WO2013189282A1 - 多肽药物缓释微球制剂及其制备方法 - Google Patents

多肽药物缓释微球制剂及其制备方法 Download PDF

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WO2013189282A1
WO2013189282A1 PCT/CN2013/077403 CN2013077403W WO2013189282A1 WO 2013189282 A1 WO2013189282 A1 WO 2013189282A1 CN 2013077403 W CN2013077403 W CN 2013077403W WO 2013189282 A1 WO2013189282 A1 WO 2013189282A1
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polylactic acid
microspheres
polypeptide drug
oil
release
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PCT/CN2013/077403
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English (en)
French (fr)
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刘锋
曹付春
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广州帝奇医药技术有限公司
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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 invention belongs to the field of biomedical polymer materials and biologically active drug controlled release preparations, and in particular to a polypeptide drug sustained release microsphere preparation and a preparation method thereof. Background technique
  • the bioactive degradable material (for example, polymer material) is used to wrap the active ingredient of the drug to form a microsphere preparation, and the biodegradable biopolymer material is gradually degraded in the body to control drug release and maintain an effective blood concentration.
  • most microsphere preparations have a high drug burst and subsequent low release, resulting in a high or low blood concentration.
  • it is easy to cause a decrease or degradation of the activity of the bioactive pharmaceutical ingredient during the production of the microsphere preparation, especially for polypeptide drugs. Therefore, there is a need for a new formulation and process to improve the burst release and effective blood concentration during sustained release of such sustained release formulations.
  • Exenatide is a synthetic North American exendin-4 consisting of 39 amino acid residues with the molecular formula C 184 H 28 . 0 6 . S, relative molecular weight 4186. 57, its amino acid sequence is as follows: Hi s-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser
  • GLP-1 human glucagon-like peptide-1
  • GLP-1 mammalian glucagon-like polypeptide-1
  • GLP-1 has the same amino acid sequence, and its main biological functions are: 1 increase insulin biosynthesis and glucose-dependent insulin secretion; 2 stimulate ⁇ -cell proliferation and regeneration, inhibit ⁇ -cell apoptosis and increase the number of ⁇ -cells; 3 inhibition of glucagon secretion; 4 inhibition of glycogen production, but does not cause severe hypoglycemia; 5 inhibition of postprandial gastrointestinal motility and secretion function; 6 reduce appetite, reduce food intake; 7 pairs of nerve cells have Protective effects.
  • Exenatide injection was approved by the US Food and Drug Administration (FDA) in April 2005.
  • the trade name is Byetta (Exenatide Injection), which is not ideal for improving the use of metformin and sulfonylureas.
  • Blood glucose control in patients with type 2 diabetes is used to control weight.
  • Clinical results show that exenatide is effective in the treatment of diabetes. The half-life of exenatide is only 2.4 hours, which requires two injections per day.
  • the FDA approved a weekly injection of the exenatide sustained release dosage form, under the trade name Byd Ure0 n.
  • exenatide sustained release preparation produced by El i Li l ly and Company, Amy 1 in, Alkermes Company uses the principle of phase coagulation to prepare exenatide microspheres, and the formulation of the preparation includes 5% exenatide, 2% sucrose and 93% PLGA (50: 50).
  • the shape of the microspheres prepared by this method is irregular, not a regular sphere, the surface is uneven, the particle size distribution is uneven, and the average particle size is between 2 ( ⁇ 40 ⁇ m.
  • the average particle size and particle size distribution of the microspheres greatly reduce the yield, and at the same time increase the complexity of the preparation process and the difficulty of the aseptic processing.
  • Domestic research institutes also use the double emulsion method (W/0/W) to prepare similar products.
  • Microspheres, according to the double emulsification method there are two methods of direct mechanical stirring and membrane emulsification.
  • the membrane emulsification method can produce microspheres with relatively uniform particle size
  • the polypeptide drugs are mostly soluble in water, and the drug is easy to be used in the preparation process.
  • the diffusion of the external water phase results in a low encapsulation efficiency and an increase in production costs, and the cost is also a key factor in the production of microsphere preparations.
  • the bioactive polypeptide drug and the polylactic acid-glycolic acid copolymer are respectively dissolved in water and an organic solvent, and belong to mutually incompatible two phases: oil phase and water phase, drug and polylactic acid-
  • the glycolic acid copolymer is a heterogeneous system.
  • a method for preparing a polypeptide drug sustained release microsphere comprises the following steps:
  • an oil phase containing 0.5 to 5 wt% stabilizer the oil phase selected from the group consisting of soybean oil, peanut oil, corn oil, sesame oil, mineral oil, dimethicone, cottonseed oil, olive oil, coconut oil, One or more of orange oil, aliphatic hydrocarbon, cycloaliphatic hydrocarbon or aromatic hydrocarbon; the stabilizer is selected from the group consisting of lecithin, sp an 80, glyceryl monostearate or polyglyceryl distearate; The effect is as a continuous phase of the emulsion.
  • a certain amount of stabilizer is added to the oil phase. The amount of stabilizer affects the size of the final microspheres. The higher the stabilizer content, the smaller the emulsion droplets formed during emulsification, and the smaller the particle size of the resulting microspheres.
  • the mixture of the step (3) and the oil phase of the step (4) are homogenized to form a 0/0 type emulsion according to a volume ratio of 1: 2 to 50; the method for forming the emulsion is mechanical stirring (time; Tl5min), high pressure Homogenization or high shear homogenization.
  • the surface tension between the two phases and the two phases affects the final microsphere size.
  • the organic solvent can be removed by stirring at 30 minutes (T3000 r pm stirring speed) for 6 to 15 hours. The same effect can be achieved by heating, evaporation under reduced pressure, etc.
  • the rate of solvent evaporation affects the surface morphology of the microspheres, and the evaporation rate of the organic solvent is slow.
  • the surface of the microspheres is denser and the microspheres have a small porosity.
  • the polypeptide drug is exenatide, glucagon-like peptide (GLP_1), luteinizing hormone releasing hormone (LHRH), cytokine, tumor necrosis factor, growth hormone, descending Calcium, epidermal growth factor (EGF), nerve growth factor (NGF), interferon, growth hormone, enzyme, interleukin, erythropoietin, immunoglobulin, antibody, colony stimulating factor, insulin or its analogue , a derivative, a modification or a salt.
  • GLP_1 glucagon-like peptide
  • LHRH luteinizing hormone releasing hormone
  • cytokine cytokine
  • tumor necrosis factor growth hormone
  • growth hormone descending Calcium
  • EGF epidermal growth factor
  • NGF nerve growth factor
  • interferon growth hormone
  • enzyme enzyme, interleukin, erythropoietin, immunoglobulin, antibody, colony stimulating factor, insulin or its analogue , a derivative, a modification or
  • the polypeptide drug is exenatide, liraglutide or a pharmaceutically acceptable salt thereof.
  • the concentration of the polylactic acid-glycolic acid copolymer solution or the polylactic acid solution is not limited
  • the protective agent is human serum albumin, zinc salts such as zinc chloride, zinc carbonate, zinc sulfate, and vinegar. Zinc, sucrose or gelatin.
  • the protective agent acts to maintain the stability of the bioactive polypeptide drug and prevent the polypeptide drug from forming a polymer that is inactivated or unable to be released.
  • the concentration of Exenatide or Liraglutide is 5 (T 200 mg/ml and the concentration of the protective agent is 50 to 100 mg/ml.
  • the volume ratio of the polylactic acid-glycolic acid copolymer solution to the exenatide solution is
  • the content of the stabilizer is 0.5% ⁇ 3%.
  • the volume ratio of the mixed liquid to the oil phase is 1: 2 to 10.
  • the polylactic acid-glycolic acid copolymer has a molecular weight of 1000 ( ⁇ 50000).
  • the viscosity of the oil phase is 1 (T500 cp.
  • the viscosity of the oil phase has a large effect on the particle size of the microspheres, and the greater the viscosity, the larger the droplet size formed during emulsification.
  • the viscosity of the oil phase is 3 (Tl00 Cp .
  • the polylactic acid-glycolic acid copolymer has an intrinsic viscosity of 0. ⁇ 0.
  • the viscous viscosity of the polylactic acid-glycolic acid copolymer is 0. 3 ⁇ 0. 5.
  • the invention also provides a polypeptide drug sustained-release microsphere preparation prepared by the above preparation method.
  • the preparation mainly comprises a biodegradable high molecular polymer, a polypeptide drug and a protective agent, and the biodegradable high molecular polymer is one of polylactic acid-glycolic acid copolymer, polylactic acid or modified modification thereof. kind or several.
  • the polypeptide drug sustained release microsphere preparation is subcutaneously or intramuscularly injected, mainly comprising a sustained release composition (microsphere) composed of a bioactive polypeptide drug and a biodegradable polymer, and the biologically active polypeptide drug is uniformly dispersed in the biological Degraded polymer.
  • a sustained release composition composed of a bioactive polypeptide drug and a biodegradable polymer
  • the biologically active polypeptide drug is uniformly dispersed in the biological Degraded polymer.
  • Preferred microspheres have a particle size of 2 ( ⁇ 100 ⁇ m, more preferably 3 ( ⁇ 60 ⁇ m).
  • the average particle size of the microspheres also affects the release of the bioactive polypeptide drug from the microspheres. As the particle size of the microspheres increases Large, bioactive peptide drugs are gradually slowed down from the microspheres, and the corresponding release time is also prolonged. The larger the size of the microspheres, the more difficult the injection, and the more painful the patient
  • the raw materials of the sustained release microspheres of the present invention are as follows:
  • Exenatide (exemplified by exenatide, other bioactive peptide drugs such as GLP-1, LHRH, liraglutide, calcitonin, cytokines, tumor necrosis factor, growth) Hormone, EGF, NGF, interferon, growth hormone, enzyme, interleukin, erythropoietin, immunoglobulin, antibody, colony stimulating factor, insulin, and the above-mentioned proteins, peptide analogs, derivatives, modifications and Salt, the above polypeptide drug may be obtained by natural extraction, chemical synthesis or Genetic engineering method)
  • Exenatide is a 39 amino acid peptide, and exenatide is an analog of human glucagon-like peptide-1 (GLP-1), a receptor agonist of GLP-1, and GLP-1. With the same physiological function, exenatide is shown to bind and activate the known human GLP-1 receptor in vitro. This means that glucose-dependent insulin synthesis and islet beta cells secrete insulin in the body by including cAMP and/or other intracellular signaling mechanisms. In the case of elevated glucose levels, exenatide promotes the release of insulin from beta cells. After in vivo administration, exenatide mimics the anti-hyperglycemic effect of GLP-1.
  • the role of the protective agent is to prevent the biological activity (such as exenatide) from decreasing in the process of preparing the microspheres, on the one hand, contributing to the stability of the protein, preventing the protein from forming a polymer inside the microsphere and being unable to release, and On the one hand, it can reduce the drug burst effect.
  • biological activity such as exenatide
  • the protective agents that can be used in the present invention mainly include sugars and sugar alcohols: mainly small molecule sugars.
  • sugars and sugar alcohols mainly small molecule sugars.
  • protein human albumin, fibrin, etc.
  • Polymer stabilizer gelatin, gum arabic, peach gum, xanthan gum, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, povidone, dextran; zinc salt Class: Zinc acetate, zinc carbonate, zinc sulfate, zinc chloride.
  • the protective agent selected in the present invention is one or more of the above.
  • the high molecular weight polymer used in the present invention is determined by a person skilled in the art in consideration of the degradation rate, physical properties, terminal chemistry, and the like of the polymer.
  • the molecular weight and composition of the polylactic acid-glycolic acid copolymer and polylactic acid affect the release of the bioactive polypeptide drug from the microspheres. ⁇ 0. 5 ⁇
  • the polylactic acid-glycolic acid copolymer in the lactide: lactide 25 : 75 0: 10
  • the molecular weight of polylactic acid is 4000 to 50000.
  • Polylactic acid-glycolic acid copolymer, polylactic acid may be end-blocked, unblocked (terminal carboxyl group) and modified by other groups
  • the resulting microspheres have a lower burst rate than those with terminally blocked PLGA.
  • the preparation method of the polypeptide drug sustained-release microsphere of the invention adopts the 0/0 method, and the polylactic acid-glycolic acid copolymer and the protective agent and the polypeptide drug are co-dissolved in an organic solvent to form a completely uniform mixed solution, and the mixed solution is added to the oil.
  • Phase vegetable oil
  • the continuous phase is the oil phase during the preparation process, which eliminates the problem of diffusion of the drug to the outer aqueous phase during the preparation of the double emulsion method, and improves the drug embedding rate, and the drug embedding rate is 60% ⁇ 95%.
  • Polypeptide drug and protective agent are uniformly embedded in polylactic acid-hydroxyl Acid copolymer inside the microspheres.
  • the bioactive peptide drug is slowly released through the surface pores of the microspheres and the degradation of the polymer material of the microspheres in vivo, and the release time can be as long as one week to several months.
  • the in vitro release test results show that the release conforms to the near zero-order release.
  • the preparation method of the polypeptide drug sustained release microsphere preparation of the invention only needs to emulsify and volatilize the organic solvent to obtain regular microspheres and drugs uniformly distributed in the microspheres, the process is simple, the operation is simple, the preparation repeatability is good, the batch There was no significant difference between them.
  • the obtained microspheres were uniform in particle size, narrow in distribution, controllable in particle size, rounded on the surface of the microspheres, and low in microbubble burst rate.
  • the exenatide sustained/lilastin peptide prepared by the invention has no micro-spheres and agglomeration between the microspheres, the microspheres are not broken, the drug release is stable and sustained, and the drug activity is maintained at more than 90% during the release in vivo. It can be used to treat type 2 diabetes and control weight.
  • Figure 1 is a scanning electron micrograph of Example 4 Exenatide sustained-release microspheres
  • Example 2 is an in vitro cumulative release curve of Example 2 Exenatide sustained-release microspheres
  • Figure 3 is a cumulative release curve of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 4 is a graph showing the blood glucose concentration-time curve on the first day after administration of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 5 is a graph showing the blood glucose concentration-time curve on the 5th day after administration of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 6 is a graph showing the blood glucose concentration-time curve on the 10th day after the administration of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 7 is a graph showing the blood glucose concentration-time curve on the 15th day after administration of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 8 is a graph showing the blood glucose concentration-time curve on the 20th day after administration of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 9 is a graph showing blood glucose concentration-time on the 30th day after administration of the sustained release microsphere preparations of Examples 4 and 14;
  • Figure 10 is a graph showing the time-blood concentration of exenatide and liraglutide sustained release microspheres of Examples 4 and 14 in vivo. detailed description
  • a method for preparing exenatide sustained-release microspheres comprising the steps of:
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, washed with cyclohexane, and the residual organic solvent in the microspheres is removed to obtain PLGA microspheres, which are collected.
  • the granules are in a range of 5 to 30 ⁇ m, the drug loading is 4.12%, and the encapsulation efficiency is 89.17%.
  • the encapsulation of the exenatide sustained-release microspheres is in the range of 5 to 30 ⁇ m, and the drug loading is 4.58%, encapsulated, except that the PLGA is replaced by the terminal carboxyl group PLGA. The rate is 90. 49%.
  • the specific step is the same as in Example 1, except that the PLGA is replaced by the MPEG-PLGA and the end-capped PLGA.
  • the particle size of the exenatide sustained-release microspheres is in the range of 5 to 30 ⁇ m, and the drug loading is 4. 22%. The rate is 88.45%.
  • the protein particles easily migrate to the surface of the microspheres, resulting in a higher burst effect, but due to poor hydrophilicity, The water uptake rate is low during the release process, the microsphere skeleton is slowly degraded, and the terminal carboxyl group PLGA and MPEG-PLGA are highly hydrophilic and have a high biodegradation rate.
  • Example 4
  • a preparation method of exenatide sustained-release microspheres comprising the following steps:
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, washed with cyclohexane, and the residual organic solvent in the microspheres is removed to obtain PLGA microspheres, which are collected.
  • the microspheres were dried in a lyophilizer to obtain exenatide sustained-release microspheres having a particle size of 2 ( ⁇ 50 ⁇ m, a drug loading of 7.18%, and an encapsulation efficiency of 94.14%.
  • Example 5 Compared with Example 1, the degreasing phase changed from liquid paraffin to peanut oil, the other conditions were unchanged, and the particle size of the microspheres became significantly larger. This is because the viscosity of peanut oil is greater than that of liquid paraffin, which forms larger droplets of soybean oil. The same results can be obtained when the viscosity of sesame oil, corn oil, cottonseed oil, methyl silicone oil, etc. is greater than that of liquid paraffin.
  • step 4 The specific steps are the same as those in Example 4, except that the content of lecithin in step 4 is reduced from 2% to 0.5%, and the particle size of the exenatide sustained-release microspheres is 4 ( ⁇ 100 ⁇ m, the drug loading is 4.25). %, Encapsulation rate 83. 23%.
  • Example 4 The specific steps are the same as those in Example 4, except that the content of lecithin in step 4 is changed from 2% to 5%, and the particle size of the exenatide sustained-release microspheres is 2 ( ⁇ 60 ⁇ m, the drug loading is 7.32%, The encapsulation efficiency was 92.16%.
  • Example 7 Compared with Example 4, the lecithin content was increased, the particle diameter of the microspheres was decreased, the lecithin content was decreased, and the particle diameter of the microspheres was increased. 5 ⁇ 3% The microspheres conforming to the particle size range can be obtained from the above examples. Example 7
  • Example 8 The specific steps are the same as those in Example 4, except that the peanut oil content in step 4 is reduced from 50 mL to 10 mL, and the particle size of the exenatide sustained-release microspheres is 6 ( ⁇ 100 ⁇ m, the drug loading is 3.14%, and the encapsulation efficiency is 67. 13%.
  • Example 8 the peanut oil content in step 4 is reduced from 50 mL to 10 mL, and the particle size of the exenatide sustained-release microspheres is 6 ( ⁇ 100 ⁇ m, the drug loading is 3.14%, and the encapsulation efficiency is 67. 13%.
  • Example 8 Example 8
  • Example 9 The specific steps are the same as those in Example 4, except that the peanut oil content in step 4 is increased from 50 mL to 200 mL, and the particle size of the exenatide sustained-release microspheres is 3 ( ⁇ 60 ⁇ m, the drug loading is 6.54%, and the encapsulation efficiency is 93. 63%. From the above examples, it is concluded that the oil phase is relatively small in volume of the drug and the protective agent solution, the stability of the two phases of the emulsion is poor, the particle size distribution of the microspheres is not uniform, the amount of the oil phase is excessive, and the production cost is increased.
  • the preferred ratio of oil phase to mixed liquor is from 5 to 10:1.
  • Example 10 The specific procedure is the same as in Example 4, except that the solvent in the step 1 is changed from acetic acid to acetonitrile, and the particle size of the exenatide sustained-release microspheres is 3 ( ⁇ 60 ⁇ m, the drug loading is 5.36%, and the encapsulation efficiency is 73. 26%.
  • Example 10 The specific procedure is the same as in Example 4, except that the solvent in the step 1 is changed from acetic acid to acetonitrile, and the particle size of the exenatide sustained-release microspheres is 3 ( ⁇ 60 ⁇ m, the drug loading is 5.36%, and the encapsulation efficiency is 73. 26%.
  • Example 10 The specific procedure is the same as in Example 4, except that the solvent in the step 1 is changed from acetic acid to acetonitrile, and the particle size of the exenatide sustained-release microspheres is 3 ( ⁇ 60 ⁇ m, the drug loading is 5.36%, and the encapsulation efficiency is 73. 26%.
  • a preparation method of exenatide sustained-release microspheres comprising the following steps:
  • step (3) homogenizing the mixture of step (3) with the oil phase of step (4) to form a 0/0 type emulsion
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, washed with cyclohexane, and the residual organic solvent in the microspheres is removed to obtain PLGA microspheres, which are collected.
  • the microspheres were dried in a freeze dryer to obtain exenatide sustained-release microspheres having a particle diameter of 3 ( ⁇ 60 ⁇ m, a drug loading of 3.89%, and an encapsulation efficiency of 85.20%. 11
  • a preparation method of exenatide sustained-release microspheres comprising the following steps:
  • step (5) mixing the mixed liquid of the step (3) with the oil phase of the step (4) to form a 0/0 type emulsion; (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, washed with cyclohexane, and the residual organic solvent in the microspheres is removed to obtain PLGA microspheres, which are collected.
  • the microspheres were dried in a freeze dryer to obtain exenatide sustained-release microspheres having a particle diameter of 3 ( ⁇ 60 ⁇ m, a drug loading of 3.93%, and an encapsulation efficiency of 86.60%. 12
  • a preparation method of exenatide sustained-release microspheres comprising the following steps:
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, washed with cyclohexane, and the residual organic solvent in the microspheres is removed to obtain PLGA microspheres, which are collected.
  • the microspheres were dried in a freeze dryer to obtain exenatide sustained-release microspheres having a particle diameter of 3 ( ⁇ 60 ⁇ m, a drug loading of 6.34%, and an encapsulation efficiency of 84.89%. 13
  • a preparation method of exenatide sustained-release microspheres comprising the following steps:
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, and After washing with cyclohexane, the organic solvent remaining in the microspheres is removed to obtain PLA microspheres, and the collected microspheres are placed in a freeze dryer to be dried to obtain exenatide sustained-release microspheres having a particle diameter of 3 ( 1480 ⁇ The drug loading of 8.4%, the encapsulation efficiency of 82. 34%.
  • a method for preparing liraglutide sustained-release microspheres comprising the following steps:
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, washed with cyclohexane, and the residual organic solvent in the microspheres is removed to obtain PLGA microspheres, which are collected.
  • the microspheres were dried in a freeze dryer to obtain liraglutide sustained-release microspheres having a particle diameter of 3 ( ⁇ 60 ⁇ m, a drug loading of 4.49%, and an encapsulation efficiency of 93.54%. 15
  • a method for preparing liraglutide sustained-release microspheres comprising the following steps:
  • step (6) stirring and volatilizing the emulsion of step (5) to remove acetic acid; after the organic solvent is completely evaporated, the microspheres are collected by centrifugation, and After washing with cyclohexane, the organic solvent remaining in the microspheres is removed to obtain PLA microspheres, and the collected microspheres are placed in a freeze dryer to be dried to obtain liraglutide sustained-release microspheres having a particle diameter of 3 ( 1660 ⁇ The ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16
  • the specific procedure is the same as in Example 13, except that the particle size is in the range of 3 ( ⁇ 60 ⁇ m, the drug loading is 9.87%, and the encapsulation efficiency is 91.73%.
  • the particle size of the sustained release microspheres of the present invention is preferably 2 ( ⁇ 50).
  • the particle size of the microspheres is smaller, less than 20 micrometers.
  • the vegetable oil peanut oil, soybean oil, sesame oil, etc.
  • the content of stabilizer is selected at about 2%, preferably lecithin. Determination of particle size distribution of microspheres:
  • the microsphere size distribution was measured using a Malvern laser particle size analyzer (Mastersizer 2000, Malvern). The 5 mg microsphere freeze-dried powder was weighed, added to 50 mL of purified water, and shaken with a vortex shaker for 5 min to uniformly disperse the microspheres, and the measurement was performed by a laser particle size analyzer.
  • the particle size distribution coefficient is obtained by the following formula:
  • Di is the particle size of a single microsphere
  • cU is the average particle size of the microsphere
  • N is the total number of microspheres, N>300.
  • microspheres The morphology and surface properties of the microspheres were observed by SEM.
  • the microsphere lyophilized powder was lightly applied to the conductive paste attached to the sample stage using a sampling rod. After spraying gold (120 s) under vacuum, it was observed with a scanning electron microscope (S-3700N Japan).
  • S-3700N Japan a scanning electron microscope
  • the NaOH-SDS method reported in the literature was used to determine the drug loading and encapsulation efficiency of the microspheres.
  • the specific operation was as follows: Accurately weigh 10 mg of microspheres with 1 ml of 0.1 mol ⁇ L- 1 NaOH (containing 5% SDS) solution. The suspension was shaken in a 100 rpm, 37 ° C water bath shaker for 24 h, centrifuged at SOOO rpm for 10 min, and the supernatant was taken. The content of exenatide in the supernatant was determined by high performance liquid chromatography. The weight of the drug contained in the microsphere
  • exenatide/lilastuide was measured by an ELISA method and assayed according to the method of the active GLP-1 (7-36)-specific enzyme-free kit (US/EDI).
  • US/EDI active GLP-1 (7-36)-specific enzyme-free kit
  • the phosphate buffer of the release medium is pH 7.4, and the method is as follows: 50 mg of the microspheres are placed in a 10 mL centrifuge tube, and the release medium is pH 7.4. Liquid (containing 0.02% sodium azide as bacteriostatic agent, 0. 05% soil temperature 80 as a wetting agent), placed at a temperature of 37 ° C ⁇ 0. 5 ° C for 1 h, lh The sample was taken out, and then centrifuged at 5000 rpm for 15 min. The supernatant was taken out, and the concentration of exenatide/lilastuide in the supernatant was determined by HPLC to calculate the release percentage. Table 1 lists the in vitro burst release and retention of drug activity using the polypeptide drug microsphere formulations of the different examples. Table 2 shows the content of each component in the different examples. Table 1
  • Example 1 5-30 25 2. 05 93. 9
  • Example 2 5-40 36 1. 16 95. 6
  • Example 3 5-30 26 5.
  • 32 92. 2 Example 4 30-60 12 1. 36 96 4
  • Example 5 40-100 25 2. 06 92. 6
  • Example 6 20-60 18 2.
  • 32 93. 4 Example ⁇ 60-100 30 2. 46 92. 9
  • Example 8 30-80 15 1. 25 93. 6
  • Example 9 50-100 24 1. 84 92. 2
  • Example 10 30-60 27 1. 30 95. 9
  • Example 11 30-60 23 1. 56 95. 6
  • Example 12 30-80 26 1. 23 96. 2
  • Example 13 30-80 25 3. 36 96 4
  • Example 15 30-60 20 1. 32 93. 4
  • Example 16 30-60 23 1. 12 92. 7 From Table 1, prepared by the present invention
  • the particle size of the sustained release microspheres is between 5 and 100 nm.
  • the burst release rate of the drug in vitro is mostly below 5%, and the particle size distribution coefficient is 1 ( ⁇ 30, the activity of the drug remains above 92%.
  • the viscosity of the oil phase is on the microsphere
  • the particle size has a large influence, and when the liquid paraffin having a relatively high viscosity is used, the obtained microspheres have a small particle diameter; conversely, when a low-viscosity oil phase (vegetable oil such as peanut oil) is used, a microsphere having a larger particle diameter can be obtained.
  • a low-viscosity oil phase vegetable oil such as peanut oil
  • Example 1 Feed 0 / 0 Example 1 4. 12 2. 51 92. 25 1. 12 Example 2 4. 58 2. 53 91. 75 1. 14 Example 3 4. 22 2. 53 1. 13 Example 4 7 18 2. 46 89. 76 0. 6 Example 5 4. 25 2. 65 89. 16 3. 94 Example 6 7. 32 2. 47 89. 71 0. 5 Example ⁇ 3. 14 2. 86 90. 01 3. 99 Example 8 6. 54 2. 84 89. 56 1. 06 Example 9 5. 36 2. 96 89. 56 2. 12 Example 10 3. 89 3. 33 0. 65 Example 11 3. 93 2. 75 92. 56 0. 76 Example 12 6. 34 5. 74 86. 65 1. 27
  • Example 13 8. 14 3. 57 86. 87 1. 42
  • Example 15 9. 18 0. 67 88. 97 1. 18
  • Example 16 9. 5 ⁇ 6% ⁇ The singularity of the present invention, the content of the protective agent is 0. 5 ⁇ 6%, bio The degradation polymer is between 85% and 95%. Determination of in vitro release of Exenatide sustained-release microspheres:
  • the 50 mg microspheres were placed in a 10 mL centrifuge tube, and the release medium was pH 7.4 phosphate buffer (containing 0.22% sodium azide as a bacteriostatic agent, and 0. 05% soil temperature 80 as a wetting agent).
  • the in vitro release of the microspheres was carried out in a constant temperature water bath shaker at an oscillation speed of 100 rpm and a temperature of 37 ° C ⁇ 0.5 ° C. 5 ⁇ Example 2, respectively, on the first, 2, 3, 4, 5, 6 and 7 days, respectively, on the first, 2, 4, 8, 12, 16, 20, 30 days to remove 0. 5mL release medium
  • the content of the drug is determined by high performance liquid chromatography and supplemented with fresh release medium.
  • the sustained release microspheres of Examples 2, 4 and 14 have a good sustained release effect.
  • the release time can be from one week to as long as one month.
  • the drug group and the blank control group were randomly divided into two groups.
  • the drug group was subcutaneously injected with an appropriate amount of the microspheres prepared in Example 4 and Example 14, and the blank group was subcutaneously injected with the same amount of physiological saline.
  • 18 mmol/kg of glucose was intraperitoneally injected on the 1st, 5th, 10th, 15th, 20th, and 30th day after administration.
  • a blank blood sample was taken from each mouse, and then blood was taken at 5, 10, 30, and 60 minutes after the injection. The blood glucose concentration before and after the injection was measured.
  • SD rats were randomly divided into three groups, and the appropriate amount of microspheres of Examples 4 and 14 were injected subcutaneously, respectively, at 1, 2, 3, 4, 6, 8, 10, 12, 15, 18, 22 after administration.
  • Blood was taken from the tail vein at 26 and 30 days. Immediately, the blood was placed in an anticoagulation tube, centrifuged at 15000 g for 3 min, and the plasma was transferred to a clean centrifuge tube, and -80 was frozen for testing. The content of exenatide in plasma was detected by enzyme-linked immunosorbent assay, and the detection method was carried out according to the instructions of rat glucagon-like peptide 1 (GLP-1) ELISA kit (Shanghai Yaji Bio-Biotechnology Co., Ltd.).
  • GLP-1 rat glucagon-like peptide 1

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Abstract

一种多肽药物缓释微球制剂。该制剂的制备方法包括步骤：将聚乳酸一羟基乙酸共聚物或聚乳酸和保护剂、多肽药物共同溶解在有机溶剂中，形成均一的混合溶液，混合溶液加入到油相形成乳液，除去有机溶剂，离心，洗涤，冷冻干燥，即得多肽药物缓释微球。采取0/0法，杜绝了药物向外水相扩散，药物包埋率提高到60%〜95%。生物活性多肽药物释放时间可长达数周至数月，体外释放符合近似零级释放。

Description

多肽药物缓释微球制剂及其制备方法 技术领域
本发明属于生物医用高分子材料与生物活性药物控释制剂研究领域， 具体地说， 本发明 涉及一种多肽药物缓释微球制剂及其制备方法。 背景技术
大多数的蛋白、 多肽类药物， 口服生物利用度很低， 以致口服后不能产生足够高的有效 血药浓度， 这类药物不能通过口服途径给药。 皮下注射时由于体内蛋白酶的存在， 药物在体 内的半衰期很短， 需要频繁注射， 增加了患者的痛苦， 降低患者依从性。
利用生物相容性可降解材料 （例如高分子材料） 包裹药物活性成分， 制成微球制剂， 通 过可降解的生物高分子材料在体内逐步降解来控制药物释放， 维持有效的血药浓度。 然而， 大多数微球制剂都存在很高的药物突释现象以及此后的低释， 造成血药浓度过高或者低于有 效血药浓度。 此外， 在微球制剂生产过程中很容易造成生物活性药物成分活性降低或降解， 对于多肽类药物来说尤其如此。 因此， 需要一种新的制剂和工艺来改善这类缓释制剂的突释 及维持释放期间的有效血药浓度。
艾塞那肽 （exenatide ) 是人工合成的北美毒蜥外泌肽 （exendin-4)， 由 39个氨基酸残 基构成， 分子式为 C184H28 。06。S， 相对分子量 4186. 57， 其氨基酸序列如下： Hi s-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser
Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-I l e-Glu-Trp-Leu-Lys-Asn-Gly-Gly -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2。 艾塞 ¾贩为人胰高血糖素样肽 _1 (GLP-1 ) 的类似物， 与 GLP-1具有相同的生理功能， 其 53%的氨基酸顺序与哺乳动物胰高血糖素样多 肽 -1 (GLP-1 ) 的氨基酸顺序相同， 其主要生物功能为： ①增加胰岛素的生物合成及葡萄糖依 赖性促胰岛素分泌； ②剌激 β 细胞增殖和再生， 抑制 β 细胞凋亡从而增加 β 细胞的数量； ③抑制胰高血糖素的分泌； ④抑制肝糖生成， 但不会引起严重低血糖； ⑤抑制餐后胃肠道动 力及分泌功能； ⑥降低食欲， 减少食物的摄入； ⑦对神经细胞具有保护作用。 可促进葡萄糖 依赖的胰岛素分泌， 抑制不适当的葡萄糖依赖的胰高血糖素的分泌， 减慢胃排空， 改善外周 组织对胰岛素的敏感性， 充分控制血糖。 艾塞那肽注射剂已于 2005年 4月获美国食品药品监督管理局 （FDA) 批准上市， 商品名 为百泌达 （Byetta, Exenatide Injection) , 用于改善使用二甲双胍和磺酰脲类药物不理想 的 2型糖尿病患者的血糖控制或者用来控制体重。 临床结果显示， 艾塞那肽用于糖尿病治疗 效果明显。 艾塞那肽的半衰期仅 2. 4小时， 需每日注射两次。 2012年 1月 27日， FDA批准了 每周注射一次的艾塞那肽缓释剂型， 商品名 BydUre0n。
美国礼来公司 （El i Li l ly and Company )、 Amy 1 in, Alkermes公司生产的艾塞那肽缓释 制剂 （Bydureon) 采用相凝聚原理来制备艾塞那肽微球， 该制剂其配方包括 5%的艾塞那肽、 2%蔗糖及 93%PLGA ( 50： 50 )。
此方法制备的微球形状不规整， 不是规则圆球， 表面凹凸不平， 粒径分布不均匀， 平均粒径在 2(Γ40微米之间。 制备过程中需要筛选出不符合要求的部分， 来控制微球的 平均粒径和粒径分布， 使产率大大下降， 同时增加了制备工艺的复杂性和无菌工艺操作 的难度。 国内也有研究机构采用复乳法 （W/0/W) 制备类似微球， 按照复乳化方法不同 有直接机械搅拌和膜乳化两种方法。膜乳化法虽然可制得粒径相对均一的微球， 但多肽 药物大都易溶于水， 在制备过程中药物易于向外水相扩散， 造成药物包封率不高， 生产 成本上升， 而成本亦是微球制剂生产过程中的一个关键因素。
现有技术在制备微球过程中， 生物活性多肽药物与聚乳酸-羟基乙酸共聚物分别溶 于水和有机溶剂， 分属互不相容两相： 油相和水相， 药物和聚乳酸 -羟基乙酸共聚物是 非均相体系。 发明内容
基于此， 有必要提供一种多肽药物缓释微球制剂的制备方法， 其制备得到的缓释微球制 剂能有效延长在体内的作用时间。
一种多肽药物缓释微球的制备方法， 包括以下步骤：
(1) 将聚乳酸-羟基乙酸共聚物或聚乳酸溶于有机溶剂中， 形成浓度为 10(T800mg/mL的 溶液； 所述聚乳酸-羟基乙酸共聚物中乙交酯： 丙交酯 =15 : 85 0 : 10， 所述聚乳酸-羟基乳酸 共聚物的分子量为 2000〜65000; 所述聚乳酸的分子量为 4000〜50000; 所述有机溶剂为二氯甲 烷、 三氯甲烷、 乙酸乙酯、 丙酮、 乙酸、 乙腈中的一种或几种；
(2) 分别按 l(T500mg/ml和 l(T200mg/ml的浓度， 将多肽药物和保护剂溶于无菌水中， 得到多肽药物溶液， 所述保护剂为糖、 糖醇类、 蛋白类、 无机盐、 高分子稳定剂中的一种或 几种；
(3)按体积比为 1 : 5〜50将步骤（1)的聚乳酸-羟基乙酸共聚物溶液或聚乳酸溶液与步骤 (2) 的多肽药物溶液混合， 搅拌至形成均一、 澄清、 透明的混合液；
(4) 配制含 0. 5〜5wt%稳定剂的油相， 所述油相选自大豆油、 花生油、 玉米油、 芝麻油、 矿物油、 二甲基硅油、 棉籽油、 橄榄油、 椰子油、 橘油、 脂肪烃、 环脂烃或芳香烃中的一种 或几种； 所述稳定剂选自卵磷脂、 span80、 单硬脂酸甘油酯或双硬脂酸聚甘油酯； 油相作用 是作为乳液连续相， 为了保持乳液两相稳定， 油相中需加入一定量的稳定剂。 稳定剂的含量 影响最终微球粒径的大小。 稳定剂含量越高， 乳化时形成的乳滴越小， 最终得到的微球粒径 越小。
(5)按体积比 1 : 2〜50将步骤 (3)的混合液与步骤 (4)的油相均质形成 0/0型乳液； 形成乳 液的方法有机械搅拌 （时间; Tl5min)、 高压均质或高剪切均质等。 两相比及两相间的表面张 力影响最终微球粒径大小。 有机相与油相体积比越大， 对应得到的微球粒径越大。
(6)挥发步骤 (5)的乳液除去有机溶剂， 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤，除去微球中残留的有机溶剂，把收集到的微球放入真空冷冻干燥器干燥 i0h， 得到多肽药物缓释微球制剂， 置于 -20°C保存。
可以在 30(T3000rpm搅拌速度下搅拌 6〜15h除去有机溶剂， 也可以采取加热、 减压蒸发 等方法达到相同效果。 溶剂挥发的速度影响微球的表面形态， 有机溶剂挥发速度慢， 得到的 微球表面更加致密， 微球孔隙率小。
在其中一些实施例中，所述多肽药物为艾塞那肽（exenatide )、胰高血糖素样肽（GLP_1 )、 促黄体激素释放激素 （LHRH)、 细胞因子、 肿瘤坏死因子、 生长激素、 降钙素、 表皮生长因子 (EGF)、 神经生长因子 （NGF)、 干扰素、 生长激素、 酶、 白细胞介素、 促红细胞生长素、 免 疫球蛋白、 抗体、 集落剌激因子、 胰岛素或其类似物、 衍生物、 修饰物或盐。
在其中一个实施例中， 所述多肽药物为艾塞那肽、 利拉鲁肽或其药学上可接受盐。
在其中一些实施例中， 所述聚乳酸-羟基乙酸共聚物溶液或聚乳酸溶液的浓度为
10(T500mg/mL。聚乳酸 -羟基乳酸共聚物或聚乳酸在有机溶剂中的浓度越大， 乳化时形成的液 滴越大， 对应最后形成微球的粒径越大。
在其中一些实施例中， 所述保护剂为人血白蛋白、 锌盐如氯化锌、 碳酸锌、 硫酸锌和醋 酸锌、 蔗糖或明胶。 保护剂的作用是保持生物活性多肽药物的稳定性， 防止多肽药物形成多 聚体失活或无法释放。
在其中一些实施例中， 艾塞那肽或利拉鲁肽的浓度为 5(T200mg/ml， 保护剂的浓度为 50〜100 mg/ml。
在其中一些实施例中， 所述聚乳酸-羟基乙酸共聚物溶液与艾塞那肽溶液的体积比为
1 : 5〜10。
在其中一些实施例中， 所述稳定剂的含量为 0. 5%〜3%。
在其中一些实施例中， 所述混合液与油相的体积比为 1 : 2〜10。
在其中一些实施例中， 聚乳酸-羟基乙酸共聚物的分子量为 1000(Γ50000。
在其中一些实施例中， 油相的粘度为 l(T500cp。 油相的粘度对微球粒径有较大影响， 粘 度越大， 乳化时形成的液滴尺寸越大。
在其中一些实施例中， 油相的粘度为 3(Tl00Cp。
在其中一些实施例中， 聚乳酸-羟基乙酸共聚物的特性粘数为 0. Γ0. 5。
在其中一个实施例中， 所述聚乳酸-羟基乙酸共聚物的特性粘数为 0. 3^0. 5。 本发明还提供了一种由上述制备方法制得的多肽药物缓释微球制剂。 所述制剂主要包括 可生物降解的高分子聚合物、多肽药物和保护剂， 所述可生物降解的高分子聚合物为聚乳酸- 羟基乙酸共聚物、 聚乳酸或其改性修饰物中的一种或几种。 该多肽药物缓释微球制剂是经皮 下或肌肉注射的， 主要是由生物活性多肽药物与可生物降解的聚合物构成的缓释组合物 （微 球）， 生物活性多肽药物均匀分散在可生物降解的聚合物中。优选的微球粒径在 2(Γ100微米， 更优选的为 3(Γ60微米。 微球平均粒径的大小也影响生物活性多肽药物从微球中的释放。 随 着微球粒径的逐渐增大， 生物活性多肽药物从微球中释放速度也逐渐减慢， 相应释放时间也 随之延长。 微球粒径越大， 注射时越困难， 病人的疼痛感越强。
本发明的缓释微球的原料如下：
1. 艾塞那肽（以艾塞那肽为例说明，也可以是其它的生物活性多肽药物，如 GLP-1、 LHRH、 利拉鲁肽、 降钙素、 细胞因子、 肿瘤坏死因子、 生长激素、 EGF、 NGF、 干扰素、 生长激素、 酶、 白细胞介素、 促红细胞生长素、 免疫球蛋白、 抗体、 集落剌激因子、 胰岛素以及上述蛋 白、 多肽的类似物、 衍生物、 修饰物及盐， 上述多肽药物可以是通过天然提取、 化学合成或 者基因工程方法得到）
艾塞那肽是一种含有 39个氨基酸的多肽， 艾塞那肽为人胰高血糖素样肽 -1 ( GLP-1 ) 的 类似物， 是 GLP-1的受体激动剂， 与 GLP-1具有相同的生理功能， 艾塞那肽在体外显示可以 结合并活化已知的人类 GLP-1受体。 这就意味着通过包括 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。体内给药后艾塞那肽模拟 GLP-1的某种抗高 血糖药作用。
2. 保护剂
保护剂的作用是防止生物活性物 （例如艾塞那肽） 在制备微球的过程中活性降低， 一方 面有助于蛋白的稳定， 防止蛋白在微球内部形成多聚体而无法释放， 另一方面可以减小药物 突释作用。
本发明可以选用的保护剂主要有糖、 糖醇类： 主要是小分子的糖。 例如： 蔗糖、 乳糖、 海藻糖、 纤维二糖、 甘露糖、 麦芽糖、 肌糖、 绵白糖、 菊糖、 右旋糖苷、 麦芽糖糊精、 甘露 醇等； 蛋白类： 人血白蛋白、 纤维蛋白等； 高分子稳定剂： 明胶、 阿拉伯胶、 桃胶、 黄原胶、 甲基纤维素、 羧甲基纤维素钠、 羟丙基纤维素、 卡波姆、 聚维酮、 葡聚糖； 锌盐类： 醋 酸锌、 碳酸锌、 硫酸锌、 氯化锌。 本发明选用的保护剂为上述中的一种或几种。
3. 聚乳酸 -羟基乙酸共聚物或聚乳酸
本发明中所用到的高分子聚物合由本领域技术人员综合考虑聚合物的降解率、物理性质、 端基化学等确定的。聚乳酸 -羟基乙酸共聚物、聚乳酸的分子量及组成影响生物活性多肽药物 从微球中的释放。 聚乳酸-羟基乙酸共聚物中乙交酯： 丙交酯=25 : 75 0 : 10， 所述聚乳酸-羟 基乳酸共聚物的分子量为 2000〜65000，特性粘数为 0. Γ0. 5。聚乳酸的分子量为 4000〜50000。 聚乳酸 -羟基乙酸共聚物、 聚乳酸可以是端基封闭、 未封闭 （端羧基）及经过其他基团修饰的
(例如 MPEG-PLGA、 MPEG_PLGA)。 聚合物采用端羧基 PLGA (末端未封闭） 或 MPEG-PLGA时， 所得微球比采用端基封闭 PLGA具有更低的突释率。
本发明的多肽药物缓释微球制备方法采取 0/0法， 将聚乳酸-羟基乙酸共聚物和保护剂、 多肽药物共同溶解在有机溶剂中， 形成完全均一的混合溶液， 混合溶液加入到油相（植物油） 形成乳液。 制备过程中连续相为油相， 杜绝了复乳法制备过程中药物向外水相扩散的问题， 提高了药物包埋率， 药物包埋率在 60%〜95%。 多肽药物和保护剂均匀地包埋在聚乳酸-羟基乙 酸共聚物微球内。 生物活性多肽药物通过微球表面孔隙和随着微球的聚合物材料在体内降解 缓慢释放出来， 释放时间可长达一周至数月， 体外释放试验结果表明释放符合近似零级释放。
本发明的多肽药物缓释微球制剂的制备方法只需要乳化、 挥发有机溶剂即可得到规整的 微球和在微球中均匀分布的药物， 工序简单， 操作简单， 制备重复性好， 批次之间无显著 差别， 得到的微球粒径均一、 分布窄、 粒径可控， 微球表面圆整， 微球突释率低。
本发明制备的艾塞那肽缓 /利拉鲁肽释微球， 微球之间无粘连和团聚， 微球无破裂， 药物 释放平稳、 持续， 药物活性在体内释放期间保持在 90%以上， 可适用于治疗 II型糖尿病和控 制体重。 附图说明
图 1为实施例 4艾塞那肽缓释微球的扫描电镜照片；
图 2为实施例 2艾塞那肽缓释微球体外累计释放曲线；
图 3为实施例 4、 14缓释微球制剂体外累计释放曲线；
图 4为实施例 4、 14缓释微球制剂给药后第 1天血糖浓度-时间曲线图；
图 5为实施例 4、 14缓释微球制剂给药后第 5天血糖浓度-时间曲线图；
图 6为实施例 4、 14缓释微球制剂给药后第 10天血糖浓度-时间曲线图；
图 7为实施例 4、 14缓释微球制剂给药后第 15天血糖浓度-时间曲线图；
图 8为实施例 4、 14缓释微球制剂给药后第 20天血糖浓度-时间曲线图；
图 9为实施例 4、 14缓释微球制剂给药后第 30天血糖浓度-时间曲线图；
图 10为实施例 4、 14的艾塞那肽及利拉鲁肽缓释微球在体内时间-血药浓度图。 具体实施方式
以下结合具体实施例来详细说明本发明。
实施例 1
—种艾塞那肽缓释微球的制备方法， 包括以下步骤：
(1) 将 l. Og聚乳酸-羟基乙酸共聚物（分子量 15000， 乙交酯： 丙交酯 =75 : 25， 端羧基) 溶于 5ml无水乙酸中， 形成聚乳酸-羟基乙酸共聚物溶液；
(2) 称取 50mg艾塞那肽， 30mg蔗糖溶于 0. 5mL无菌水中； (3)将步骤（1)的聚乳酸-羟基乙酸共聚物溶液与步骤 (2)的艾塞那肽溶液混合， 用磁力搅 拌器搅拌至形成完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 2wt%卵磷脂的液体石蜡， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相均质乳化处理 5-15min形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLGA微球， 把收集到的微球放入冷冻干燥 机干燥，得到艾塞那肽缓释微球，微球粒径在 5〜30微米范围内，载药量 4. 12%，包封率 89. 17%。 实施例 2
除 PLGA由端羧基 PLGA替换为端基封闭 PLGA外， 具体步骤同实施例 1， 得到艾塞那肽缓 释微球粒径在 5〜30微米范围内， 载药量 4. 58%, 包封率 90. 49%。 实施例 3
除 PLGA由 MPEG-PLGA替换为端基封闭 PLGA外， 具体步骤同实施例 1， 得到艾塞那肽缓 释微球粒径在 5〜30微米范围内， 载药量 4. 22%， 包封率 88. 45%。
实施例 1、 2、 3比较， 聚合物采用端羧基 PLGA (末端未封闭） 或 MPEG-PLGA时， 所得微 球比采用端基封闭 PLGA具有更低的突释率， 端基封闭 PLGA分子是烷基末端， 而未封闭的则 是羧基末端， MPEG-PLGA具有 PEG链， 后两者聚合物具有更佳亲水性。 由于烷基末端的存在， 端基封闭 PLGA在有机溶剂中溶解性能好， 在微球制备过程中蛋白颗粒易迁移到微球表面， 造 成了较高的突释效应， 然而由于亲水性差， 在释放过程中水分摄取率低， 微球骨架降解缓慢， 而端羧基 PLGA和 MPEG-PLGA亲水性高， 生物降解速度快。 实施例 4
一种艾塞那肽缓释微球的制备方法， 包括以下步骤：
(1) 将 l. Og聚乳酸-羟基乙酸共聚物（分子量 15000， 乙交酯： 丙交酯 =75 : 25)溶于 5ml 乙酸中， 形成聚乳酸-羟基乙酸共聚物溶液；
(2) 称取 80mg艾塞那肽， 30mg人血白蛋白溶于 0. 5mL无菌水中；
(3)将步骤（1)的聚乳酸-羟基乙酸共聚物溶液与步骤 (2)的艾塞那肽溶液混合， 用磁力搅 拌器搅拌至形成完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 2wt%卵磷脂的花生油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相均质乳化， 均质形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLGA微球， 把收集到的微球放入冷冻干燥 机干燥，得到艾塞那肽缓释微球，微球粒径在 2(Γ50微米范围内，载药量 7. 18%，包封率 94. 14%。
与实施例 1相比， 除油相由液体石蜡变为花生油外， 其他条件不变， 微球粒径明显变大， 这是由于花生油的粘度大于液体石蜡， 形成乳滴更大， 用大豆油、 芝麻油、 玉米油、 棉籽油、 甲基硅油等粘度大于液体石蜡的油相时， 可得到相同结果。 实施例 5
具体步骤同实施例 4， 除步骤 4中卵磷脂的含量由 2%降为 0. 5%， 得到艾塞那肽缓释微球 粒径在 4(Γ100微米范围内， 载药量 4. 25%, 包封率 83. 23%。
实施例 6
具体步骤同实施例 4， 除步骤 4中卵磷脂的含量由 2%变为 5%， 得到艾塞那肽缓释微球粒 径在 2(Γ60微米范围内， 载药量 7. 32%, 包封率 92. 16%。
与实施例 4相比， 增加卵磷脂含量， 微球粒径减小， 减少卵磷脂含量， 微球粒径增大。 从上述实施例得出稳定剂卵磷脂的含量优选在 0. 5〜3%可得到符合粒径范围的微球。 实施例 7
具体步骤同实施例 4， 除步骤 4中花生油含量由 50mL降为 10mL， 得到艾塞那肽缓释微球 粒径在 6(Γ100微米范围内， 载药量 3. 14%, 包封率 67. 13%。 实施例 8
具体步骤同实施例 4， 除步骤 4中花生油含量由 50mL增加为 200mL， 得到艾塞那肽缓释 微球粒径在 3(Γ60微米范围内， 载药量 6. 54%， 包封率 93. 63%。 从上述实施例得出， 油相与 药物、 保护剂溶液体积比较小时， 形成乳液两相稳定性较差， 微球粒径分布不均匀， 油相用 量过多， 生产成本增加， 优选的油相与混合液的比例在 5〜10 : 1。 实施例 9
具体步骤同实施例 4， 除步骤 1 中溶剂由乙酸变为乙腈， 得到艾塞那肽缓释微球粒径在 3(Γ60微米范围内， 载药量 5. 36%， 包封率 73. 26%。 实施例 10
一种艾塞那肽缓释微球的制备方法， 包括以下步骤：
(1) 将 l. Og聚乳酸-羟基乙酸共聚物（分子量 5000， 乙交酯： 丙交酯 =25 : 75)溶于 5ml无 水乙酸中， 形成聚乳酸-羟基乙酸共聚物溶液；
(2) 称取 50mg艾塞那肽， 30mg明胶溶于 0. 5mL无菌水中；
(3)将步骤（1)的聚乳酸-羟基乙酸共聚物溶液与步骤 (2)的艾塞那肽溶液混合， 用磁力搅 拌器搅拌至形成完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 2wt%span80的花生油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相均质形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLGA微球， 把收集到的微球放入冷冻干燥 机干燥， 得到艾塞那肽缓释微球， 微球粒径在 3(Γ60 微米范围内， 载药量 3. 89%， 包封率 85. 20%。 实施例 11
一种艾塞那肽缓释微球的制备方法， 包括以下步骤：
(1) 将 l. Og聚乳酸-羟基乙酸共聚物（分子量 65000， 乙交酯： 丙交酯 =85 : 15)溶于 5ml 无水乙酸中， 形成聚乳酸-羟基乙酸共聚物溶液；
(2) 称取 50mg艾塞那肽， 30mg碳酸锌溶于 0. 5mL无菌水中；
(3)将步骤（1)的聚乳酸-羟基乙酸共聚物溶液与步骤 (2)的艾塞那肽溶液混合， 用磁力搅 拌器搅拌至形成完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 2wt%span60的玉米油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相混合均质形成 0/0型乳液； (6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLGA微球， 把收集到的微球放入冷冻干燥 机干燥， 得到艾塞那肽缓释微球， 微球粒径在 3(Γ60 微米范围内， 载药量 3. 93%， 包封率 86. 60%。 实施例 12
一种艾塞那肽缓释微球的制备方法， 包括以下步骤：
(1) 将 l. Og聚乳酸-羟基乙酸共聚物（分子量 15000， 乙交酯： 丙交酯 =90 : 10)溶于 5ml 无水乙酸中， 形成聚乳酸-羟基乙酸共聚物溶液；
(2) 称取 80mg艾塞那肽， 65mg醋酸锌溶于 0. 5mL无菌水中；
(3)将步骤（1)的聚乳酸-羟基乙酸共聚物溶液与步骤 (2)的艾塞那肽溶液混合， 用磁力搅 拌器搅拌至形成完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 lwt%卵磷脂的大豆油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相机械搅拌， 均质形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLGA微球， 把收集到的微球放入冷冻干燥 机干燥， 得到艾塞那肽缓释微球， 微球粒径在 3(Γ60 微米范围内， 载药量 6. 34%， 包封率 84. 89%。 实施例 13
一种艾塞那肽缓释微球的制备方法， 包括以下步骤：
(1) 将 0. 5g聚乳酸 （分子量 15000)溶于 5ml无水乙酸中， 形成聚乳酸溶液；
(2) 称取 50mg艾塞那肽， 20mg硫酸锌溶于 0. 5mL无菌水中；
(3) 将步骤（1)的聚乳酸溶液与步骤 (2)的艾塞那肽溶液混合， 用磁力搅拌器搅拌至形成 完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 0. 5wt%卵磷脂的芝麻油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相机械搅拌， 均质形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLA微球， 把收集到的微球放入冷冻干燥 机干燥， 得到艾塞那肽缓释微球， 微球粒径在 3(Γ80 微米范围内， 载药量 8. 14%， 包封率 82. 34%。 实施例 14
一种利拉鲁肽缓释微球的制备方法， 包括以下步骤：
(1) 将 l. Og聚乳酸-羟基乙酸共聚物（分子量 15000， 乙交酯： 丙交酯 =75 : 25)溶于 5ml 无水乙酸中， 形成聚乳酸-羟基乙酸共聚物溶液；
(2) 称取 50mg艾塞那肽， 10mg氯化锌溶于 0. 5mL无菌水中；
(3)将步骤（1)的聚乳酸-羟基乙酸共聚物溶液与步骤 (2)的利拉鲁肽溶液混合， 用磁力搅 拌器搅拌至形成完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 2wt%卵磷脂的大豆油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相混合均质均质形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLGA微球， 把收集到的微球放入冷冻干燥 机干燥， 得到利拉鲁肽缓释微球， 微球粒径在 3(Γ60 微米范围内， 载药量 4. 49%， 包封率 93. 54%。 实施例 15
一种利拉鲁肽缓释微球的制备方法， 包括以下步骤：
(1)将 l. Og聚乳酸 （端羧基 PLA， 分子量 50000)溶于 5ml无水乙酸中， 形成聚乳酸共聚 物溶液；
(2) 称取 150mg利拉鲁肽肽， 50mg甘露糖溶于 0. 5mL无菌水中；
(3) 将步骤（1)的聚乳酸溶液与步骤 (2)的利拉鲁肽溶液混合， 用磁力搅拌器搅拌至形成 完全均一、 澄清、 透明的混合液， 作为有机相；
(4) 配制 50ml含 2wt%卵磷脂的花生油， 作为油相；
(5) 将步骤 (3)的混合液与步骤 (4)的油相混合均质形成 0/0型乳液；
(6)搅拌挥发步骤 (5)的乳液除去乙酸； 待有机溶剂完全挥发干净后， 离心收集微球， 加 入环己烷洗涤， 除去微球中残留的有机溶剂， 得到 PLA微球， 把收集到的微球放入冷冻干燥 机干燥，得到利拉鲁肽缓释微球，微球粒径在 3(Γ60微米范围内，载药量 9. 18%，包封率 84. 73%。 实施例 16
除由 MPEG-PLA替换 PLA外， 具体操作步骤同实施例 13， 微球粒径在 3(Γ60微米范围内， 载药量 9. 87%, 包封率 91. 73%。 从上述实施例得出， 通过选用不同的油相、 两相比例及稳定剂含量， 可以得到不同粒径 的微球， 粒径范围在 5〜100纳米间。 本发明的缓释微球粒径优选在 2(Γ50微米间， 用液体石 蜡做油相时， 得到微球粒径较小， 小于 20微米。 采用植物油 （花生油、 大豆油、 芝麻油等） 能得到粒径较大的微球， 粒径范围在 2(Γ100微米间。 稳定剂的含量选择在 2%左右， 优选卵 磷脂。 微球粒径分布测定：
微球粒径分布用马尔文激光粒度测定仪（Mastersizer 2000, Malvern)进行测定。称取 5mg 微球冻干粉， 加入到 50mL纯化水中， 用漩涡振荡器震荡 5min， 使微球分散均匀， 采用激光 粒度测定仪进行测定。 粒径分布系数通过下式得出：
Figure imgf000013_0001
di为单个微球的粒径， cU为微球平均粒径， N为微球的总数， N>300.
微球形态观察：
微球的形态、 表面特性用 SEM来观察。 用取样棒把微球冻干粉末轻涂于贴在样品台上的 导电胶上。 真空条件下喷金 （120s) 后， 用扫描电子显微镜观察 （S-3700N 日本）。 实施例 4 得到的微球的形态如图 1所示， 微球表面圆整。 微球载药量和包封率的测定
采用文献中报道的 NaOH-SDS法来测定微球的载药量和包封率， 具体操作如下： 准确称量 lOmg微球， 用 lml 0. Imol · L- 1 NaOH (含 5 %SDS) 溶液混悬， 在 lOOrpm, 37°C水浴摇床中震 荡 24h， SOOOrpm离心 10min， 取上清液， 高效液相色谱法测定上清液中的艾塞那肽含量。 微球中所含药物重量
载药量％ = X 100%
微球的总重量
微球的实际载药量
微球的包封率％ = X 100%
微球的理论载药量
艾塞那肽 /利拉鲁肽活性测定：
通过 ELISA方法检测艾塞那肽 /利拉鲁肽的活性， 按照活性 GLP-1 ( 7-36 )特异性酶免试 剂盒 （美国 /EDI ) 说明书方法测定。 艾塞那肽 /利拉鲁肽体外初始释放测定：
将上述实施例制备的艾塞那肽 /利拉鲁肽缓释微球进行体外释放的测定， 测定方法为分 别称取 50mg微球置于 10mL离心管中， 释放介质为 pH7. 4的磷酸缓冲液 （含 0. 02%的叠氮化 钠作为抑菌剂， 0. 05%的土温 80作为润湿剂）， 置于温度 37°C ± 0. 5°C条件下放置 lh， lh后， 取出样品， 接着 5000rpm离心 15min， 取出上清液， HPLC测定上清液中艾塞那肽 /利拉鲁肽浓 度， 计算出释放百分比。 表 1列出了使用不同实施例的多肽药物微球制剂的体外突释及药物 活性保留。 表 2为不同实施例各组分含量。 表 1
粒径分布 体外突 活性保
微球 粒径
释0 /0 留0 /。
实施例 1 5-30 25 2. 05 93. 9 实施例 2 5-40 36 1. 16 95. 6 实施例 3 5-30 26 5. 32 92. 2 实施例 4 30—60 12 1. 36 96. 4 实施例 5 40—100 25 2. 06 92. 6 实施例 6 20—60 18 2. 32 93. 4 实施例 Ί 60-100 30 2. 46 92. 9 实施例 8 30-80 15 1. 25 93. 6 实施例 9 50-100 24 1. 84 92. 2
实施例 10 30-60 27 1. 30 95. 9 实施例 11 30-60 23 1. 56 95. 6 实施例 12 30-80 26 1. 23 96. 2 实施例 13 30-80 25 3. 36 96. 4 实施例 14 30-60 24 2. 06 95. 6 实施例 15 30-60 20 1. 32 93. 4 实施例 16 30-60 23 1. 12 92. 7 从表 1中，本发明制备的缓释微球粒径在 5〜100nm间，体外药物的突释率大多在 5%以下， 粒径分布系数在 1(Γ30间， 药物的活性保留在 92%以上。 油相的粘度对微球粒径影响较大， 用粘度较大的液体石蜡时， 得到的微球粒径较小； 相反的， 采用低粘度油相时 （花生油等植 物油） 可以得到粒径较大微球。
表 2 高分子
多肽药
微球 保护剂％ 聚合物材 水分％
物0 /0
料0 /0 实施例 1 4. 12 2. 51 92. 25 1. 12 实施例 2 4. 58 2. 53 91. 75 1. 14 实施例 3 4. 22 2. 53 1. 13 实施例 4 7. 18 2. 46 89. 76 0. 6 实施例 5 4. 25 2. 65 89. 16 3. 94 实施例 6 7. 32 2. 47 89. 71 0. 5 实施例 Ί 3. 14 2. 86 90. 01 3. 99 实施例 8 6. 54 2. 84 89. 56 1. 06 实施例 9 5. 36 2. 96 89. 56 2. 12 实施例 10 3. 89 3. 33 0. 65 实施例 11 3. 93 2. 75 92. 56 0. 76 实施例 12 6. 34 5. 74 86. 65 1. 27
实施例 13 8. 14 3. 57 86. 87 1. 42 实施例 14 4. 49 1. 06 93. 56 0. 89 实施例 15 9. 18 0. 67 88. 97 1. 18 实施例 16 9. 87 3. 54 85. 49 1. 1 从表 2中看出， 本发明制备的多肽药物缓释微球， 药物含量在 ; Γ10%间， 保护剂的含量 在 0. 5〜6%， 可生物降解高分子聚合物在 85%〜95%间。 艾塞那肽缓释微球体外释放的测定：
将上述实施例 2、 4、 14 制备的艾塞那肽缓释微球进行体外释放的测定， 测定方法为将
50mg微球置于 10mL离心管中， 释放介质为 pH7. 4的磷酸缓冲液（含 0. 02%的叠氮化钠作为抑 菌剂， 0. 05%的土温 80 作为润湿剂）， 置于恒温水浴摇床中， 在震荡速度 lOOrpm, 温度 37 °C ± 0. 5 °C条件下进行微球的体外释放测定。 实施例 2分别在 1、 2、 3、 4、 5、 6、 7天， 实 施例 4、 14分别在第 1、 2、 4、 8、 12、 16、 20、 30天取出 0. 5mL释放介质用高效液相色谱法 测定药物的含量， 并补充新鲜的释放介质。 从图 2、 3中可以看出， 实施例 2、 4、 14的缓释 微球具有很好的缓释效果。通过使用不同分子量、组成的聚乳酸-羟基乙酸共聚物制成的缓释 微球制剂， 释放时间可以从一周至长达一月。 动物实验：
血糖浓度检测
取 SD大鼠 16只， 雌性， 体重 200g左右。 随机分为药物组和空白对照组， 药物组皮下注 射适量实施例 4、 实施例 14制备得到的微球， 空白组皮下注射等量生理盐水。 分别于给药后 的第 1、 5、 10、 15、 20、 30天腹腔注射 18mmol/kg 的葡萄糖， 注射前每鼠先取空白血样， 然后在注射后的 5、 10、 30、 60min 取血， 测定注射前后的血糖浓度。 葡萄糖的测定参照葡 萄糖测定试剂盒（广州阳普医疗科技有限公司）说明书进行。 制作时间和血糖浓度的曲线图。 结果见图 4-9， 从图中可以看出， 注射葡萄糖后， lOmin后药物组血糖浓度 （实施例 4， 实施 例 14 ) 均较对照组显著降低， 说明 30天后血液中还存在药物， 微球具有明显的缓释作用。 体内释放：
SD大鼠随机分为三组， 分别皮下注射实施例 4、 14的适量微球， 分别于给药后的第 1、 2、 3、 4、 6、 8、 10、 12、 15、 18、 22、 26、 30天尾静脉取血， 立刻将血液置于抗凝管中， 15000g 离心 3min， 将血浆转移至干净的离心管中， -80冻存， 以备检测。 血浆中的艾塞那肽含量采 用酶联免疫法检测， 检测方法按照大鼠胰高血糖素样肽 1 (GLP-1 ) ELISA检测试剂盒 （上海 雅吉生物生物科技有限公司） 说明书进行。
实验结果如图 10所示， 由图 10可以看出， 实施例 4， 14制剂在开始有一个较低的突释， 然后在 5-30天内维持一个相对稳定的血药浓度。 以上所述实施例仅表达了本发明的几种实施方式， 其描述较为具体和详细， 但并不能因 此而理解为对本发明专利范围的限制。 应当指出的是， 对于本领域的普通技术人员来说， 在 不脱离本发明构思的前提下， 还可以做出若干变形和改进， 这些都属于本发明的保护范围。 因此， 本发明专利的保护范围应以所附权利要求为准。

Claims

权 利 要 求
1、 一种多肽药物缓释微球制剂， 其特征在于， 所述制剂主要包括可生物降解的高分子聚 合物、 多肽药物和保护剂， 所述可生物降解的高分子聚合物为聚乳酸 -羟基乙酸共聚物、聚乳 酸或其改性修饰物中的一种或几种。
2、 一种权利要求 1所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 包括以下步 骤：
(1) 将聚乳酸-羟基乙酸共聚物或聚乳酸溶于有机溶剂中， 形成浓度为 10(T800mg/mL的 聚乳酸-羟基乙酸共聚物溶液或聚乳酸溶液； 所述聚乳酸-羟基乙酸共聚物中乙交酯： 丙交酯 =25 : 75^90 : 10, 所述聚乳酸-羟基乙酸共聚物的分子量为 200(Γ65000 ; 所述聚乳酸的分子量 为 400(Γ50000; 所述有机溶剂为二氯甲烷、 三氯甲烷、 乙酸乙酯、 丙酮、 乙酸、 乙腈中的一 种或几种；
(2) 分别按 l(T500mg/ml和 l(T200mg/ml的浓度， 将多肽药物和保护剂溶于无菌水中， 得到多肽药物溶液， 所述保护剂为糖、 糖醇类、 蛋白类、 高分子类、 锌盐类稳定剂中的一种 或几种；
(3) 按体积比为 50: 1将步骤（1)的聚乳酸-羟基乙酸共聚物溶液或聚乳酸溶液与步骤
(2)的多肽药物溶液混合， 搅拌至形成均一、 澄清、 透明的混合液；
(4) 配制含 0. 5〜5wt%稳定剂的油相， 所述油相选自大豆油、 花生油、 玉米油、 芝麻油、 矿物油、 二甲基硅油、 棉籽油、 橄榄油、 椰子油、 橘油、 脂肪烃、 环脂烃或芳香烃中的一种 或几种； 所述稳定剂选自卵磷脂、 sPan80、 span60, 单硬脂酸甘油酯、 双硬脂酸聚甘油酯中 的一种或几种；
(5) 按体积比 1 : 2 〜50将步骤 (3)的混合液与步骤 (4)的油相均质形成乳液；
(6)挥发步骤 (5)的乳液除去有机溶剂， 离心洗涤， 冷冻干燥， 即得多肽药物缓释微球制 剂。
3、根据权利要求 2所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 所述多肽药 物为艾塞那肽、 利拉鲁肽、 胰高血糖素样肽、 促黄体激素释放激素、 降钙素、 细胞因子、 肿 瘤坏死因子、 生长激素、 表皮生长因子、 神经生长因子、 干扰素、 生长激素、 酶、 白细胞介 素、 促红细胞生长素、 免疫球蛋白、 抗体、 集落剌激因子、 胰岛素或上述多肽药物的类似物、 衍生物、 修饰物或药学可接受盐。 4、根据权利要求 3所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 所述多肽药 物为艾塞那肽、 利拉鲁肽或其药学上可接受盐。
5、根据权利要求 4所述的多肽药物缓释微球制剂的制备方法，其特征在于，所述步骤（1) 中聚乳酸 -羟基乙酸共聚物或聚乳酸溶液的浓度为 10(T500mg/ml ; 所述步骤 (2)中艾塞那肽或 利拉鲁肽的浓度为 5(T200mg/ml ; 所述保护剂的浓度为 5(Tl00mg/ml。
6、根据权利要求 2-5任一项所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 所 述步骤 (3)中聚乳酸-羟基乙酸共聚物或聚乳酸溶液与多肽药物溶液的体积比为 5〜10： 1。
7、根据权利要求 2-5任一项所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 所 述步骤 (4)中稳定剂的含量为 0. 5%〜3wt%。
8、根据权利要求 2-5任一项所述的多肽药物缓释微球的制备方法， 其特征在于， 所述步 骤 (5)中混合液与油相的体积比为 1 : 5〜10。
9、根据权利要求 2-5任一项所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 所 述保护剂为明胶、 人血白蛋白、 蔗糖、 甘露糖、 醋酸锌、 碳酸锌、 硫酸锌氯化锌。
10、 根据权利要求 2-5任一项所述的多肽药物缓释微球制剂的制备方法， 其特征在于， 所述聚乳酸-羟基乙酸共聚物的分子量为 1000(Γ50000， 所述聚乳酸-羟基乙酸共聚物中乙交 酯： 丙交酯 =50 : 50^75 : 25 , 聚乳酸的分子量为 4000-15000。
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