CN101668535B

CN101668535B - The pharmaceutical compositions of GLP 1

Info

Publication number: CN101668535B
Application number: CN200780051882.2A
Authority: CN
Inventors: 董正欣; R·谢里夫-谢赫; J-A·科尔德罗里戈尔; R·阿略萨米拉韦特; F·拉孔贝; M·D·托马利纳马埃斯特雷
Original assignee: Ipsen Pharma SAS
Current assignee: Ipsen Pharma SAS
Priority date: 2006-12-29
Filing date: 2007-12-31
Publication date: 2017-07-28
Anticipated expiration: 2027-12-31
Also published as: WO2008082656A1; KR101247665B1; AU2007340369C1; KR20090096739A; EP2109454A4; AU2007340369B2; CN101668535A; AU2007340369A1; JP2010523473A; EP2109454A1

Abstract

The present invention relates to the peptide analogues of glucagon-like peptide 1, its officinal salt, the method using such analogue treatment mammal and the therefore useful pharmaceutical composition for including the analog.

Description

GLP-1 pharmaceutical compositions

Background technology

The present invention relates to changing for the composition containing the peptide analogues of glucagon-like-peptide-1 and/or its officinal salt Enter, prepare the method, pharmaceutical composition and the method that mammal is treated using such composition of such composition.

Glucagon-like-peptide-1 (7-36) acid amides (GLP-1) passes through the high blood of hyperglycemic factor precursor pancreas in intestines L- cells The synthesis of tissue specificity post translational processing (Vamdell, J.M. etc., J.Histochem of sugared element former (preproglucagon) Cytochem, 1985：33：1080-6), and in response to food it is released into the circulatory system.GLP-1 plasma concentration from About 15pmol/L fasting levels rise to 40pmol/L peak level after the meal.Have confirmed, just in given blood sugar concentration It is about high 3 times when when oral glucose administration, the increase of plasma insulin is than intravenous glucose administration for rising (Kreymann, B. etc., Lancet 1987：2,1300-4).The diet of this insulin releasing for being referred to as duodenin effect Property increase it is mainly antibody mediated, and think that GLP-1 is the maximally effective physiology duodenin of people.Except pancreotropic hormone effect Outside, GLP-1 also glucagon suppressions are secreted, and delay gastric emptying (Wettergren A. etc., Dig Dis Sci 1993：38： 665-73), and the processing of periphery glucose (D ' Alessio, D.A. etc., J.Clin Invest 1994 can be promoted：93： 2293-6)。

In 1994, it can make completely with non-insulin-depending type sugar in single SC (s/c) dosage for observing GLP-1 GLP-1 treatment potential has been suggested that after this normal result of postprandial glucose levels of the patient of urine sick (NIDDM) (Gutniak, M.K. etc., Diabetes Care 1994：17：1039-44).Think that the effect was both increased by insulin releasing to be situated between Lead, and mediation is reduced by glucagon secretion.In addition, it has already been proven that intravenous infusion GLP-1 can postpone the meal of NIDDM patient Gastric emptying (Williams, B. etc., J.Clin Endo Metab 1996 afterwards：81：327-32).Different from sulfonylurea, GLP-1 Pancreotropic hormone effect depend on blood sugar concentration (Holz, G.G.4^thDeng Nature 1993：361：362-5).Therefore, in low blood The insulin releasing loss that GLP-1- is mediated under sugared concentration can prevent serious hypoglycemia.This synergy causes GLP-1 to have Have more than unique potential treatment advantage of other activating agents currently used for treatment NIDDM.

Numerous studies have demonstrated that when giving healthy individuals, GLP-1 effectively influence blood sugar level and insulin and Glucagon concentrations (Orskov, C, Diabetologia 35：701-711,1992；Hoist, J.J. etc.,Potential of GLP-1 in diabetes management, Glucagon III, Handbook of Experimental Pharmacology, Lefevbre PJ are edited, Berlin, Springer Verlag, 1996, the 311-326 pages), these effects It is glucose dependency (Kreymann, B. etc., Lancet ii：1300-1304,1987；Weir, G.C. etc., Diabetes38：338-342,1989).In addition, it is also effective (Gutniak, M., N.Engl J in diabetic Med 226：1316-1322,1992；Nathan, D.M. etc., Diabetes Care15：270-276,1992), 2 types sugar can be made Blood sugar level normal (Nauck, M.A. etc., Diabetologia 36 of urine disease individual：741-744,1993) and improvement 1 type trouble Glycemic control (Creutzfeldt, W.O. etc., Diabetes Care 19 of person：580-586,1996) so that it is outstanding to indicate its It can increase the ability of insulin sensitivity/reduction insulin resistance.Propose GLP-1 and its activator being used in hair Give birth to the individual (referring to WO 00/07617) in Non-Insulin Dependent Diabetes Mellitus risk and (beautiful for treating gestational diabetes mellitus State's patent discloses No.20040266670).

In addition to the above, the treatment use also in the presence of many in mammal such as people, for them, The application of display GLP-1 and its activator includes but is not limited to：Improve study, promote neuroprotection and/or alleviate nervous centralis The disease or obstacle (such as by adjusting nerve to occur) and such as Parkinson's, Alzheimer disease, Huntington chorea of system Disease, ALS, apoplexy, the symptom of ADD and neuropsychopathy syndrome (U.S. Patent Publication No.20050009742 and 20020115605)；Liver stem cells/progenitor cells are changed into functional pancreatic cells (WO03/033697)；Prevent beta cell Fail (deterioration) (U.S. Patent Publication No.20040053819 and 20030220251) and stimulation beta-cell proliferation (U.S. Patent Publication No.20030224983)；Treat obesity (U.S. Patent Publication No.20040018975；WO98/19698)； Appetite-suppressing and induction full sense (U.S. Patent Publication No.20030232754)；Treat irritable bowel syndrome (WO 99/ 64060)；The reduction incidence of disease and/or the death rate (U.S. Patent Publication No.20040162241, WO98/ related to myocardial infarction 08531) with apoplexy (referring to WO 00/16797)；Treatment is comprehensive with the acute coronary being characterized in the absence of Q- ripple myocardial infarctions Simulator sickness (U.S. Patent Publication No.20040002454)；Weaken post-surgical catabolic and change (United States Patent (USP) No.6,006,753)； Treat hibernating myocardium or diabetes cardiomyopathy (U.S. Patent Publication No.20050096276)；Suppress the blood of norepinephrine Pulp-water puts down (U.S. Patent Publication No.20050096276)；Increase natruresis, reduction urine potassium concn (U.S. Patent Publication No.20050037958)；The treatment illness related to Poisoning hypervolemia (toxic hypervolemia) or obstacle, example Such as kidney failure, congestive heart failure, nephrotic syndrome, hepatic sclerosis, pulmonary edema and hypertension (U.S. Patent Publication No.20050037958)；Induce variable force reaction (inotropic response) and increase cardiac contractile force (U.S. Patent Publication No.20050037958)；Treat Stein-Leventhal syndrome (U.S. Patent Publication No.20040266678 ＆ 20040029784)； Treat respiratory distress (U.S. Patent Publication No.20040235726)；By non-diet by way of, i.e. pass through intravenous, subcutaneous, flesh Interior, peritonaeum or other injections are transfused by way of improvement nutrition (U.S. Patent Publication No.20040209814)；Treat the nephrosis (U.S. Patent discloses No.20040209803)；Treat left ventricular systolic dysfunction, such as left side with abnormal left ventricular ejection fraction Ventricular systolic dysfunction (U.S. Patent Publication No.20040097411)；Suppress antroduodenal motility, for example for Treat or prevent gastrointestinal tract disorder such as diarrhoea, postoperative dumping syndrome and irritable bowel syndrome and operated as endoscopy In premedication (U.S. Patent Publication No.20030216292)；Treat Critical Illness Polyneuropathy Patients (critical Illness polyneuropathy, CIPN) and systemic inflammatory response syndrome (SIRS) (U.S. Patent Publication No.20030199445)；Adjust triglyceride levels and treatment dyslipidemia (dyslipidemia) (U.S. Patent Publication No.20030036504 and 20030143183)；Organ-tissue damage (the U.S. caused by blood flow Reperfu- sion after treatment ischaemic Patent discloses No.20020147131)；Treat the coronary heart disease risk factor (coronary heart disease risk Factor, CHDRF) syndrome (U.S. Patent Publication No.20020045636) etc..

However, GLP-1 is metabolized unstable, plasma half-life (t in vivo_1/2) it is only 1-2 minutes.Exogenous administration GLP-1 also fast degradation (Deacon, C.F. etc., Diabetes 44：1126-1131,1995).This metabolism unstability limit Natural GLP-1 treatment potential is made.Have been carried out largely attempting improving GLP-1 with the improvement by preparation similar with its The treatment potential of thing.For example, International Patent Publication No.WO 01/57084 describes the crystal for producing GLP-1 analogs Method, it is stated that these analogs can be used for pharmaceutical composition of the preparation comprising the crystal and pharmaceutically acceptable carrier such as may be used Inject medicine.GLP-1 (7-37) OH non-homogeneous crystallite cluster has been grown from saline solution and with zinc and/or M-cresol is checked (Kim and Haren, Pharma.Res. volume 12, o. 11th after carrying out crystal immersion processing (1995)).The GLP containing acicular crystal and amorphous sediment is prepared for via the phosphate solution containing zinc or nucleoprotamine (7-36)NH₂Coarse crystallization suspension (Pridal etc., International Journal of Pharmaceutics the 136th Volume, the 53-59 pages (1996)).European Patent Publication No.EP 0619322A2 are described by mixed protein in pH 7-8.5 Some combinations of solution and salt and low molecular poly (PEG) in buffer solution prepare GLP-1 (7-37) OH crystallite shape The method of formula.United States Patent (USP) No.6,566,490 describe especially GLP-1 crystallite kind crystalline substance, it is stated that it contributes to production to purify Peptide product.The GLP-1 with the smooth bar in four directions or plate-like shape is disclosed in United States Patent (USP) 6,555,521 (US ' 521) brilliant Body, it is stated that they have improved purity and show the activity in vivo of extension.US ' 521 teaches this kind of crystal phase to equal It is even and for longer periods keep suspension than existing haptophore and imperfect crystal formation suspension, it is stated that existing haptophore With imperfect crystal formation suspension rapid subsidence, aggregation or cluster each other, block syringe needle and typically aggravate unpredictable Dosage.

It has been suggested that by it is biodegradable it is poly- [(dl- lactide-co-glycolides)-β-ethylene glycol-β-(- lactide- Co- glycolide)] triblock copolymer be used for GLP-1 controlled release preparation.However, similar to other polymer systems, three block The scheme and inconsistent particle that the preparation of copolymer is related to complexity are formed.

Similarly, also point out by Biodegradable polymeric for example it is poly- [(lactic-co-glycolic acid) (PLGA) be used for peptide Sustained delivery formulations.However, not agreeing with applying this kind of biodegradable polymer in the art, because these polymer Typically there is poor water solubility, it is necessary to the organic solvent such as dichloromethane unmixing with water and/or need in process of production Want harsh preparation condition.Induction peptide or egg of interest can be increased by thinking the preparation condition of this kind of organic solvent and/or harshness The risk of white matter conformational change, thus cause structural intergrity decline and bioactivity it is impaired (Choi etc., Pharm.Research, volume 21, the 5th phase (2004)).Poloxamer equally exists defect.(Id.)

GLP-1 compositions described in above-mentioned bibliography are not ideal in terms of the pharmaceutical preparation for preparing GLP, because it Be intended to trap impurity and be also difficult to reproducibly produce and apply.Furthermore it is known that GLP analogs are when concentration is raised Induce nausea, it is therefore desirable to provide with reduction initial plasma concentration Sustained drug effect (Ritzel etc., Diabetologia, 38：720-725(1995)；Gutniak etc., Diabetes Care, 17 (9)：1039-1044(1994)； Deacon etc., Diabetes, 44：1126-1131(1995)).Accordingly, it would be desirable to be easier to and reliably produce, be easier to and can It is applied to patient and provides the initial plasma concentration of reduction to reduce or eliminate the GLP-1 systems of undesirable side effect with reproduces Agent.

Summary of the invention

The present invention can be summarised in following paragraph and claim.Therefore, the present invention, which is provided, includes GLP-1 analogs Pharmaceutical composition.Especially preferably according to the GLP-1 analogs of formula (I)：

(Aib^8,35)hGLP-1(7-36)NH₂

(I)

Or its officinal salt, wherein the preparation of the composition excellent production is provided, it is special using, pharmacokinetics and pharmacodynamics The adverse side effect of property and decrease.The pharmaceutical composition of the present invention is not preferably by the ZnCl of the clarification with pH4₂Aqueous solution group Into wherein described [Aib^8,35]hGLP-1(7-36)NH₂Presence concentration be 4mg/ml and described ZnCl₂Presence concentration For 0.5mg/ml.

A preferred embodiment of the present invention is there is provided first with improved drug release characteristics, preferably with what is reduced Begin the prominent pharmaceutical composition for releasing (burst).

Present invention also offers the pharmaceutical composition for including formula (I) compound of the acting duration with extension.

In preferred feature, the present invention also provides following pharmaceutical compositions, and it is precipitated under physiological pH in vivo, and formation is held The in-situ precipitate thing of continuous release drug characteristic.

Another embodiment of the invention provides include formula (I) compound or pharmaceutically acceptable salt thereof and pharmaceutically acceptable The pharmaceutical composition of carrier or diluent.Described carrier or diluent preferably comprises water.

In preferred feature, the present invention provides the pharmaceutical composition of inclusion compound or GLP-1 peptide analogues, the medicine The salt of compositions and GLP-1 peptides, or the mixture of GLP-1 peptides and its salt are prepared together.

Preferably, the salt of GLP-1 peptide analogues is selected from and organic acid (such as acetic acid, lactic acid, apple in described pharmaceutical composition Tartaric acid, ascorbic acid, butanedioic acid, benzoic acid, citric acid, methanesulfonic acid or toluenesulfonic acid) formed officinal salt and inorganic acid The officinal salt of (such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid or phosphoric acid) formation.The officinal salt of the strong acid of such as hydrochloric acid is It is especially preferred.Strong acid is defined as the acid with the pKA less than 4.5.Other preferred peptide salt in described pharmaceutical composition are that have The salt of machine acid (such as acetic acid or trifluoroacetic acid, lactic acid, malic acid, ascorbic acid, butanedioic acid, benzoic acid or citric acid).

In a preferred embodiment, the release characteristics (release of solubility, pH and pharmaceutical composition Profile) can be by the way that the mol ratio of the GLP-1 analogs of salt form and the GLP-1 analogs of salt-independent shape be adjusted to super Release characteristics are crossed, and reduce the initial peak (initial spike) in GLP-1 analog concentration to adjust.

In preferred embodiments, pharmaceutical composition also includes divalent metal, to reduce the water solubility of composition, thus Extending release characteristics and reduce initial burst or initial peak in plasma concentration simultaneously.It is preferred that divalent metal include zinc and copper. The salt form of divalent metal is especially preferred, and it includes but is not limited to the villaumite and acetate of divalent metal.CuAc₂、CuCl₂、 ZnAc₂And/or ZnCl₂It is most preferred.Preferably, in described pharmaceutical composition divalent metal and/or divalent metal salt it is dense Degree is about 0.0005mg/ml to about 50mg/m.Even further preferably, divalent metal and/or divalence in described pharmaceutical composition The concentration of metal salt is about 0.01mg/ml to about 0.50mg/ml.It is highly preferred that described pharmaceutical composition includes dilution, wherein The dilution includes pharmaceutically acceptable water solution.Dilution includes sterilized water.

In another embodiment, described pharmaceutical composition also includes divalent metal and/or divalent metal salt, wherein described GLP-1 analogs described in pharmaceutical composition and the divalent metal and/or divalent metal salt mol ratio are about 6: 1 to about 1: 1.Preferably, the ratio is about 5.5: 1 to about 1: 1.It is highly preferred that 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.The party of the present invention The ratio of 1.5: 1 ± 10% each desired value is about meant that in face, it is therefore desirable for ratio covers such as 1.35-1.65: 0.85- 1.15。

Preferably, described pharmaceutical composition includes aqueous mixture, suspension or solution, wherein the GLP-1 analogs, The concentration of formula (I) compound or its salt is about 0.5%-30% (w/w)., should in the aqueous mixture, suspension or solution The concentration of GLP-1 analogs and/or its salt is even more preferably about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%th, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%th, 27%, 28%, 29% or 30% (w/w).It is highly preferred that GLP-1 analogs described in the aqueous solution and/or its The concentration of salt is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 14%, 15%, 16%, 19%th, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 29% or 30% (w/w).It is highly preferred that the aqueous solution Described in GLP-1 analogs and/or its salt concentration be about 1%, 2%, 3%, 4%, 5%, 6%, 9%, 10%, 11%, 22%th, 23%, 24%, 25% or 26% (w/w).It is highly preferred that GLP-1 analogs described in the aqueous solution and/or its The concentration of salt is about 1%, 2%, 3%, 4%, 5%, 6%, 10%, 22%, 23%, 24%, 25% or 26% (w/w).It is more excellent Selection of land, the concentration of GLP-1 analogs and/or its salt described in the aqueous solution be about 1%, 2%, 5%, 10%, 23% or 25% (w/w)." about " following implication is meant：For the concentration of about 0.5% to about 4%, ± 0.5% desired value is desired Scope (such as 0.5% to 1.5% i.e. about 1%)；For about 5% and more than 5% aimed concn, 20% desired value is the phase The scope (such as 8% to 12% i.e. about 10%) of prestige.

Preferably, [Aib^8,35]hGLP-1(7-36)NH₂, the concentration of GLP-1 analogs or its salt in pharmaceutical composition be About 1% (weight/volume), and [Aib^8,35]hGLP-1(7-36)NH₂With the divalent metal and/or mole of divalent metal salt Than for about 1.5: 1.It is highly preferred that [Aib^8,35]hGLP-1(7-36)NH₂Or concentration of its salt in described pharmaceutical composition is about 2% (weight/volume), and [Aib^8,35]hGLP-1(7-36)NH₂Or its salt and the divalent metal and/or divalent metal salt Mol ratio is about 1.5: 1.It is further preferred that [Aib^8,35]hGLP-1(7-36)NH₂Or its salt is in described pharmaceutical composition Concentration is about 10% (weight/volume), and [Aib^8,35]hGLP-1(7-36)NH₂Or its salt and the divalent metal and/or divalence The mol ratio of metal salt is about 1.5: 1.Most preferably, [Aib^8,35]hGLP-1(7-36)NH₂Or its salt is in described pharmaceutical composition In concentration be about 23% or about 25% (weight/volume), and [Aib^8,35]hGLP-1(7-36)NH₂Or its salt and the divalence The mol ratio of metal and/or divalent metal salt is about 1.5: 1.

In preferred embodiments, GLP-1 analogs, [Aib^8,35]hGLP-1(7-36)NH₂Or its salt is in drug regimen Concentration in thing is about 5% (weight/volume), and the mol ratio of the peptide and the divalent metal and/or divalent metal salt is about 5.4∶1.It is highly preferred that [Aib^8,35]hGLP-1(7-36)NH₂Or the concentration of its salt in the composition be about 5% (weight/ Volume) and described ratio is about 4.0: 1.Additionally preferably, [Aib^8,35]hGLP-1(7-36)NH₂Or its salt is in the combination Concentration in thing is about 10% (weight/volume) and described ratio is about 5.4: 1.Still further preferably, [Aib^8,35] hGLP-1(7-36)NH₂Or the concentration of its salt in the composition is about 10% (weight/volume) and described ratio is about 4.0∶1。

Preferably, described divalent metal and/or divalent metal salt are provided with zinc chloride or zinc acetate.It is highly preferred that Described zinc acetate is with ZnAc₂·2H₂O form is provided.

In alternate embodiment, described divalent metal and/or divalent metal salt is carried with copper chloride or copper acetate For.

In one embodiment, the pH of described pharmaceutical composition is adjusted upward using alkali.It is highly preferred that being entered using NaOH The described pH adjustment of row.It is more preferred still that using the pH of NaOH adjustment described pharmaceutical compositions so that as 0.9% NaCl of use When being diluted to about 1/2 initial concentration, use direct potential determination method (direct potentiometric determination) Obtain about 5.0-5.5 pH value.

The preferred embodiments of the invention are characterised by pharmaceutical composition, wherein being prepared to composition so as to such as formula (I) the GLP-1 peptide analogues or its salt of compound or its salt are needing to discharge in its individual such as mammal, preferably human body The time of extension.Preferably, the release of the compound extends at least 1 hour, more preferably extends at least 4,6,12 or 24 Hour.It is more preferred still that the composition is prepared so as to formula (I) compound individual release at least 36 in vivo, 48, 60th, 72,84 or 96 hours.It is more preferred still that being prepared to the composition so that formula (I) compound is in individual release in vivo At least about 5,6,7,8,9,10,11,12,13 or 14 days.It is more preferred still that being prepared to the composition so that formula (I) is changed Compound individual release at least about 2 in vivo, 3 or 4 weeks.Even further preferably, being prepared to the composition so that formula (I) is changed Compound is in individual release at least about 1,1.5,2 in vivo or 3 months or longer time.

In one aspect of the invention, the salt content of GLP-1 peptide analogues is adjusted in described pharmaceutical composition to be improved Solubility and stability of the GLP-1 peptide analogues in pharmaceutical composition, and also provide improved by reducing initial burst Internal release characteristics.

Term " regulation " in this aspect of the present invention means GLP-1 analogs by adjusting salt form and salt-independent shape The mol ratios of GLP-1 analogs adjusts salt content.

Even further preferably, the peptide salt in described pharmaceutical composition is the hydrochloric acid of the formula (I) peptide or the salt of acetic acid, or chlorine Compound or acetate (acetates).In described pharmaceutical composition, acetate or chloride and the formula (I) compound are with about 0.5: 1 to about 10: 1 final mol ratio is present.More preferably described ratio is about 0.8: 1 to about 9: 1.Even further preferably, institute It is about 1: 1 to about 6: 1 to state ratio.Most preferably, the ratio is about 3.0: 1, especially 3.2: 1.

In this aspect of the invention, the mol ratio of acetate or chloride and peptide means acetate in pharmaceutical composition (CH3COO^-) or chlorine (Cl^-) molar ratio and pharmaceutical composition in peptide molar ratio.For example, 3: 1 in pharmaceutical composition Mol ratio, acetate is three times of peptide molar content in proportion.This is the stoichiometric proportion of compound and other materials.

Middle word " about " is it is intended that the ratio of 1.5: 1 ± 10% each desired value in this aspect of the invention, therefore the phase The ratio of prestige includes the ratio for covering such as 1.35-1.65: 0.85-1.15.

In another preferred aspect of the present invention, the pH of pharmaceutical composition is adjusted by the Determination of Ac of regulation composition. Preferably, the pH scopes of described pharmaceutical composition are pH 3 to pH 6.The pH scopes of the more preferably pharmaceutical composition are pH 3.5 to 5.5.The pH scopes of the most preferably pharmaceutical composition are pH 4.2 to pH 4.6.

It is preferred that, in order to be acidified pharmaceutical composition, the content of acetate can be increased by adding acetic acid.

In one embodiment, the pH of described pharmaceutical composition can be by adjusting the content of acetate from low acetate The peptide salt of radical content or GLP-1 analogs without Determination of Ac starts increase.

In preferred embodiments, the pH in the final pharmaceutical composition of content adjustment by adjusting acetate or chloride Such as peptide concentration, zinc concentration, chemical stability, physical stability and internal release characteristics can be adjusted by reducing initial burst Etc. parameter.

Zn or Cu content is fixed in one aspect of the invention, and pH is controlled by the content for adjusting acetate. Increased Determination of Ac shows improved solubility and physical stability, and the Determination of Ac reduced shows what pH was acted on Increase and to C_maxThe reduction of effect.

In preferred embodiments, described pharmaceutical composition includes aqueous mixture, suspension or solution.

The present invention also provides the method for triggering GLP-1 rule-activator effects, and methods described includes making GLP-1 (7-36) NH₂Match somebody with somebody The acceptor of body is directly or indirectly contacted with GLP-1 analogs or its salt.

In preceding method, GLP-1 (7-36) NH₂The acceptor of part is present in animal individual, is preferably in In primate, it is more preferably the presence of in people.Therefore, in this embodiment, the invention provides draw in the individual for needing it The method for sending the rule-activator effect from GLP-1 acceptors, this method includes applying the individual composition of the present invention, wherein The composition includes the GLP-1 analogs or its officinal salt of effective dose.

In the preferred aspect of preceding method, the individual is with disease or illness or in generation disease or illness Risk in people, the disease or illness be selected from type i diabetes, type ii diabetes, gestational diabetes mellitus, obesity, bulimia nerovsa (excessive appetite), full sense deficiency (insufficientsatiety) and metabolic disorder.It is preferred that described disease is Type i diabetes or type ii diabetes.

In another preferred aspect of preceding method, the individual is with disease or in the wind for occurring disease People in danger, the disease is selected from：Type i diabetes；Type ii diabetes；It is fat；Glucagonoma；Airway secretion obstacle；Joint It is scorching；Osteoporosis；Central nervous system disease；ISR；Neurodegenerative disease；Kidney failure；Congestive heart failure；Nephrosis is comprehensive Simulator sickness；Hepatic sclerosis；Pulmonary edema；Hypertension；Wherein need the obstacle of reduction food intake；Central nervous system disease or obstacle (such as by adjusting nerve to occur, and for example Parkinson's, Alzheimer disease, Huntington's chorea, ALS, apoplexy, ADD and Neuropsychopathy syndrome (neuropsychiatricsyndromes))；Irritable bowel syndrome；Myocardial infarction (is for example reduced The relative incidence of disease and/or the death rate)；Apoplexy；Acute coronary syndrome is (such as with the absence of Q- ripple myocardial infarctions The acute coronary syndrome being characterized)；Post-surgical catabolic changes；Hibernating myocardium or diabetes cardiomyopathy；Urinate sodium row Let out not enough (insufficient urinary sodium excretion)；Urinate too high (the excessive urinary of potassium concn potassium concentration)；The illness related to Poisoning hypervolemia or obstacle (such as kidney failure, congested Heart failure, nephrotic syndrome, hepatic sclerosis, pulmonary edema and hypertension)；Stein-Leventhal syndrome；Respiratory distress；Nephrosis；Zuo Xin Room contractile dysfunction (such as the left ventricular systolic dysfunction with abnormal left ventricular ejection fraction)；Gastrointestinal tract disorder such as abdomen Rush down, postoperative dumping syndrome and irritable bowel syndrome (i.e. by suppressing antroduodenal motility)；Critical characteristic of disease is multiple Nerve disease (CIPN)；Systemic inflammatory response syndrome (SIRS)；Dyslipidemia；After ischaemic caused by blood flow Reperfu- sion Organ-tissue is damaged；With the coronary heart disease risk factor (CHDRF) syndrome.

In another aspect of the present invention, it is a feature of the present invention that by liver stem cells/ancestral in the individual for needing it Cell transformation functional pancreatic cell, the decline of prevention beta cell and stimulation beta-cell proliferation, suppression norepinephrine blood plasma Level, induction variable force react and increase cardiac contractile force, (for example pass through intravenous, subcutaneous, intramuscular, abdomen by non-diet approach Film or other injections or infusion by way of) improve nutrition, pretreatment carry out endoscopy operation individual and regulation triglycerides The method of level, methods described includes formula (I) compound comprising effective dose that the present invention is applied to the individual or it can medicine With the preparation of salt.The individual is preferably mammal, more preferably primate, even more preferably from being people.

Brief description

Fig. 1 is depicted applies about 1mg [Aib to dog single SC (s.c.)^8,35]hGLP-1(7-36)NH₂The blood obtained afterwards Starch characteristic pattern (median).In each case, by peptide with the peptide comprising about 1% (weight/volume) and with about 1.5 peptide : the form of the aqueous Zn composition of Zn mol ratios is administered.Filled square and hollow square are represented wherein such as this paper institutes State the composition that pH is adjusted with NaOH；Black triangle represents the composition that wherein unused NaOH adjusts pH；Solid circles are represented The composition buffered with AcOH/AcO-.

Fig. 2 is depicted applies about 15mg [Aib to dog single SC (s.c.)^8,35]hGLP-1(7-36)NH₂The blood obtained afterwards Starch characteristic pattern (median).In each case, by peptide with the peptide comprising about 10% (weight/volume) and with about 1.5 Peptide: the form of the aqueous Zn composition of Zn mol ratios is administered.Filled square and hollow square are represented wherein as herein The composition that pH is adjusted with NaOH；Black triangle represents the composition that wherein unused NaOH adjusts pH；Solid circles generation The composition that table is buffered with AcOH/AcO-.

Fig. 3 is depicted applies about 1mg [Aib to dog single SC (s.c.)^8,35]hGLP-1(7-36)NH₂The blood obtained afterwards Starch characteristic pattern (median).In each case, peptide is administered in following semi-solid aqueous Zn composition form：It is solid Circle：The peptide of about 5% (weight/volume), peptide: Zn mol ratios are about 5.4: 1, no pH adjustment；Empty circles：About 10% (weight/ Volume) peptide, peptide: Zn mol ratios are about 5.4: 1, no pH adjustment；Hollow square：The peptide of about 10% (weight/volume), peptide: Zn mol ratios are about 5.4: 1, and pH is adjusted with NaOH；Filled square：The peptide of about 10% (weight/volume), peptide: Zn mol ratios are About 4: 1, adjust pH with NaOH.

Fig. 4 provides the schematic diagram for the various devices that can be used for preparing some preparations of the present invention.

Fig. 5 is depicted applies about 1mg [Aib to dog single SC (s.c.)^8,35]hGLP-1(7-36)NH₂The blood obtained afterwards Starch characteristic pattern (median).By peptide using peptide concentration as about 2% and peptide: the aqueous Zn composition form that Zn mol ratios are about 1.5: 1 It is administered.

Fig. 6 is depicted applies about 15mg [Aib to dog single SC (s.c.)^8,35]hGLP-1(7-36)NH₂The blood obtained afterwards Starch characteristic pattern (median).By peptide using peptide concentration as about 25% and peptide: the semi-solid Zn composition form that Zn mol ratios are about 4: 1 It is administered.

Fig. 7 is depicted applies about 15mg [Aib to dog single SC (s.c.)^8,35]hGLP-1(7-36)NH₂The blood obtained afterwards Starch characteristic pattern (median).By peptide using peptide concentration as about 23% and peptide: the semi-solid Zn composition shape that Zn mol ratios are about 1.5: 1 Formula is administered.

Fig. 8 is depicted applies 0.3mg (3 μ L 10% solution) following GLP-1 analogs to rat single SC (s.c.) The full-time journey plasma profile figure (median) obtained after hydrochloride test formulation：

(1)(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloride and CuCl₂:(Aib^8,35)hGLP-1(7-36)NH₂/CuCl₂'s Mol ratio is 1.5: 1.The peptide concentration is 10% (w/w) (30mM) in pH about 5.5 water.

(2)(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloride and ZnCl₂:(Aib^8,35)hGLP-1(7-36)NH₂/ZnCl₂'s Mol ratio is 1.5: 1.The peptide concentration is 10% (w/w) (30mM) in pH about 5.5 water.

Fig. 9 is depicted applies 0.3mg (3 μ L 10% solution) following GLP-1 analogs to rat single SC (s.c.) The full-time journey plasma profile figure (median) obtained after acetate test formulation：

(Aib^8,35)hGLP-1(7-36)NH₂Acetate and ZnCl₂:(Aib^8,35)hGLP-1(7-36)NH₂/ZnCl₂Rub Your ratio is 1.5: 1.The peptide concentration is 10% (w/w) (30mM) in pH about 5.5 water.

Figure 10 depicts the test applied to rat single SC (s.c.) shown in 0.3mg (3 μ L 10% solution) Fig. 8 The early stage plasma profile figure (median) obtained after preparation.

Figure 11 depicts the test applied to rat single SC (s.c.) shown in 0.3mg (3 μ L 10% solution) Fig. 9 The early stage plasma profile figure (median) obtained after preparation.

Figure 12 depicts three kinds applied to rat single SC (s.c.) shown in 0.3mg (3 μ L 10% solution) Fig. 8 Remaining (the Aib of injection position after test formulation^8,35)hGLP-1(7-36)NH₂Estimation percentage.

Detailed description of the invention

The preferred GLP-1 peptides of peptide salt as the present invention are expressed from the next herein, such as (Aib^8,35)hGLP-1 (7-36)NH₂, wherein the amino acid being replaced from native sequences is located at (such as Aib between first group of round parentheses^8,35Represent The Ala in hGLP-1 is replaced with Aib⁸And Gly³⁵).Aib is the abbreviation of α-aminoacid.Abbreviation GLP-1 means pancreas hyperglycaemia Plain sample peptide -1；HGLP-1 means human glucagon-like-peptide-1.Numeral between second group of round parentheses refers to being present in peptide Amino acid numbering (such as hGLP-1 (7-36) refers to the 7th to the 36th amino acid of people GLP-1 peptide sequence).hGLP-1 The sequence of (7-37) is listed in Mojsov, S., Int.J.Peptide Protein Res .40,1992, in the 333-342 pages. hGLP-1(7-36)NH₂In name " NH₂" represent that the C- ends of peptide are amidated.HGLP-1 (7-36) means that C- ends are trips From acid.In hGLP-1 (7-38), unless otherwise stated, the residue on the 37th and 38 is respectively Gly and Arg.

GLP-1 peptide analogues used are particularly preferably the form of officinal salt in the present invention.The example of this kind of salt includes But it is not limited to those and organic acid (such as acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, butanedioic acid, benzene first Acid, methanesulfonic acid, toluenesulfonic acid or pamoicacid), inorganic acid (such as hydrochloric acid, sulfuric acid or phosphoric acid) and polymeric acid (such as tannic acid, carboxylic Methylcellulose, PLA, polyglycolic acid or polylactic-co-glycolic acid) formed salt.Prepare the salt of the peptide of the present invention Standard method that typical method is well known in the art and can exchanged by salt is carried out.It therefore, it can lead to Cross and peptide is dissolved in a small amount of 0.25N acetic acid aqueous solutions and by the tfa salt of the peptide of the present invention (by being obtained using preparation HPLC purified peptide To tfa salt, eluted with the cushioning liquid containing TFA) change into another salt, such as acetate.The solution of gained is applied half- On preparation HPLC post (Zorbax, 300SB, C-8).Post is eluted with following solution：(1) 0.1N ammonium acetate solutions, 0.5 is small When；(2) 0.25N acetic acid aqueous solutions, 0.5 hour；(3) linear gradient (20% to 100% solution B, in 30 minutes), flow velocity (solution A is 0.25N acetic acid aqueous solutions within 4ml/ minutes；Solution B is acetic acid of the 0.25N in acetonitrile/water 80: 20).Collection contains The fraction of peptide and it is refrigerated to dry.

As well known to those skilled in the art, GLP-1 known and potential use can change and be a variety of It is various (referring to Todd, J.F. etc., Clinical Science, 1998,95, the 325-329 pages；And Todd, J.F. etc., European Journal of Clinical Investigation, 1997,27, the 533-536 pages).Therefore, it is sharp to cause The administration of compound of the invention for the purpose of dynamic agent effect can have and the effect of GLP-1 identicals itself and purposes.Can be with GLP-1 these variable purposes are summarized as follows, treated：Type i diabetes, type ii diabetes, obesity, glucagonoma, gas Road paracrisis, metabolic disorder, arthritis, osteoporosis, central nervous system disease, ISR, neurodegenerative disease, renal failure Exhaust, congestive heart failure, nephrotic syndrome, hepatic sclerosis, pulmonary edema, hypertension, wherein need reduce food intake obstacle And various other illnesss as described herein and obstacle.Therefore, the present invention includes formula in the range of it including defined herein (I) compound as active component pharmaceutical composition.

The dosage of active component can change in the preparation of the present invention；But, it is necessary that the amount of active component causes can Obtain suitable dosage.The dosage of selection depends on required therapeutic action, the duration using approach and treatment, and one As determined by attending doctor.In general, the active effective dose for the present invention is 1 × 10^-7To 200mg/kg/ days, it is excellent Select 1 × 10^-4To in the range of 100mg/kg/ days, as single dose or multiple dosage can be divided into it is administered.

The preferred parenteral administration of preparation of the present invention, such as intramuscular, intraperitoneal, intravenous, subcutaneous administration.

Preparation of the invention for parenteral administration includes sterile aqueous or non-aqueous solution, supensoid agent, gel Agent or emulsion, condition are to can reach required internal release characteristics.The example of non-aqueous solvent or medium has propane diols, poly- second two The organosilane ester such as ethyl oleate of alcohol, vegetable oil such as olive oil and corn oil, gelatin and injectable.This kind of formulation can also contain Assistant agent (adjuvant) such as preservative, wetting agent, emulsifying agent and dispersant.Can be for example, by the filter mistake through retaining bacterium Filter, by mixing bactericidal agent into composition, by irradiating composition or they being sterilized by heating combination.Also may be used So that they are made can be using the preceding sterile solid being dissolved at once in sterilized water or some other sterile injectable solvents Composition forms.

The synthesis of peptide

Synthetically prepared by standard solid phase peptide or it can be passed through standard solid-phase available for the peptide for implementing the present invention Peptide symthesis is made.For example, with reference to Stewart, J.M. etc.,Solid Phase Synthesis(Pierce Chemical Co., Second edition .1984).

The following example describes to can be used for or have been used for prepare can be advantageously carried out the synthesis of the peptide of the present invention with it Method, the synthetic method is well known to the skilled person.Other methods are also those skilled in the art's many institute's weeks Know.The purpose for providing embodiment is to be illustrated, and is not meant to limit the scope of the present invention in any way.

The peptide of such as GLP-1 analogs can be obtained by different synthesis well known by persons skilled in the art, described to close Into the final precipitation including peptide, freeze-drying process, vacuum drying or other drying processes known in the art.Ion in the present invention Displacement chromatography, the permeation-exchange of buffer solution and the peptide that desalination (difiltration) can be purifying or the different salt forms of selection Appropriate method.

Boc- β Ala-OH, Boc-D-Arg (Tos)-OH and Boc-D-Asp (OcHex) are purchased from NovaBiochem, and the Holy Land is sub- Brother, California.Boc-Aun-OH is purchased from Bachem, King ofPrussia, PA.Boc-Ava-OH and Boc-Ado-OH purchases From Chem-lmpex International, Wood Dale, IL.Boc-2NaI-OH is purchased from Synthetech, Inc. Austria Bhujerba Buddhist nun, OR.

The full name of other abbreviations used herein is as follows：Boc：Tertbutyloxycarbonyl；HF：Hydrogen fluoride；Fm：Formoxyl； Xan：Xanthyl；Bzl：Benzyl；Tos：Tosyl；DNP：2,4- dinitrophenyls；DMF：Dimethylformamide；DCM：Two Chloromethanes；HBTU：2- (1H- BTA -1- bases) -1,1,3,3- tetramethylurea hexafluorophosphate；DIEA：Diisopropyl second Base amine；HOAc：Acetic acid；TFA：Trifluoroacetic acid；2ClZ：2- benzyloxycarbonylchloride bases；2BrZ：2- bromo-benzyloxycarbonyls；OcHex：O-ring oneself Base；Fmoc：9- fluorenylmethyloxycarbonyls；HOBt：N- hydroxybenzotriazoles；PAM resins：4- hydroxymethyl phenyl acetylamino methyl trees Fat；Tris：Three (methylol) aminomethanes；And Bis-Tris：(i.e. 2- is double for double (2- ethoxys) amino-three (methylol) methane (2- ethoxys) amino -2- (methylol) -1,3-PD).Term " halo " or " halogen " include fluorine, chlorine, bromine and iodine.

Unless otherwise defined, otherwise all technologies used herein and scientific terminology have and the technical field of the invention The identical implication that those of ordinary skill is generally understood that.In addition, by all publications being mentioned above, patent application, patent and Other bibliography are incorporated herein by reference.

Embodiment 1

(Aib^8,35)hGLP-1(7-36)NH₂

(Aib^8,35)hGLP-1(7-36)NH₂Detailed synthetic procedure step in International Patent Publication No.WO 00/ There is provided in 34331 (PCT/EP99/09660), the content intact of the document is incorporated herein by reference.In short, Compound is synthesized on Applied Biosystems (Foster City, CA) 430A type peptide synthesizers, adjusts the instrument to enter Row accelerates the synthesis of Boc- chemical solid phases.Referring to Schnolzer etc., Int.J.Peptide Protein Res., 40：180 (1992).Using replace with 0.91mmol/g 4- methylbenzhydrylamines (MBHA) resin (Peninsula, Belmont, CA).Use Boc amino acid (Bachem, CA, Torrance, the CA protected with following side chain；NovaBiochem., 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-lle-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.Handled 2 × 1 minutes by using 100%TFA and remove Boc groups.Using in 4ml HBTU (2.0mmol) and DIEA (1.0ml) pre-activate Boc amino acid (2.5mmol) in DMF and not to peptide-resin TFA Salt be coupled in the case of formerly neutralizing.Coupling time is 5 minutes, except Boc-Aib-OH residues and following residue： Boc-Lys (2ClZ)-OH and Boc-His (DNP)-OH, wherein coupling time are 2 hours.

At the end of peptide chain is assembled, with solution of the 20% mercaptoethanol/10%DIEA in DMF by resin treatment 2 × 30 Minute to remove the DNP groups on His side chains.Then handled 2 × 2 minutes by using 100%TFA and remove N- terminal Boc groups. With in the DMF containing 10%DIEA and peptide-resin (1 × 1 minute) after, by using 15% monoethanolamine/15% water/70%DMF's Solution handles the formoxyl on the side chain for removing Trp for 2 × 30 minutes.Peptide-resin is washed with DMF and DCM and is done under reduced pressure It is dry.By the way that peptide-resin in the 10ml HF comprising 1ml anisoles and dithiothreitol (DTT) (24mg) is stirred to enter for 75 minutes at 0 DEG C The final cracking of row.HF is removed with nitrogen stream.Extract with ether (6 × 10ml) debris and with 4N HOAc (6 × 10ml) Take.

It is anti-phase using Reverse phase preparative high performance liquid chromatography (HPLC) applicationC₁₈Post (Nest Group, Southborough, MA) peptide mixer in purified aqueous extract.With linear gradient (20% to 50% solution B, 105 points Clock) with (solution A=water containing 0.1%TFA of the flow velocitys of 10ml/ minutes；Solution B=the acetonitrile containing 0.1%TFA) elution post.Receive Collect fraction and examined with analytic type HPLC.Merge fraction that those contain pure products and be refrigerated to dry.In the compound In one example of synthesis, 135mg white solids are obtained.It is 98.6% based on the purity that analytic type HPLC is analyzed.Electron spray Mass spectrograph (MS (ES)) S analyses obtain molecular weight for 3339.7 (consistent with the molecular weight 3339.7 of calculating).

Embodiment 2

Formulation operations step I

2.1 materials, stock solution is calculated

A) material：ZnCl₂, NaOH tablets and 35% hydrochloric acid are obtained from Panreac Quimica, Barcelona, western class Tooth.WFI (aseptic injection/filling scouring water) is obtained from B.Braun Medical, Barcelona, Spain.

B) stock solution

(i)ZnCl₂PH=3：

1. under agitation, 35% HCl is added in WFI to reach pH=3.

2. in volumetric flask, shift the ZnCl of weighed amount₂.Under agitation, pH=3HCl is added to reach about 1-4mg ZnCl₂/ ml final concentration.

(ii)ZnCl₂, pH=2

1. under agitation, 35% HCl is added in WFI to reach pH=2.

2. in volumetric flask, shift the ZnCl of weighed amount₂.Under agitation, pH=2HCl is added to reach about 4-12mg ZnCl₂/ ml final concentration.

(iii) NaOH, 0.1 to 10mg/ml：

1. in volumetric flask, shift the NaOH of weighed amount.Under agitation, WFII is added to reach about 0.1-10mg NaOH/ Ml final concentration.

(iv)Lyophilized 20mg aliquots (Aib^8,35)HGLP-1(7-36)NH₂/ bottle：

1. prepare 0.04% (v/v) acetic acid and WFI dilutions.

2. in volumetric flask, shift (the Aib of weighed amount^8,35)HGLP-1(7-36)NH₂(acetate).Under agitation, add 0.04% enough acetic acid is so that final concentration reaches 20mg (Aib^8,35)HGLP-1(7-36)NH₂/ml.Using 0.45 micron of filter After device is sterile filtered, the aliquot of the 1ml solution is transferred to lyophilized bottle, is freeze-dried, by dry product storage At -22 DEG C.

(v) the 50mg aliquots (Aib of freeze-drying^8,35)HGLP-1(7-36)NH₂/ bottle：

1. prepare 0.1% (v/v) acetic acid and WFI dilutions.

2. in volumetric flask, shift (the Aib of weighed amount^8,35)hGLP-1(7-36)NH₂(acetate).Under agitation, add 0.1% enough acetic acid is so that final concentration reaches 50mg (Aib^8,35)HGLP-1(7-36)NH₂/ml.After being sterile filtered, The aliquot of the 1ml solution is transferred to lyophilized bottle and cold dry.

C) calculate

(i) gross weight/volume of the excipient (E) for composition is determined：

E=(A × 100/T)-(A/P)

Wherein：

Excipient of the E=in terms of mg；

The content (mg) of the pure peptides of A=；

The aimed concn of T=compositions；For example, if target is 2%, the value is 2；And

The concentration (mg peptides/100mg preparations) of the pure peptides of P=.

On the cumulative volume of excipient, 1ml=1g hypothesis is used.

(ii) ZnCl added into every ml or g composition solutions is determined₂Volume/weight (W)：

A) for the composition wherein adjusted without pH, W=100%E；

B) for wherein peptide is about 1% or about 2% or up to about 10% and use alkali adjusts pH liquid preparation, W =80%E；

C) it is about 1% or about 2% or up to about 10% and using alkali adjustment pH semisolid or gel system for wherein peptide For agent, W=50%E；

D) for wherein peptide is about 25% and use alkali adjusts pH semisolid or gel preparation, W=66.66%E；

E) for wherein peptide is preparation reconstructed by lyophilized formulations and using alkali adjustment pH, W=90%E.

(iii) the NaOH volume/weight (W) added into every ml or g composition solutions is determined：

A) for wherein peptide is about 1% or about 2% or up to about 10% and use alkali adjusts pH preparation, W= 20%E；

B) it is about 1% or about 2% or up to about 10% and using alkali adjustment pH semisolid or gel system for wherein peptide For agent, W=50%E；

C) for wherein peptide is about 25% and use alkali adjusts pH semisolid or gel preparation, W=33.33%E；

D) for wherein peptide is preparation reconstructed by lyophilized formulations and using alkali adjustment pH, W=10%E.

(iv) is determined for the ZnCl in each composition₂Concentration (mg/ml or mg/g)：

[ZnCl₂136.29 × A of]=()/(W × 3339.76 × R)

Wherein：

The content (mg) of the pure peptides of A=

R=peptides/Zn mol ratio

For wherein peptide is about 1% or about 2% or about 10% or up to about 23% preparation, R=1.5；

For wherein peptide is about 25% preparation, R=4.0；And

The ZnCl added in the every g or ml composition solutions of W=₂The weight (g) or volume (ml) of solution.

2.2 have the peptide and ZnCl that 1-10% is freezed₂The preparation without the pH compositions adjusted

As it used herein, the preparation of the peptide comprising certain percentage, which describes each composition total weight, includes one Determine the preparation of the peptide of weight, such as 1% peptide describes the preparation that every 100g total compositions include 1g peptides.

It is following to prepare the preparation for including about 1% or about 2% up to about 10% peptide.By (the Aib prepared as described⁸ ^{, 35})HGLP-1(7-36)NH₂Lyophilized sample and pH 3 ZnCl₂Stock solution with 100% total excipient volume and [peptide: Zn]=be sufficiently mixed at 1.5: 1.

A the) (Aib lyophilized by mixing 20mg^8,35)HGLP-1(7-36)NH₂(seeing above 2.1B (iv)) and 2ml ZnCl₂Solution (0.272mg/ml；See above 2.1B (i)) prepare 1% composition；

B the) (Aib lyophilized by mixing 20mg^8,35)HGLP-1(7-36)NH₂(seeing above 2.1B (iv)) and 1ml ZnCl₂Solution (0.544mg/ml；See above 2.1B (i)) prepare 2% composition；

C the) (Aib lyophilized by mixing 50mg^8,35)HGLP-1(7-36)NH₂(seeing above 2.1B (v)) and 0.45ml ZnCl₂Solution (3.023mg/ml；See above 2.1B (i)) prepare 10% composition.

Make lyophilized peptide and solution equilibria to room temperature.By the ZnCl of designated volume₂Solution injection contains the small of lyophilized peptide Bottle, makes 1% or 2% peptide combinations aquation about 2 minutes, makes 10% peptide combinations aquation about 60 minutes, or until all lyophilized The agglomerate of peptide is free of in the complete aquation of peptide and solution.After aquation, the peptide of reconstruct is shaken about 1 minute.

The peptide that can be taken off appropriate dissolving is administered, for example, 100ul is equal to according to 1% peptide solutions prepared of A above 1mg dosage, 50ul is equal to 1mg dosage according to 2% peptide solutions prepared of B above, and 10% peptide that 150ul is prepared according to C above is molten Liquid is equal to 15mg dosage etc..

Using the teaching in the application, those skilled in the art can change peptide and ZnCl₂Amount to be detailed below 1%, 2% and 10% composition beyond composition and required dosage.

2.3 have the peptide and ZnCl that 1-10% is freezed₂Progress pH adjustment composition preparation

It is following to prepare the preparation for including about 1% or about 2% up to about 10% peptide.By (the Aib prepared as described⁸ ^{, 35})HGLP-1(7-36)NH₂Lyophilized sample and pH 3 ZnCl₂Stock solution is sufficiently mixed with 90% total excipient volume. Required pH value of solution is reached by adding dilute NaOH solution.

A the) (Aib lyophilized by mixing 20mg^8,35)HGLP-1(7-36)NH₂(seeing above 2.1B (iv)) and 1.8ml ZnCl₂Solution (seeing above 2.1B (i)) prepares 1% composition；

B the) (Aib lyophilized by mixing 20mg^8,35)HGLP-1(7-36)NH₂(seeing above 2.1B (iv)) and 0.9ml ZnCl₂Solution (seeing above 2.1B (i)) prepares 2% composition；

C the) (Aib lyophilized by mixing 50mg^8,35)HGLP-1(7-36)NH₂(seeing above 2.1B (v)) and 0.40ml ZnCl₂Solution (seeing above 2.1B (i)) prepares 10% composition.

Added into above-mentioned solution must volume (10% excipient cumulative volume) dilute NaOH solution to reach target rich Degree and pH.For example, for each case：

1% composition：Add the NaOH solution of 0.2ml suitable concns

2% composition：Add the NaOH solution of 0.1ml suitable concns

10% composition：Add the NaOH solution of 0.05ml suitable concns

Using the teaching in the application, those skilled in the art can change peptide and ZnCl₂Amount to be detailed below 1%, 2% and 10% composition beyond composition.

2.4 have 1-10% peptides and ZnCl₂The preparation without the pH fluid compositions adjusted

It is following to prepare the liquid preparation for including about 1% or about 2% up to about 10% peptide.Weigh (Aib^8,35)HGLP-1(7- 36)NH₂Sample and with pH 3 ZnCl₂Stock solution mixes to reach the aimed concn of 1%, 2%, up to 10% peptide.Mixed After conjunction, composition is sterile filtered, stored for future use.

2.5 have 1-10% peptides and ZnCl₂Progress pH adjustment fluid composition preparation

It is following to prepare the liquid preparation for including about 1% or about 2% up to about 10% peptide.Weigh (Aib^8,35)HGLP-1(7- 36)NH₂Sample and with pH 3 ZnCl₂Stock solution is sufficiently mixed with 80% total excipient volume.Zinc solution can be ZnCl₂Or ZnAc2.2H2O.The required pH of solution is reached by adding dilute NaOH solution.Make in this way that preparation of preparation C5 is extremely C13。

Using the teaching in the application, those skilled in the art can change peptide and ZnCl₂Amount it is described herein to obtain 1%, 2% and 10% beyond composition.

2.6 have 25% peptide and ZnCl₂The preparation without the pH semisolid/gel combinations adjusted

It is following to prepare the semisolid or gel preparation for including about 25% peptide.Weigh (Aib^8,35)HGLP-1(7-36)NH₂Sample And with pH 2 ZnCl₂Stock solution is sufficiently mixed with 66.66% total excipient volume.Zinc solution can be ZnCl₂Or ZnAc₂.2H₂O.Make preparation of preparation C1 and C2 in this way.

More specifically, preparing semi-solid or gel combination using " push-pull (push-pull) " mixing method：

A) the desired amount of peptide is weighed into the disposable note for being mounted with special two-way hand-operated valve HV (I.D.=0.5mm) in advance Emitter S1 post bucket and put the tube into syringe Luer holes；

B) syringe plunger is fixed using stainless steel SR；

C) HV in S1 is connected with pumping source and open HV.After 10 minutes, HV is closed；

D) zinc solution precise is entered to second disposable syringe S2 post bucket；

E) and then connect S2 and HV free portion；

F) HV is opened, solvent is pumped into the post bucket comprising peptide powder S1 with vacuum；

G) HV is closed, solvent injection device S2 is taken out, so that the peptide powder in aquation S1；

H) SR is taken out, syringe plunger is slowly discharged；

I) the mobile syringe plunger (push and pull) in the case where not opening HV, so that powder material is soaked completely by solvent It is wet；

J) two-way stainless steel connector SC (I.D.=1.0mm) is put into the note with the pipe being located in syringe Luer holes End is pushed into emitter S2 and by its plunger；

K) HV in S1 is opened to discharge vacuum, then takes out HV.Make syringe plunger movement so as to by syringe post bucket In air reduce to bottom line；With

L) by SC connection S1 and S2 and composition is made to be mediated from S1 by SC to S2.

Using the teaching in the application, those skilled in the art can change peptide and ZnCl₂Amount it is described herein to obtain 25% beyond composition.

2.7 have 25% peptide and ZnCl₂Progress pH adjustment semisolid/gel combination preparation

It is following to prepare the semisolid or gel preparation for including about 25% peptide.Weigh (Aib^8,35)HGLP-1(7-36)NH₂Sample And with pH 2 ZnCl₂Stock solution is sufficiently mixed with 66.66% total excipient volume.Zinc solution can be ZnCl₂Or ZnAc₂·2H₂O.Required pH value of solution is reached by adding dilute NaOH solution.In the present embodiment, it is necessary in zinc and NaOH solution Between segmentation be added to total liquid volume in powder.Therefore, the concentration of zinc solution is adjusted, so as to the totality of required zinc solution Product is down to 50% (the step d) added to total liquid product in peptide powder.As described detail below to being added in peptide powder Residue 50% total liquid volume in add NaOH solution.Make preparation of preparation C3 and C4 in this way.

The semisolid or gel combination for carrying out pH adjustment are prepared using " push-pull " mixing method：

B) syringe plunger is fixed using stainless steel SR；

E) and then connect S2 and HV free portion；

H) SR is taken out, syringe plunger is slowly discharged；

K) HV in S1 is opened to discharge vacuum, then takes out HV.Make syringe plunger movement so as to by syringe post bucket In air reduce to bottom line；

M) after homogenizing, take out the aliquot of mix products to determine the concentration of peptide；

N) amount of the NaOH solution needed for the remaining intermediate host product of precise and calculating reaches needed for pH；

O) NaOH solution precise is entered into the 3rd disposable syringe S3；With

P) syringe plunger is slowly compressed to reduce the air in syringe cavity to bottom line.Pass through SC connections two Branch syringe and composition is mediated by SC.

Table 1

^*It show desired value.In all situations, actual value is in the range of the 5% of desired value.

^*It show desired value.In all situations, actual value is in the range of the 10% of desired value.

The measure of 3.0GLP-1 receptor affinities

Following operative step test can be used to can be used for the ability for implementing the compound combination GLP-1 acceptors of the present invention.

Cell culture：

The culture expression GLP-1 acceptors in the Dulbecco improvement Eagle culture mediums (DMEM) containing 10% hyclone RIN 5F rat insulins oncocyte (ATCC-#CRL-2058, American Type Culture collection (American Type Culture Collection), Manassas, VA), it is maintained into 5%CO in about 37 DEG C₂The humidification atmosphere of/95% air In.

Radioligand is combined：

By using Brinkman Polytron (Westbury, NY) (setting 6,15 seconds) in 50mM ice-cold 20ml RIN cells are homogenized in Tris-HCl and prepare film for radioligand binding.By centrifuging (39,000g/10 minutes) Thing will be homogenized to wash twice and final precipitation is suspended in containing 2.5mM MgCl again₂, 0.1mg/ml bacitracins In (Sigma Chemical, Saint Louis, MO) and 0.1%BSA 50mM Tris-HCl.In order to be measured, by aliquot (0.4ml) and 0.05nM (¹²⁵I) GLP-1 (7-36) (~2200Ci/mmol, New England Nuclear, Boston, MA) It is incubated together in the presence of the unlabelled competitive test peptides with and without 0.05ml.After 100 minutes are incubated (25 DEG C), pass through GF/C filters (Brandel, Gaithersburg, MD) fast filtering through being soaked in advance in 0.5% polyethyleneimine will be combined (¹²⁵I) GLP-1 (7-36) with it is free (¹²⁵I) GLP-1 (7-36) is separated.Then with 50mM Tris-HCl's ice-cold 5ml Aliquot washs filter 3 times and by γ spectrometries (Wallac LKB, Gaithersburg, MD) to being trapped in filter On the radioactivity of combination counted.By specifically bind be defined as combine it is total (¹²⁵I) GLP-1 (7-36) is subtracted and had Combined in the presence of 1000nM GLP1 (7-36) (Bachem, Torrence, CA) (¹²⁵I)GLP-1(7-36)。

4. the measure of solubility and pH relations

4.1. in phosphate buffered saline solution (PBS) compound solubility and pH relations measure

Following operating procedure test can be used to be advantageously used for implementing the compound of the present invention determining it in difference Solubility of the pH with a temperature of in PBS.

By being pre-mixed powder (SIGMA, production code member by 1 bag：P-3813 1 liter of deionized water) is dissolved in be included 138mM NaCl, 2.7mM KCl and pH prepare PBS buffer stock solution for 7.4 10mM phosphate buffered salines.By using phosphorus Acid and/or sodium hydroxide adjust the pH of the stock solution to prepare the PBS with different pH value.

The compound of compound sample to be tested 2mg, such as 2mg embodiments 1 can be weighed in vial.To The aliquot of the 50ul PBSs added in each bottle under certain pH.Solution is vortexed, if necessary, entered Row sonication, until clarification.To each test pH, the cumulative volume of the buffer solution needed for record dissolving 2mg compounds and calculating Solubility.

The peptide solution clarified under room temperature (20-25 DEG C) is put into refrigerating box (4 DEG C) overnight, then checks peptide at 4 DEG C Solubility.

4.2. in salt solution compound solubility and pH relations measure

Following operating procedure test can be used to be advantageously used for implementing the compound of the present invention determining it in difference Solubility of the pH value with a temperature of in salt solution.

Salt solution stock solution is prepared by the way that 9g NaCl are dissolved in into 1 liter of deionized water.It can be adjusted by using HCl and/or NaOH The pH of the whole stock solution prepares the saline solution with different pH value.

The compound of compound sample to be tested 2mg, such as 2mg embodiments 1 can be weighed in vial.To The aliquot of the 50ul saline solutions added in each bottle under certain pH.Bottle is vortexed, if necessary, carried out Sonication, until clarification.To each test pH, the cumulative volume of the salt solution needed for record dissolving 2mg compounds and dissolving is calculated Degree.

4.3. the measure of solubility of the compound in pH 7 salt solution

Following operating procedure test can be used to be advantageously used for implementing the compound of the present invention determining it in room temperature Under solubility in pH=7 salt solution.

Saline solution is prepared by the way that 9g NaCl are dissolved in into 1 liter of deionized water.Can be by compound sample to be tested 2mg The compound of product, such as embodiment 1 weighs into vial and adds the aliquot of 1ml salt solution, at the same carry out be vortexed and Sonication, until clarification.Record the cumulative volume of salt solution for dissolving 2mg peptides and calculate solubility at room temperature.

4.4. the measure of solubility of the compound in different pH salt solution

Following operating procedure test can be used to be advantageously used for implementing the compound of the present invention determining it in room temperature Under solubility in the saline solution with different pH.

Salt solution stock solution is prepared by the way that 9g NaCl are dissolved in into 1 liter of deionized water.Should by using HCl and NaOH processing The aliquot of salt solution stock solution obtains the saline solution with different pH value.

The compound of compound sample to be tested 2mg, such as embodiment 1 can be weighed into vial.Add The aliquot of brine buffer solutions of the 50ul under certain pH.Solution is vortexed and sonication is carried out, until clarification.Note Record dissolves the cumulative volume of the buffer solution used in 2mg peptides and calculates solubility.

5. the water solubility of compound and the measure of zinc concentration relation

Following operating procedure test can be used to be advantageously used for implementing the compound of the present invention determining it in difference Solubility under zinc concentration in pH 7 water.

By by ZnCl₂Deionized water is dissolved in adjust pH to 2.7 to 100mg/ml concentration and using HCl to prepare Zinc stock solution.Prepared by preparing the suitable dilution of stock solution with different ZnCl₂Solution (" Zn tests of concentration Solution ").

The compound of compound to be tested 1mg, such as 1mg embodiments 1 is dissolved in each Zn test solution of 250ul to obtain Solution with 4mg/ml compounds.Then the pH of the solution is adjusted using 0.2N NaOH, until observing white precipitate shape Into.Centrifugation solution and use HPLC analysis mother liquors.The UV absorption areas at test compound peak are measured, by bent with correction Line compares the concentration for determining test compound in mother liquor.

As the representative example available for the compound for implementing the present invention, tested in the determination method being described immediately above The compound of embodiment 1, obtains following result (saline solution, pH 7.0, room temperature)：

Table 2 ZnCl₂Concentration solubility

(μg/ml) (mg/ml)

0 5.788

80 0.0770

500 0.0579

1000 0.0487

1500 0.0668

2500 0.1131

6. use IEF gel determinations isoelectric point (pI)

Invitrogen ' s Novex IEF pH3-10 gels can be used for determining GLP-1 peptide (such as chemical combination of embodiment 1 Thing) pI.It is 0.5mg/ml that Peptidyl compounds to be tested are dissolved in into water to concentration.For each this kind of compound, by 5ul Resulting solution and 5ulSample buffer 2X is (by 20mM arginine free alkali, 20mM lysine free-bases and 15% Glycerine constitute) mixing, by the 10ul sample solutions of gained together with protein standards sample load on gel.

Electrophoretic buffer is obtained also from Invitrogen and runs gel according to the explanation of manufacturer, general as follows：100V Constant 1 hour, constant 1 hour of subsequent 200V, constant 30 minutes of subsequent 500V.

Then gel is fixed 30 minutes in the 12%TCA comprising 3.5% sulfosalicylic acid, then uses colloidal Comassie Hereafter blue (Colloidal Coomassie Blue) according to usingThe explanation dye that ColloidalBlue Kit are set up Color 2 hours, then takes off stained over night in water.

Gel is scanned and analyzed with program Fragment Analysis 1.2.Relative to as follows The pI of the n-compound of pI values calculates the pI of unknown peptide：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 of the compound determination of embodiment 1 is 7.60.

7. the in vivoassay of rat

The composition of the following determination method test present invention can be used to promote in vivo and humidification to determine them Ability.

7.1. laboratory operating procedures：

In experiment the previous day, to the bull Sprague- for weighing about 300-350g under chlorohydrate anesthesia Dawley rats (Taconic, Germantown, NY) are implanted into atrium dextrum jugular vein sleeve pipe.Then by Rat Fast 18 hours, this Appropriate test composition or vehicle-control is injected in 0 time afterwards.Continue to make Rat Fast in whole experiment process.

By by 100mg/ml ZnCl₂Solution is diluted to prepare 0.5mg/ in the HCl/water solution with pH 2.7 ml ZnCl₂Solution.By 1mg formulas (I) compound ((Aib^8,35)hGLP-1(7-36)NH₂) be dissolved in 250 μ l solution to obtain 4 times settled solutions with 4mg/ml compounds and 0.5mg/ml Zn of pH.

In 0 time, subcutaneous rat (sc) injection is given：(a) (the Aib being described immediately above^8,35)hGLP-1(7-36)NH₂It is molten Liquid or vehicle-control.In both cases, volume injected is very small (4-6 μ l), is applied to the agent of the GLP-1 compounds of individual Measure as 75 μ g/kg.The reasonable time after being injected with sc, by the intravenous μ l blood samples of (iv) cannula withdrawal 500 and gives rat Intravenous glucose is attacked to test whether to exist enhanced insulin secretion.The time of glucose challenge is after compound injection 0.25th, 1,6,12 and 24 hours.After initial blood sample is extracted, intravenous injection glucose (1g/kg) and with 500 μ l heparin Salt dissolving water (10U/ml) is rinsed.500 μ l blood samples are extracted when hereafter, 2.5 after glucose injection, 5,10 and 20 minutes.At these 500 μ l heparinized salines (10U/ml) are injected intravenously by sleeve pipe at once after each.Centrifugation of blood samples, is adopted from each sample Collect blood plasma and sample is stored in 20 DEG C, in case detecting their insulin content.Inhaled using rat insulin enzyme linked immunological Attached measure (ELISA) kit (American Laboratory Products Co., Windham, NH) determines each sample In amount of insulin.

7.1.1. result：

It was observed that lasting insulin enhancing activity, it can be lured by the glucose injection during whole 24 hours of this experiment Lead.

8. the in vivoassay of dog

There are many in vivoassay methods known in the art to enable those skilled in the art to determine composition and promote activation Compound extends the ability of release in vivo.

8.1.1% peptide combinations：

As example, prepare in ZnCl₂Cushioning liquid in include the aqueous test formulation of 1% (w/w) formula (I) compound (peptide: Zn ratio=1.5: 1.0).

Maintain the age the 14-21kg body weight of 42-78 months 6 males of total than brother (Beagle) dog free water simultaneously And once (the dry standard diets of about 400g (SAFE 125) are fed daily.By dog fasting 18 hours, test composition is then applied.

Test compound is applied in interscapular region by subcutaneous route.The Terumo for having 0.33-12mm with 0.3ml is injected Applied volume (about 20 microlitres/animal) is made in device (BS=30M2913).Thus the theoretical dose of about 0.2mg peptides is reached.

After application about=0, the time of 8,15,30,45 minutes and 1,2,4,8 and 12 hour and 1,2,3,4,5 and 6 day Taken at regular intervals blood sample.Snap frozen blood after sampling, in case centrifugation, decanted plasma and the snap frozen during determining.From Peptide plasma concentration is measured after line solid phase extractions, then carries out extracting with the united online phases of LC-MS/MS, uses Analyst The data that v1.2 software processings are obtained.

Composition shows the extension release of the active peptide of at least 2 days.

8.2.1% (Aib^8,35)hGLP-1(7-36)NH₂) solution：

Using the identical in vivoassay operating procedure described in 8.1 sections substantially with more than, check that following combination thing is prolonging The ability of tested peptide is discharged in the long time.For each in following 4 kinds of compositions, the concentration of peptide is about 1% (wt/wt), the ratio of peptide and zinc is about 1.5: 1, and the peptide dosage of administration is about 1mg.

Solution 8.2.A：Containing (i) 90%ZnCl₂(0.298mg/ml) and (ii) 10%NaOH's (0.975mg/ml) (Aib in solution^8,35)hGLP-1(7-36)NH₂；

Solution 8.2.B：In ZnCl₂(Aib in solution (0.286mg/ml)^8,35)hGLP-1(7-36)NH₂；

Solution 8.2.C：It is substantially similar with solution 8.2.B and use AcOH/AcO^-Buffering

Solution 8.2.D：It is substantially similar with solution 8.2.A.

Composition provides (Aib^8,35)hGLP-1(7-36)NH₂Extension release, as shown in fig. 1.

8.3.1% (Aib^8,35)hGLP-1(7-36)NH₂) solution：

Using the identical in vivoassay operating procedure described in 8.1 sections substantially with more than, check that following combination thing is prolonging The ability of tested peptide is discharged in the long time.For following composition, the concentration of peptide is about 2% (wt/wt), peptide and zinc ratio It is about 1.5: 1, the peptide dosage of administration is about 1mg.

Solution 8.3.：Containing (i) 80%ZnCl₂(0.695mg/ml) and (ii) 20%NaOH's (1.75mg/ml) is molten (Aib in liquid^8,35)hGLP-1(7-36)NH₂。

Composition provides (Aib^8,35)hGLP-1(7-36)NH₂Extension release, as shown in Figure 5.

8.4.10% peptide solution：

Using the identical in vivoassay operating procedure described in 8.1 sections substantially with more than, check that following combination thing is prolonging The ability of tested peptide is discharged in the long time.For each in following 4 kinds of compositions, the concentration of peptide is about 10% (wt/wt), the ratio of peptide and zinc is about 1.5: 1, and the peptide dosage of administration is about 15mg.

Solution 8.4.A：Containing (i) 90%ZnCl₂(3.367mg/ml) and (ii) 10%NaOH's (5.01mg/ml) is molten (Aib in liquid^8,35)hGLP-1(7-36)NH₂；

Solution 8.4.B：In ZnCl₂(Aib in solution (2.993mg/ml)^8,35)hGLP-1(7-36)NH₂；

Solution 8.4.C：It is substantially similar with solution 8.4.B and use AcOH/AcO^-Buffering

Solution 8.4.D：It is substantially similar with solution 8.4.A.

Composition provides (Aib^8,35)hGLP-1(7-36)NH₂Extension release, as shown in Figure 2.

8.5. semi-solid combination：

Using the identical in vivoassay operating procedure described in 8.1 sections substantially with more than, following semi-solid composition is checked Thing discharges the ability of tested peptide in the time of extension.For composition 8.5.A., the concentration of peptide is about 5%, and is just combined For thing 8.5.B, 8.4.C and 8.5.D., the concentration of peptide is about 10% (wt/wt).Composition 8.5.A, 8.5.B and 8.5.C's The ratio of peptide and zinc is about 5.4: 1, and for composition 8.5.D, the ratio is about 4.0: 1.For all 4 kinds of compositions, The peptide dosage of administration is about 1mg.

Solution 8.5.A：Containing the ZnCl in WFI₂(Aib in the semi-solid combination of (0.40mg/ml)^8,35) hGLP-1(7-36)NH₂。

Solution 8.5.B：It is substantially similar with composition 8.5.A, wherein ZnCL2 concentration is adjusted upward to maintain peptide: Zn Ratio be about 5.4: 1.

Solution 8.5.C：Containing (i) 50%ZnCl₂(1.69mg/ml) and (ii) 50%NaOH (1mg/ml) semisolid In (Aib^8,35)hGLP-1(7-36)NH₂。

Solution 8.5.D：Containing (i) 50%ZnCl₂(2.28mg/ml) and (ii) 50%NaOH (1mg/ml) semisolid In (Aib^8,35)hGLP-1(7-36)NH₂。

Composition provides (Aib^8,35)hGLP-1(7-36)NH₂Extension release, as shown in Figure 3.

8.6. semi-solid combination：

Using the identical in vivoassay operating procedure described in 8.1 sections substantially with more than, following semi-solid composition is checked Thing discharges the ability of tested peptide in the time of extension.Use 5.22mg/ml ZnCl₂Solution prepares combination under pH=2.0 Thing.Enough peptides are provided to produce the semi-solid combination of the peptide and 25% peptide of the ratio between zinc with about 4: 1.As provided herein Like that, the pH of composition is adjusted using 10mg/ml NaOH.The peptide dosage of administration is about 15mg.

Composition 8.6 provides (Aib^8,35)hGLP-1(7-36)NH₂Extension release, as shown in Figure 6.

8.7. semi-solid combination：

Using the identical in vivoassay operating procedure described in 8.1 sections substantially with more than, following semi-solid composition is checked Thing discharges the ability of tested peptide in the time of extension.Use 8.5mg/ml ZnCl₂Solution compositions formulated under pH=2.0. Enough peptides are provided to produce the semi-solid combination of the peptide and 23% peptide of the ratio between zinc with about 1.5: 1.2.6 save more than The method compositions formulated of middle detailed description.The peptide dosage of administration is about 15mg (equivalent to about 65 microlitres of compositions).

Composition 8.6 provides (Aib^8,35)hGLP-1(7-36)NH₂Extension release, as shown in Figure 7.

The other measure carried out with the various variants of disclosed preparation equally carries out in vivoassay and had been acknowledged The composition of the present invention provides useful medicine delivery platform for formula (I) compound.Use teachings of the present application, this area skill Art personnel can change peptide, ZnCl₂Amount and pH to prepare the composition of invention as described herein.

Embodiment 9

1. PK distribution characters are adjusted by the Determination of Ac in 10% peptide solution.

It this embodiment disclose and applied with the stoichiometric level single SC of 15mg/ dogs comprising 10% (Aib^8,35)hGLP1 (7-36)NH₂With zinc chloride [(Aib^8,35)hGLP1(7-36)NH₂: two kinds of occasional combination things Zn=1.5: 1] After (extemporaneous composition), male is than (Aib in brother (Beagle) dog^8,35)hGLP1(7-36)NH₂Medicine Thing dynamics research.

The method for carrying out internal test is identical with disclosed in the 8.1st section.

This example illustrates PK distribution characters are adjusted by the Determination of Ac in pharmaceutical composition, thus in pH The ratio of [acetate/peptide] in upper influence pharmaceutical composition.

PH regulation is controlled by way of adjusting Determination of Ac, the Determination of Ac of reduction shows make increased to pH With.

The change of acetate is also shown to C_maxEffect.In general, the Determination of Ac reduction C of reduction_maxValue.

Increased Determination of Ac shows the improvement to solubility and physical stability.

According to the selection of preparation, by adjusting the ratio of acetate/peptide to solubility or stability (such as C_max) improvement Compensated by the ratio for adjusting peptide/Zn.This, which can see, adjusts stability, solubility, pH or C_maxSystem, the system have three Individual variable.

In this embodiment, abbreviation SD represents standard deviation.AUC means the sweet wormwood vegetarian noodles under plasma concentration v. time curve Product.

Abbreviation MRT be meant that average residence time (MRT), this be assess bioavailability speed parameter, with than Compared with MRT and tmax (time of peak drug concentration).MRTt uses data during 0 from last loading time to calculate.

Table 3 has concentrated the result of the 10% peptide combinations batch with different [acetate/peptide] ratios and more subcutaneous than brother dog The result of administration.For [acetate/peptide] mol ratio of [3.7: 1], Peak drug plasma concentration value (C_max) it is 8.10ng/ml (SD=1.80ng/ml), and with the C of the batch offer 5.65ng/ml (SD=2.61ng/ml) compared with low-ratio [3.2: 1]_max Value.

Table 3

Preparation 10%15mg 10%15mg

Peptide/Zn ratios 1.5: 1 1.5: 1

10.GLP-1 peptides salt/divalent metal preparation

10.1. method

Prepare (Aib^8,35)hGLP-1(7-36)NH₂The 1mg/mL aqueous solution and PBS solution, and pH is adjusted to 7.0.System Standby CaCl₂、CuCl₂、MgCl₂And ZnCl₂10mg/mL liquid storages in water.CaCl₂、MgCl₂And ZnCl₂The pH of solution adjust to 7.0。CuCl₂The pH of solution can not alkalize, because Cu is precipitated.Therefore, using pH 4.4 CuCl₂Solution.

4 μ L aqueous metallic ions or PBS solution are added to 200 μ L (Aib^8,35)hGLP-1(7-36)NH₂1mg/mL it is molten In liquid, to produce 200 μ g/mL final concentration of metal ions.The solution that is mixed to get simultaneously checks precipitation.If forming precipitation, Then centrifuged suspension.(Aib in suspension^8,35)hGLP-1(7-36)NH₂Concentration determined by HPLC.

10.2. result

There is (the Aib of bivalent metal ion in table 4.^8,35)hGLP-1(7-36)NH₂Solubility

	The 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.(Aib^8,35)hGLP-1(7-36)NH₂The pharmacokinetic study of/divalent metal pH5.5 settled solution preparations

Three kinds of different (Aib are prepared using following steps^8,35)hGLP-1(7-36)NH₂Preparation：

(1)(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloride and CuCl₂

(2)(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloride and ZnCl₂

(3)(Aib^8,35)hGLP-1(7-36)NH₂Acetate and ZnCl₂

(tfa salt is obtained from the peptide purified using preparation HPLC to the tfa salt of GLP-1 analogs, with the buffer solution containing TFA Elution) another salt, such as acetate can be converted into, this is by the way that peptide is dissolved in a small amount of 0.25N acetic acid aqueous solutions.Obtain Solution is applied in half preparation HPLC column (Zorbax, 300SB, C-8).The post is small with the elution 0.5 of (1) 0.1N ammonium acetate solutions When, (2) 0.25N acetic acid aqueous solutions are eluted 0.5 hour, and (20% to 100% solution B in 30 minutes, solution A is with (3) 0.25N acetic acid aqueous solutions；Solution B is the 0.25N acetic acid in acetonitrile/water, 80: 20) is washed with the linear gradient of 4ml/ minutes flow velocitys It is de-.Collect the fraction containing peptide and freeze to drying.

(Aib is prepared by step of freeze drying^8,35)hGLP-1(7-36)NH₂Hydrochloride.20mg(Aib^8,35)hGLP-1(7-36) NH₂Acetic acid is in 4mL 20mM HCl/water solution and incubation at room temperature 10 minutes.Frozen samples are simultaneously freezed overnight.Freeze again Carry out twice and determine the chloride content of final product.The chloride content of determination is 5.38%.

(1)(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloride and CuCl₂：

(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloric acid 5.3mg (peptide content is 95%) is dissolved in 50 μ L 20mM CuCl₂Water In solution.With about1N NaOH regulation pH to about 5.5.

(Aib^8,35)hGLP-1(7-36)NH₂/CuCl₂Mol ratio be 1.5: 1.The concentration of peptide is 10% (w/w) in water (30mM), pH is about 5.5.

(2)(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloride and ZnCl₂：

(Aib^8,35)hGLP-1(7-36)NH₂Hydrochloric acid 5.3mg (peptide content is 95%) is dissolved in 50 μ L 20mM ZnCl₂Water In solution.With about1N NaOH regulation pH to about 5.5.

(Aib^8,35)hGLP-1(7-36)NH₂/ZnCl₂Mol ratio be 1.5: 1.The concentration of peptide is 10% (w/w) in water (30mM), pH is about 5.5.

(3)(Aib^8,35)hGLP-1(7-36)NH₂Acetate and ZnCl₂：

(Aib^8,35)hGLP-1(7-36)NH₂Acetic acid 5.5mg (peptide content is 92%) is dissolved in 50 μ L 20mM ZnCl₂Water In solution.Obtained solution lyophilized overnight is simultaneously re-dissolved in 50 μ L water.PH to about 5.5 is adjusted with about 1 μ L 1N NaOH. (Aib^8,35)hGLP-1(7-36)NH₂/ZnCl₂Mol ratio be 1.5: 1.The concentration of peptide is 10% (w/w) (30mM), pH in water It is about 5.5.

10.4. apply and blood sample collection

These three (Aib are applied to subcutaneous rat with 0.3mg/ rats (3 μ L 10% solution)^8,35)hGLP-1(7-36)NH₂ Preparation.The 5th, 10,15,30 minutes；1st, 2,4,8 hours and 1,2,3,4,7,10 days collect blood sample.By centrifuging from blood Collect blood plasma and be stored in -80 DEG C.Also collect the tissue of injection site, be homogenized in 5x methanol, and be stored in -80 DEG C.

Two rats are used for the 5th, 10,15,30 minutes and 1,2,4, the data point of 8 hours.One rat is used for 1,2,3, 4th, 7, the data point of 10 days.

10.5.LC-MS/MS sample preparation

With 10 μ L formic acid acidifying blood plasma (200 μ L) and with 600 μ L acetonitrile precipitations.Supernatant is collected by centrifugation and is concentrated in vacuo to dry It is dry.30% acetonitrile that residue is dissolved in 150 μ L water is simultaneously centrifuged.50 μ L of supernatant are injected to analyze for LC-MS/MS.

30% acetonitrile and 50 μ L of injection that tissue methanolic extract (10 μ L) is diluted in 1mL water are for LC-MS/MS points Analysis.

10.6.LC-MS/MS analyze

LC-MS/MS analyses are carried out using the API4000 mass spectrometer systems for being equipped with Turbo Ionspray detectors.Use The molecular ion monitoring of MRM patterns with ion pair 668.9 and 136.1.

The HPLC separation μ posts of (2) 2 × 30mm of Luna C8 3, were transported for 0.30mL/ minutes with flow velocity in 10 minutes from 10%B Go to 90%B and carry out.Buffer A is 1% formic acid in water, and buffer B is 1% formic acid in acetonitrile.

LOQ is 0.5ng/mL.

10.7. result and summary

Its plasma concentration is calculated with the standard calibration curve figure of peptide.0.06mg/mL(Aib^8,35)hGLP-1(7-36)NH₂ (0.3mg/ rats, in 5mL methanolic extracts) is used as 100% to calculate the remaining percentage of injection site.

(the Aib of table 5.^8,35)hGLP-1(7-36)NH₂Plasma concentration

(Aib^8,35)hGLP-1(7-36)NH₂The full-time journeys of HCl preparation pharmacokinetic properties be illustrated in Fig. 8. (Aib^8,35)hGLP-1(7-36)NH₂Early stages of HCl preparation pharmacokinetic properties be illustrated in Fig. 9.(Aib^8,35)hGLP- 1(7-36)NH₂The full-time journeys of acetic acid salt preparation pharmacokinetic properties be illustrated in Figure 10.(Aib^8,35)hGLP-1(7-36)NH₂ Early stages of acetic acid salt preparation pharmacokinetic properties be illustrated in Figure 11.

The injection site of table 6. residue (Aib^8,35)hGLP-1(7-36)NH₂Estimation percentage

(Aib^8,35)hGLP-1(7-36)NH₂Tissue accumulation feature in injection site is further illustrated in Figure 12.

Table 7.PK parameters

As a result show that there is provided with reduction for the salt form of GLP-1 analogs, the especially salt form of joint divalent metal salt Initial plasma concentration acceptable extended release preparation, it can reduce or eliminate undesired side effect.

The hydrochloride of data display strong acid salt, such as GLP-1 analogs, shows the initial plasma concentration further reduced. It is not bound by this theoretical, it is believed that the preferably initial plasma concentration of reduction GLP-1 analog hydrochlorides and internal neutralization Cheng Xiangguan.It is more than pH 5.5 in composition (1) and (2), 100% acid is chloride form, not free acid.Therefore, After hypodermic injection, body fluid can neutralize the solution faster, and the solution is thus precipitated faster.These reductions for neutralizing the time are led Cause smaller, more inapparent initial plasma concentration or initial plasma peak.

The publication of above-mentioned reference is incorporated herein by reference.Other embodiments of the present invention from foregoing disclosure be it is aobvious and It is clear to, and is intended to completely be described to cover with the invention that appended claim is defined herein.

Claims

1. comprising GLP-1 analogs [Aib^8,35]hGLP-1(7-36)NH₂Pharmaceutical composition, it can medicine by the analog Prepared together with the mixture of salt and analog, described pharmaceutical composition is also included in divalent metal salt, described pharmaceutical composition The officinal salt of analog and the mol ratio of analog, and the like can be adjusted with the mol ratio of the divalent metal salt, With the releasing effect of the solubility, pH and the internal release characteristics that adjust the peptide of this in described pharmaceutical composition,

The scope of the mol ratio of GLP-1 analogs and the divalent metal salt is 5.4: 1 wherein described in described pharmaceutical composition To 1.5: 1,

Wherein described [Aib^8,35]hGLP-1(7-36)NH₂The molar ratio ranges of officinal salt and GLP-1 peptide analogues be 0.5: 1 to 10: 1,

Wherein described [Aib^8,35]hGLP-1(7-36)NH₂Officinal salt be acetic acid or hydrochloric acid officinal salt, it is and wherein described Divalent metal salt is selected from CuAc₂、CuCl₂、ZnAc₂And ZnCl₂。

2. the pharmaceutical composition of claim 1, wherein the officinal salt is [Aib^8,35]hGLP-1(7-36)NH₂·HCl· Zn。

3. the pharmaceutical composition of claim 1, wherein the officinal salt is [Aib^8,35]hGLP-1(7-36)NH₂Acetic acid Zn。

4. the pharmaceutical composition of claim 1, wherein the officinal salt is [Aib^8,35]hGLP-1(7-36)NH₂·HCl· Copper.

5. the pharmaceutical composition of claim 1, GLP-1 analogs and the divalent metal wherein described in described pharmaceutical composition The mol ratio of salt is 5.4: 1,4.0: 1 or 1.5: 1.

6. the pharmaceutical composition of claim 1, GLP-1 analogs and the divalent metal wherein described in described pharmaceutical composition The mol ratio of salt is 1.5: 1.

7. the pharmaceutical composition of claim 1, wherein the officinal salt of the analog is the hydrochloride or acetate of the peptide.

8. the final mol ratio of the pharmaceutical composition of claim 7, wherein acetate or hydrochloride and the analog for 1: 1 to 6∶1。

9. the pharmaceutical composition of claim 7, wherein acetate or hydrochloride and the final mol ratio of the analog are 3.0: 1。

10. the pharmaceutical composition of claim 1, wherein the peptide analogues discharge at least 84 or 96 hours in subject.

11. the pharmaceutical composition of claim 1, wherein the peptide analogues discharge at least 5 in subject, 6,7,8,9,10, 11st, 12,13 or 14 days.

12. the pharmaceutical composition of claim 1, wherein the peptide analogues discharge at least 2 in subject, 3 or 4 weeks.

13. the pharmaceutical composition of claim 1, wherein the peptide analogues discharge at least 1 in subject, 1.5,2 or 3 Month.

14. the pharmaceutical composition of claim 1, wherein the composition has 3.5 to 5.5 pH value.

15. the pharmaceutical composition of claim 1, wherein the composition has 4.2 to 4.6 pH value.