 2% or about up to 10% and is a liquid formulation adjusted to pH with base;

c) W = 50% E when the peptide is about 1%, about 2% or about up to 10% and is a semisolid or gel formulation with pH adjusted with base;

d) W = 66.66% E for about 25% peptide and a semisolid or gel formulation with pH adjusted with base;

e) W = 90% E for formulations whose peptides are reconstituted from a freeze-dried formulation and pH adjusted with base.

(iii) to determine the volume / weight (W) of NaOH added to each ml or g of the composition solution:

a) W = 20% E when the peptide is about 1%, about 2% or about up to 10% and the pH adjusted formulation with base;

b) W = 50% E when the peptide is about 1%, about 2% or about up to 10% and is a semisolid or gel formulation with pH adjusted with a base;

c) W = 33.33% E for about 25% peptide and a semisolid or gel formulation with pH adjusted with base;

d) W = 10% E for formulations whose peptides are reconstituted from a freeze-dried preparation and pH adjusted with base.

(iv). To determine the concentration of ZnCl ₂ (mg / ml or mg / g) used in each composition:

[ZnCl ₂ ] = (136.29 XA) / (W x 3339.76 x R)

In the above,

A = amount of pure peptide in mg.

R = molar ratio of peptide / Zn

R = 1.5: a formulation wherein the peptide is about 1%, about 2% or about 10%, or up to about 23%;

R = 4.0: a formulation with about 25% peptide; And

W = weight (g) or volume (ml) of ZnCl ₂ added to each ml or g of the composition solution.

2.2 1-10% freeze-dried peptides and ZnCl ₂ Containing pH Preparation of the composition without control

Herein, the formulations described as peptide percentages are formulations of peptide weight per total weight of the composition. Eg 1% peptide means a formulation comprising 1 g of peptide per 100 g of total composition. Formulations containing about 1%, about 2%, or up to 10% peptides were prepared as follows. The one produced as described in the freeze-dried sample of ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 to 100% of the ZnCl ₂ stock solution to the total volume of excipient pH3 and peptides: a 1: Zn = 1.5 Mix thoroughly.

A) 1% compositions are freeze-dried ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 20 mg ( the 2.1 B (iv) reference) the ZnCl ₂ solution (0.272 mg / ㎖; the 2.1 B (i Prepared by mixing with 2 ml).

B) 2% compositions are freeze-dried ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 20 mg ( the 2.1 B (iv) reference) the ZnCl ₂ solution (0.544 mg / ㎖; the 2.1 B (i Prepared).

C) 10% compositions are freeze-dried ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 50 mg ( the 2.1 B (iv) reference) the ZnCl ₂ solution (3.023 mg / ㎖; the 2.1 B (i ) And 0.45 ml.

Freeze-dried peptides and solutions were equilibrated to room temperature. ZnCl _{2 at} stated capacity The solution is injected into a vial containing the freeze-dried peptide, and about 2 minutes for 1% or 2% peptide composition, about 60 minutes for 10% peptide composition, or both freeze-dried peptides are all hydrated Hydration was continued until there was no lump. After hydration, the reconstituted peptide was stirred for about 1 minute.

As a dose, an appropriate amount of the dissolved peptide was taken, for example 100 ul of 1% peptide prepared as A was equivalent to 1 mg dose, 50 ul of 2% peptide prepared as B was equal to 1 mg dose 150 ul of 10% peptide prepared as C is equivalent to the 15 mg dose.

In the context of the present invention, one skilled in the art can alter the amounts of peptide and ZnCl ₂ to make desired dosages as well as compositions other than the 1%, 2% and 10% compositions detailed below.

2.3 1-10% freeze-dried peptides and ZnCl ₂ Containing pH Preparation of the composition adjusted

A formulation comprising about 1%, about 2%, or about 10% peptide was prepared as follows. Described by ^{[Aib 8, 35] HGLP-} 1 (7-36) it was fully mixed with freeze-dried sample of the NH ₂ to 90% of the ZnCl ₂ stock solution to the total volume of the excipient prepared as pH3. The desired pH of the solution was achieved by adding NaOH diluent.

A) 1% compositions are freeze-dried ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 20 mg ( the 2.1 B (iv) reference) the ZnCl ₂ solution (see the 2.1 B (i)) 1.8 ㎖ It is prepared by mixing with.

B) 2% compositions are freeze-dried ^{[Aib 8, 35] HGLP-} 1 (7-36) NH 2 20 mg ( the 2.1 B (iv) reference) the ZnCl ₂ solution (see the 2.1 B (i)) 0.9 ㎖ It is prepared by mixing with.

C) 10% compositions are freeze-dried ^{[Aib 8, 35] HGLP-} 1 , see (7-36) NH ₂ 50 mg (the 2.1 B (iv) reference) the ZnCl ₂ solution (the 2.1 B (i)) 0.40 ㎖ It is prepared by mixing with.

To this solution, the required amount of NaOH dilute solution (10% of the total volume of excipient) is added to make the target concentration and pH. For example, in each case:

1% Composition: Add 0.2 ml of NaOH solution in proper concentration

2% Composition: Add 0.1 ml NaOH solution at appropriate concentration

10% Composition: Add 0.05 ml NaOH solution at appropriate concentration

In the context of the present invention, one skilled in the art can alter the amounts of peptide and ZnCl ₂ to make compositions other than the 1%, 2% and 10% compositions detailed below.

2.4 1-10% freeze-dried peptides and ZnCl ₂ Containing pH Preparation of the liquid composition without control

Liquid formulations comprising about 1%, or about 2%, or up to about 10% peptides were prepared as follows. Sample [Aib ^{8, 35]} were weighed _{HGLP-1 (7-36) NH 2} , in combination with ZnCl ₂ stock solution of pH3, he made a target concentration of 1%, 2%, or up to 10% peptide. After mixing, the composition was filtered sterilized and stored until use.

2.5 1-10% freeze-dried peptides and ZnCl ₂ Containing pH Preparation of the liquid composition adjusted

Liquid formulations comprising about 1%, or about 2%, or up to about 10% peptides were prepared as follows. Sample [Aib ^{8, 35]} were weighed _{HGLP-1 (7-36) NH 2} , was thoroughly mixed with 80% of the ZnCl ₂ stock solution to the total volume of excipient pH3. The zinc solution may be ZnCl ₂ or ZnAc ₂ H ₂ 0. The desired pH of the solution was titrated by addition of dilute NaOH solution. Formulations C5 to C13 were prepared in this manner.

In the context of the present invention, one skilled in the art can alter the amounts of peptide and ZnCl ₂ to make compositions other than the 1%, 2% and 10% compositions shown herein.

2.6 25% peptide and ZnCl ₂ Containing pH Unregulated Semisolid / Gel composition

Semi-solid or gel formulations containing about 25% peptide were prepared as follows. Sample [Aib ^{8, 35]} were weighed _{HGLP-1 (7-36) NH 2} , it was thoroughly mixed with 66.66% of a ZnCl ₂ stock solution to the total volume of excipient pH2. The zinc solution may be ZnCl ₂ or ZnAc ₂ .2H ₂ 0. Formulations C1 and C2 were prepared in this manner.

More specifically, the semisolid or gel composition was prepared by the "push-pull" mixing method:

 a) Weigh the desired amount of peptide into a disposable syringe S1 pail preloaded with a special binary hand valve HV (I. D. = 0.5 mm) and place the tubing into the syringe luer hole;

b) secure the syringe plunger with stainless steel rod SR;

c) Connect HV in S1 to the vacuum source and open HV. Close HV after 10 minutes;

d) Accurately weigh the zinc solution into the second disposable syringe S2;

e) connect S2 with the unconnected portion of the HV;

f) open the HV and allow the solvent to enter the barrel S1 with the peptide powder in vacuo;

g) Close the HV and remove the solvent syringe S2 to hydrate the peptide powder in S1;

h) subtract the SR, leaving the syringe plunger alone;

i) the syringe plunger is moved (pushed and pulled) without opening the HV, so that the powder is completely wet with the solvent;

j) Mount a binary stainless connector SC (I.D. = 1.0 mm) with a tube inside the syringe luer hole in the syringe S and push the plunge to the end;

k) Open HV at S1, apply vacuum, and remove HV. Push the syringe plunge to minimize the air in the syringe barrel; And

l) S1 and S2 are joined by SC and the composition is mixed through SC from S1 to S2.

In the context of the present invention, those skilled in the art can alter the amounts of peptide and ZnCl ₂ to make compositions other than the 25% compositions shown herein.

2.7 25% peptide and ZnCl ₂ Containing pH Adjusted Semisolid / Gel composition

Semi-solid or gel formulations containing about 25% peptide were prepared as follows. Sample [Aib ^{8, 35]} were weighed _{HGLP-1 (7-36) NH 2} , it was thoroughly mixed with 66.66% of a ZnCl ₂ stock solution to the total volume of excipient pH2. The zinc solution may be ZnCl ₂ or ZnAc ₂ .2H ₂ 0. The desired pH of the solution is titrated by addition of dilute NaOH solution. In this example, the total volume of liquid added to the powder must be divided into zinc and NaOH solutions. Therefore, the concentration of the zinc solution was adjusted to reduce the total volume of zinc solution required to 50% of the total liquid volume added to the peptide powder (step d). The remaining 50% of the total liquid volume added to the peptide powder was added as NaOH solution as described in detail below. Formulations C3 and C4 were prepared in this manner.

pH-adjusted semisolid or gel compositions were prepared by the "push-pull" mixing method:

a) Weigh the desired amount of peptide into a disposable syringe S1 pail preloaded with a special binary hand valve HV (I. D. = 0.5 mm) and place the tube into the syringe luer hole;

b) secure the syringe plunger with stainless steel rod SR;

c) Connect the HV of S1 with the vacuum source and open the HV. Close HV after 10 minutes;

d) Accurately weigh the zinc solution into a second disposable syringe S2 canister;

e) connect S2 with the unconnected portion of the HV;

f) open the HV and allow the solvent to enter the barrel S1 with the peptide powder in vacuo;

g) Close the HV and remove the solvent syringe S2 to hydrate the peptide powder in S1;

h) the SR is removed so that the syringe plunger is not secured;

i) the syringe plunger is moved (pushed and pulled) without opening the HV, so that the powder is completely wet with the solvent;

j) Mount a binary stainless connector SC (I.D. = 1.0 mm) with a tube inside the syringe luer hole in the syringe S and push the plunge to the end;

k) Open the HV of S1, apply vacuum and remove the HV. Move the syringe plunge to minimize the air in the syringe barrel;

l) S1 and S2 are joined by SC and the composition is mixed through SC from S1 to S2;

m) after homogenization is performed, an aliquot of the hybrid product is taken to determine the peptide concentration;

n) Accurately weigh the remaining intermediate bulk product and calculate the amount of NaOH solution needed to titrate the desired pH;

o) Accurately weigh the NaOH solution into a third disposable syringe S3;

p) Slowly compress the syringe plunge to minimize air in the syringe chamber. Two syringes are connected to the SC and the composition is mixed through the SC.

In the context of the present invention, those skilled in the art can alter the amounts of peptide and ZnCl ₂ to make compositions other than the 25% compositions shown herein.

Table 1

Example ^* Peptide% solution ^** Peptide: Zinc Non Peptide dosage 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% NaOH 1 mg / ml 5.4: 1 1 mg C4 10 50% ZnCl ₂ 2.28 mg / ml, 50% NaOH 1 mg / ml 4: 1 1 mg C5 5 80% ZnCl ₂ 0.674 mg / ml, 20% NaOH 3.81 mg / ml 4: 1 1 mg C6 2 80% ZnCl ₂ 0.26 mg / ml, 20% NaOH 2.15 mg / ml 5.4: 1 1 mg C7 10 80% ZnCl ₂ 3.81 mg / ml, 20% NaOH 4.47 mg / ml 1.5: 1 1 mg C8 10 80% ZnAc ₂ .2H ₂ 0 2.3 mg / ml, 20% NaOH 6.1 mg / ml 4: 1 1 mg C9 2 80% ZnCl ₂ 0.695 mg / ml, 20% NaOH 1.75 mg / ml 1.5: 1 1 mg C10 2 80% ZnAc ₂ .2H ₂ 0 1.12 mg / ml, 20% NaOH 1.44 mg / ml 1.5: 1 1 mg C11 2 80% ZnCl ₂ 0.695 mg / ml, 20% NaOH 1.75 mg / ml 1.5: 1 1 mg C12 One 80% ZnCl ₂ 0.384 mg / ml, 20% NaOH 0.875 mg / ml 1.5: 1 1 mg C13 10 80% ZnCl ₂ 3.85 mg / ml, 20% NaOH 4.47 mg / ml 1.5: 1 15 mg

^* : Indicates a target value. The actual value is around 5% of the target in all cases.

^** : indicates the target value. The actual value is around 10% of the target in all cases.

3.0 GLP -One Receptor  Affinity determination

Compounds usable in the practice of the present invention can be tested for their ability to bind GLP-1 receptors in the following manner.

Cell culture:

RIN 5F rat insulinoma cells expressing GLP-1 receptor (ATCC- # CRL-2058, American Type Culture Collection, Manassas, VA) were Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum. Incubated in a humidified 5% CO ₂ /95% air at about 37 ° C.

radiation Labeled  Ligand ( Radioligand ) For

RNI cells were homogenized with Brinkman Polytron (Westbury, NY) (set to 6, 15 sec) in 20 ml of ice-cold 50 mM Tris-HCl to prepare a membrane for binding studies on radiolabeled ligands. The homogenate is washed twice by centrifugation (39,000 g / 10 min) and the final pellet contains 2.5 mM MgCl ₂ , 0.1 mg / ml baccitracin (Sigma Chemical, St. Louis, Mo.) and 0.1% BSA Suspended in 50 mM Tris-HCl. For analysis, aliquots (0.4 mL) were dispensed with 0.05 nM ( ¹²⁵ I) GLP-1 (7-36) (˜2200 Ci / mmol, New England Nuclear, Boston, Mass.) And the unlabeled competitive test peptide 0.05 Incubate with and without addition of ml. After incubation for 100 minutes (25 ° C.), the combined ( ¹²⁵ I) GLP-1 (7-36) was rapidly filtered with a GF / C filter (Brandel, Gaithersburg, MD), previously wetted with 0.5% polyethyleneimine, Separate from unbound. The filter was then washed three times with 5 ml of ice-cold 50 mM Tris-HCl and then the bound radioactivity captured in the filter was measured with a gamma meter (Wallac LKB, Gaithersburg, MD). Specific binding was defined as limiting binding in the presence of 1000 nM GLP-1 (7-36) (Bachem, Torrence, CA) at the total binding of ( ¹²⁵ I) GLP-1 (7-36).

4. Solubility vs pH  decision

4.1 Phosphoric Acid Buffered  Brine PBS Solubility of the compound in pH Decision

Compounds that can be usefully used in the practice of the present invention can be tested in the following ways to determine PBS solubility at various pHs and temperatures.

PBS stock was prepared by dissolving one nucleus of pre-mix powder (SIGMA, Product No .: P-3813) in 1 L of deionized water to prepare 10 mM phosphate buffered saline, pH 7.4, containing 138 mM NaCl, 2.7 mM KCl. Buffer was prepared. The pH of this stock solution can be adjusted with phosphoric acid and / or sodium hydroxide to prepare PBS buffers of various pH.

2 mg of the compound to be tested, such as 2 mg of the compound of Example 1, were measured and placed in a glass vial. To each vial 50 μl of PBS buffer at a specific pH was added. This solution is vortexed and sonicated until it is transparent as necessary. At each pH tested, the total volume of buffer needed to dissolve 2 mg of compound was recorded and solubility was calculated.

The clear peptide solution at room temperature (20-25 ° C.) was placed in the refrigerator (4 ° C.) overnight, after which the solubility at 4 ° C. was evaluated.

4.2 Solubility of Compounds in Brine Versus pH  decision

Compounds that can be usefully used in the practice of the present invention can be tested by the following methods to determine the solubility in saline at various pH and temperatures.

Stock saline is prepared by dissolving 9 g of NaCl in 1 L of deionized water. The pH of this stock solution is adjusted with HCl and / or NaOH to prepare saline at various pHs.

2 mg of the compound to be tested, such as 2 mg of the compound of Example 1, were measured and placed in a glass vial. To each vial was added 50 [mu] l of saline at a particular pH. This solution is vortexed and sonicated until it is transparent as necessary. At each pH tested, the total volume of saline needed to dissolve 2 mg of compound was recorded and solubility was calculated.

The clear peptide solution at room temperature (20-25 ° C.) was placed in the refrigerator (4 ° C.) overnight, after which the solubility at 4 ° C. was evaluated.

4.3 pH  Solubility Determination of Compounds in Brine of 7.0

Compounds that can be usefully used in the practice of the present invention can be tested in the following manner to determine the solubility in saline at pH 7 at room temperature.

Stock saline is prepared by dissolving 9 g of NaCl in 1 L of deionized water. 2 mg of the compound to be tested, such as the compound of Example 1, are measured and placed in a glass vial, vortexed with the addition of 1 ml of brine and sonicated until clear. The total volume of saline needed to dissolve 2 mg of compound is recorded and the solubility is calculated.

4.4 various pH Determination of Solubility of Compounds in Saline

Compounds that can be usefully used in the practice of the present invention can be tested by the following methods to determine their solubility in saline at various pH at room temperature.

Stock saline is prepared by dissolving 9 g of NaCl in 1 L of deionized water. Fractions of this stock solution were treated with HCl and NaOH to prepare saline at various pHs.

2 mg of the compound to be tested, such as the compound of Example 1, are measured and placed in a glass vial. Add 50 [mu] l of saline buffer at a specific pH. This solution is vortexed and sonicated until clear. The total volume of buffer required to dissolve 2 mg of compound is recorded and the solubility is calculated.

5. Determination of Solubility of Compounds versus Zinc Concentration

Compounds that can be usefully used in the practice of the present invention can be tested in the following manner to determine the solubility in water of pH 7 at various zinc concentrations.

ZnCl ₂ is dissolved in deionized water at a concentration of 100 mg / ml, and the pH is adjusted to 2.7 with HCl to prepare a zinc stock solution. Dilute this stock solution appropriately to obtain ZnCl ₂ Prepare solutions of varying concentrations ("Zn test solutions").

1 mg of the compound to be tested, such as 1 mg of the compound of Example 1, is dissolved in 250 [mu] l of each Zn test solution to make a solution containing 4 mg / ml of compound. The pH is then adjusted with 0.2 N NaOH until white precipitate formation is observed in this solution. The precipitated solution was centrifuged and the mother liquor was analyzed using HPLC. The UV absorbance area of the test compound peak is calculated and the concentration of the test compound in the parent solution is determined by comparison with a calibration curve.

As a representative compound that can be used in the practice of the present invention, the compound of Example 1 was investigated by the above analytical method, and the following result was obtained (aqueous saline, pH 7.0, room temperature):

TABLE 2

ZnCl ₂ Concentration (µg / ml) Water Soluble (mg / mL) 0 5.788 80 0.0770 500 0.0579 1000 0.0487 1500 0.0668 2500 0.1131

6. IEF Gel  Used pI  decision

Invitrogen's Novex IEF pH3-10 gel can be used to determine the pi of GLP-1 peptide. The peptide compound to be tested is dissolved in water at a concentration of 0.5 mg / ml. In each compound, 5 μl of the solution prepared is mixed with 5 μl of Novex ^® sample buffer 2X (consisting of 20 mM arginine free base, 20 mM lysine free base and 15% glycerol) and the resulting 10 μl sample solution is added to the gel. Load with protein standard samples.

Development buffer was also purchased from Invitrogen, and was generally run on the gel according to the manufacturer's instructions as follows: constant at 100 V for 1 hour then 200 V for 1 hour, again 500 V for 30 minutes Stay with.

The gel was then fixed in 12% TCA with 3.5% sulfosalicyclic acid for 30 minutes, then stained for 2 hours with Colloidal Coomassie Blue according to the instructions in the Novex ^® Colloidal Blue Kit and then desalted with water overnight.

The gel was scanned and analyzed with the fragment analysis 1.2 program. The pi of unknown peptides was calculated by comparing the pi of standard compounds with pi values of 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.

The pI measurement of the compound of Example 1 was 7.60.

7. In the rat In vivo  analysis

Compounds of the invention can be tested to determine their ability to promote and enhance in vivo effects with the following assay methods.

7.1 Experimental Process:

The day before the experiment, the cannula was implanted into the right atrial jugular vein with anesthesia of approximately 300-35 g g Sprague-Dawley male adult rats (Taconic, Germantown, NY) with chlorohydrate. The rats were then fasted for 18 hours before injecting the appropriate test compound or vehicle control at time zero. Rats were fasted continuously during the entire experiment.

100 mg / mL solution of ZnCl _{2 in} aqueous HCl solution at pH 2.7 was diluted with water, 0.5 mg / mL ZnCl ₂ The solution was prepared. The compounds of the formula 250 ㎕ of the solution ^{(I) [Aib 8, 35} ] hGLP- 1 (7-36) was dissolved in NH ₂₎ 1 mg, the concentration of the compound is 4 mg / ㎖ and Zinc 0.5 mg / A clear solution of pH 4, ml, was obtained.

In time 0, the rats in (a) the ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2) or the solution (b) were injected subcutaneously (sc) with vehicle control group. In both cases, the injection volume was minimal (4-6 μl) and the dose of GLP-1 compound administered to the subject was 75 μg / kg. At an appropriate time after sc injection, 500 μl of blood was taken via an intravenous (iv) cannula and glucose was injected into iv to test insulin secretion potentiation. The time of glucose infusion is 0.25, 1, 6, 12 and 24 hours after compound injection. After the first blood sample was taken, glucose (1 g / kg) was injected intravenously and 500 μl of heparinized saline (10 U / ml) was flushed. Thereafter, 500 μl of blood samples were collected 2.5, 5, 10 and 20 minutes after glucose injection. Immediately after each of these, 500 μl of heparinized saline (10 U / mL) was iv injected directly through the cannula. Blood samples were centrifuged to collect plasma from each sample and the samples were stored at −20 ° C. until the insulin content was analyzed. Insulin content in each sample was determined using an enzyme-linked immunosorbent assay (ELISA) kit (American Laboratory Products Co., Windham, NH) in rats.

7.1.1. result:

Long-term insulin-enhanced activity was observed to be inducible by glucose injection during the entire 24-hour experiment.

8. In dogs In vivo  analysis

In order to determine the ability of a composition to promote long-term secretion of the active compound in vivo, there are several in vivo assay methods known in the art that can be performed by those skilled in the art.

8.1 1% Peptide Composition:

For example, compound 1% (w / w) of formula (I) is ZnCl ₂ Test aqueous formulations were prepared comprising in buffer (peptide: Zn ratio is 1.5: 1.0).

Six male Beagle dogs, ages 14-21 kg, 42-78 months of age, were freely accessible to water and provided a meal once daily (about 400 g of standard dry food (SAFE 125)). The dog was fasted for 18 hours before administering the test composition.

Test compounds were administered subcutaneously in the interscapular region. Dosages (approximately 20 μl per animal) were administered with a 0.3 ml Terumo syringe equipped with 0.33-12 mm (BS = 30M2913). That is, about 0.2 mg of peptide was used at the theoretical dose.

Blood samples were periodically drawn after administration at about 0, 8, 15, 30 and 45 minutes, 1, 2, 4, 8 and 12 hours, and after 1, 2, 3, 4, 5 and 6 days. Blood was sampled until centrifuged and then rapidly cooled, plasma was taken and subjected to a rapid frozen pending assay. Peptide plasma concentrations were determined after offline solid phase extraction and subsequent on-line phase extraction coupled with LC-MS / MS, and the data obtained were managed with Analyst v1.2 software.

The composition was found to prolong the release of the active peptide for at least 2 days.

8.2 1% [ Aib ^{8 , 35} ] hGLP -1 (7-36) NH ₂ A) solution:

Using the in vivo assay method substantially the same as that shown in section 8.1 above, the following compositions were examined for their ability to release the subject peptide for an extended period of time. In each of the four compositions below, the peptide concentration is about 1% (wt / wt), the ratio of peptide to zinc is about 1.5: 1, and the dose of peptide administered is about 1 mg.

Solution 8.2.A: [Aib ^{8 , 35} ] hGLP-1 (7-36) in a solution comprising (i) 90% ZnCl ₂ (0.298 mg / ml) and (ii) 10% NaOH (0.975 mg / ml) NH ₂

Solution 8.2.B: [Aib ^{8 , 35} ] hGLP-1 (7-36) NH _{2 in} ZnCl ₂ (0.286 mg / mL) solution

Solution 8.2.C: Solution 8.2. Substantially similar to B, buffered with AcOH / AcO ⁻

Solution 8.2. D: substantially similar to solution 8.2.A.

1, the above composition have long-term release of ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2.

8.3. One% [ Aib ^{8 , 35} ] hGLP -1 (7-36) NH ₂  solution:

Using the in vivo assay method substantially the same as shown in section 8.1 above, the following compositions were investigated for their ability to release the subject peptide over an extended period of time. In the compositions below, the peptide concentration is about 2% (wt / wt), the ratio of peptide to zinc is about 1.5: 1, and the dose of peptide administered is about 1 mg.

Solution 8.3 .: [ ^{iib 8,35} ] hGLP-1 (7-36) NH in a solution comprising (i) 80% ZnCl ₂ (0.695 mg / ml) and (ii) 20% NaOH (1.78 mg / ml) ₂

As shown in Figure 5, the composition [Aib ^{8, 35]} were long-term release of _{hGLP-1 (7-36) NH 2} .

8.4. 10% peptide solution:

Using the in vivo assay method substantially the same as shown in section 8.1 above, the following compositions were investigated for their ability to release the subject peptide over an extended period of time. In each of the four compositions below, the peptide concentration is about 10% (wt / wt), the ratio of peptide to zinc is about 1.5: 1, and the dose of peptide administered is about 15 mg.

Solution 8.4.A: [Aib ^{8 , 35} ] hGLP-1 (7-36) in a solution comprising (i) 90% ZnCl ₂ (3.367 mg / ml) and (ii) 10% NaOH (5.01 mg / ml) NH ₂

Solution 8.4.B: [Aib ^{8 , 35} ] hGLP-1 (7-36) NH _{2 in} ZnCl ₂ (2.993 mg / mL) solution

Solution 8.4.C: substantially similar to Solution 8.4.B, buffered with AcOH / AcO ⁻

Solution 8.4. D: substantially similar to solution 8.4.A.

Also, the compositions are [Aib ^{8, 35]} were long-term release of hGLP-1 (7-36) NH ₂ as shown in Fig.

8.5. Semisolid  Composition:

Using the in vivo assay method substantially the same as shown in section 8.1 above, the following semisolid compositions were investigated for their ability to release peptides of interest over an extended period of time. The peptide concentration of composition 8.5.A. is about 5%, but composition 8.5.B., 8.5.C. And the peptide concentration of 8.5.D. is about 10% (wt / wt). 8.5.A., 8.5.B. And 8.5.C. The ratio of peptide to zinc in the composition is about 5.4: 1, and 8.5.D. The ratio in the composition is about 4.0: 1. In all four compositions, the dosage of peptide administered is about 1 mg.

Composition 8.5.A: [Aib ^8,35 ] hGLP-1 (7-36) NH ₂ in a solution comprising ZnCl ₂ (0.40 mg / ml) in WFI

Composition 8.5.B: Substantially the same as Composition 8.5.A., the concentration of ZnCl ₂ was upregulated to maintain the ratio of peptide to zinc at about 5.4: 1.

Composition 8.5.C: [Aib ^{8 , 35} ] hGLP-1 (7-) in semi-solid form comprising (i) 50% ZnCl ₂ (1.69 mg / ml) and (ii) 50% NaOH (1 mg / ml) 36) NH ₂

Composition 8.5. D: (i) 50% ZnCl 2 (2.28 mg / ㎖) and (ii) 50% NaOH (1 mg / ㎖) Four half box of [Aib ^{8, 35]} The solid forms of the hGLP-1 (7-36) NH ₂

As shown in FIG. 3, the compositions released long term [Aib ^8,35 ] hGLP-1 (7-36) NH ₂ .

8.6. Semi-solid composition:

Using the in vivo assay method substantially the same as shown in section 8.1 above, the following semisolid compositions were investigated for their ability to release peptides of interest over an extended period of time. The composition was formulated with a 5.22 mg / ml ZnCl ₂ solution at pH 2.0. Using a sufficient amount of peptide, a 25% peptide semisolid composition with a peptide: zinc ratio of about 4: 1 was prepared. The pH of the composition was adjusted as described herein with 10 mg / ml NaOH. The dose of peptide administered is about 15 mg.

6, wherein the composition is a 8.6 ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 were discharged for long periods of time.

8.7. Semisolid  Composition:

Using the in vivo assay method substantially the same as shown in section 8.1 above, the following semisolid compositions were investigated for their ability to release peptides of interest over an extended period of time. The composition was formulated with a 8.5 mg / ml ZnCl ₂ solution at pH 2.0. A sufficient amount of peptide was used to prepare a 23% peptide semi-solid composition having a peptide: zinc ratio of about 1.51. The composition was formulated according to the method detailed in section 2.6 above. The dose of peptide administered is about 15 mg (corresponding to about 65 μl of the composition).

As shown in FIG. 7, the 8.7 composition prolonged release of [Aib ^8,35 ] hGLP-1 (7-36) NH ₂ .

Additional assays for various substitutions of the disclosed formulations were performed in vivo, and it was found that the compositions of the present invention provide a useful drug delivery platform for the compounds of formula (I). Using the teachings of the present invention, one skilled in the art can alter the amounts and pH of peptides and ZnCl ₂ to prepare the compositions of the present invention as disclosed herein.

Example 9

1.PK Profile Control by Acetate Content in 10% Peptide Solution

Two kinds including a present embodiment is ^{10% (Aib 8, 35)} hGLP1 (7-36) NH 2 and _{^{ZnCl 2 [1 (Aib 8,}} 35) hGLP1 (7-36) NH 2:: Zn = 1.5] Pharmacokinetic studies of (Aib ^8,35 ) hGLP1 (7-36) NH ₂ in Beagle male dogs after a single subcutaneous injection of the instant composition at 15 mg / dog are ^disclosed .

The method for performing the in vivo assay is the same as described in paragraph 8.1.

In this example, the control of the PK profile by the acetate content in the pharmaceutical composition and thus the effect of the [acetate / peptide] ratio of the pharmaceutical composition on the pH is shown.

The pH adjustment is made by controlling the acetate content, and decreasing acetate content shows an increased effect on pH.

Acetate fluctuations also affect C _max . In general, decreasing acetate content decreases the C _max value.

Increasing the content of acetate appears to improve solubility and physical stability.

In the formulation of choice, solubility or stability improvement by controlling the ratio of acetate / peptide is offset by control of peptide / Zn, such as C _max . This can be seen as a system with three possible variables for controlling solubility, stability, pH, or C _max .

For example, the abbreviation SD stands for standard deviation. AUC stands for Artemisinin area under the plasma concentration-time curve.

The abbreviation MRT means mean residence time (MRT) and is a parameter for estimating bioavailability in order to compare MRT with tmax, which is the drug peak concentration time. MRT was calculated using data from the entire last sampling period at 0 hours.

Table 3 summarizes the results of subcutaneous administration of 10% peptide composition batches with various [acetate / peptide] ratios to Beagle dogs. The drug peak value (C _max ) at the plasma concentration was 8.10 ng / ml (SD = 1.80 ng / ml) when the [acetate / peptide] molar ratio was [3.7: 1] and the molar ratio was lower than [3.2: 1]. The C _max value in the case was 5.65 ng / ml (SD = 2.61 ng / ml).

TABLE 3

Formulation 10% 15mg 10% 15mg Peptide / Zn Ratio 1.5: 1 1.5: 1 parameter unit MEAN (n = 5) S.D. MEAN (n = 4) S.D. Dosage Μg kg ^-One 857.7 131.0 694.8 46.5 t _max d 0.208 0.167 0.111 0.068 C _max ng · ml ^-One 8.10 1.80 5.65 2.61 t _{1/2 app} d 3.32 0.66 6.77 2.04 AUC _t ng · ml ^-One D 53.5 14.3 38.2 9.2 AUC ng · ml ^-One D 55.4 15.7 41.6 8.9 AUC _extrap. % 2.99 1.83 8.44 5.00 MRT _t d 9.31 2.25 7.48 1.39 MRT d 9.96 2.60 9.85 2.54 [Acetate: peptide] 3.7: 1 3.2: 1

10. GLP-1 Peptide Salts / Vayl Metal Formulations

10.1. Way

(Aib ^8,35 ) hGLP-1 (7-36) NH ₂ 1 mg / mL water and PBS solution were prepared and the pH was adjusted to 7.0. A 10 mg / mL Sotok solution of CaCl ₂ , CuCl ₂ , MgCl ₂ , and ZnCl ₂ was prepared in water. Since Cu precipitated, the pH of CaCl ₂ , MgCl ₂ , and ZnCl ₂ solutions could not be basic. Thus, a solution of CuCl _{2 at} pH 4.4 was used.

4 ㎕ metal ion water or PBS solution was added to ^{200 ㎕ (Aib 8, 35)} hGLP-1 (7-36) NH 2 1 mg / mL solution, and the resulting metal ion concentration is to be 200 ㎍ / mL. The prepared solution was mixed and the precipitation was checked. If precipitation occurred, the suspension was centrifuged. The concentration of (Aib ^8,35 ) hGLP-1 (7-36) NH _{2 in} the supernatant was determined by HPLC.

10.2. result

Table 4.2 in the presence of a metal ion ^{(Aib 8, 35) hGLP-} 1 (7-36) NH ₂ The solubility of

Aqueous solution, mg / mL PBS solution, mg / mL CaCl ₂ > 1 (pH 7.1) > 1 (pH 6.8) CuCl ₂ 0.058 (pH 7.1) 0.039 (pH 6.8) MgCl ₂ > 1 (pH 7.2) > 1 (pH 6.9) ZnCl ₂ 0.108 (pH 6.9) 0.056 (pH 6.8)

10.3. (Aib ^8,35 hGLP-1 (7-36) NH ₂ Pharmacokinetic Study of the Bivalent Metal pH 5.5 Clear Solution Formulations

The ^{(Aib 8, 35) hGLP-} 1 (7-36) 3 of different formulations of NH _2, was prepared by the following method.

^{(1) (Aib 8, 35} ) hGLP-1 (7-36) NH 2 HCl salt and CuCl ₂

^{(2) (Aib 8, 35} ) hGLP-1 (7-36) NH 2 HCl salt and ZnCl ₂

(3) (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ acetate salt and ZnCl ₂

Acetate salts obtained by dissolving the TFA salt of the GLP-1 analog (TFA salt is obtained by eluting with TFA containing buffer using preparative HPLC to purify the peptide) in a small amount of 0.25 N acetate solution Can be converted to other salts such as The resulting solution is applied to a semi-preparative HPLC column (Zorbax, 300 SB, C-8). The column was (1) 0.5 hour with 0.1 N ammonium acetate solution, (2) 0.5 hour with 0.25 N acetic acid solution, and (3) linear concentration gradient (solution B from 20% to 100% for 30 minutes), flow rate 4 ml / Elution with min (solution A is 0.25N acetic acid aqueous solution; solution B is 0.25N acetic acid in acetonitrile / water 80:20). Fractions containing peptides were combined and dried by freeze drying.

Depending on the drying process (Aib ^{8, 35)} for _{hGLP-1 (7-36) NH 2} HCl salt was prepared. 20 mg (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ acetate was dissolved in 4 mL of 20 mM HCl aqueous solution and incubated for 10 minutes at room temperature. Samples were frozen and lyophilized overnight. Freeze drying was performed two more times and the chloride content of the final product was determined. The chloride content determined was 5.38%.

^{(1) (Aib 8, 35} ) hGLP -1 (7-36) NH 2 HCl salt and CuCl ₂ :

5.3 mg (peptide content 95%) of (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ HCl was dissolved in 50 μl of 20 mM CuCl ₂ aqueous solution. The pH was adjusted to about 5.5 using about 2 μl of 1 N NaOH. The molar ratio of (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ / CuCl ₂ was 1.5: 1. Peptide concentration in water (w / w) was 10% (30 mM) and pH was about 5.5.

(Aib (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ HCl salt and ZnCl ₂ :

The ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl 5.3 mg ( peptide content 95%) was dissolved in 20 mM ZnCl ₂ aqueous solution of 50 ㎕. The pH was adjusted to about 5.5 using about 2 μl of 1 N NaOH. The molar ratio of (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ / ZnCl ₂ was 1.5: 1. Peptide concentration in water (w / w) was 10% (30 mM) and pH was about 5.5.

(Aib ^8,35 ) hGLP-1 (7-36) NH ₂ acetate salt with ZnCl ₂ :

5.5 mg (peptide content 92%) of (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ acetate were dissolved in 50 μl of 20 mM ZnCl ₂ aqueous solution. The resulting solution was lyophilized overnight and then dissolved again in 50 μl of water. The pH was adjusted to about 5.5 using about 1 μl of 1 N NaOH. The molar ratio of (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ / ZnCl ₂ was 1.5: 1. Peptide concentration in water (w / w) was 10% (30 mM) and pH was about 5.5.

10.4. Dosing and Blood Sample Collection

Three (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ formulations were administered subcutaneously at 0.3 mg / rat (3 μl of 10% solution) in rats. Blood samples were collected at 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 7 days and 10 days. It was centrifuged to obtain plasma from blood and stored at -80 ° C. Tissues of the injected site were also extracted, homogenized with 5x methanol and stored at -80 ° C. Two rats took data at 5, 10, 15, 30, 1, 2, 4 and 8 hours. The other rat took data at 1, 2, 3, 4, 7, 10 hours.

10.5. LC - MS Of MS  Sample manufacturing

Plasma (200 μl) was acidified with 10 μl formic acid and precipitated using 600 μl of acetonitrile. The supernatant was collected by centrifugation, concentrated in vacuo and dried. The residue was dissolved in 150 μl 30% acetonitrile in water and centrifuged. 50 μl of supernatant was added to LC-MS / MS analysis.

10.6. LC - MS Of MS analysis

LC-MS / MS analysis was performed with an API4000 mass spectrometry system equipped with a Turbo Ionspray probe. MRM mode of molecular ion detection and ion pairs 668.9 and 136.1 were used.

HPLC separation was carried out using a Luna C8 (2) 2 × 30 mm 3μ column at 10% B-> 90% B for 10 min at 0.30 mL / min flow rate. Buffer A is a 1% formic acid aqueous solution and Buffer B is a 1% formic acid acetonitrile solution.

LOQ was 0.5 ng / mL.

10.7. Results and synthesis

Plasma concentrations of peptides were calculated using its standard calibration plot. A (0.3 mg / rat of 5 mL of methanol ^{extract) 0.06 mg / mL (Aib 8} , 35) hGLP-1 (7-36) NH 2 with a 100%, and calculated the% remaining around the injection.

Table ^{5. (Aib 8, 35) hGLP} -1 (7-36) NH 2 plasma concentration

Hour h ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 Plasma concentrations of HCl and CuCl ₂ Dosage (ng / mL) ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 Plasma concentrations of HCl and ZnCl ₂ Dosage (ng / mL) ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 plasma levels of acetate and ZnCl ₂ Dosage (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 One 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

^{(Aib 8, 35) hGLP-} 1 (7-36) shows a graph of the total lapse of time of the pharmacokinetic profile of the formulations of the HCl salt NH ₂ in Fig. ^{(Aib 8, 35) hGLP-} 1 initial time trend graph of the pharmacokinetic profile of the formulations of the HCl salt (7-36) NH ₂ is shown in Fig. A graph over the time course of the pharmacokinetic profile of the acetate salt formulation of (Aib ^8,35 ) hGLP-1 (7-36) NH ₂ is shown in FIG. 10. ^{(Aib 8, 35) hGLP-} 1 (7-36) graphs for an initial transition between the pharmacokinetic profile of the acetate salt formulations of the NH ₂ is shown in Fig.

Table 6. (Aib ^{8, 35)} at the injection site hGLP-1 (7-36) NH ₂ residue estimate%

Hours, days ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl and CuCl ₂ % Estimated residual at injection site ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl and ZnCl ₂ % Estimated residual at injection site ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 acetate and ZnCl ₂ % Estimated residual at injection site One 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

At the injection site ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 indicates additional tissue accumulation profile in Fig.

Table 7. PK Parameters

^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl and CuCl plasma concentration of the _second dose (ng / mL) ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 HCl , and the plasma concentration of ZnCl ₂ Dose (ng / mL) ^{(Aib 8, 35) hGLP-} 1 (7-36) NH 2 acetate and ZnCl plasma concentration of the _second dose (ng / mL) T _max , h One 0.5 0.25 C _max , ng / ml 11.8 13.8 32.2 AUC ng-hr / ml 204 514 394

As a result, the salt form of the GLP-1 analog, especially in combination with a divalent metal salt, provides an acceptable long-term release formulation with reduced initial plasma concentrations, which can alleviate or eliminate unwanted side effects.

The data indicate that strong acid salts, such as the HCl salt of the GLP-1 analog, further reduce the initial plasma concentration. Without being bound by theory, it is believed that an excellent reduction in the initial plasma concentration of the HCl salt of the GLP-1 analog is related to the in vivo neutralization process. In compositions (1) and (2) at pH 5.5, 100% of the acid is in chloride form, with no free acid. Thus, after subcutaneous injection, body fluids can neutralize the solution more quickly and precipitate the solution more quickly. This reduction in neutralization time results in lower and less apparent initial plasma concentrations or spikes.

The publications cited above are hereby incorporated by reference. Further examples of the present invention will be apparent from the foregoing, and are intended to be included in the present invention as fully described herein and specified in the following claims.

Claims (17)

A pharmaceutical composition comprising a GLP-1 analog, Formulated with a pharmaceutically acceptable salt of GLP-1 analog or a mixture of the analog and a salt thereof, provides a molar ratio of analog salt: peptide analog in the pharmaceutical composition, Wherein said molar ratio is adjustable to modulate the release effect on the solubility, pH, and in vivo release profile of the peptide in the pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the GLP-1 analogue is [Aib ^{8 , 35} ] hGLP-1 (7-36) NH ₂ . The pharmaceutical composition of claim 2, further comprising a divalent metal and / or a divalent metal salt. The pharmaceutical composition according to claim 3, wherein the divalent metal is zinc or copper. The pharmaceutical composition of claim 3, wherein the composition comprises a divalent metal salt selected from the group consisting of CuAc ₂ , CuCl ₂ , ZnAc ₂ , and ZnCl ₂ . According to claim 2 or 5, wherein said ^{[Aib 8, 35] hGLP-} 1 (7-36) is characterized in that the pharmaceutical composition as a pharmaceutically acceptable salt thereof with the organic acid salt of NH _2. 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. 8. A pharmaceutical composition according to claim 7, wherein said acid is acetic acid. According to claim 2 or 5, wherein said ^{[Aib 8, 35] hGLP-} 1 (7-36) NH ₂ is a salt of a pharmaceutical composition, characterized in that the acceptable salt of an inorganic acid with a pharmaceutical. The pharmaceutical composition according to claim 9, wherein the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid. The pharmaceutical composition of claim 10, wherein the acid is hydrochloric acid. The pharmaceutical composition of claim 3, wherein the molar ratio of the [Aib ^{8 , 35} ] hGLP-1 (7-36) NH ₂ salt to the GLP-1 peptide analog is about 0.5: 1 to about 10.:1. . The method of claim 3, wherein the molar ratio of the [Aib ^{8 , 35} ] hGLP-1 (7-36) NH ₂ salt to the divalent metal and / or divalent metal salt in the pharmaceutical composition is from about 6: 1 to about 1: Pharmaceutical composition, characterized in that 1. The molar ratio of claim 13, wherein the molar ratio of the [Aib ^{8 , 35} ] hGLP-1 (7-36) NH ₂ salt to the divalent metal and / or divalent metal salt in the pharmaceutical composition is from about 5.4: 1 to about 1.5: Pharmaceutical composition, characterized in that 1. 15. The method of claim 14, wherein the pharmaceutically acceptable salt thereof ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 · A pharmaceutical composition, characterized in that HCl · Zn. 15. The method of claim 14, wherein the pharmaceutically acceptable salt thereof ^{[Aib 8, 35] hGLP-} 1 (7-36) NH 2 · A pharmaceutical composition, characterized in that the acetate · Zn. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable salt is [Aib ^8,35 ] hGLP-1 (7-36) NH ₂ · HCl · copper.

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