WO2015158270A1 - 一种含有艾塞那肽的组合物及其制备方法 - Google Patents

一种含有艾塞那肽的组合物及其制备方法 Download PDF

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Publication number
WO2015158270A1
WO2015158270A1 PCT/CN2015/076688 CN2015076688W WO2015158270A1 WO 2015158270 A1 WO2015158270 A1 WO 2015158270A1 CN 2015076688 W CN2015076688 W CN 2015076688W WO 2015158270 A1 WO2015158270 A1 WO 2015158270A1
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Prior art keywords
microspheres
solution
exenatide
plga
polyvinyl alcohol
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PCT/CN2015/076688
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English (en)
French (fr)
Inventor
王涛
王麒麟
徐钱钱
孙丽芳
孟莹
李菊
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Shandong Luye Pharmaceutical Co Ltd
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Shandong Luye Pharmaceutical Co Ltd
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Priority claimed from CN201410153581.2A external-priority patent/CN103932993B/zh
Priority claimed from CN201410153089.5A external-priority patent/CN103932992B/zh
Application filed by Shandong Luye Pharmaceutical Co Ltd filed Critical Shandong Luye Pharmaceutical Co Ltd
Priority to US15/304,528 priority Critical patent/US20170035856A1/en
Priority to JP2016562940A priority patent/JP2017511371A/ja
Priority to EP15779307.6A priority patent/EP3132790A4/en
Publication of WO2015158270A1 publication Critical patent/WO2015158270A1/zh
Anticipated expiration legal-status Critical
Priority to US15/803,807 priority patent/US20180117123A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention is in the field of medicine and relates to a composition containing exenatide in the form of microspheres.
  • the invention also relates to the preparation of the microspheres
  • Exenatide is the first member of the GLP-1 family of synthetic incretin analogues. It mimics the biological pattern of GLP-1 secreted by human body. It can enhance glucose dependence after being released into the circulation from the intestine. Insulin secretion.
  • the amino acid sequence of Exenatide partially overlaps with human GLP-1 and is shown to bind and activate the known human GLP-1 receptor, allowing glucose-dependent insulin synthesis by including cAMP and/or other intracellular signaling mechanisms.
  • islet beta cells secrete insulin secretion in the body. In the case of elevated glucose levels, exenatide promotes the release of insulin from beta cells while exenatide improves glycemic control by reducing fasting and postprandial blood glucose concentrations in patients with type 2 diabetes.
  • the products that exenatide has been marketed mainly include injections, which are only used for subcutaneous injection.
  • the recommended starting dose is 5 ⁇ g twice daily, and after 1 month of treatment, the dose can be increased to 10 ⁇ g depending on the clinical response.
  • the preparation was injected twice daily (bid) with an average peak time of 2.1 h and an average elimination half-life of 2.4 h, which was mainly eliminated by the kidneys.
  • Such injections require frequent administration and do not continue to activate the GLP-1 receptor, and are less compliant for diabetic patients who require long-term administration.
  • the phase separation method has been used to prepare exenatide microspheres, and the phase separation method has many production steps, high requirements on equipment, high quality requirements for sterilizing and solvent residues of pharmaceutical products, and also Separation method
  • the microspheres prepared by the preparation process also require product processing in multiple processes; because of the above-mentioned deficiencies in the phase separation of exenatide microspheres, some research institutes have carried out research on the production of exenatide microspheres by solvent evaporation method.
  • the equipment requirements are low, the working steps are less, and the products can be processed with fewer post-processing steps to meet the relevant quality standards.
  • the commercial product of Exenatide is the acetate of exenatide.
  • the product standard requires: 3% ⁇ acetic acid content ⁇ 12%.
  • the prior art has no relevant acetic acid content control for the production of exenatide microspheres.
  • the acetic acid content of exenatide microspheres has an effect on the bioavailability of exenatide during storage.
  • the content of acetic acid exceeds 0.01%, the bioavailability of exenatide in the microspheres during storage is produced. The problem of falling.
  • the inventors conducted intensive studies to provide a composition containing exenatide, which is a microsphere, and the starting material is exenatide acetate (commercially available).
  • the product 3 ⁇ acetic acid content ⁇ 12%) and polylactide glycolide (PLGA), and the prepared exenatide microspheres controlled the acetic acid content to be 0.01% or less.
  • the present invention provides a composition containing exenatide, the composition is in the form of microspheres, and the microsphere starting material is exenatide acetate and polylactide glycolide (PLGA), after preparation
  • the composition has an acetic acid content of less than 0.01%.
  • the content of the exenatide is from 2 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, based on the total weight of the composition.
  • the content of polylactide glycolide (PLGA) is from 90 to 98%, preferably from 92 to 97%, more preferably from 94 to 96%, based on the total weight of the composition.
  • the lactide-glycolide copolymer has the English name Poly(lactide-co-glycolide), abbreviated as PLGA.
  • the polylactide glycolide (PLGA) of the present invention has a molecular weight of from 6000 to 45,000 Daltons, preferably from 10,000 to 30,000 Daltons, more preferably from 10,000 to 25,000 Daltons, said molecular weight being referred to as "weight average molecular weight", Referred to as "molecular weight”.
  • the molar ratio of lactide to glycolide of PLGA is from 90:10 to 10:90, preferably from 75:25 to 25:75, more preferably from 60:40 to 40:60, especially from 50:50.
  • the intrinsic viscosity of PLGA is from 0.1 to 0.40 dL/g, preferably from 0.10 to 0.35 dL/g, more preferably from 0.10 to 0.30 dL/g.
  • Inherent viscosity measurement method of PLGA PLGA was prepared into a solution of about 0.5% (w/v) with chloroform, and its intrinsic viscosity was measured at 30 ° C using a Cannon-Fenske glass capillary viscometer.
  • PLGA 50/50, 0.20, 16000
  • the molar ratio of lactide to glycolide is 50:50
  • the intrinsic viscosity is 0.20 dl/g
  • the molecular weight is 16,000 daltons. Ester copolymer.
  • compositions provided herein are prepared by solvent evaporation. Accordingly, the present invention also provides a process for the preparation of a composition comprising exenatide.
  • the preparation process of the microspheres comprises: preparing PLGA into an organic solution with an organic solvent, mixing exenatide acetate with an organic solution of PLGA to form colostrum; adding colostrum to the external aqueous phase to emulsify, solidifying and volatilizing the organic solvent , after washing, lyophilization to obtain microspheres.
  • the organic solvent may dissolve lactide glycolide (PLGA), which may be an organic solvent such as dichloromethane, acetone, acetonitrile or the like which can dissolve PLGA, preferably dichloromethane.
  • PLGA lactide glycolide
  • the washing liquid is distilled water or an alkaline buffer, preferably distilled water, and the weight ratio of the microspheres to the washing liquid is 1:250 and above, and preferably the weight ratio is 1:330 or more.
  • the lyophilized microspheres were set to a re-drying temperature below the glass transition temperature of the exenatide acetate and PLGA mixture. By this adjustment, the present invention enables to obtain an exenatide microsphere product having an extremely low acetic acid content and meeting quality standards and having good properties.
  • the concentration and pH of the external aqueous phase can be adjusted during the preparation process.
  • the outer aqueous phase is a polyvinyl alcohol solution, the average molecular weight of the polyvinyl alcohol is 13000-23000; the weight percent concentration of the polyvinyl alcohol of the solution (unit g/100 ml) is 0.5-1.5%, preferably the weight of the polyvinyl alcohol.
  • the volume percent concentration is 1%; the pH of the solution is 5.6 ⁇ 1.0, preferably the pH of the solution is 5.6 ⁇ 0.5, and more preferably the pH of the solution is 5.6 ⁇ 0.2.
  • the polyvinyl alcohol in the present invention is The residual amount in the provided composition should be no more than 0.008% by weight.
  • the microspheres of the present invention can be obtained by the following exemplary operation of the solvent evaporation method: weighing a certain amount of exenatide or a salt thereof and the like, and simultaneously weighing a known amount of the auxiliary solvent , protein protectants, surfactants, porogens and other auxiliary materials, you can first dissolve exenatide or its salts and analogues with distilled water, the selected excipients can also be dissolved in aqueous solution; weigh a certain amount of PLGA, use organic solvents Dissolving, the organic solvent may be dichloromethane, acetone, acetonitrile or the like to dissolve PLGA in an organic solvent, preferably dichloromethane; an aqueous solution containing exenatide and an auxiliary material is mixed with a PLGA organic solution to form a uniform colostrum; The colostrum containing exenatide or a salt thereof and the like is added to an external aqueous phase (such as a polyviny),
  • exenatide acetate may be prepared without first preparing an aqueous solution, and exenatide acetate may be directly mixed with a PLGA organic solution to form colostrum.
  • the outer aqueous phase of the process for preparing the microspheres is a polyvinyl alcohol solution; the weight percent concentration of the polyvinyl alcohol of the solution is 0.5-1.5%, preferably 1%, and the pH of the solution is 5.6 ⁇ 1.0, preferably 5.6 ⁇ 0.5. More preferably, it is 5.6 ⁇ 0.2.
  • the pH of the external aqueous phase plays a key role in the characteristics of the prepared microspheres.
  • the present invention can adjust the pH of the external aqueous phase with an acid, a base or a buffer to make an oil containing exenatide or a salt thereof and the like.
  • the phase (colostrum) is solidified in the outer aqueous phase of the selected pH after emulsification, laying the foundation for obtaining good properties of exenatide or its salts and analog microspheres.
  • the pH of the external aqueous phase can be stabilized within a certain range by using an acid, a base or a buffer.
  • the acid used may be a water-soluble acid such as hydrochloric acid, sulfuric acid, acetic acid or amino acid, and the commonly used one is 0.1 mol/L hydrochloric acid;
  • the base used may be a water-soluble base such as sodium hydroxide, potassium hydroxide, sodium hydrogencarbonate or sodium sulfite.
  • the commonly used one is, for example, 0.1 mol/L sodium hydroxide, and the buffer may be a citrate buffer or phosphoric acid.
  • the weight percent concentration of polyvinyl alcohol in the outer aqueous phase plays an important role in the properties of the prepared microspheres, and the present invention adopts At a concentration of 0.5-1.5%, preferably 1%, the microspheres are cured in a selected concentration of the outer aqueous phase to obtain a microsphere product that meets quality standards and has good properties.
  • the polyvinyl alcohol of the present invention has an average molecular weight of 13,000-23000, and the average molecular weight of the polyvinyl alcohol is matched.
  • the aqueous solution of polyvinyl alcohol plays an important role in the characteristics of the prepared microspheres, and the microspheres are solidified in the outer aqueous phase of the selected average molecular weight polyvinyl alcohol, and the microsphere products satisfying the quality standard and having good characteristics can be obtained. .
  • the invention prepares exenatide or a salt thereof and the like microspheres by adjusting the polyvinyl alcohol concentration and the pH value of the outer aqueous phase during the preparation of the microspheres, so that the microspheres have high encapsulation efficiency and uniform particle size, and the polyethylene is obtained. A property of less alcohol residue.
  • the invention can use distilled water or buffer as the cleaning liquid to clean the microspheres, the weight ratio of the microspheres to the cleaning liquid is 1:250 and above, preferably the weight ratio is 1:330 and above; cleaning the micro at a selected weight ratio
  • the ball lays the foundation for obtaining exenatide microspheres with very low acetic acid content.
  • the buffer used can be a buffer of citrate buffer, phosphate buffer or acetate.
  • a buffer such as a liquid or a phthalate buffer is preferably distilled water.
  • lyophilization is abbreviated as freeze-drying, and generally includes pre-freezing, sublimation drying, repeated freezing sublimation, and re-drying process.
  • Lyophilization is a drying method in which a solution is frozen into a solid, and the liquid is directly sublimated from the frozen state without being subjected to a liquid state under low temperature and low pressure conditions.
  • the invention defines the temperature of the re-drying process, and other processes can be carried out by a conventional method.
  • the re-drying temperature of the present invention is below the glass transition temperature of the mixture of exenatide acetate and PLGA (emulsified), so that Obtain microsphere products that meet the quality standards and have good characteristics.
  • the present invention prepares exenatide microspheres by controlling the content of acetic acid in the process of preparing microspheres, so that the microspheres reach a long storage period and the bioavailability is constant, and the storage period of the products is prolonged. .
  • the invention adopts exenatide acetate as a starting material to prepare microspheres, and by reducing the concentration of acetic acid to a very low content, the stability of the microspheres is improved, and the storage time of the medicine is prolonged; the preparation method is determined by forming a ball.
  • the selection of the amount of the cleaning solution and the time and temperature of the freeze-dried microspheres finally reach the concentration of the acetic acid for controlling the microspheres; the preparation method can achieve high encapsulation efficiency by adjusting the concentration and pH of the aqueous phase during the emulsification process. Uniform, less polyvinyl alcohol residue.
  • PVA solution Preparation of polyvinyl alcohol solution: precision weighing of polyvinyl alcohol (average molecular weight 13000-23000) Body, measure the volume of the aqueous solution, use one-half of the volume of water to dissolve the weighed polyvinyl alcohol solids, after the polyvinyl alcohol solids are dissolved, add the remaining water to the solution, stir and mix, determine the solution The pH value was adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution was stabilized at 6.5.
  • Preparation method Weigh the formula amount of exenatide, add distilled water to stir and dissolve, prepare a solution, weigh the formula amount of glycolide-lactide copolymer (PLGA), add dichloromethane (CH 2 Cl 2 ) to dissolve and dissolve The two solutions are uniformly mixed to obtain colostrum; the prepared PVA solution is sterilized and filtered, and then added to a vacuum emulsification mixer (hereinafter referred to as a microsphere preparation kettle) and cooled to 7 to 13 ° C as an external aqueous phase. The colostrum was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm.
  • a vacuum emulsification mixer hereinafter referred to as a microsphere preparation kettle
  • the homogenization stirring speed was reduced to 150 rpm for 5 h.
  • the microspheres were collected by filtration, and the aqueous solution was washed with microspheres. (Specific method can be found in Example 11), remove PVA residue, transfer to lyophilization tray, add mannitol injection and appropriate amount of aqueous solution, freeze-dry in freeze dryer (see Example 11 for specific method); freeze-dried product After sieving, the microspheres are obtained.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. In 5.1.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, and the pH of the solution is measured, and 0.1 mol/L hydrochloric acid and 0.1 mol/L are used. The pH of the aqueous solution of polyvinyl alcohol was adjusted with sodium hydroxide until the pH of the aqueous solution stabilized at 6.1.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 4.6.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 6.5.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 4.7.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 9.0.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 3.0.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 5.8.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • Preparation of polyvinyl alcohol solution accurately weigh polyvinyl alcohol (average molecular weight 13000-23000) solid, measure the volume of aqueous solution, use one-half of the volume of the aqueous solution to dissolve the weighed polyvinyl alcohol solid, etc. After the polyvinyl alcohol solid is dissolved, the remaining water is added to the solution, stirred and mixed, the pH value of the solution is measured, and the pH value of the polyvinyl alcohol aqueous solution is adjusted with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide until the pH of the aqueous solution is stable. At 5.6.
  • the encapsulation efficiency, particle size, and polymer residue of the microspheres are determined, as shown in Table 1-3.
  • the encapsulation efficiency of exenatide or a salt thereof and the like microspheres was determined as follows: 20 mg of exenatide or a salt thereof and the like microspheres prepared in the examples were placed in a 10 ml volumetric flask, and added.
  • the encapsulation efficiency is expressed as a percentage of the actual encapsulation amount as a percentage of the theoretical encapsulation amount.
  • the measurement results are shown in Table 1.
  • Example 1 85.4%
  • Example 2 91.6%
  • Example 3 99.1%
  • Example 4 90.3%
  • Example 5 88.3%
  • Example 6 93.1%
  • Example 7 92.5%
  • Example 8 85.8% Comparative example 1 64.2% Comparative example 2 69.1% Comparative example 3 68.2% Comparative example 4 66.4%
  • Test Example 2 Detecting the particle size and particle size distribution of exenatide or a salt thereof and the like microspheres
  • Chromatographic conditions a gel column (TSK-GEL G2000SWXL) was used as the stationary phase, and a pH 7.0 potassium dihydrogen phosphate buffer was used as the mobile phase at a flow rate of 0.8 ml/min, which was detected by a refractive index detector.
  • the preparation of the standard curve is taken from the appropriate amount of PVA, accurately weighed, and formulated into a PVA mother liquor of about 1.0 mg/ml, and then diluted to prepare solutions of 0.01, 0.02, 0.05, 0.1, 0.2, and 0.3 mg/ml, respectively, as standard solutions, respectively.
  • Take 100 ⁇ l of the standard solution inject into the liquid chromatograph, and record the chromatogram; take the sample concentration as the abscissa, the peak area as the ordinate, and make a standard curve for linear regression.
  • the correlation coefficient should not be less than 0.999.
  • Determination method take about 500mg of microspheres, accurately weighed, first degrade the microspheres with 50ml of 2mol/L NaOH solution for 36 hours, neutralize with 2mol/L HCl solution, and neutralize the solution to retain the relative molecular mass of 3500.
  • the bag was dialyzed for 36 hours, the PLGA degradation product was removed, and the resulting solution was lyophilized in vacuo. After reconstitution with water and dilution to 2 ml, as a test solution, 100 ⁇ l was injected into a liquid chromatograph, and a chromatogram was recorded; the obtained peak area was calculated by a standard curve.
  • the polyvinyl alcohol residue is expressed in terms of the actual residual weight as a percentage of the total weight of the microspheres.
  • the measurement results are shown in Table 3.
  • the residual of polyvinyl alcohol in the microspheres is related to the concentration and pH of the aqueous phase, and when controlling the pH and concentration of the aqueous phase,
  • the polyvinyl alcohol residue data was determined, and the polyvinyl alcohol of Example 1-7 remained below 0.008%, and the microspheres had less residual polyvinyl alcohol, which was safe for clinical application; the residual polyvinyl alcohol residue data in the comparative example exceeded 0.01%, and the microspheres More polyvinyl alcohol remains.
  • Preparation method Weigh 7.5g of exenatide acetate raw material, add 25ml of distilled water and stir to dissolve, prepare a solution, weigh 142.5g of glycolide-lactide copolymer (PLGA 5050 2A 16000), add dichloromethane (CH 2 Cl 2 ) 675ml stir and dissolve, the two solutions are mixed uniformly to obtain colostrum; 75L of the prepared 1% PVA solution (for the specific preparation of the solution method, see Example 3), sterilized and filtered, and then added to the vacuum emulsification mixer (hereinafter referred to as The microsphere preparation kettle was cooled to 7 to 13 ° C as an external aqueous phase.
  • the colostrum was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm. After the emulsification was continued for 60 s, the homogenization stirring speed was reduced to 150 rpm for 5 h. After the microspheres were solidified, the microspheres were collected by filtration and weighed 37.5 kg.
  • the distilled water is used as a washing liquid to rinse the microspheres, transferred to a lyophilized tray, added with mannitol injection and an appropriate amount of aqueous solution, and lyophilized in a freeze dryer; the drying temperature is set to 40 ° C (determination (exenatide acetate + PLGA)
  • the mixture had a glass transition temperature of 43 ° C and a drying time of 36 hours, and the lyophilized product was sieved to obtain microspheres.
  • the acetic acid content of the microspheres was measured, as shown in Table 4.
  • Preparation method Weigh 7.5g of exenatide acetate raw material, add 25ml of distilled water and stir to dissolve, prepare a solution, weigh 142.5g of glycolide-lactide copolymer (PLGA 5050 2A 17000), add dichloromethane (CH 2 Cl 2 ) 675ml stir and dissolve, the two solutions are mixed uniformly to obtain colostrum; 75L of the prepared 1% PVA solution (for the specific preparation of the solution method, see Example 3), sterilized and filtered, and then added to the vacuum emulsification mixer (hereinafter referred to as The microsphere preparation kettle was cooled to 7 to 13 ° C as an external aqueous phase.
  • the colostrum was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm. After the emulsification was continued for 60 s, the homogenization stirring speed was reduced to 150 rpm for 4 h. After the microspheres were solidified, the microspheres were collected by filtration, and 45 kg of distilled water was weighed.
  • microspheres were rinsed as a cleaning solution, transferred to a lyophilized tray, mannitol injection and an appropriate amount of aqueous solution were added, and lyophilized in a freeze dryer; the drying temperature was set to 40 ° C (determination of exenatide acetate + PLGA mixture) The glass transition temperature was 43 ° C), the drying time was 36 hours, and the lyophilized product was sieved to obtain microspheres.
  • the acetic acid content of the microspheres was measured, as shown in Table 4.
  • Preparation method Weigh 7.5g of exenatide acetate raw material, add 25ml of distilled water and stir to dissolve, prepare a solution, weigh 142.5g of glycolide-lactide copolymer (PLGA 5050 2A 16000), add dichloromethane (CH 2 Cl 2 ) 675ml stir and dissolve, the two solutions are mixed uniformly to obtain colostrum; 75L of the prepared 1% PVA solution (for the specific preparation of the solution method, see Example 3), sterilized and filtered, and then added to the vacuum emulsification mixer (hereinafter referred to as The microsphere preparation kettle was cooled to 7 to 13 ° C as an external aqueous phase.
  • the colostrum was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm. After the emulsification was continued for 60 s, the homogenization stirring speed was reduced to 150 rpm for 4 h. After the microspheres were solidified, the microspheres were collected by filtration and weighed 49.5 kg.
  • the acetic acid content of the microspheres was measured, as shown in Table 4.
  • Preparation method Weigh 7.5g of exenatide acetate raw material, add 25ml of distilled water and stir to dissolve, prepare a solution, weigh 142.5g of glycolide-lactide copolymer (PLGA 5050 2A 16000), add dichloromethane (CH 2 Cl 2 ) 675ml stir and dissolve, the two solutions are mixed uniformly to obtain colostrum; 75L of the prepared 1% PVA solution (for the specific preparation of the solution method, see Example 3), sterilized and filtered, and then added to the vacuum emulsification mixer (hereinafter referred to as The microsphere preparation kettle was cooled to 7 to 13 ° C as an external aqueous phase.
  • the colostrum was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm. After the emulsification was continued for 60 s, the homogenization stirring speed was reduced to 150 rpm for 4 h. After the microspheres were solidified, the microspheres were collected by filtration, and 60 kg of distilled water was weighed.
  • microspheres were rinsed as a cleaning solution, transferred to a lyophilized tray, mannitol injection and an appropriate amount of aqueous solution were added, and lyophilized in a freeze dryer; the drying temperature was set to 40 ° C (determination of exenatide acetate + PLGA mixture) The glass transition temperature was 43 ° C), the drying time was 36 hours, and the lyophilized product was sieved to obtain microspheres.
  • the acetic acid content of the microspheres was measured, as shown in Table 4.
  • Example 13 Preparation of Exenatide Microspheres by Re-drying Temperature Set to 38 ° C
  • Preparation method Weigh 7.5g of exenatide acetate raw material, add 25ml of distilled water and stir to dissolve, prepare a solution, weigh 142.5g of glycolide-lactide copolymer (PLGA 5050 2A 16000), add dichloromethane (CH 2 Cl 2 ) 675ml stir and dissolve, the two solutions are mixed uniformly to obtain colostrum; 75L of the prepared 1% PVA solution (for the specific preparation of the solution method, see Example 3), sterilized and filtered, and then added to the vacuum emulsification mixer (hereinafter referred to as The microsphere preparation kettle was cooled to 7 to 13 ° C as an external aqueous phase.
  • the colostrum was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm. After the emulsification was continued for 60 s, the homogenization stirring speed was reduced to 150 rpm for 4 h. After the microspheres were solidified, the microspheres were collected by filtration and weighed 49.5 kg.
  • the distilled water is used as a washing solution to rinse the microspheres, transferred to a lyophilized tray, added with mannitol injection and an appropriate amount of aqueous solution, and lyophilized in a freeze dryer; the drying temperature is set to 38 ° C (determination of exenatide + PLGA mixture) The glass transition temperature was 43 ° C), the drying time was 96 hours, and the lyophilized product was sieved to obtain microspheres.
  • the acetic acid content of the microspheres was measured, as shown in Table 4.
  • the sterilized oil phase was added to the microsphere preparation kettle, and the homogenization emulsification speed was 400 rpm. After the emulsification was continued for 60 s, the homogenization stirring speed was reduced to 150 rpm for 4 h. After the microspheres were solidified, the microspheres were collected by filtration.
  • the acetic acid content of the microspheres was measured, as shown in Table 4.
  • the column is 10 meters long and has an inner diameter of 0.32 mm.
  • the inner layer is coated with a 0.33 ⁇ m FFAP-CB fused silica capillary column.
  • Inlet temperature 220 ° C; detector temperature: 250 ° C; split ratio 100:1;
  • column temperature program starting temperature 50 ° C, residence time 0.10 minutes, heating rate 30 ° C / min, final temperature 230 ° C, residence time 5 minutes; the injection volume is 1 ⁇ l; the number of theoretical plates should be no less than 5000 according to the peak of acetic acid, and the separation between the peak of acetic acid and the internal standard should meet the requirements.
  • Correction factor determination Take 1.0 ml of n-hexadecane in a 50 ml volumetric flask, add 30 ml of dimethylformamide to dissolve and dilute to the mark, shake well, and use as an internal standard solution.
  • Another acetic acid reference substance is about 625mg, accurately weighed, placed in a 100ml volumetric flask, dissolved in dimethylformamide and diluted to the mark, shaken, and set aside.
  • test solution According to the preparation of the Goserelin microspheres prepared in Example 1, about 50mg, accurately weighed, placed in a 2ml volumetric flask, add 1ml of dimethylformamide to dissolve, precision addition of 100 ⁇ l internal standard The solution was diluted to the mark with dimethylformamide and shaken. Take 1 ⁇ l into the gas chromatograph and calculate according to the internal standard method. The measurement results are shown in Table 4.
  • Test drug Comparative Example 5, Exerin peptide microspheres prepared in Examples 11 and 13 were prepared and tested for stability (40 ° C temperature, 75% humidity condition for 6 months). The test results are shown in Table 5.
  • SD rat (Shandong Green Leaf Pharmaceutical Co., Ltd. animal house).
  • time of blood collection before administration (0 hours) and 1h, 6h, 1d, 2d, 3d, 5d, 7d, 9d, 11d, 14d, 16d, 18d, 21d, 23d, 25d, 28d after administration;
  • the present invention provides a composition containing exenatide, the composition is in the form of microspheres, and the microsphere starting material is exenatide acetate and polylactide glycolide (PLGA), and the prepared composition is acetic acid.
  • the content is less than 0.01%.
  • select the appropriate cleaning solution and its amount select appropriate re-drying temperature curing microspheres during the freeze-drying process of the microspheres, and obtain microsphere products that meet the quality standards and have good characteristics.
  • the suitable outer aqueous phase is selected, the prepared microspheres have uniform particle size and less polymer residue, and can provide better quality products for clinical application.
  • the invention is suitable for industrial applications.

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Abstract

一种含有艾塞那肽的组合物，所述组合物是微球形式，所述微球采用醋酸艾塞那肽和聚丙交酯乙交酯（PLGA）作为原料，制备完成后的组合物中的醋酸含量小于0.01%。一种制备艾塞那肽微球的方法，所述方法包括选择选择适合的外水相，适当用量的清洗液清洗微球，以及在微球冻干后，选择适当的再干燥温度固化微球。所述微球稳定性提高，延长药物的储存时间，粒径均匀，聚合物残留少。

Description

一种含有艾塞那肽的组合物及其制备方法 技术领域
本发明属于医药领域，涉及到一种含有艾塞那肽的组合物，所述组合物是微球形式。本发明还涉及该微球的制备
背景技术
艾塞那肽是人工合成的肠促胰岛素类似物GLP-1家族的第一个成员，它模拟人体生理状态下分泌GLP-1的生物模式，从肠内释放入循环中后可以增强葡萄糖依赖性胰岛素分泌。艾塞那肽的氨基酸序列与人类GLP-1部分重叠，在体显示可以结合并活化已知的人类GLP-1受体，通过包括cAMP和/或其他细胞内信号传导机制使葡萄糖依赖性胰岛素合成及胰岛β细胞在体内分泌胰岛素增加。在葡萄糖浓度升高的情况下，艾塞那肽可促进胰岛素从β细胞中释放同时艾塞那肽通过减少2型糖尿病患者空腹和餐后血糖浓度，从而改善血糖控制。
艾塞那肽已上市的产品主要有注射剂，仅用于皮下注射。推荐起始剂量为5μg，每日两次，治疗1个月后，可根据临床反应将剂量增加至10μg。该制剂每日两次(bid)注射，平均达峰时间为2.1h，平均消除半衰期为2.4h，主要由肾脏消除。这种注射剂需要频繁给药，且不能持续活化GLP-1受体，对于需要长期给药的糖尿病患者而言依从性较差。
现有技术已有采用相分离方法制备艾塞那肽微球，采用相分离方法生产操作步骤多，对设备工艺要求高，医药产品相关无菌及溶剂残留等质量标准要求，也会使的相分离方法制备工艺制备的微球还需要多个工序的产品处理；因为相分离生产艾塞那肽微球存在以上不足，一些研究机构开展采用溶剂挥发法生产艾塞那肽微球研究，其生产设备要求低，工作操作步骤较少，采用较少的后处理工序处理产品即可达到相关质量标准要求。艾塞那肽市售产品为艾塞那肽的醋酸盐，产品标准要求：3％≤醋酸含量≤12％，现有技术对生产的艾塞那肽微球过程未有相关醋酸含量控制，但是通过研究发现艾塞那肽微球醋酸含量对储存期内艾塞那肽生物利用度有影响，当醋酸含量超过0.01％，会产生使储存期内微球中的艾塞那肽生物利用度下降的问题。
发明内容
针对现有技术存在的上述缺陷，本发明人进行深入研究，提供一种含有艾塞那肽的组合物，所述组合物是微球，起始原料采用艾塞那肽醋酸盐(市售产品，3≤醋酸含量≤12％)和聚丙交酯乙交酯(PLGA)，且所述制备的艾塞那肽微球控制醋酸含量为0.01％以下。
本发明提供一种含有艾塞那肽的组合物，所述组合物是微球形式，所述微球起始原料采用醋酸艾塞那肽和聚丙交酯乙交酯(PLGA)，制备完成后的组合物醋酸含量小于0.01％。
所述艾塞那肽的含量，相对于组合物总重量为2-10％，优选3-8％，更优选4-6％。聚丙交酯乙交酯(PLGA)的含量，相对于组合物总重量为90-98％，优选92-97％，更优选94-96％。
所述丙交酯-乙交酯共聚物，英文名称为Poly(lactide-co-glycolide)，简称PLGA。本发明中聚丙交酯乙交酯(PLGA)的分子量为6000-45000道尔顿，优选为10000-30000道尔顿，更优选10000-25000道尔顿，所述分子量指“重均分子量”，简称为“分子量”。PLGA的丙交酯和乙交酯的摩尔比为90∶10-10∶90，优选75∶25-25∶75，更优选60∶40-40∶60，尤其是50∶50。PLGA的特性粘度为0.1-0.40dL/g，优选范围0.10-0.35dL/g，更优选为0.10-0.30dL/g。PLGA的特性粘度(inherent viscosity)测定方法：将PLGA用氯仿配制成约0.5％(w/v)的溶液，于30℃采用Cannon-Fenske玻璃毛细管粘度计测定其特性粘度。
为方便描述，下文对丙交酯和乙交酯的摩尔比以及特性粘度在其括号中进行表示。如“PLGA(50/50，0.20，16000)”表示丙交酯和乙交酯的摩尔比为50∶50，特性粘度为0.20dl/g，分子量为16000道尔顿的丙交酯-乙交酯共聚物。
本发明提供的组合物采用溶剂挥发法制备。因此，本发明还提供一种含有艾塞那肽的组合物的制备方法。
本发明制备微球过程包括：将PLGA用有机溶剂制成有机溶液，将醋酸艾塞那肽与PLGA有机溶液混合，形成初乳；将初乳添加至外水相中乳化，固化、挥发有机溶剂，再经清洗，冻干获得微球。
所述有机溶剂可以溶解丙交酯乙交酯(PLGA)，其可以是二氯甲烷、丙酮、乙腈等可以溶解PLGA的有机溶剂，优选二氯甲烷。
在制备过程中可以选用适当比例的清洗液，同时设定规定的再干燥温度。所述清洗液采用蒸馏水或碱性缓冲液，优选蒸馏水，微球与清洗液的重量比为1∶250及以上，优选重量比为1∶330及以上。所述冻干微球设定再干燥温度为醋酸艾塞那肽与PLGA混合物玻璃化转变温度以下。通过该调整使本发明能够获得极低醋酸含量且符合质量标准且特性良好的艾塞那肽微球产品。
在制备过程中可以调整外水相的浓度和pH值。所述外水相为聚乙烯醇溶液，所述聚乙烯醇平均分子量为13000-23000；溶液的聚乙烯醇重量体积百分浓度(单位g/100ml)为0.5-1.5％，优选聚乙烯醇重量体积百分浓度为1％；溶液的PH值为5.6±1.0，优选溶液的PH值为5.6±0.5，更优选溶液的PH值为5.6±0.2。通过该调整，使本发明中聚乙烯醇在 所提供的组合物中残留量应不大于0.008％(重量百分比)。
具体的，本发明所述微球可通过溶剂挥发法下述示例性操作制备获得：称量一定量的艾塞那肽或其盐及类似物，可以同时称量已知的一定量的助溶剂、蛋白保护剂、表面活性剂、致孔剂等辅料，可以先用蒸馏水溶解艾塞那肽或其盐及类似物，选用的辅料也可以溶解于水溶液；称量一定量的PLGA，用有机溶剂溶解，所述有机溶剂可以是二氯甲烷、丙酮、乙腈等可以溶解PLGA的有机溶剂，优选二氯甲烷；将含有艾塞那肽和辅料的水溶液与PLGA有机溶液混合，形成均匀的初乳；将含有艾塞那肽或其盐及类似物的初乳添加至外水相(如聚乙烯醇溶液)中乳化，固化、挥发有机溶剂，清洗，冻干获得微球。
以上操作中，醋酸艾塞那肽可以不预先配制成水溶液，将醋酸艾塞那肽直接与PLGA有机溶液混合而形成初乳。
制备微球过程的所述外水相为聚乙烯醇溶液；溶液的聚乙烯醇重量体积百分浓度为0.5-1.5％，优选为1％，溶液的pH值为5.6±1.0，优选5.6±0.5，更优选5.6±0.2。
外水相的pH值对制备的微球的特性起到关键作用，本发明可采用酸、碱或缓冲液调整外水相的pH值，使含有艾塞那肽或其盐及类似物的油相(初乳)在乳化后，在选定的pH值的外水相中固化成型，为获得良好特性的艾塞那肽或其盐及类似物微球打下基础。
采用酸、碱或缓冲液，可以使外水相的pH值稳定在一定的范围，采用的酸可以是盐酸、硫酸、醋酸、氨基酸等水溶性的酸，比较常用的如0.1mol/L盐酸；采用的碱可以是氢氧化钠、氢氧化钾、碳酸氢钠、亚硫酸钠等水溶性的碱，比较常用的如0.1mol/L氢氧化钠，所述缓冲液可以是枸橼酸盐缓冲液、磷酸盐缓冲液、醋酸盐缓冲液、邻苯二甲酸盐缓冲液等缓冲液，比较常用的如邻苯二甲酸盐缓冲液(pH＝5.6)。
如上所述酸、碱或缓冲液的配制，可以按照《中国药典2010》第二部附录的记录配制，如0.1mol/L盐酸、0.1mol/L氢氧化钠按照附录XV F滴定液记载配制，邻苯二甲酸盐缓冲液(pH＝5.6)按照XV D缓冲液记载配制。
在制备艾塞那肽或其盐及类似物微球的过程中，外水相的聚乙烯醇重量体积百分浓度(g/100ml)对制备的微球的特性起到重要作用，本发明采用浓度为0.5-1.5％，优选为1％，在选定的浓度的外水相中固化微球，可以获得符合质量标准且特性良好的微球产品。
本发明所述聚乙烯醇的平均分子量为13000-23000，以此平均分子量的聚乙烯醇配 制的聚乙烯醇水溶液，在制备的微球的特性起到重要作用，在选定的平均分子量的聚乙烯醇的外水相中固化微球，可以获得符合质量标准且特性良好的微球产品。
本发明通过在制备微球过程中调整外水相的聚乙烯醇浓度和pH值制备艾塞那肽或其盐及类似物微球，使微球达到包封率高，粒径均匀，聚乙烯醇残留少的特性。
在制备艾塞那肽微球的过程中，成球后的清洗液用量的选择及冻干微球时间和温度对制备的微球的醋酸含量减少起到关键作用。
本发明可采用蒸馏水或缓冲液作为清洗液对微球清洗，微球与清洗液的重量比为1∶250及以上，优选重量比为1∶330及以上；在选定的重量比下清洗微球，为获得极低醋酸含量的艾塞那肽微球打下基础。
采用蒸馏水或碱性缓冲液作为清洗液对微球清洗，可以使清洗液的pH值稳定在一定的范围，采用的缓冲液可以是枸橼酸盐缓冲液、磷酸盐缓冲液、醋酸盐缓冲液、邻苯二甲酸盐缓冲液等缓冲液，优选蒸馏水。
本发明在制备艾塞那肽微球的过程中，冻干为冷冻干燥的简称，通常包括预冻、升华干燥、反复冷冻升华、再干燥过程。冻干是溶液冻结成固体，在低温低压条件下，从冻结状态不经过液态而直接升华除去液体的一种干燥方式。本发明对再干燥过程温度进行限定，其他过程采用常规方法即可。
再干燥为冻干过程当升华干燥阶段完成后，为尽可能除去残余的液体需要进一步的干燥的工艺。冻干过程中的再干燥温度对制备的微球醋酸的含量起到重要作用，本发明采用再干燥温度为在醋酸艾塞那肽与PLGA混合物(乳化完成)的玻璃化转变温度以下，如此可以获得符合质量标准且特性良好的微球产品。
采用以上技术措施，本发明通过在制备微球过程中控制醋酸的含量制备艾塞那肽微球，使微球达到较长的储存期内，生物利用度不变的特性，延长产品的储存期。
本发明采用醋酸艾塞那肽作为起始原料制备微球，通过降低醋酸的浓度至极低含量，使微球的稳定性提高，延长药物的储存时间；所述制备方法是通过成球后确定清洗液用量的选择及冻干微球时间和温度，最终达到控制微球醋酸的浓度；所述制备方法在乳化过程中通过调整水相的浓度和pH值，可以实现包封率高，粒径均匀，聚乙烯醇残留少的特性。
具体实施方式
下面通过实施例对本发明加以进一步的说明，但不以任何形式限制本发明。
实施例1：外水相为0.5％聚乙烯醇水溶液(pH＝6.5)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液(PVA溶液)样配制：精密称量聚乙烯醇(平均分子量13000-23000)固 体，量取计算体积的水溶液，用量取好的水体积的二分之一溶解称量的聚乙烯醇固体，待聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在6.5。
制备方法：称取配方量艾塞那肽，加蒸馏水搅拌溶解，配制成溶液，称取配方量乙交酯-丙交酯共聚物(PLGA)，加二氯甲烷(CH2Cl2)搅拌溶解，将二个溶液混合均匀得初乳；将配制好的PVA溶液除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将初乳加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳化60s后，降低均质搅拌速度为150rpm，维持5h，待微球固化后过滤收集微球，水溶液冲洗微球(具体方法可参见实施例11)，去除PVA残留，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干(具体方法可参见实施例11)；冻干品经过筛混匀，得到微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例2：外水相为0.8％聚乙烯醇水溶液(pH＝5.1)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在5.1。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例3：外水相为1.0％聚乙烯醇水溶液(pH＝5.6)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用邻苯二甲酸盐缓冲液(pH＝5.6)调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在5.6。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例4：外水相为1.2％聚乙烯醇水溶液(pH＝6.1)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L 氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在6.1。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例5：外水相为1.5％聚乙烯醇水溶液(pH＝4.6)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在4.6。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例6：外水相为1.0％聚乙烯醇水溶液(pH＝6.5)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在6.5。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例7：外水相为1.0％聚乙烯醇水溶液(pH＝4.7)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在4.7。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
实施例8：外水相为1.0％聚乙烯醇水溶液(pH＝5.6)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量110000-200000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用邻苯二甲酸盐缓冲液(pH＝5.6)调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在5.6。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
对比例1：外水相为1.0％聚乙烯醇水溶液(pH＝9.0)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在9.0。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
对比例2：外水相为1.0％聚乙烯醇水溶液(pH＝3.0)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在3.0。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
对比例3：外水相为5.0％聚乙烯醇水溶液(pH＝5.8)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在5.8。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
对比例4：外水相为0.1％聚乙烯醇水溶液(pH＝5.6)制备艾塞那肽或其盐及类似物微球
聚乙烯醇溶液样配制：精密称量聚乙烯醇(平均分子量13000-23000)固体，量取计算体积的水溶液，用量取好的水溶液体积的二分之一溶解称量的聚乙烯醇固体，等聚乙烯醇固体溶解后，将剩余的水加入溶液，搅拌混匀，测定溶液pH值，用0.1mol/L盐酸、0.1mol/L氢氧化钠调整聚乙烯醇水溶液pH值，直到水溶液pH值稳定在5.6。
制备方法：采用与实施例1相同的方法制备微球。
对微球的包封率，粒径，聚合物残留测定，具体见表1-3。
试验例1 检测艾塞那肽或其盐及类似物微球的包封率
艾塞那肽或其盐及类似物微球的包封率测定按如下方法进行：取20mg在实施例中制备的艾塞那肽或其盐及类似物微球，置10ml量瓶中，加2ml冰醋酸超声使溶解，至澄清透明，缓慢加入水，用水稀释至刻度，摇匀，放置，使不溶性物质下沉，取上清液为供试品溶液；另取艾塞那肽对照品适量，精密称定，用水溶解并定量稀释至每1ml中约含0.1mg艾塞那肽的溶液，作为对照品溶液；精密吸取供试品溶液及对照品溶液各20μl，注入液相色谱仪，记录色谱图；按标法以峰面积计算供试品中艾塞那肽的含量，以计算包封量。
包封率以实际包封量占理论包封量的百分比的方式表达。测量结果见表1。
表1艾塞那肽或其盐及类似物微球的包封率
实验编号 包封率
实施例1 85.4％
实施例2 91.6％
实施例3 99.1％
实施例4 90.3％
实施例5 88.3％
实施例6 93.1％
实施例7 92.5％
实施例8 85.8％
对比例1 64.2％
对比例2 69.1％
对比例3 68.2％
对比例4 66.4％
结果分析：实施例1-8制备的艾塞那肽或其盐及类似物微球，药物与溶液的添加方法对微球的包封率没有影响，包封率与水相的浓度和pH值相关，控制水相的pH值和浓度时；微球的包封率均在85％以上；对比例中采用相同的方法，水相的pH值和浓度不在控制的范围内时，微球的包封率都低于70％。
试验例2 检测艾塞那肽或其盐及类似物微球的粒度及粒度分布
取本品照粒度与粒度分布测定法(中国药典2010年版二部附录IX E第三法)测定。以0.1％吐温20溶液作为分散剂，将分散剂约120ml置于粒度仪(采用马尔文Mastersizer2000粒度分析仪)测定的样品分散装置中，调节转速控制装置，以每分钟2100转的转速搅拌。首先测量分散剂背景，然后取本品1瓶，倒入分散剂中，待本品分散均匀后，测其D90、 D50、D10粒径。平行测量3次，测量结果取平均值。计算其跨距，采用的方法为《中国药典》2010版第二部附录XIX微囊、微球与脂质体制剂指导原则计算方法
跨距＝(D90-D10)/D50
跨距以上述公式计算，测量结果见表2。
表2艾塞那肽或其盐及类似物微球的粒度跨距
实验编号 跨距
实施例1 0.76
实施例2 0.74
实施例3 0.24
实施例4 0.78
实施例5 0.64
实施例6 0.44
实施例7 0.41
实施例8 0.79
对比例1 2.01
对比例2 2.29
对比例3 2.04
对比例4 2.07
结果分析：实施例1-8制备的艾塞那肽或其盐及类似物微球，药物与溶液的添加方法对微球的跨距没有影响，微球的跨距与水相的浓度和pH值相关，控制水相的pH值和浓度时，测定微球的跨距，数据显示跨距均在0.8以下，表明制备的微球粒径分布均匀；对比例中水相的pH值和浓度不在控制的范围内时，微球的跨距大于2.0，微球粒径不均一。
试验例3 检测艾塞那肽或其盐及类似物微球的聚乙烯醇残留
聚乙烯醇残留检测方法：
色谱条件：以凝胶色谱柱(TSK-GEL G2000SWXL)为固定相，以pH 7.0的磷酸二氢钾缓冲液为流动相，流速为0.8ml/min，示差折光检测器检测。
标准曲线的制备取PVA适量，精密称定，配成约1.0mg/ml的PVA母液，再分别稀释制成0.01、0.02、0.05、0.1、0.2和0.3mg/ml的溶液，作为标准溶液，分别取标准溶液100μl，注入液相色谱仪，记录色谱图；以样品浓度为横坐标，峰面积为纵坐标，作标准曲线，进行线性回归，相关系数不得小于0.999。
测定法：取微球约500mg，精密称定，先以2mol/L NaOH溶液50ml降解微球36小时，以2mol/L HCl溶液中和，中和后的溶液以截留相对分子质量为3500的透析袋透析36小时，除去PLGA降解产物，所得溶液真空冻干。加水复溶并稀释至2ml，作为供试品溶液，取100μl注入液相色谱仪，记录色谱图；将所得峰面积用标准曲线计算其含量。
聚乙烯醇残留以实际残留重量占微球总重量的百分比的方式表达。测量结果见表3。
表3艾塞那肽或其盐及类似物微球的聚乙烯醇残留
实验编号 聚乙烯醇残留
实施例1 0.0067％
实施例2 0.0054％
实施例3 0.0019％
实施例4 0.0068％
实施例5 0.0079％
实施例6 0.0074％
实施例7 0.0043％
实施例8 0.0072％
对比例1 0.0281％
对比例2 0.0263％
对比例3 0.0397％
对比例4 0.0157％
结果分析：实施例1-8制备的艾塞那肽或其盐及类似物微球，微球中聚乙烯醇残留与水相的浓度和pH值相关，控制水相的pH值和浓度时，测定聚乙烯醇残留数据，实施例1-7的聚乙烯醇残留在0.008％以下，微球聚乙烯醇残留少，临床应用安全；对比例中相关聚乙烯醇残留数据超过0.01％，微球的聚乙烯醇残留较多。
实施例9：采用(醋酸艾塞那肽与PLGA)∶清洗液＝1∶250(按重量)制备艾塞那肽微球
制备方法：称取7.5g醋酸艾塞那肽原料，加蒸馏水25ml搅拌溶解，配制成溶液，称取142.5g乙交酯-丙交酯共聚物(PLGA 5050 2A 16000)，加二氯甲烷(CH2Cl2)675ml搅拌溶解，将二个溶液混合均匀得初乳；将配制好的1％PVA溶液75L(具体配制溶液方法可以参见实施例3)除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将初乳加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳 化60s后，降低均质搅拌速度为150rpm，维持5h，待微球固化后过滤收集微球，称量37.5kg蒸馏水作为清洗液冲洗微球，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干；再干燥温度设定为40℃(测定(醋酸艾塞那肽+PLGA混合物)的玻璃化转变温度为43℃)，干燥时间为36小时，冻干品经过筛混匀，得到微球。
对微球的醋酸含量进行测定，具体见表4。
实施例10：采用(醋酸艾塞那肽与PLGA)∶清洗液＝1∶300(按重量)制备艾塞那肽微球
制备方法：称取7.5g醋酸艾塞那肽原料，加蒸馏水25ml搅拌溶解，配制成溶液，称取142.5g乙交酯-丙交酯共聚物(PLGA 5050 2A 17000)，加二氯甲烷(CH2Cl2)675ml搅拌溶解，将二个溶液混合均匀得初乳；将配制好的1％PVA溶液75L(具体配制溶液方法可以参见实施例3)除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将初乳加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳化60s后，降低均质搅拌速度为150rpm，维持4h，待微球固化后过滤收集微球，称量45kg蒸馏水作为清洗液冲洗微球，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干；再干燥温度设定为40℃(测定醋酸艾塞那肽+PLGA混合物的玻璃化转变温度为43℃)，干燥时间为36小时，冻干品经过筛混匀，得到微球。
对微球的醋酸含量进行测定，具体见表4。
实施例11：采用(醋酸艾塞那肽与PLGA)∶清洗液＝1∶330(按重量)制备艾塞那肽微球
制备方法：称取7.5g醋酸艾塞那肽原料，加蒸馏水25ml搅拌溶解，配制成溶液，称取142.5g乙交酯-丙交酯共聚物(PLGA 5050 2A 16000)，加二氯甲烷(CH2Cl2)675ml搅拌溶解，将二个溶液混合均匀得初乳；将配制好的1％PVA溶液75L(具体配制溶液方法可以参见实施例3)除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将初乳加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳化60s后，降低均质搅拌速度为150rpm，维持4h，待微球固化后过滤收集微球，称量49.5kg蒸馏水作为清洗液冲洗微球，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干；再干燥温度设定为40℃(测定醋酸艾塞那肽+PLGA混合物的玻璃化转变温度为43℃)，干燥时间为36小时，冻干品经过筛混匀，得到微球。
对微球的醋酸含量进行测定，具体见表4。
实施例12：采用(醋酸艾塞那肽与PLGA)∶清洗液＝1∶400(按重量)制备艾塞那肽微球
制备方法：称取7.5g醋酸艾塞那肽原料，加蒸馏水25ml搅拌溶解，配制成溶液，称取142.5g乙交酯-丙交酯共聚物(PLGA 5050 2A 16000)，加二氯甲烷(CH2Cl2)675ml搅拌 溶解，将二个溶液混合均匀得初乳；将配制好的1％PVA溶液75L(具体配制溶液方法可以参见实施例3)除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将初乳加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳化60s后，降低均质搅拌速度为150rpm，维持4h，待微球固化后过滤收集微球，称量60kg蒸馏水作为清洗液冲洗微球，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干；再干燥温度设定为40℃(测定醋酸艾塞那肽+PLGA混合物的玻璃化转变温度为43℃)，干燥时间为36小时，冻干品经过筛混匀，得到微球。
对微球的醋酸含量进行测定，具体见表4。
实施例13：采用再干燥温度设定为38℃制备艾塞那肽微球
制备方法：称取7.5g醋酸艾塞那肽原料，加蒸馏水25ml搅拌溶解，配制成溶液，称取142.5g乙交酯-丙交酯共聚物(PLGA 5050 2A 16000)，加二氯甲烷(CH2Cl2)675ml搅拌溶解，将二个溶液混合均匀得初乳；将配制好的1％PVA溶液75L(具体配制溶液方法可以参见实施例3)除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将初乳加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳化60s后，降低均质搅拌速度为150rpm，维持4h，待微球固化后过滤收集微球，称量49.5kg蒸馏水作为清洗液冲洗微球，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干；再干燥温度设定为38℃(测定醋酸艾塞那肽+PLGA混合物的玻璃化转变温度为43℃)，干燥时间为96小时，冻干品经过筛混匀，得到微球。
对微球的醋酸含量进行测定，具体见表4。
对比例5：制备醋酸含量超过0.01％的艾塞那肽微球
称取7.5g醋酸艾塞那肽原料，加蒸馏水25ml搅拌溶解，配制成溶液，称取142.5g乙交酯-丙交酯共聚物(PLGA 5050 2A)，加二氯甲烷(CH2Cl2)675ml搅拌溶解，将二个溶液混合均匀得初乳；将配制好的1％PVA溶液75L(具体配制溶液方法可以参见实施例3)除菌过滤后加入至真空乳化搅拌机(以下简称微球制备釜)中并冷却至7～13℃，作为外水相。将已除菌过滤的油相加入至微球制备釜中，均质乳化速度为400rpm，加料结束继续乳化60s后，降低均质搅拌速度为150rpm，维持4h，待微球固化后过滤收集微球，称量30kg蒸馏水作为清洗液冲洗微球，转移至冻干盘中，加入甘露醇注射液和适量水溶液，置冷冻干燥机中冻干；再干燥温度设定为38℃(测定醋酸艾塞那肽+PLGA混合物的玻璃化转变温度为43℃)，干燥时间为36小时，冻干品经过筛混匀，得到微球。
对微球的醋酸含量进行测定，具体见表4。
试验例4 不同艾塞那肽微球醋酸含量检测
检测方法：照气相色谱法[中国药典2010年版二部附录V E第(3)法]测定。
色谱条件与系统适用性试验：色谱柱为长10米，内径0.32mm，内层涂有0.33μm的FFAP-CB熔融石英毛细管柱。进样口温度：220℃；检测器温度：250℃；分流比100∶1；柱温程序：起始温度50℃，停留时间0.10分钟，升温速率30℃/分钟，最终温度230℃，停留时间5分钟；进样量1μl；理论塔板数按乙酸峰计算应不低于5000，乙酸峰与内标峰的分离度应符合规定。
校正因子测定：取正十六烷1.0ml置50ml量瓶中，加二甲基甲酰胺30ml溶解并稀释至刻度，摇匀，作为内标溶液。另取乙酸对照品约625mg，精密称定，置100ml量瓶中，用二甲基甲酰胺溶解并稀释至刻度，摇匀，备用。精密量取上述溶液10ml至100ml量瓶中，精密加入内标溶液5ml，用二甲基甲酰胺溶解并稀释至刻度，摇匀。取1μl注入气相色谱仪，连续进样3-5次，按平均峰面积计算校正因子。
供试品溶液的制备与测定：按实施例1、制备的戈舍瑞林微球取约50mg，精密称定，置2ml量瓶中，加二甲基甲酰胺1ml溶解，精密加入100μl内标溶液，用二甲基甲酰胺稀释至刻度，摇匀。取1μl注入气相色谱仪，按内标法计算。测定结果见表4。
表4艾塞那肽微球醋酸含量检测
实例名称 醋酸含量
实施例9 0.0094％
实施例10 0.0036％
实施例11 0.0017％
实施例12 0.0021％
实施例13 0.0045％
对比例5 0.0732％
结果分析：实施例9-13制备的艾塞那肽微球，采用(醋酸艾塞那肽+PLGA)∶清洗液＝1∶250以上清洗艾塞那肽微球，冻干再干燥温度为醋酸艾塞那肽与PLGA混合物的玻璃化转变温度以下，微球的醋酸含量均在0.01％以下；对比例中采用相同的方法，采用清洗液低于(醋酸艾塞那肽+PLGA)250倍清洗艾塞那肽微球，微球的醋酸含量高于0.01％。
试验例5 稳定性实验前后艾塞那肽微球体内释放对比试验
试验材料：
试验药物：对比例5、实施例11、13制备的艾塞那肽微球进行制备完成及稳定性考察(40℃温度，75％的湿度条件放置六个月)完成后样品体内释放对比试验，检测结果见表5。
实验动物：
SD大鼠(山东绿叶制药有限公司动物房)。
实验仪器：
QTRAP5500型质谱仪，配有离子喷雾离子化源，美国Applied Biosystem公司；Agilent1290高效液相色谱系统，包括二元输液泵，自动进样器，柱温箱；Anke TGL-16G飞鸽台式离心机，上海安亭科学仪器厂；Tμrbo Vap LV氮吹仪，Biotage公司生产。
实验方法：
a)实验动物：SD大鼠，体重240±20g，雄性，每组16只；
b)给药途径和剂量：给药剂量2mg/kg，腹部皮下注射；
c)采血时间点：于给药前(0小时)及给药后1h、6h、1d、2d、3d、5d、7d、9d、11d、14d、16d、18d、21d、23d、25d、28d；
d)生物样品测定：酶联免疫法。
结果见表5。
表5大鼠肌肉注射微球后不同时间的艾塞那肽浓度(ng/mL)
Figure PCTCN2015076688-appb-000001
结果显示：醋酸艾塞那肽在制备微球过程中，醋酸的含量对产品储存期的生物利用度有较大影响，在稳定性考察后(40℃温度，75％的湿度条件放置六个月)，体内释放有变化，醋酸含量小于0.01％的微球体内释放没有变化，而醋酸含量没有控制的微球的生物利用度下降20％以上。
工业应用性
本发明提供含有艾塞那肽的组合物，所述组合物是微球形式，微球起始原料采用醋酸艾塞那肽和聚丙交酯乙交酯(PLGA)，制备完成后的组合物醋酸含量小于0.01％。在制备艾塞那肽微球的过程中，选择适当的清洗液及其用量，在微球冻干过程中，选择适当的再干燥温度固化微球，获得符合质量标准且特性良好的微球产品，使微球的稳定性提高，延长药物的储存时间。在制备艾塞那肽微球的过程中，选择适合的外水相，制备的微球粒径均匀，聚合物残留少的特性，可为临床应用提供质量更优的产品。本发明适于工业应用。

Claims (20)

  1. 一种含有艾塞那肽的组合物，所述组合物是微球形式，所述微球起始原料采用醋酸艾塞那肽或其盐及类似物和聚丙交酯乙交酯(PLGA)，制备完成后的组合物醋酸含量小于0.01％。
  2. 根据权利要求1所述的组合物，所述艾塞那肽的含量，相对于组合物总重量为2-10％，优选3-8％，更优选4-6％。
  3. 根据权利要求2所述的组合物，所述聚丙交酯乙交酯(PLGA)的含量，相对于组合物总重量为90-98％，优选92-97％，更优选94-96％。
  4. 根据权利要求3所述的组合物，所述聚丙交酯乙交酯(PLGA)的分子量为6000-45000道尔顿，优选为10000-30000道尔顿，更优选10000-25000道尔顿。
  5. 根据权利要求3所述的组合物，PLGA的丙交酯和乙交酯的摩尔比为90：10－10：90，优选75：25－25：75，更优选60：40-40：60，尤其是50:50。
  6. 根据权利要求4所述的组合物，PLGA的特性粘度为0.1-0.40dL/g，优选范围0.10-0.35dL/g，更优选为0.10-0.30dL/g。
  7. 根据权利要求1至6任一所述的组合物，采用溶剂挥发法制备成球，制备微球过程包括：将PLGA用有机溶剂制成有机溶液，将醋酸艾塞那肽与PLGA有机溶液混合，形成初乳；将初乳添加至外水相中乳化，固化、挥发有机溶剂，再经清洗，冻干获得微球。
  8. 根据权利要求7所述的组合物，通过选用适当比例的清洗液，同时设定规定的再干燥温度得到微球。
  9. 根据权利要求8所述的组合物，所述有机溶剂可以溶解丙交酯乙交酯(PLGA)，其可以是二氯甲烷、丙酮、乙腈等可以溶解PLGA的有机溶剂，优选二氯甲烷；所述清洗采用蒸馏水或碱性缓冲液作为清洗液，优选蒸馏水，微球与清洗液的重量比为1：250及以上，优选重量比为1：330及以上。
  10. 根据权利要求8或9所述的组合物，所述冻干中再干燥温度设定为醋酸艾塞那肽与PLGA混合物的玻璃化转变温度以下。
  11. 根据权利要求7所述的组合物，所述外水相为聚乙烯醇溶液，所述组合物中聚乙烯醇重量百分比不大于0.008％。
  12. 根据权利要求11所述的组合物，所述外水相聚乙烯醇溶液的聚乙烯醇重量 体积百分浓度(g/100ml)为0.5-1.5％，优选聚乙烯醇重量体积百分浓度为1％；溶液的PH值为5.6±1.0，优选溶液的PH值为5.6±0.5，更优选溶液的PH值为5.6±0.2。
  13. 根据权利要求12所述的组合物，用酸、碱或缓冲液调整外水相的PH值，所述酸、碱或缓冲液为水溶性的酸、碱或缓冲液，优选所述酸为0.1mol/L盐酸，所述碱为0.1mol/L氢氧化钠，所述缓冲液为邻苯二甲酸盐缓冲液(PH＝5.6)。
  14. 根据权利要求11或12或13所述的组合物，所述组合物中聚乙烯醇平均分子量为13000-23000。
  15. 制备微球形式含有艾塞那肽的组合物的方法，以醋酸艾塞那肽或其盐及类似物和聚丙交酯乙交酯(PLGA)为初始原料，采用溶剂挥发法制备成球；制备微球过程包括：将PLGA用有机溶剂制成有机溶液，将艾塞那肽与PLGA有机溶液混合，形成初乳；将初乳添加至外水相中乳化，固化、挥发有机溶剂，再经清洗，冻干获得微球。
  16. 根据权利要求15所述的方法，通过选用适当比例的清洗液，同时设定规定的再干燥温度得到微球。
  17. 根据权利要求16所述的方法，所述有机溶剂可以溶解丙交酯乙交酯(PLGA)，其可以是二氯甲烷、丙酮、乙腈等可以溶解PLGA的有机溶剂，优选二氯甲烷；所述清洗采用蒸馏水或碱性缓冲液作为清洗液，优选蒸馏水；微球与清洗液的重量比为1：250及以上，优选重量比为1：330及以上。
  18. 根据权利要求16或17所述的方法，所述冻干中再干燥温度设定为醋酸艾塞那肽与PLGA混合物的玻璃化转变温度以下。
  19. 根据权利要求15所述的方法，所述外水相为聚乙烯醇溶液；溶液的聚乙烯醇重量体积百分浓度(g/100ml)为0.5-1.5％，优选聚乙烯醇重量体积百分浓度为1％；溶液的PH值为5.6±1.0，优选溶液的PH值为5.6±0.5，更优选溶液的PH值为5.6±0.2；且聚乙烯醇在所述组合物中残留重量百分比不大于0.008％。
  20. 根据权利要求19所述的方法，用酸、碱或缓冲液调整外水相的PH值，所述酸、碱或缓冲液为水溶性的酸、碱或缓冲液，优选所述酸为0.1mol/L盐酸，所述碱为0.1mol/L氢氧化钠，所述缓冲液为邻苯二甲酸盐缓冲液(PH＝5.6)。
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