CN110038118A - The medicinal composition for injections of Gluca Gen sample peptide-1 receptor stimulant - Google Patents

Abstract

The present invention relates to a kind of medicinal composition for injections containing Gluca Gen sample peptide-1 receptor stimulant, wherein Gluca Gen sample peptide-1 receptor stimulant is purified by Bacillus coli expression and is made, with the sequence as shown in Seq ID No.2, medicinal composition for injections contains Gluca Gen sample peptide-1 receptor stimulant, citric acid-sodium citrate buffer salt and polyalcohol.The invention further relates to injections and freeze drying powder injection containing Gluca Gen sample peptide-1 receptor stimulant.Gluca Gen sample peptide-1 receptor stimulant medicinal composition for injections of the present invention have many advantages, such as it is safe and stable, be readily transported and store, be suitable for industrialized production.

Description

The medicinal composition for injections of Gluca Gen sample peptide-1 receptor stimulant

Technical field

The invention belongs to field of pharmaceutical preparations, and in particular to a kind of note of Gluca Gen sample peptide-1 receptor stimulant Penetrate pharmaceutical composition.

Background technique

Natural glucagon-like peptide-1 receptor stimulant (Exendin-4) be South America Monster saliva in separate A kind of biologically active polypeptide (Eng, J etc., J.Biol.Chem., 267:7402- being made of 39 amino acid residues out 05,1992).Following [SEQ.ID.NO.1]: the His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp- of its amino acid sequence 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-Ala-Pro-Pro-Pro-Ser-NH₂。

Exendin-4 and the intracorporal glucagon-like-peptide-1 of people (GLP-1) have 53% homology (Goke etc., J.Biol Chem., 268:19650-55,1993), the study found that Exendin-4 is generated and GLP-1 phase in animal and human body As biological activity, and the action time of Exendin-4 is obviously prolonged that (Eng J., exendin-4 is to db/db compared with GLP-1 The prolonged action of mouse hyperglycemia, Diabetes, 45 (supplementary issues 2): 152A (abstract 554), 1996), show that pancreotropic hormone is living Property, US5424286 proposes Exendin-4 for treating diabetes and preventing the purposes of hyperglycemia.

Peace Milin company and Li Lai company have developed chemically synthesized Exendin-4 injection, FDA approval in 2005 cooperatively It is listed in the U.S., trade name Byetta, for exclusive use melbine, sulfonylurea drugs or melbine and sulfonylurea The treatment of class drug combination fails to obtain the type-2 diabetes mellitus well controlled.

Gluca Gen sample peptide-1 receptor stimulant (rExendin-4 is abbreviated as rE4) is to utilize genetic engineering hand The polypeptide of section production, molecular weight 4187Da, following [SEQ.ID.NO.2]: the His-Gly-Glu-Gly- of amino acid sequence 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-Ala-Pro-Pro-Pro-Ser。

It can satisfy scale using technique for gene engineering preparation and reorganization glucagon-like peptide-1 receptor stimulant (rE4) The demand of mass production, at the same can with the cost that reduces of high degree, CN1693459, CN1635117, CN1455001, The applications such as CN102618552A disclose the method for preparing rE4 using technique for gene engineering, this method have it is easy to operate, at This cheap, advantages of environment protection.

Technique for gene engineering production rE4 may remain the foreign proteins such as coli somatic albumen and external source residual DNA, rE4 The stability of preparation is influenced by factors above, and the preparation of E4 is prepared different from chemical synthesis.E4 preparation in the prior art Prescription is not able to satisfy the demands such as the stability of rE4 preparation, safety.Injection has many advantages, such as easy to use；Freeze drying powder injection Have many advantages, such as to be easy to transport, store, stability is preferable；The dosage form that can be used as rE4 is researched and developed.

The patent CN1384755A of Anmilin Mediciens Co., Ltd discloses a kind of pharmaceutical formulation, it include exendin or Exendin agonist peptide, buffer, etc. osmolality modifiers, the pH of the pharmaceutical formulation be about 3.0- 7.0.Liquid and freeze-drying multi-dose formulation also include the preservative of 0.005%-1.0%, which is selected from metacresol, phenol, benzyl Alcohol, methyl p-hydroxybenzoate, propylparaben and butyl p-hydroxybenzoate, buffer be acetate, phosphate, Citrate or glutamate.Etc. weight molar concentrations bleeding agent be carbohydrate or polyalcohol, carbohydrate be selected from gala Sugar, arabinose and lactose, polyalcohol are selected from sorbierite, mannitol, inositol, glycerol, xylitol and polyethylene glycol.The patent Pharmaceutical preparation contains preservative, which is applied to Gluca Gen sample peptide-1 receptor stimulant preparation, note Accidental rE4 aggregation and fibrillation when liquid is placed are penetrated, to can become sticky or muddy in several hours, thus becomes unsuitable for injecting. And when being prepared as freeze-dried, with foreign protein or exogenous DNA physical-chemical reaction may occur for auxiliary material or preservative, then exist It is dissolved in during water forms injection and precipitating, influence preparation stability.

CN101444618A discloses a kind of pharmaceutical preparation containing Exenatide, it is characterised in that: the drug system Agent contains Exenatide, is able to maintain that buffer and enhancing Exenatide that preparation pH value under aqueous solution state is 3.0-7.0 The auxiliary material of stability, the auxiliary material can be optionally from one kind of glucose, sucrose, methionine, mannitol or glycine or several Kind.The buffer can be optionally from disodium hydrogen phosphate-citrate buffer solution, phosphate buffer, acetate buffer solution, barbital buffering Liquid or citrate buffer solution.It can be prepared as freeze drying powder injection.The program stresses low dosage E4 preparation, and finds under study for action, Its auxiliary material used is applied to the phenomenon that accidental precipitation precipitates when rE4 preparation, influences preparation stability.

Summary of the invention

There is operation using the method for technique for gene engineering preparation and reorganization glucagon-like peptide-1 receptor stimulant (rE4) Simply, low in cost, advantages of environment protection can be widely applied in the industrialized production of rE4.Due to gene work Impurity composition is different from chemically synthesized Exenatide in the rE4 of journey technology preparation, and the rE4 product of Bacillus coli expression is purified Afterwards there may be a small amount of foreign protein, external source residual DNA, preparation stability is influenced.The drug containing Exenatide of the prior art It is impossible to meet Gluca Gen sample peptide-1 receptor stimulant preparation securities, the demand of stability for preparation, to limit The listings of rE4 related preparations.

The present inventor attempts various buffer systems and cosolvent prescription, has screened one kind by largely testing The medicinal composition for injections of Gluca Gen sample peptide-1 receptor stimulant (rE4) suitable for Bacillus coli expression, should Composition has good safety and stability, is suitable for large-scale production and post storage transports.

The present invention provides a kind of medicinal composition for injections containing Gluca Gen sample peptide-1 receptor stimulant, Middle Gluca Gen sample peptide-1 receptor stimulant is purified by Bacillus coli expression and is made, and is had as shown in Seq ID No.2 Sequence, it is characterised in that contain Gluca Gen sample peptide-1 receptor stimulant, citric acid-sodium citrate buffer salt and more First alcohol.

Biological products generally have complicated structure, need to select suitable buffer system, and strict control buffer PH value, it is ensured that the concentration of biological products, purity and activity will not because of external environment variation and change.Hair of the invention Bright people respectively screens a large amount of buffer system at research initial stage, is found by a large amount of stability tests, and citric acid is slow The system of punching can largely keep the stability of rE4.

Medicinal composition for injections of the present invention citric acid-sodium citrate buffer salinity optional 10 in configuration process ~30mM, preferably 20mM.

The present inventor has found that citric acid buffer salinity is for rE4 medicinal composition for injections under study for action Stability also has certain influence, is tested by buffer salinity, it was demonstrated that buffer is citric acid-sodium citrate buffer solution, dense Stability of the rE4 pharmaceutical composition under solution state is optimized when degree is 20mM.

Polyalcohol is one of sorbierite, mannitol or combinations thereof in medicinal composition for injections of the present invention.

Carbohydrate, polyalcohol, amino acid and its derivative, inorganic salts, glycerol, polymer are as polyethylene glycol (PEG) etc. Referred to as protein cosolvent (isolate and purify the unstability and its countermeasure of middle protein, Feng little Li etc., bioengineering into Exhibition, 2000, Vol.20,67~71).Sugar and polyalcohol can increase the stability of pharmaceutical grade protein in water, common carbohydrate Including sucrose, glucose, trehalose and maltose, and common polyalcohol has glycerol, mannitol, sorbierite, PEG and inositol Deng.Some amino acid such as glycine, arginine, aspartic acid and paddy ammonia amine acyl etc., can increase pharmaceutical grade protein in given pH Under solubility, and its stability can be improved, can also be used as the stabilizer of polypeptide drug.The inventors found that polynary The addition of alcohol can be improved the stability of rE4, and rE4 injection can be improved in the addition of a large number of experiments discovery sorbierite or mannitol Stability；Meanwhile the addition of sorbierite or mannitol can prevent temperature from increasing or being lyophilized the denaturation of rE4 during redissolution.

The present inventor in the course of the research carries out buffer system-cosolvent (carbohydrate, polyalcohol, amino acid) As a result orthogonal test confirms that citric acid buffer system and sorbierite or mannitol is used to combine as the injectable drug of cosolvent Object can utmostly keep the stability of rE4 preparation.

The present invention provides a kind of medicinal composition for injections containing Gluca Gen sample peptide-1 receptor stimulant, institute Stating medicinal composition for injections is injection, and the injection is composed of the following components:

Preferably, the injection is composed of the following components:

In specific implementation process, injection provided by the invention can be made of the following components:

In specific implementation process, it is preferable that the injection can be composed of the following components:

The above specific embodiment should not be construed as limiting the scope of the present invention.

The present inventor studies the content of rE4 in preparation, finds rE4 excessive concentration or too low can make At protein crystal, precipitating is precipitated, influences rE4 stability and biological activity.

Injection of the present invention, pH are 4.0~6.0, preferably 5.0~5.8；Further preferably 5.6.

The present inventor has found under study for action, the stability of citric acid buffer salinity and pH for rE4 injection Also there is certain influence, tested by pH, it was demonstrated that buffer is citric acid-sodium citrate buffer salt, and pH is at 4.0~6.0, note Liquid is penetrated to have good stability；For pH at 5.0~5.8, injection stability is preferable；The stability of injection obtains most when pH is 5.6 Optimization.

Injection of the present invention, wherein can also contain preservative, preservative is phenol 2000-3000 parts, concentration For 0.2~0.3% (weight/liquor capacity, w/v)；Preferably 2500 parts, concentration 0.25%.

The present invention also provides a kind of medicinal composition for injections containing Gluca Gen sample peptide-1 receptor stimulant, The medicinal composition for injections is freeze drying powder injection, and every freeze drying powder injection is composed of the following components:

Preferably, every freeze drying powder injection is composed of the following components:

In specific implementation process, freeze drying powder injection provided by the invention can be composed of the following components:

Component	Content	Weight (mg/ branch)
			Gluca Gen sample peptide-1 receptor stimulant	50~200 parts	0.05~0.20
Citric acid	862~4129 parts	0.862~4.129
			Sodium citrate	1015~5206 parts	1.015~5.206
Polyalcohol	10000~50000 parts	10~50

；

In specific implementation process, it is preferable that the freeze drying powder injection is composed of the following components:

Component	Content	Weight (mg/ branch)
			Gluca Gen sample peptide-1 receptor stimulant	120 parts	0.12
Citric acid	2450 parts	2.45
			Sodium citrate	2360 parts	2.36
Polyalcohol	30000 parts	30

；

The above specific embodiment should not be construed as limiting the scope of the present invention.

Freeze drying powder injection provided by the invention pH under solution state is 4.0~6.0, preferably 4.5~5.5.

The present inventor has found that citric acid buffer salinity and pH are freezing rE4 freeze drying powder injection under study for action Stability during dry-redissolution also has certain influence, passes through buffer salinity test and pH is tested, it was demonstrated that buffer is lemon Lemon acid-buffered sodium citrate salt, in preparing solution processes, citric acid buffer salinity is 20mM, and pH freezes at 4.0~6.0 RE4 concentration and biological activity do not change during dry-redissolution；Stability is most in the citrate buffer solution of pH4.5~5.5 Excellent, freeze-drying-redissolution and long-term storage will not influence preparation stability.

The present inventor has found that preservative may be with external source residual DNA present in rE4 stoste during the experiment Or foreign protein generates interaction, thus there is certain influence to stability of rE4 preparation during freeze-drying-redissolution.Freeze-drying When be added without preservative can avoid rE4 preparation be lyophilized-redissolution process generate crystallization or precipitating.If you need to short-term preservation when use, then It can select freeze-dried powder being dissolved in the water for injection containing preservative when redissolving.

Using rE4 freeze drying powder injection made from the preparation method, rE4 stablizes without denaturation, and long-term character of placing is multiple without change It is molten for without albumen precipitation, injection protein content is unchanged during injection, biological activity is without substantially changeing, meet preparation Related request.The freeze-dried powder can be dissolved in sterile water for injection when use or contain 0.2~0.3% (weight/liquor capacity, w/ V) in the Injectable sterile water of phenol；Preferably, 1 freeze-dried powder is dissolved in 480 μ L Injectable sterile water or 480 μ L contains In the Injectable sterile water of 0.25% (weight/liquor capacity, w/v) phenol.

The present invention also provides a kind of injectable drug groups prepared containing Gluca Gen sample peptide-1 receptor stimulant The method for closing object, which comprises the following steps:

1. weighing appropriate citric acid, sodium citrate, polyalcohol respectively, is dissolved with Injectable sterile water, be configured to dilution buffer；

2. assay approval Gluca Gen sample peptide-1 receptor stimulant stoste is diluted to final concentration using dilution buffer；

3. the filter membrane degerming for being 0.22 μm with aperture；

4. dispensing；

5. optionally, finished product is freezed and is lyophilized in time after packing, freeze drying powder injection is obtained, products temperature in overall process is lyophilized Not higher than 25 DEG C；

Optionally, it when medicinal composition for injections is prepared as injection, can weigh molten in appropriate phenol addition Injectable sterile water Solution.When being prepared as injection, 4. step can be packed as 1.2mL/ branch.

The present invention also provides the medicinal composition for injections containing Gluca Gen sample peptide-1 receptor stimulant to make The purposes being ready for use in the drug for the treatment of type-2 diabetes mellitus.

The present invention is using technique for gene engineering preparation and reorganization glucagon-like peptide-1 receptor stimulant (rE4) as effectively Ingredient studies auxiliary material and its proportion by a large number of experiments, and final choice is suitable for the medicinal composition for injections of rE4.The life of rE4 Produce have at low cost, advantages of environment protection, rE4 composition of the present invention have it is safe and stable, be readily transported and store The advantages that, it is suitable for industrialized production, can satisfy relevant market demand.

Specific embodiment

Following embodiment is illustrated to of the invention, should not be construed as limiting to the scope of the present invention.

RE4 used is with reference in the application such as CN1693459, CN1635117, CN1455001, CN102618552A in embodiment Disclosed scheme utilizes technique for gene engineering construction of expression vector by the present inventor, and fermented, purifying is made.RE4 stoste used It is detected through HPLC, concentration > 98%；Using ELISA method living is surveyed based on cell, generated by measurement sample stimulus cell CAMP concentration comes the activity of test sample, rE4 stoste activity >=1.12 × 10⁶AU/mg；Impurity uses " Chinese Pharmacopoeia " 2010 Method detection in version third portion annex IX, XII, meets regulation.

Detection method

With reference to:

Three annex of " Chinese Pharmacopoeia " version in 2010, III B high performance liquid chromatography；

Instrument: high performance liquid chromatograph, ultrapure water machine, ultrasonic cleaner；

Reagent consumptive material: 5 chromatographic column of Source, micropore filtering film, acetonitrile, trifluoroacetic acid；

Mobile phase A: taking 50mL acetonitrile, and 1mL trifluoroacetic acid is settled to 1000mL with the above ultrapure water of 18.2M Ω ㎝, after mixing Ultrasonic degassing after being filtered with 0.22 μm of organic film；

Mobile phase B: taking 950mL acetonitrile, and 1mL trifluoroacetic acid is settled to 1000mL with the above ultrapure water of 18.2M Ω ㎝, after mixing Ultrasonic degassing after being filtered with 0.22 μm of organic film；

Sample treatment: rE4 stoste need to be diluted to suitable concentration by the detection of product stoste, and finished product detection need to be by rE4 finished product preparation 1mL It is directly placed into sample introduction bottle, if sample size answers selection of casing to be detected less than 500 μ l；

Loading and detection: using autosampler sample introduction, and each sample is into 20 μ L, and column temperature is 40 DEG C, flow velocity 1.0mL/min, inspection Wavelength 215nm is surveyed, pillar is balanced to baseline with mobile phase A, with 100%A+0%B to 30%A+70%B gradient elution 35min, Record map.The area for measuring its main peak calculates corresponding content.

Bioactivity detection

Principle: in conjunction with receptor on cells, stimulation intracellular biological active material increases rE4, shows as intracellular cAMP concentration Increase.Intracellular cAMP concentration increases related in dosage to rE4；

Instrument: Biohazard Safety Equipment, CO₂Incubator, MM-I type micro oscillator, microplate reader, desk centrifuge；

Reagent consumptive material: rExendin-4 standard items, cell line: RIN-m5F cell (rat Langerhans islet oncocyte, ATCC, CRL- 11605⁺), 1640 basal medium of RPMI, fetal calf serum, 0.25% trypsase, DMSO, BSA, IBMX, cAMP are enzyme-linked to exempt from Epidemic disease assay kit, 96 porocyte culture plates, pipettor；1640 complete medium of RPMI: 1640 basal medium of RPMI+ 10% fetal calf serum；RE4 dilution: RPMI 1640 basal medium+0.1%BSA (bovine serum albumin(BSA))+1mM IBMX, it is existing With current；IBMX concentrate (100mM): it is dissolved in DMSO (dimethyl sulfoxide)；Phosphate buffer (PBS): NaCl 8.0g, NaHPO₄ 1.44g、KCl 0.2g、KH₂PO₄The dissolution of 0.2g, 1L pure water, high pressure sterilization 25 minutes；Cell dissociation buffer: 0.25% Trypsase, 4 DEG C of preservations；

Detection method:

1. logarithmic growth phase cell, digestion, counting, adjustment cell density are 1.5 × 10⁵/ mL is inoculated in 96 with 200 holes μ L/ Porocyte plates, 37 DEG C of 5%CO₂Culture is sticked to cell；

2. replacing 96 porocyte plates cell liquid is 1640 basal medium of RPMI, 100 holes μ L/, 37 DEG C of 5%CO₂Continue culture 12 Hour；

3. with freshly prepared rE4 dilution pre-dilution rE4 standard items, product to be tested；Simultaneously with rE4 dilution as negative control (referred to as IBMX control)；

4. removing culture medium, each sample and negative control (100 hole μ L/) is added, 37 DEG C are reacted 5 minutes；

5. removing rE4 reaction solution, PBS is washed cell 1 time, removes residual liquid；

6. every hole is added the cell pyrolysis liquid that 120 μ l kits provide, 37 DEG C lytic cell 10 minutes；96 porocyte plates 600g from The heart 10 minutes, supernatant is taken to carry out ELISA experiment；

7. the cAMP concentration generated using ELISA Reverse transcriptase method measurement cytositimulation, experimental implementation are illustrated according to kit It carries out；

8. carrying out data processing and analysis with Origin8.5 software or other softwares；

9. calculating: using cAMP concentration as abscissa, drawing standard curve by ordinate of OD value, calculated according to regression curve negative CAMP concentration in control wells and experimental group hole (various concentration standard items, stoste or preparation)；

The cAMP concentration that various concentration standard items, product to be tested stimulation generate is that corresponding experimental group cAMP concentration subtracts negative control hole The value of cAMP concentration；

Using the logarithm of standard items or product to be tested extension rate as abscissa, it is vertical sit that corresponding aperture cytositimulation, which generates cAMP concentration, Koji-making line is marked and drawed, standard items and product to be tested median effective dose extension rate are calculated separately out by linear equation；

The calculating of rExendin-4 sample specific activity:

The preparation of embodiment 1:rE4 freeze drying powder injection

1. weighing citric acid, sodium citrate, mannitol respectively according to the above recipe quantity, add 800mL water for injection predissolve；

2. appropriate rE4 stoste is added；

3. water for injection is settled to 1000mL, pH value of solution 4.5, citric acid-sodium citrate buffer salinity is 20mM；

4. with 0.22 μm of filter membrane aseptic filtration；

5. according to being lyophilized after the packing of 1mL/ branch.

The preparation of embodiment 2:rE4 injection

1. weighing citric acid, sodium citrate, mannitol, phenol respectively according to the above recipe quantity, 800mL water for injection is added to be pre-dissolved Solution；

2. appropriate rE4 stoste is added；

3. water for injection is settled to 1000mL, pH value of solution 5.6, citric acid-sodium citrate buffer salinity is 20mM；

4. with 0.22 μm of filter membrane aseptic filtration；

5. being dispensed according to 1.2mL/ branch.

Embodiment 3~7:rE4 concentration is investigated

Referring to the prescription and its preparation process of embodiment 1, freeze drying powder injection specification is only changed to 0.03 respectively, 0.05,0.10, 0.15, freeze drying powder injection is prepared in 0.20mg/ branch.

Embodiment 8~12:rE4 concentration is investigated

Referring to the prescription and its preparation process of embodiment 2, rE4 concentration in injection is only changed to 0.05 respectively, 0.1,0.2, 0.3, injection is prepared in 0.4mg/mL.

Embodiment 13~17: the investigation of different buffer systems

Buffer system, is only changed to the phosphoric acid of same concentrations by the prescription and its preparation process for respectively referring to embodiment 1,2 respectively Buffer (pH5.8), acetate buffer solution (pH4.5), glutamate buffers (pH5.5), disodium hydrogen phosphate-citrate buffer solution (pH4.5), freeze-dried powder and injection is prepared in disodium hydrogen phosphate-potassium phosphate buffer (pH5.0).

Embodiment 18~21: the investigation of different citric acid salt concentrations

The prescription and its preparation process of embodiment 1,2 are respectively referred to, only by citric acid-sodium citrate in preparing solution processes Buffer system concentration is changed to 5mM, 10mM, 30mM, 50mM, and freeze-dried powder and injection is prepared.

Embodiment 22~29: the investigation of different cosolvent

Respectively refer to the prescription and its preparation process of embodiment 1,2, only by cosolvent be changed to the sorbierite of isoconcentration, glycerol, Freeze-dried powder and injection is prepared in polyethylene glycol, glucose, lactose, xylitol, glycine, methionine.

Embodiment 30~33: the investigation of cosolvent concentration

Respectively refer to the prescription and its preparation process of embodiment 1,2, only by mannitol concentration be changed to 5mg/ branch, 10mg/ branch, 50mg/ branch, 100mg/ branch, are prepared freeze-dried powder and injection.

Embodiment 34: buffer system and cosolvent orthogonal test

Referring to the prescription and its preparation process of embodiment 2, buffer system (20mM) and cosolvent (30mg/mL) are matched according to table 1, RE4 injection is prepared, the stabilization gender gap of the rE4 injection of different prescriptions is investigated

Experimental facilities: simulation transport test machine (production of Dongguan City Yuan Ke detecting instrument Co., Ltd)

Parameter: speed: 100-300rpm；Amplitude: 1 inch 2 of 5.4mm, maximum load: 100Kg；Working time: 9999H/M/S； Power of motor: 1HP；Maximum current: 4A；Bed dimension: 100cm × 120cm

Experimental method: prescription preparation gained rE4 injection in table 1 at 2~8 DEG C of temperature, is placed on simulation transport test machine Carry out simulation transport test, amplitude 25.4mm, frequency 4Hz, test speed 240rpm, the testing time 1 hour

Before experiment and experiment terminates to stand 30min, takes supernatant to detect rE4 concentration after sample 10000g centrifugation.RE4 concentration uses HPLC detection

Experimental result: it is shown in Table 1

The different prescription injection stability of table 1 (in terms of rE4 concentration (μ g/mL))

Conclusion: the rE4 injection prepared using citric acid-sodium citrate as buffer system, mannitol or sorbierite as cosolvent Liquid has preferable stability, and citric acid-sodium citrate is used to inject as buffer system, mannitol as the rE4 of cosolvent Liquid has optimal stability.

Embodiment 35~41: the investigation of injection difference pH

The prescription and its preparation process of embodiment 1,2 are respectively referred to, citric acid-trisodium citrate ratio is only adjusted, by pH 3.0,4.0,5.0,5.5,5.8,6.0,6.6 are changed to, freeze-dried powder and injection is prepared.

Embodiment 42~44: the investigation of preservative

Referring to 1 prescription of embodiment and its preparation process, 0.25% phenol, metacresol, benzylalcohol are only added wherein as anti-corrosion Agent prepares freeze-dried powder.

Embodiment 45: the influence that preservative redissolves freeze-dried powder

Following groups injection is prepared using the freeze-dried powder of embodiment 1 and the freeze-dried powder of embodiment 42~44

A: rE4 freeze-dried powder prepared by embodiment 1 is dissolved in 480 μ L sterile water for injection；

B: rE4 freeze-dried powder prepared by embodiment 1 is dissolved in the sterile phenol-aqueous solution of 480 μ L, phenol content 0.25%；

C~E: rE4 freeze-dried powder prepared by embodiment 42~44 is dissolved in 480 μ L sterile water for injection respectively

Preparation terminate stand 30min, sampling, the appearance of sample survey, sample be placed in centrifuge tube 10000g centrifugation after observe it is visible Foreign matter takes supernatant HPLC to measure rE4 concentration

Experimental result: 2 are shown in Table

The influence that 2 preservative of table redissolves freeze-dried powder

Number	Appearance	Visible foreign matters	RE4 concentration (μ g/mL)
				A (embodiment 1 is dissolved in sterile water)	Colourless clear liquid	Meet regulation	250
B (embodiment 1 is dissolved in phenol-aqueous solution)	Colourless clear liquid	Meet regulation	249
				C (embodiment 42 is dissolved in sterile water)	Colourless clear liquid	CentrifugationTube bottom is visible afterwardsMinute quantity white precipitate	230
D (embodiment 43 is dissolved in sterile water)	White suspension	CentrifugationTube bottom is visible afterwardsA small amount of white precipitate	175
				E (embodiment 44 is dissolved in sterile water)	White suspension	CentrifugationTube bottom is visible afterwardsA small amount of white precipitate	170

As it can be seen that preservative is added during preparing freeze-dried powder, preservative may in freeze-drying process with rE4 and/or its Foreign protein, external source residual DNA react, and lead to polypeptide denaturation during redissolving, are precipitated, influence preparation stability.

Comparative example 1: listing has been developed in referencePreparation prescription

Respectively according to the preparation process of embodiment 1,2, freeze drying powder injection and injection is prepared.

2) preparation prescription 2 of comparative example 2:CN101444618A example 2

With reference to the prescription of the 2) preparation prescription 2 of CN101444618A example 2, wherein it will be changed to rE4 by Exenatide, other operations are same 2) preparation prescription 2 of CN101444618A example 2, prepare freeze drying powder injection and injection respectively.

Embodiment 46:rE4 freeze drying powder injection stability experiment

Experiment purpose: the stabilization gender gap of the rE4 freeze drying powder injection of different prescriptions is investigated；

Experimental drug: embodiment 1,3~7,13~33,35~41,1~2 gained rE4 freeze drying powder injection of comparative example；

Experimental facilities: simulation transport test machine (production of Dongguan City Yuan Ke detecting instrument Co., Ltd)；

Experimental method: embodiment 1,3~7,13~33,35~41,1~2 gained rE4 freeze drying powder injection of comparative example are dissolved in respectively 480 μ L Injectable sterile water are placed on simulation transport test machine at 2~8 DEG C of temperature and carry out simulation transport test, amplitude is 25.4mm, frequency 4Hz, test speed 240rpm, the testing time 1 hour.Before experiment and experiment terminates to stand 30min, samples, The appearance of sample survey, visible foreign matters take supernatant to detect biological activity, rE4 concentration after sample 10000g centrifugation.Biology is living Property using ELISA method living is surveyed based on cell, the cAMP concentration generated by measurement sample stimulus cell is come test sample Activity；RE4 concentration is detected using HPLC；

Experimental result: 3 are shown in Table

The stability of table 3:rE4 freeze drying powder injection

Conclusion:

1, rE4 concentration has certain influence to the stability of rE4 freeze drying powder injection, and every freeze-dried powder contains 50~200 parts of rE4 (0.05~0.20mg is dissolved in 480 μ L waters for injection) preparation stability is qualified, and freeze-drying-redissolution polypeptide nature is without change；Every jelly Dry powder contains that 120 parts of rE4 (0.12mg is dissolved in 480 μ L waters for injection) preparation stability is more excellent, and freeze-drying-redissolution will not be to rE4 Concentration, biological activity have an impact.The various aspects such as facilitate to comprehensively consider from stability, preparation cost, patient medication, every jelly Freeze drying powder injection optimal stability when amount in dry powder containing rE4 is 120 parts (0.12mg is dissolved in 480 μ L waters for injection)；

2, buffer system type also has certain influence to the stability of rE4 freeze drying powder injection.When buffer system is citric acid systems When, preparation stability is best.Preparation stability is preferable when preparing solution, citric acid-sodium citrate concentration is 10~30mM, Physicochemical property, biological activity are without change when freeze-drying-redissolution, and citric acid-sodium citrate buffer salinity is when preparing solution Preparation stability is best when 20mM.That is, freeze drying powder injection contains 862~4129 parts of citric acid, 1015~5206 parts of sodium citrate When freeze drying powder injection stability it is more excellent, optimal stability when containing 2450 parts of citric acid, 360 parts of sodium citrate；

3, the type of cosolvent also has certain influence to the stability of rE4 injection.When cosolvent is sorbierite or mannitol, Preparation stability is preferable；When cosolvent is mannitol, preparation stability is best；When cosolvent is other sugar, polyalcohol or ammonia When base acid, the stability of rE4 injection is reduced, the visible crystallization of when freeze-drying-redissolution or precipitating.Polyalcohol is in freeze drying powder injection Preparation stability is preferable when 10000~50000 parts (10mg/ branch~50mg/ branch), and polyalcohol is 30000 parts of (30mg/ branch) when systems Agent optimal stability；

4, rE4 freeze drying powder injection when preparing solution, pH is 4.0~6.0 preparation it is more stable, rE4 freeze-dried powder when pH4.5~5.5 Injection is the most stable, and physicochemical property, solution ph, biological activity are without change when freeze-drying-redissolution.

Embodiment 46:rE4 injection stability experiment

Experiment purpose: the stabilization gender gap of the rE4 injection of different prescriptions is investigated；

Experimental drug: embodiment 2,8~33,35~41,1~2 gained rE4 injection of comparative example；

Experimental facilities: simulation transport test machine (production of Dongguan City Yuan Ke detecting instrument Co., Ltd)；

Parameter: speed: 100-300rpm；Amplitude: 1 inch 2 of 5.4mm, maximum load: 100Kg；Working time: 9999H/M/S； Power of motor: 1HP；Maximum current: 4A；Bed dimension: 100cm × 120cm；

Experimental method: embodiment 2,8~33,35~41,1~2 gained rE4 injection of comparative example are set at 2~8 DEG C of temperature In carrying out simulation transport test on simulation transport test machine, amplitude 25.4mm, frequency 4Hz, test speed 240rpm, when test Between 1 hour.Before experiment and experiment terminate stand 30min, sampling, the appearance of sample survey, visible foreign matters, sample 10000g from Supernatant is taken to detect biological activity, rE4 concentration after the heart.Biological activity, which is used, surveys ELISA method living based on cell, passes through survey The cAMP concentration for determining the generation of sample stimulus cell carrys out the activity of test sample；RE4 concentration is detected using HPLC；

Experimental result: 4 are shown in Table

The stability of table 4:rE4 injection

Conclusion:

1, rE4 concentration has certain influence to the stability of rE4 injection, and the amount containing rE4 is 100~300 parts in injection When (0.1~0.3mg/mL), preparation stability is qualified.Many-sided synthesis such as facilitate to examine from stability, preparation cost, patient medication Consider, is optimum protein quality when the amount containing rE4 is 250 parts (0.25mg/mL) in injection；

2, buffer system type also has certain influence to the stability of rE4 injection.When buffer system is citric acid systems, system Agent optimal stability.When citric acid-sodium citrate concentration is 10~30mM, preparation stability is preferable, preparation when concentration is 20mM Optimal stability.I.e. in injection contain 60~4130 parts of citric acid, 1020~5210 parts of sodium citrate when preparation stability compared with It is excellent；Preparation stability is best when containing 1160 parts of citric acid, 4260 parts of sodium citrate；

3, the type of cosolvent also has certain influence to the stability of rE4 injection.When cosolvent is sorbierite or mannitol, Preparation stability is preferable；When cosolvent is mannitol, preparation stability is best；When cosolvent is other cosolvent, rE4 note The stability for penetrating liquid reduces.In injection polyol amount be 10000 parts~50000 parts (10mg/mL~50mg/mL) when preparation Stability is preferable, and preparation stability is best when content is 30000 parts (30mg/mL)；

4, rE4 preparation pH preparation at 4.0~6.0 is more stable, and rE4 injection formulation stability is preferable when pH5.0~5.8, Preparation is the most stable when pH5.6.

Embodiment 47: long-term stable experiment

The resulting rE4 freeze drying powder injection of embodiment 1, the resulting injection of embodiment 2 are stored in refrigerator, temperature is maintained at It 2~8 DEG C, in 0 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, is sampled within 36 months, freeze-dried powder takes It is redissolved in 480 μ L Injectable sterile water after out, stands 30min, examine appearance, visible foreign matters, pH value, biological activity, pure (HPLC method), rE4 concentration are spent, experimental result is as follows:

5 rE4 freeze drying powder injection long-time stability of table

6 rE4 injection long-time stability of table

Conclusion: 2~8 DEG C of rE4 freeze drying powder injection provided by the present invention are stored 3 years, and 2~8 DEG C of injection are stored 18 months, every Index has good stability without significant change.

Claims (9)

1. a kind of medicinal composition for injections containing Gluca Gen sample peptide-1 receptor stimulant, wherein the recombination high blood of pancreas Sugared element sample peptide-1 receptor stimulant is purified by Bacillus coli expression to be made, and has the sequence as shown in Seq ID No.2, feature It is containing Gluca Gen sample peptide-1 receptor stimulant, citric acid-sodium citrate buffer salt and polyalcohol.

2. pharmaceutical composition as described in claim 1, it is characterised in that the polyalcohol is one of sorbierite, mannitol Or combinations thereof.

3. pharmaceutical composition as claimed in claim 1 or 2, it is characterised in that described pharmaceutical composition is injection, the injection Liquid is composed of the following components:

Preferably, the injection is composed of the following components:

4. injection as claimed in claim 3, wherein also containing Injectable sterile water, injection pH is 4.0~6.0, excellent Select 5.0~5.8；Further preferably 5.6.

5. injection as claimed in claim 4, it is characterised in that: also contain preservative in injection, preservative is phenol 2000~3000 parts, preferably 2500 parts.

6. pharmaceutical composition as claimed in claim 1 or 2, it is characterised in that described pharmaceutical composition is freeze drying powder injection, Be characterized in that: every freeze drying powder injection is composed of the following components:

Preferably, every freeze drying powder injection is composed of the following components:

7. pharmaceutical composition as claimed in claim 6, it is characterised in that: described pharmaceutical composition is freeze drying powder injection, the jelly Dry powder injection pH under solution state is 4.0~6.0, preferably 4.5~5.5.

8. a kind of method for preparing pharmaceutical composition as described in any one of claims 1 to 7, which is characterized in that including following Step:

1. weighing appropriate citric acid, sodium citrate, polyalcohol respectively, is dissolved with Injectable sterile water, be configured to dilution buffer；

2. assay approval Gluca Gen sample peptide-1 receptor stimulant stoste is diluted to final concentration using dilution buffer；

3. the filter membrane degerming for being 0.22 μm with aperture；

4. dispensing；

5. optionally, finished product is freezed and is lyophilized in time after packing, freeze drying powder injection is obtained, products temperature in overall process is lyophilized Not higher than 25 DEG C；

Optionally, it when medicinal composition for injections is prepared as injection, can weigh molten in appropriate phenol addition Injectable sterile water Solution.

9. pharmaceutical composition described in any one of the claims is in preparing the drug for treating type-2 diabetes mellitus Purposes.