WO2009024015A1

WO2009024015A1 - A stable formulation of exenatide

Info

Publication number: WO2009024015A1
Application number: PCT/CN2008/001387
Authority: WO
Inventors: Huijuan Zhong; Yuxia Wu; Yuru Li
Original assignee: Jiangsu Hansen Pharmaceutical Co., Ltd.
Priority date: 2007-08-15
Filing date: 2008-07-29
Publication date: 2009-02-26
Also published as: CN101366692A; TW200950802A

Abstract

The invention provides a stable formulation of exenatide, preparation method and use thereof, and a method for content determination of exenatide in the formulation. The formulation of present invention has increased significantly stability in a long term, and is useful in treating diabetes and conditions that would be benefited by lowering plasma glucose or delaying and/or slowing gastric emptying or inhibiting food intake.

Description

A stable exenatide preparation technical field

The present invention relates to exendin agonist preparations, in particular to a stable exenatide preparation, a preparation method thereof and use thereof, which can be administered by injection and non-injection routes, and are used for prevention and treatment including type I and type II. Diabetes, including diabetes, and for the prevention and treatment of conditions that benefit from lowering plasma glucose levels, effectively delaying and/or slowing gastric emptying or reducing food intake. Background technique

According to the WHO report in 1997, there were approximately 135 million diabetic patients worldwide in 1995, and it is predicted to surge to 199 million by 2025. Diabetes (type II diabetes, NIDDM) has become a tumor, cardiovascular and cerebrovascular The third chronic disease that seriously endangers human health after the disease. In recent years, the incidence of diabetes in China has been increasing, and the situation of younger patients has become increasingly prominent. In 1997, the survey results of 11 provinces and cities in China showed that the prevalence of diabetes (DM) standardization was 321%, and the prevalence of impaired glucose tolerance (IGT) was 4.76%. It is speculated that China has II Type 2 diabetes patients and impaired glucose tolerance are 50 million people, at least 1/4-1/2 of the diabetic patients are not diagnosed, and only 1/3 of the diagnosed patients are ideally controlled. Therefore, it is very important to research and develop ideal diabetes clinical drugs.

Exenatide (code AC-2993, chemical name exenatide, also known as exenatide) is an Exendin-4 agonist, which is a pancreas contained in saliva produced by the poisonous lizard (produced in the southwestern United States). The artificial synthesis of Glucagon-like Peptide-1 (GLP-1) substance can function in the body by simulating the action of incretin, thereby achieving the effect of controlling blood sugar.

The amino acid sequence of exenatide is -

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu- Ala-Val-Arg-Leu-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Al a - Pro-Pro- Pro- Ser- NH ₂ is a polypeptide of 39 nitrogen acids with a molecular weight of 4186. 6 Daoer It is soluble in water, methanol or ethanol with a specific rotation of 6Γ~- 68 °.

Exenatide can be prepared by standard solid phase peptide synthesis techniques (such as automatic or semi-automated peptide synthesis). It was first developed by Amylin and Reliance Pharmaceuticals of the United States. It is a polypeptide containing 39 amino acids and is the first approved intestinal drop. The incretin mimetics drug, its new drug marketing application was approved by the US FM in April 2005, the trade name Byetta®, can effectively control blood sugar, stimulate insulin secretion, inhibit hyperglycemia secretion and slow down the gastric emptying rate. It reduces appetite, reduces food intake and reduces body weight. It is very suitable for the treatment of type 2 diabetes, for example, to improve and control the blood sugar of patients with type 2 diabetes who are not ideal for treatment with metformin and sulfonylureas.

The known pharmacological effects of Exenatide include: (1) Significantly increased glucose-dependent insulin secretion, does not stimulate insulin secretion when blood glucose levels are normal or hypoglycemic, and stimulates islets only when blood glucose levels are high (2) inhibit the secretion of glucagon in type 2 diabetic patients, and reduce the concentration of glucagon in serum in hyperglycemia, but does not reduce the response of normal glucagon to hypoglycemia; (3) inhibition Postprandial gastrointestinal motility and secretion function, delay gastric emptying, which is conducive to postprandial blood glucose control; (4) reduce appetite, reduce food intake; (5) stimulate e-cell proliferation or embryonic insulin maturation differentiation, inhibition e Apoptosis, thereby increasing the number of beta cells; (6) Restoring the first phase response of insulin lost in type 2 diabetic patients, significantly increasing the secretion of insulin in the first phase and the second phase, improving the fasting of patients with type 2 diabetes And postprandial blood glucose levels.

In a 30-week, randomized, double-blind, placebo-controlled study, 733 patients were treated with metformin and a sulfonylurea hypoglycemic agent in patients who were unable to effectively control blood glucose [HbA^ baseline levels were (8. 2 ± 1. 0) %], first subcutaneous injection of exenatide 5 g or placebo 2 times/day; after 4 weeks, exenatide components were group A and B, group A continued to maintain the dose of exenatide unchanged Group B was switched to 2 times/day, 10 g each time; placebo group was used twice per day in the whole study; during the study period, the metformin treatment group still maintained the original dose, and the sulfonylurea treatment group was randomized. Divided into the effective dose (MAX) and the lowest recommended dose (MIN) group. The results showed that at 30 weeks of treatment, the patient's Hb was significantly reduced, the efficacy was dose-effect relationship with exenatide, and the weight of the two dose-administered groups was significantly reduced.

In addition, the clinical study of Exenatide in patients with type 2 diabetes suggests that the patient is well tolerated, the most common dose-related adverse reaction is nausea, and the adverse reactions are mostly mild and moderate, and continue in the subsequent During treatment, the incidence of nausea and vomiting and the severity of the patients decreased.

Exenatide is a novel GLP-1 analogue that reduces postprandial and fasting blood glucose in type 2 diabetic patients and maintains HbA _le levels within normal limits and stimulates insulin only when blood glucose levels are high Secretion, can control the blood sugar level of patients who do not respond to commonly used hypoglycemic agents, reduce the weight of most patients, and the patient's tolerance is good, the adverse reactions are light, suitable for diet regulation and other types of hypoglycemic drugs can not control Diabetic patients.

Chinese Patent Application No. 0 080 484 7.9 discloses a subcutaneous injection of exenatide, an intravenous injection or an intravenous infusion preparation, the entire contents of which are hereby incorporated by reference in its entirety. Summary of the invention

It is an object of the present invention to provide a stable exenatide formulation comprising exenatide, a preservative, an antioxidant, a complexing agent, an osmotic pressure adjusting agent, a buffering agent and a pH adjusting agent.

The preparation of the present invention can effectively slow gastric emptying and reduce plasma glucose levels in diabetic patients. Further, the content of exenatide in the preparation is 0.005 to 0.4% (w/v), preferably 0.0.10 to 0.10% (w/v), more preferably 0.015 to 0.05% (w/v), most preferably It is 0.025% (w/v).

Further, the preservative is a preservative commonly used in the art, and preferably the preservative is selected from the group consisting of m-cresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-ethyl benzoate, propyl p-propyl benzoate, Any one or a combination of butyl p-hydroxybenzoate, phenyl p-hydroxybenzoate, phenol, cresol, chlorobutanol, p-chlorom-cresol, more preferably m-cresol, p-chloro-cresol Any one or a combination thereof. The preservative of the present invention is used in an amount of 0.1%. (w/v) -2% (w/v), preferably 0.2% (w/v) -0.5% (w/v).

Further, the antioxidant is an antioxidant commonly used in the art, and preferably the antioxidant is selected from the group consisting of methionine, V _c , cysteine, sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, and dibutylbenzene. Any one or a combination of acid, V _E , 2, 6-di-tert-butyl 4-methylphenol, oryzanol, flavonoids, decanoic acid, more preferably methionine, Vc:, cysteine Any one or combination of acids. The antioxidant is used in an amount of 0.2% (w/v) to 5% (w/v), preferably 0.5% (w/v) to 2% (w/v) o.

Further, the complexing agent is a complexing agent commonly used in the art, and preferably the complexing agent is selected from the group consisting of disodium ethylenediaminetetraacetate, sodium calcium edetate or 1,2-diaminocyclohexanetetraacetic acid. Any one or a combination thereof. The complexing agent of the present invention is used in an amount of 0.01% (w/v) to 0.1% (w/v), preferably 0.05% (w/v) to 0.075% (w/v).

Further, the osmotic pressure adjusting agent is an osmotic pressure regulating agent commonly used in the art, such as a carbohydrate, a polyol or a salt, and preferably the osmotic pressure adjusting agent is selected from the group consisting of galactose, arabinose, lactose or any other carbohydrate, sorbitol. , mannitol, inositol, glycerol, xylitol, polypropylene glycol/ethylene glycol copolymer, polyethylene glycol 200 (PEG200), PEG 400, PEG 1450, PEG 3350, PEG 4000, PEG 6000, PEG 8000, chlorine Any one or combination of sodium or glucose, such that the aqueous continuous phase of the formulation is an isotonic or isotonic solution, effectively stabilizes the protein in the formulation and resists protein denaturation caused by elevated temperature, freeze-thaw or lyophilization processes. The carbohydrate of the present invention has no side effects on diabetic patients and does not metabolize to form a high concentration of blood sugar; more preferably, it is any one of sorbitol, mannitol, xylitol or a combination thereof.

Further, the buffering agent is a buffer commonly used in the art, and preferably the buffering agent is selected from any one or a combination of an acetate buffer, a phosphate buffer, a citrate buffer or a glutamate buffer. .

Further, the pH adjuster is a pH adjuster commonly used in the art, and preferably the pH adjuster is selected from the group consisting of tromethamine, hydrochloric acid, citric acid, potassium hydroxide, sodium hydroxide, sodium citrate, and triethanolamine. Any one or a combination thereof is more preferably tromethamine or triethanolamine.

Further, the Exenatide preparation of the present invention may further comprise a solvent which is a solvent commonly used in the art, such as water for injection, to obtain a solution of a desired concentration.

Further, the pH of the preparation is from 3.0 to 7.0, preferably from 4.0 to 6.0, more preferably from 4.0 to 5.0, to reduce the irritation of the preparation to the patient.

Further, the preparation is selected from the group consisting of a liquid preparation, a lyophilized preparation or a modified preparation thereof, and can be administered by oral, nasal, buccal, injection, sublingual, intratracheal, pulmonary delivery or dermal patch, for example, nasal administration. a preparation, a pulmonary administration preparation, an oral administration preparation, an intratracheal administration preparation, a sublingual preparation preparation or a buccal administration preparation, preferably a liquid preparation is a parenteral liquid preparation such as an injection, and the injection preparation is optional From subcutaneous injections, intravenous injections or intravenous infusions. The lyophilized preparation of the present invention is selected from the group consisting of a lyophilized unit preparation or a frozen multi-purpose preparation. The formulations of the present invention can be prepared by formulation techniques well known in the art.

If necessary, the compositions of the present invention further comprise other pharmaceutically acceptable carriers for maintaining the stability of exenatide and its formulations, which are well known in the art for the preparation of such formulations. The usual excipients or excipients, the amount and type thereof are determined according to factors such as the physical and chemical properties and content of the active ingredients in the composition, the type of the preparation, and the like. In addition, the active ingredient may be mixed with a pharmaceutically acceptable controlled release carrier according to the preparation requirements thereof, and then according to a preparation method of a controlled release preparation well known in the art, such as adding a retarder coating or micro active ingredient. After encapsulation, the pellets are prepared into a pellet, such as a sustained release pellet or a controlled release pellet; the sustained release carrier includes, but is not limited to, an oily incorporation agent, a hydrophilic colloid or a coating retarder, The oleaginous incorporation agent is glyceryl monostearate, hydrogenated castor oil, mineral oil, polysiloxane, dimethyl siloxane; the hydrophilic colloid is sodium carboxymethyl cellulose, hydroxypropyl a cellulose derivative such as cellulose or hydroxypropylmethylcellulose, or PVP, gum arabic, scutellaria or carbopol; the coating retarder is ethyl cellulose (EC), hydroxy Propylmethylcellulose (HMPC), polyvinylpyrrolidone (PVP), cellulose acetate phthalate (CAP), acrylic resin, and the like.

The lyophilized preparation of the present invention is stable and may contain a frozen 1000 excipient to improve the formability of the preparation, to facilitate the stabilization of the preparation and/or to alleviate the pain of injection, and to include surface activity which is beneficial for agglomeration characteristics and/or reconstitution. Agent. The lyophilized excipient is selected from any one of about 1.0-10% (w/v) carbohydrate, polyol, physiological saline or a combination thereof, such as sorbitol, mannitol, xylitol, glucose Or a combination thereof; the surfactant is a surfactant commonly used in the art, preferably from about 0.1 to about 1.0% (w/v) Tween 80 or other nonionic detergent.

The present invention also encompasses solid dosage forms for oral, buccal, sublingual, intratracheal, nasal and pulmonary delivery, such as liquid formulations and/or dry powder formulations. Dry powder formulations may contain extenders and/or salts which aid in the formation of particles of a certain size and a suitable particle size distribution, as well as facilitate the morphological properties of the dry powder particles and/or promote tissue uptake of the active ingredients.

When the dosage of the present invention is based on the patient's body weight, the dosage range is from about 0.005 μg/lcg/dose to about 0.2 μg/kg/dose, preferably from about 0.02 μg/kg/dose to about 0.1 μg/kg/dose, More preferably, it is about 0.05 μg/kg/dose to about 0.1 μg/kg/dose, and the recommended dosing frequency is 1-4 times/day, preferably 1-2 times/day.

For administration by injection, the recommended dosage and manner of administration is: about Ol wg / Kg - about 0.5 wg / Kg, administered in the form of 1-3 times / day, preferably 2-3 times / day, more preferably 2 times / day.

The oral dose according to the present invention comprises from about 50 to about 100 times the active ingredient, that is, a single or multiple doses of from about 500 to about 12,000 μg per day, preferably from about 500 to about 5,000 μ per day. g. The pulmonary administration dose according to the present invention comprises from about 10 to about 100 times the active ingredient, that is, the dose administered in a single dose or multiple doses is from about 100 to about 12,000 per day, preferably from about 500 to 1000 per day. The nasal, buccal and sublingual doses according to the present invention also include from about 10 to about 100 times the active ingredient, that is, the dose administered in a single dose or multiple doses is from about 100 to about 12,000 μ per day, for example, The preferred dosage for nasal administration is from about 10 to 1000 to about 1200 to 12,000 μg per day, and the preferred dosage for buccal administration is from about 10 to 1000 to about 1200 to 12,000 μg per ounce, the preferred dosage for sublingual administration. From about 10-1000 to about 1200-8,000 per day

Another object of the present invention is to provide a method for determining the content of exenatide preparation, which is characterized in that the content of exenatide in the preparation is determined by high performance liquid chromatography, and octadecylsilane bonded silica gel is used. C ₁₈ 150X4. 6rani 300A is a column packing, gradient elution from mobile phase, flow rate is 1.0ml/min, check The water having a wavelength of 220 nm, and the theoretical plate number is not less than 2,000, and the mobile phase is composed of a mobile phase A and a mobile phase B, which is a water containing 0.5% trifluoroacetic acid. The mobile phase B is acetonitrile containing 0.05% trifluoroacetic acid.

Further, the measuring method further comprises: taking an appropriate amount of the preparation, accurately weighing, dissolving and diluting into a solution having a concentration of 0.25 mg/ml with a 0. 05% aqueous solution of trifluoroacetic acid, and shaking it as a test solution; Measure 20 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram.

Further, the mobile phase composition at the start of the test is A: Β is 78: 22, and the mobile phase composition at 30 min is A: B is 35: 65.

Another object of the present invention is to provide an application of an Exenatide formulation for the preparation of a medicament for the prevention and treatment of diabetes to enhance the sensitivity of a patient to endogenous or exogenous insulin. The diabetes is selected from the group consisting of type I diabetes, type II diabetes, obesity, and other conditions that benefit from slowing gastric emptying, lowering plasma glucose levels, and reducing food intake.

The percentages of the invention are weight percent by volume ( _w / v) unless otherwise stated.

The exendin-4 of the present invention is prepared by standard solid phase peptide synthesis techniques (such as automated or semi-automated peptide synthesis): in a base such as diisopropylethylamine and a coupling agent such as dicyclohexyl carbon In the presence of diimine and 1-hydroxybenzotriazole) in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride, at room temperature A-N-carbamoyl protected amino acid and amino acid coupling of an extended peptide chain attached to a resin. The α-Ν-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction is repeated with the next desired oxime-protected amino acid added to the peptide chain. The oxime-protecting group is a oxime-protecting group commonly used in the art, preferably t-butoxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc). detailed description

The invention is specifically described by the following examples, which are merely illustrative of the technical solutions of the invention and are not intended to limit the scope of the invention. Example 1 Preparation of Exenatide Formulation

Exenatide 250mg

Tromethamine 5. 0g

Mannitol 10. 0g

M-cresol 2. 2g

Methionine 10. Og

EDTA-Na

The water for injection is added to 1000 ral. Comparative Example 1 Preparation of Exenatide Formulation Exemin 250 mg sodium acetate 6. Og mannitol 10. Og acetic acid q% m-cresol 2. 2 g water for injection is added to 1000 ml. Example 2 Preparation of Exenatide Formulation

Exenatide 250mg tromethamine 5g mannitol lO.Og m-cresol 2.28g methionine 10g acetic acid appropriate amount water for injection appropriate amount Example 3 preparation of exenatide preparation

Exenatide 250mg tromethamine 5g mannitol 10.0g m-cresol 2.2g V _c 12g

Water for injection Example 4 Preparation of Exenatide preparation

Exenatide 250rag tromethamine 5. 0g mannitol 10. 0g m-cresol 2. 2g methionine 20. Og water for injection Example 5 Preparation of exenatide preparation

克内醇。 The exenatide 250mg. tromethamine 5. 0g mannitol 10. 0g 2. 2g

10. Og

0. 05g

Appropriate amount Example 6 Preparation stability test

The Exenatide preparation prepared in Example 1 and Comparative Example 1 was placed in a glass bottle or a vial glass bottle, and sealed with a butyl rubber stopper, and placed at 25 Torr and 2 to 8 ° C, respectively. For stability investigation, high performance liquid chromatography was used to determine the change of exenatide content in the sample during the investigation.

The content of exenatide in the preparation was determined by high performance liquid chromatography with octadecylsilane bonded silica as the column packing agent (( ₁₈ 15 («4. 6瞧30(^), with water (including 0) 05% trifluoroacetic acid) is mobile phase A; mobile phase B with acetonitrile (containing 0.05% trifluoroacetic acid), the composition of mobile phase is shown in Table 1; gradient elution; flow rate is 1.0ml/min, detection The wavelength is 220 nm, and the number of theoretical plates should be no less than 2000 by exenatide. Table 1 Composition of mobile phase

Time (min) mobile phase A mobile phase B

0 78 22

30 35 65

Take appropriate amount of the preparation, accurately weighed, dissolved in 0. 05% aqueous solution of trifluoroacetic acid and diluted to a concentration of 0.25mg / ml (based on exenatide), shake well, as a test solution; precision measurement 20 μl, injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 2. Experimental results of stability of Exenatide preparation

Explanation: D in Table 2 means "day"; M means "month".

Analysis Table 2 shows that the long-term stability of the exenatide preparation of the present invention is remarkably improved.

Claims

Claims:

A stable exenatide preparation comprising exenatide, a preservative, an antioxidant, a complexing agent, an osmotic pressure adjusting agent, a buffering agent and a pH adjusting agent.

2. The preparation according to claim 1, wherein the preparation has an exenatide content of 0.005 to 0.4% (w/v), preferably 0.010 to 0.10% (w/v), more preferably 0.015 to 0.05. %(w/v), most preferably 0.025 %

3. The preparation according to claim 1 or 2, wherein the preservative is a preservative commonly used in the art, preferably m-cresol, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, Any one or a combination of propyl hydroxybenzoate, butyl p-hydroxybenzoate, phenyl p-hydroxybenzoate, phenol, cresol, chlorobutanol, p-chlorom-cresol, more preferably m-cresol Or any one or a combination of p-chlorom-cresols, the preservative being used in an amount of 0.1% (w/v) to 2% (w/v), preferably 0.2% (w/v) to 0.5% (w) /v).

4. The formulation of claim any one of claims 1-3, according to conventional art antioxidant antioxidant, preferably methionine, V _c, cysteine, sodium sulfite, sulfite Any one or a combination of sodium hydrogen, sodium metabisulfite, dibutyl benzoic acid, V _E , 2, 6-di-tert-butyl 4-methylphenol, oryzanol, flavonoids, decanoic acid, more preferably A methionine, V _c, any one or a combination of cysteine, said antioxidant in an amount of 0.2% (w / v) -5 % (w / v), preferably 0.5% (w / v) -2% (w/v).

The preparation according to any one of claims 1 to 4, wherein the complexing agent is a complexing agent commonly used in the art, preferably disodium edetate, sodium calcium edetate or 1 Any one or a combination of 2-diaminocyclohexanetetraacetic acid, the complexing agent being used in an amount of 0.01% (w/v) to 0.1% (w/v), preferably 0.05% (w/v) ) -0.075% (w/v).

6. The preparation according to any one of claims 1 to 5, wherein the osmotic pressure adjusting agent is selected from the group consisting of carbohydrates, polyols or salts, preferably galactose, arabinose, lactose, sorbitol, mannitol, muscle Alcohol, glycerin, xylitol, polypropylene glycol/ethylene glycol copolymer, PEG200, PEG 400 PEG 1450, PEG 3350, PEG 4000, PEG 6000, PEG 8000, sodium chloride, glucose, or a combination thereof, It is preferably any one or a combination of sorbitol, xylitol, or mannitol.

7. The preparation according to any one of claims 1 to 6, wherein the buffering agent is a buffer commonly used in the art, preferably an acetate buffer, a phosphate buffer, a citrate buffer or a glutamine. Any one or combination of acid salt buffers.

8. The preparation according to any one of claims 1 to 7, wherein the pH adjusting agent is a pH adjusting agent commonly used in the art, preferably tromethamine, hydrochloric acid, citric acid, potassium hydroxide, hydrogen hydroxide. Any one or a combination of sodium, sodium citrate, and triethanolamine is more preferably any one of tromethamine or triethanolamine or a combination thereof.

A formulation according to any one of claims 1-8, further comprising a solvent which is a solvent commonly used in the art, preferably water for injection.

10. A formulation according to any one of claims 1-9, wherein the formulation has a pH of from 3.0 to 7.0, preferably from 4.0 to 6.0, more preferably from 4.0 to 5.0.

The preparation according to any one of claims 1 to 10, which is selected from the group consisting of a liquid preparation, a lyophilized preparation or a modified preparation thereof, preferably the liquid preparation is a parenteral liquid preparation, more preferably a subcutaneous injection, intravenous Injection or intravenous infusion.

The preparation according to any one of claims 1 to 11, which is selected from the group consisting of a nasal preparation, a pulmonary preparation, an oral preparation, an intratracheal preparation, a sublingual preparation or Formulated by buccal administration.

13. The preparation according to any one of claims 1 to 12, wherein the lyophilized preparation is selected from the group consisting of a lyophilized unit preparation or a lyophilized multi-purpose preparation.

14. A formulation according to any one of claims 1 to 13 which comprises 0.025% (w/v) exenatide, 0.5% (w/v) tromethamine, 1.0% (w) /v) mannitol, 0.22% (w/v) m-cresol, 1.0% (w/v) methionine, and 0.01% (w/v) EDTA-Na, with the balance being water for injection.

15. The Exenatide formulation of any of claims 1-14 for use in the manufacture of a medicament for the prevention or treatment of diabetes, obesity, a condition which benefits from slowing gastric emptying, lowering plasma glucose levels or reducing food intake. Application in .

16. The use according to claim 15, wherein the diabetes is selected from the group consisting of type I diabetes or type II diabetes.

17. A method for determining the content of an exenatide preparation, characterized in that the content of exenatide in the preparation is determined by high performance liquid chromatography, and octadecylsilicone bonded silica is used as a column packing agent. , the gradient elution by the mobile phase, the flow rate is 1.0 ml/min, the detection wavelength is 220 nm, and the theoretical plate number is not less than 2000 based on exenatide, and the mobile phase consists of mobile phase A and mobile phase B, The mobile phase A is water containing 0. 05% trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.05% trifluoroacetic acid.

The method according to claim 17, wherein the measuring method further comprises: taking an appropriate amount of the preparation, accurately weighing, dissolving and diluting into a solution having a concentration of 0.25 mg/ml with a 0. 05% aqueous solution of trifluoroacetic acid, shaking uniform, As a test solution; accurately measure 20 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram.

19. The method according to claim 17 or 18, wherein the mobile phase composition at the beginning of the detection is A: Β is 78: 22, and the mobile phase composition at 30 min is A: B is 35: 65.