Effectively blood sugar control, stimulate insulin secretion, suppress the disorderly secretion of hyperglycemia and slow down the intestine evacuation velocity of stomach, reduce appetite, reduce food intake and lose weight, be highly suitable for the treatment of type ii diabetes, for example, be used to improve and control the blood glucose that metformin and sulfonylureas are treated unfavorable type ii diabetes patient.

The known pharmacological action of Exenatide comprises: (1) significantly increases glucose dependency insulin secretion accelerating, does not stimulate secretion of insulin in blood sugar level during normal or hypoglycemia, only stimulates insulin secretion when blood sugar level is higher; (2) suppress the secretion of type ii diabetes patient glucagon, and when hyperglycemia, reduce glucagon concentration in the serum, but do not weaken normal glucagon hypoglycemic reaction; (3) suppress digestive tract power and secretory function after the meal, postpone gastric emptying, thereby help the control of post-prandial glycemia; (4) reduce appetite, reduce the absorption of food; (5) stimulate the ripe differentiation of β hyperplasia or embryo's insulin, suppress the β apoptosis, thereby increase the quantity of β cell; Phase reaction when (6) recovering the insulin the first of type ii diabetes patient forfeiture significantly increases at first o'clock and reaches second o'clock phase secretion of insulin mutually, improves type ii diabetes patient's empty stomach and level of postprandial blood sugar.

At one by a definite date in the randomized, double-blind placebo-controlled study in 30 weeks, 733 examples are united and are used the effectively patient [HbA of blood sugar control of metformin and sulfonylurea hypoglycemic agent treatment_1cBaseline values at (8.2 ± 1.0) %], 2 times/day subcutaneous injection Exenatide 5 μ g or placebo at first; After 4 weeks, the Exenatide component is A group and B group, and it is constant that the A group continues to keep Exenatide dosage, and the B group is used each 10 μ g instead 2 times/day; Placebo group is all used according to 2 times/day in whole research; During the research, metformin treatment group is still kept original dosage, and the sulfonylurea treatment group then is divided into effective dose (MAX) and minimum recommended dosage (MIN) group at random.Result's demonstration, when treating for 30 weeks, patient HbA_1cSignificantly reduce, curative effect and Exenatide present dose-effect relationship, and 2 dosed administration group weight in patients all significantly alleviate.

In addition, Exenatide is used for type ii diabetes patient's clinical research prompting, patient's toleration is good, relevant with dosage common adverse reactions is for feeling sick, untoward reaction mostly is slight and moderate, and in continued treatment subsequently, the feeling sick of patient, incidence of vomiting and the order of severity thereof all descend thereupon.

Exenatide is a kind of brand-new GLP-1 analog, can reduce the type ii diabetes patient after the meal and fasting glucose, and can make HbA_1cHorizontal dimension was held in normal range, and only when blood sugar level is higher, just stimulate insulin secretion, can control blood sugar level to antidiabetic drug no response patient commonly used, reduce most of patient's body weight, and patient's better tolerance, untoward reaction is lighter, is applicable to dietary adjustments and the out of contior type ii diabetes patient of other antidiabetic drugs.

Chinese invention patent application 00804847.9 discloses subcutaneous injection agent, intravenous injection agent or the intravenous infusion preparation of Exenatide, its in full content and the disclosed content of citing document thereof all as the application's reference.

Summary of the invention

The object of the present invention is to provide a kind of stable Exenatide formulation, described preparation comprises Exenatide, antiseptic, antioxidant, chelating agent, osmotic pressure regulator, buffer agent and pH regulator agent.

Preparation of the present invention can effectively slow down diabetics gastric emptying and reduce its plasma glucose levels.

Further, the content of Exenatide is 0.005-0.4% (w/v) in the preparation, is preferably 0.010-0.10% (w/v), and more preferably 0.015-0.05% (w/v) most preferably is 0.025% (w/v).

Further, described antiseptic is this area antiseptic commonly used, preferred preservative is selected from any or its combination of metacresol, benzylalcohol, methyl parahydroxybenzoate, ethylparaben, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, P-hydroxybenzoic acid phenyl ester, phenol, cresol, chlorobutanol, parachlorometacresol, more preferably any or its combination of metacresol, parachlorometacresol.The consumption of antiseptic of the present invention is 0.1% (w/v)-2% (w/v), preferred 0.2% (w/v)-0.5% (w/v).

Further, described antioxidant is this area antioxidant commonly used, preferred antioxidant is selected from methionine, VC, cysteine, sodium sulfite, sodium sulfite, sodium pyrosulfite, dibutyl benzoic acid, VE, 2, any or its combination of 6-di-tert-butyl-4-methy phenol, oryzanol, flavonoid, citric acid, more preferably any or its combination of methionine, VC, cysteine.The consumption of antioxidant of the present invention is 0.2% (w/v)-5% (w/v), is preferably 0.5% (w/v)-2% (w/v).

Further, described chelating agent is this area chelating agent commonly used, and preferred chelating agent is selected from disodiumedetate, calcio-disodium edetate or 1, any or its combination of 2-diamino-cyclohexane tetraacethyl.The consumption of chelating agent of the present invention is 0.01% (w/v)-0.1% (w/v), is preferably 0.05% (w/v)-0.075% (w/v).

Further, described osmotic pressure regulator is this area osmotic pressure regulator commonly used, as carbohydrate, polyhydric alcohol or salt, preferred osmotic pressure regulator is selected from galactose, arabinose, lactose or any other carbohydrate, sorbitol, mannitol, inositol, glycerol, xylitol, polypropylene glycol/glycol copolymer, Macrogol 200 (PEG200), PEG400, PEG1450, PEG3350, PEG4000, PEG6000, PEG8000, sodium chloride, any of glucose or its combination, make the water continuous phase of preparation open or isosmotic solution for waiting, the effectively protein in the stabilization formulations, and opposing temperature raises, the protein denaturation that freeze thawing or freeze-dry process cause.Carbohydrate of the present invention is free from side effects to diabetics, can metabolism form the blood glucose of high concentration; Any or its combination of sorbitol, mannitol, xylitol more preferably.

Further, described buffer agent is this area buffer agent commonly used, and preferred reducing is selected from any or its combination of acetate buffer, phosphate buffer, citrate buffer agent or glutamate, Glu buffer agent.

Further, described pH regulator agent is this area pH regulator agent commonly used, preferred pH regulator agent is selected from any or its combination, the more preferably trometamol or the triethanolamine of trometamol, hydrochloric acid, citric acid, potassium hydroxide, sodium hydroxide, sodium citrate, triethanolamine.

Further, Exenatide formulation of the present invention also can comprise solvent, and described solvent is this area solvent commonly used, as water for injection, need to obtain the solution of concentration.

Further, the pH of described preparation is 3.0-7.0, is preferably 4.0-6.0, and more preferably 4.0-5.0 is to reduce the stimulation of preparation to the patient.

Further, described preparation is selected from liquid preparation, lyophilized formulations or its improvement preparation, can be used in the per os, nose, cheek, injection, Sublingual, trachea, administrations such as lung transmission or transdermal patches, for example the nasal administration preparation, in lung drug-delivery preparation, oral Preparation, trachea drug-delivery preparation, sublingual administration preparation or through the cheek drug-delivery preparation, being preferably liquid preparation is the parenteral liquid preparation, as injection, described injection can be selected from subcutaneous injection agent, intravenous injection agent or intravenous infusion agent.Lyophilized formulations of the present invention is selected from the lyophilizing unit formulation or preparation is used in lyophilizing more.Can adopt preparation technique means well known in the art to prepare preparation of the present invention.

In case of necessity, also comprise other pharmaceutically acceptable carriers in the present composition, be used to keep Exenatide and stability of formulation thereof, described pharmaceutically acceptable carrier is usual excipients or the adjuvant that is used to prepare above-mentioned preparation well known in the art, and its consumption, kind are decided according to the physicochemical property and the factors such as content, preparation type of effective ingredient in the compositions.

In addition, also active component can be mixed by its preparation requirement with pharmaceutically acceptable slow controlled release carrier, again according to the preparation method of sustained-release preparation well known in the art, as adding the blocker coating or with making micropill after the active principle microcapsulesization again, as slow-release micro-pill or controlled release micro pill; Described slow controlled release carrier includes but are not limited to oil agent, hydrophilic colloid or the coating blocker etc. of mixing, and described oil to mix agent be glyceryl monostearate, castor oil hydrogenated, Dormant oils, polysiloxanes, dimethyl siloxane; Described hydrophilic colloid is cellulose derivatives such as sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl emthylcellulose, or PVP, arabic gum, tragcanth or carbopol etc.; Described coating blocker is ethyl cellulose (EC), hydroxypropyl methylcellulose (HMPC), polyvinylpyrrolidone (PVP), cellulose acetate-phthalate (CAP), acrylic acid resinoid etc.

Lyophilized formulations of the present invention is stable, can contain freeze-dried excipient, improving the formability of preparation, and is beneficial to the stable of preparation and/or alleviates injection pain, and include the surfactant that benefits caking characteristics and/or redissolution.Described freeze-dried excipient is selected from any or its combination of about 1.0-10% (w/v) carbohydrate, polyhydric alcohol, normal saline, for example any or its combination of sorbitol, mannitol, xylitol, glucose; Described surfactant is this area surfactant commonly used, about 1.0% (w/v) Tween 80 of preferably about 0.1-or other nonionic detergent.

The present invention also comprise be used in the per os, cheek, Sublingual, trachea, solid dosage forms that nose and lung transmit, as liquid preparation and/or dry powder formulations.Dry powder formulations can contain extender and/or salt, the granule of a certain size that helps to form and suitable particle size distribution, and be beneficial to the dry powder particle morphological characteristic and/or promote the described active component of tissue picked-up.

When preparation of the present invention is that about 0.005 μ g/kg/ agent is to about 0.2 μ g/kg/ agent based on the dosage range of weight in patients, be preferably about 0.02 μ g/kg/ agent to about 0.1 μ g/kg/ agent, more preferably about 0.05 μ g/kg/ agent is to about 0.1 μ g/kg/ agent, and recommend administration frequency 1-4 time/day, be preferably 1-2 time/day.

When being used for drug administration by injection, it recommends pharmaceutical quantities and mode is: the about 0.5 μ g/Kg of about 0.1 μ g/Kg-with 1-3 time/day form administration, is preferably 2-3 time/day, more preferably 2 times/day.

Comprise about 50 to about 100 times described active component according to oral dose of the present invention, that is to say that the dosage with single agent or multi-agent administration is that every day about 500 to about 12,000 μ g, preferred every day about 500 are to about 5,000 μ g.Comprise about 10 to about 100 times described active component according to pulmonary administration dosage of the present invention, that is to say dosage with single agent or multi-agent administration be every day about 100 to about 12,000 μ g, preferred every day about 500-1000 μ g.Also comprise about 10 to about 100 times described active component according to nose of the present invention, cheek and sublingual administration dosage, that is to say that the dosage with single agent or multi-agent administration is that every day about 100 is to about 12,000 μ g.For example, the preferred dose of nose administration be every day about 10-1000 to about 1200-12,000 μ g, through the preferred dose of cheek administration be every day about 10-1000 to about 1200-12,000 μ g, the preferred dose of sublingual administration be every day about 10-1000 to about 1200-8,000 μ g.

Another object of the present invention is to provide a kind of method of measuring Exenatide formulation content, it is characterized in that, adopt the content of Exenatide in the high effective liquid chromatography for measuring preparation, with octadecylsilane chemically bonded silica C₁₈150 X 4.6mm 300A are the chromatographic column filler, carry out gradient elution by mobile phase, flow velocity is 1.0ml/min, the detection wavelength is 220nm, in Exenatide, number of theoretical plate is not less than 2000, and described mobile phase is made up of mobile phase A and Mobile phase B, described mobile phase A is the water that contains 0.05% trifluoroacetic acid, and described Mobile phase B is the acetonitrile that contains 0.05% trifluoroacetic acid.

Further, described assay method comprises that also it is an amount of to get preparation, accurate claims surely, dissolves and is diluted to the solution that concentration is 0.25mg/ml with 0.05% trifluoroacetic acid aqueous solution, shakes up, as need testing solution; Precision is measured 20 μ l need testing solutions, injects chromatograph of liquid, the record chromatogram.

Further, it is 78:22 that the mobile phase when detecting beginning consists of A:B, and it is 35:65 that the mobile phase when detecting 30min consists of A:B.

Another object of the present invention is to provide Exenatide formulation to be used for preparing the application of the medicine of preventing and treating diabetes, to strengthen the sensitivity of patient to endogenous or exogenous insulin.Described diabetes are selected from type i diabetes, type ii diabetes, obesity, other gastric emptying that has benefited from slowing down, reduce diseases such as plasma glucose levels and minimizing pickuping food.

Except as otherwise noted, percentage ratio of the present invention is percent weight in volume (w/v).

Exendin-4 application standard solid-phase peptide synthetic technology of the present invention (as automatic or semi-automatic method of peptide synthesis) makes: in the presence of a kind of alkali (as diisopropylethylamine) and coupling agent (as dicyclohexylcarbodiimide and I-hydroxybenzotriazole); in atent solvent (as dimethyl formamide, N-methylpyrrole diketone (N-methylpyrrolidinone) or dichloromethane); under the room temperature, make the aminoacid and the aminoacid coupling that is connected the prolongation peptide chain on the resin of α-N-carbamoyl protection.Adopt reagent (as trifluoroacetic acid or piperidines) to remove α-N-carbamoyl protecting group from the peptide-resin that produces, the aminoacid of the N-protected that needs with the next one that joins described peptide chain repeats coupling reaction.Described N-protected group is this area N-protected group commonly used, is preferably tert-butoxycarbonyl (tBoc) and fluorenyl methoxy carbonyl (Fmoc).

The specific embodiment

Specify the present invention below with reference to embodiment, embodiments of the invention only are used to technical scheme of the present invention is described, and non-limiting essence of the present invention.

Embodiment 1The preparation of Exenatide formulation

Exenatide 250mg

Trometamol 5.0g

Mannitol 10.0g

Metacresol 2.2g

Methionine 10.0g

EDTA-Na 0.1g

Water for injection adds to 1000ml.

The comparative example 1The preparation of Exenatide formulation

Exenatide 250mg

Sodium acetate 6.0g

Mannitol 10.0g

Acetic acid is an amount of

Metacresol 2.2g

Water for injection adds to 1000ml.

Embodiment 2The preparation of Exenatide formulation

Exenatide 250mg

Trometamol 5g

Mannitol 10.0g

Metacresol 2.28g

Methionine 10g

Acetic acid is an amount of

Water for injection is an amount of

Embodiment 3The preparation of Exenatide formulation

Exenatide 250mg

Trometamol 5g

Mannitol 10.0g

Metacresol 2.2g

VC 12g

Water for injection is an amount of

Embodiment 4The preparation of Exenatide formulation

Exenatide 250mg

Trometamol 5.0g

Mannitol 10.0g

Metacresol 2.2g

Methionine 20.0g

Water for injection is an amount of

Embodiment 5The preparation of Exenatide formulation

Exenatide 250mg

Trometamol 5.0g

Mannitol 10.0g

Metacresol 2.2g

Methionine 10.0g

EDTA-Na 0.05g

Water for injection is an amount of

Embodiment 6Preparation stability is investigated experiment

The Exenatide formulation that embodiment 1 and comparative example 1 are made places cassette vial or tubular type vial, seal with butyl rubber plug, place respectively under 25 ℃ and 2～8 ℃ of conditions, carry out the investigation of stability, adopt the variation of Exenatide content in the investigation process in the high effective liquid chromatography for measuring sample.

Adopting the content of Exenatide in the high effective liquid chromatography for measuring preparation, is chromatographic column filler (C with the octadecylsilane chemically bonded silica₁₈150 X 4.6mm 300A), be mobile phase A with water (containing 0.05% trifluoroacetic acid); With acetonitrile (containing 0.05% trifluoroacetic acid) Mobile phase B, the composition of mobile phase is referring to table 1; Gradient elution; Flow velocity is 1.0ml/min, and the detection wavelength is 220nm, and number of theoretical plate should be not less than 2000 in Exenatide.

The composition of table 1 mobile phase

It is an amount of to get preparation, accurate claims surely, dissolves and is diluted to the solution that concentration is 0.25mg/ml (in Exenatide) with 0.05% trifluoroacetic acid aqueous solution, shakes up, as need testing solution; Precision is measured 20 μ l, injects chromatograph of liquid, the record chromatogram, and the result is referring to table 2.

The study on the stability experimental result of table 2 Exenatide formulation

Illustrate: the D in the table 2 represents " day "; M represents " month ".

Analytical table 2 learns that the long-time stability of Exenatide formulation of the present invention obviously improve.

Claims

1. stable Exenatide formulation, described preparation comprises Exenatide, antiseptic, antioxidant, chelating agent, osmotic pressure regulator, buffer agent and pH regulator agent.

2. preparation according to claim 1, the content of Exenatide is 0.005-0.4% (w/v) in the described preparation, is preferably 0.010-0.10% (w/v), more preferably 0.015-0.05% (w/v) most preferably is 0.025% (w/v).

3. preparation according to claim 1 and 2, described antiseptic is this area antiseptic commonly used, be preferably any or its combination of metacresol, benzylalcohol, methyl parahydroxybenzoate, ethylparaben, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, P-hydroxybenzoic acid phenyl ester, phenol, cresol, chlorobutanol, parachlorometacresol, any or its combination of metacresol or parachlorometacresol more preferably, the consumption of described antiseptic is 0.1% (w/v)-2% (w/v), preferred 0.2% (w/v)-0.5% (w/v).

4. according to each described preparation of claim 1-3, described antioxidant is this area antioxidant commonly used, is preferably methionine, V_C, cysteine, sodium sulfite, sodium sulfite, sodium pyrosulfite, dibutyl benzoic acid, V_E, 2, any or its combination, more preferably methionine, the V of 6-di-tert-butyl-4-methy phenol, oryzanol, flavonoid, citric acid_C, cysteine any or its combination, the consumption of described antioxidant is 0.2% (w/v)-5% (w/v), is preferably 0.5% (w/v)-2% (w/v).

5. according to each described preparation of claim 1-4, described chelating agent is this area chelating agent commonly used, be preferably disodiumedetate, calcio-disodium edetate or 1, any or its combination of 2-diamino-cyclohexane tetraacethyl, the consumption of described chelating agent is 0.01% (w/v)-0.1% (w/v), is preferably 0.05% (w/v)-0.075% (w/v).

6. according to each described preparation of claim 1-5, described osmotic pressure regulator is selected from carbohydrate, polyhydric alcohol or salt, be preferably any or its combination of galactose, arabinose, lactose, sorbitol, mannitol, inositol, glycerol, xylitol, polypropylene glycol/glycol copolymer, PEG200, PEG 400, PEG 1450, PEG 3350, PEG 4000, PEG 6000, PEG 8000, sodium chloride, glucose, more preferably any or its combination of sorbitol, xylitol or mannitol.

7. according to each described preparation of claim 1-6, described buffer agent is this area buffer agent commonly used, is preferably any or its combination of acetate buffer, phosphate buffer, citrate buffer agent or glutamate, Glu buffer agent.

8. according to each described preparation of claim 1-7, described pH regulator agent is this area pH regulator agent commonly used, be preferably any or its combination of trometamol, hydrochloric acid, citric acid, potassium hydroxide, sodium hydroxide, sodium citrate, triethanolamine, more preferably any or its combination of trometamol or triethanolamine.

9. according to each described preparation of claim 1-8, also comprise solvent, described solvent is this area solvent commonly used, is preferably water for injection.

10. according to each described preparation of claim 1-9, the pH of described preparation is 3.0-7.0, is preferably 4.0-6.0, more preferably 4.0-5.0.

11. according to each described preparation of claim 1-10, described preparation is selected from liquid preparation, lyophilized formulations or its improvement preparation, the preferred liquid preparation is the parenteral liquid preparation, subcutaneous injection agent more preferably, intravenous injection agent or intravenous infusion agent.

12. according to each described preparation of claim 1-11, described preparation is selected from pernasal preparation, drug-delivery preparation, sublingual administration preparation or through the cheek drug-delivery preparation in lung drug-delivery preparation, oral Preparation, trachea.

13. according to each described preparation of claim 1-12, described lyophilized formulations is selected from the lyophilizing unit formulation or preparation is used in lyophilizing more.

14. according to each described preparation of claim 1-13, described preparation comprises the EDTA-Na of methionine and 0.01% (w/v) of metacresol, 1.0% (w/v) of mannitol, 0.22% (w/v) of Exenatide, 0.5% (w/v) trometamol, 1.0% (w/v) of 0.025% (w/v), and surplus is a water for injection.

15. each described Exenatide formulation of claim 1-14 is used for preparing and prevents and treats diabetes, obesity, the gastric emptying that has benefited from slowing down, reduces plasma glucose levels or reduce the application of the medicine of feeding disease.

16. application according to claim 15, described diabetes are selected from type i diabetes or type ii diabetes.

17. method of measuring Exenatide formulation content, it is characterized in that, adopt the content of Exenatide in the high effective liquid chromatography for measuring preparation, with the octadecylsilane chemically bonded silica is the chromatographic column filler, carry out gradient elution by mobile phase, flow velocity is 1.0ml/min, the detection wavelength is 220nm, in Exenatide, number of theoretical plate is not less than 2000, described mobile phase is made up of mobile phase A and Mobile phase B, and described mobile phase A is the water that contains 0.05% trifluoroacetic acid, and described Mobile phase B is the acetonitrile that contains 0.05% trifluoroacetic acid.

18. method according to claim 17, described assay method comprises that also it is an amount of to get preparation, accurate claims surely, dissolves and is diluted to the solution that concentration is 0.25mg/ml with 0.05% trifluoroacetic acid aqueous solution, shakes up, as need testing solution; Precision is measured 20 μ l need testing solutions, injects chromatograph of liquid, the record chromatogram.

19. according to claim 17 or 18 described methods, it is 78:22 that the mobile phase when detecting beginning consists of A:B, it is 35:65 that the mobile phase when detecting 30min consists of A:B.