WO2017107906A1 - 一种艾塞那肽微球制剂及其制备方法 - Google Patents

一种艾塞那肽微球制剂及其制备方法 Download PDF

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WO2017107906A1
WO2017107906A1 PCT/CN2016/111215 CN2016111215W WO2017107906A1 WO 2017107906 A1 WO2017107906 A1 WO 2017107906A1 CN 2016111215 W CN2016111215 W CN 2016111215W WO 2017107906 A1 WO2017107906 A1 WO 2017107906A1
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exenatide
plga
preparation
microspheres
microsphere
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PCT/CN2016/111215
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English (en)
French (fr)
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苏正兴
刘喜明
李明
赵栋
王丹
王利春
王晶翼
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四川科伦药物研究院有限公司
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Priority to CN201680012885.4A priority Critical patent/CN107405307B/zh
Publication of WO2017107906A1 publication Critical patent/WO2017107906A1/zh

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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
    • 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

Definitions

  • the invention relates to an exenatide microsphere preparation and a preparation method thereof, and relates to the field of medicine.
  • Exenatide is a synthetic North American exendin composed of 39 amino acid residues and is a glucagon-1 (GLP-1) receptor agonist.
  • the known pharmacological effects of exenatide include: (1) increasing glucose-dependent insulin secretion; (2) inhibiting glucagon secretion in type 2 diabetic patients; and (3) inhibiting postprandial gastrointestinal motility and secretory function, Delay gastric emptying, which is conducive to postprandial blood glucose control; (4) reduce appetite and reduce food intake; (5) stimulate beta cell apoptosis, thereby increasing the number of beta cells; (6) improve type II diabetes patients Fasting and postprandial blood glucose levels.
  • Exenatide has incomparable advantages over traditional treatments for diabetes.
  • the US Food and Drug Administration (FDA) approved the listing of exenatide in the United States in April 2005. However, due to the short half-life, the product is injected twice a day, and frequent injections make the patient less compliant.
  • Amylin developed the exenatide microsphere preparation Bydureon once a week, but the initial release of the microsphere preparation in the body is obvious, the release lag period is long, C max (higher peak plasma concentration) (refer to patent CN 101065116A), it is easy to cause high blood sugar fluctuations in patients.
  • Amylin developed the exenatide microsphere preparation injected once a month. In the phase II clinical trial, due to fluctuations in peak and valley concentrations, the toxicity was severe, and phase III clinical studies were not available.
  • the object of the present invention is to provide an exenatide microsphere preparation, a preparation method thereof and a use thereof, so as to solve the problem that the existing similar products have obvious burst release, long release lag period and high C max /C ave .
  • the present invention provides an Exenatide microsphere preparation comprising the following weight Percentage of components: 3%-10% exenatide or exenatide salt, 88%-97% PLGA, wherein PLGA is a mixture of low viscosity PLGA and high viscosity PLGA, said low viscosity PLGA and high viscosity
  • the PLGA weight ratio is from 1:1 to 9:1.
  • the weight percentage of the exenatide or exenatide salt is preferably 3% to 8%, further preferably 5% to 8%, more preferably 5%. -6%, most preferably 5.4%, 5.5%, 5.6%, 5.7%, 5.8%.
  • the PLGA weight percentage is preferably from 90% to 96%, further preferably from 92% to 95%, more preferably from 93% to 94%, and most preferably from 93%. , 93.1%, 93.2%, 93.3%, 93.4%, 94%, 94.4%, 94.8%, 95%.
  • exenatide salt may be any pharmaceutically acceptable exenatide salt, including exenatide organic acid salt and exenatide inorganic acid salt, for example, exenatide acetate which is currently medicinal Also includes exenatide hydrochloride, exenatide lactate, exenatide trifluoroacetate, exenatide citrate, exenatide fumarate, esena Peptide malonate, exenatide maleate, exenatide tartrate, exenatide aspartate, exenatide benzoate, exenatide succinate or AI
  • exenatide salt can be used as a preferred exenatide salt starting material for the preparation of exenatide microsphere preparations.
  • the low viscosity PLGA has an intrinsic viscosity of from 0.05 dl/g to 0.25 dl/g.
  • the high viscosity PLGA has an intrinsic viscosity of 0.40 dl/g to 0.75 dl/g.
  • the low viscosity PLGA has a molecular weight of 3000-40000 Daltons.
  • the high viscosity PLGA has a molecular weight of 3,000 to 100,000 Daltons.
  • the intrinsic viscosity refers to the inherent viscosity of PLGA, that is, the ratio of the natural logarithm of the relative viscosity of the PLGA solution to its weight concentration.
  • the low viscosity PLGA is one or both of 5050DLG 1A PLGA and 5050DLG 2A PLGA.
  • the high viscosity PLGA is one or more of 5050DLG 4A PLGA, 5050DLG4.5A PLGA, 5050DLG 7A PLGA.
  • the weight percentage of the high viscosity PLGA is not more than 50%, which can significantly improve the performance of the microsphere preparation of the present invention in the release plate stage.
  • the low viscosity PLGA and high viscosity PLGA weight ratio is from 1.1:1 to 8:1, preferably from 1.2:1 to 6:1, more preferably from 1.5:1 to 4:1.
  • the Exenatide microsphere preparation further comprises 0% to 2% by weight of a protective agent.
  • the weight percentage of the above protective agent is preferably from 0.8% to 1.5%, further preferably from 1% to 1.5%, more preferably from 1% to 1.2%, most preferably from 1% to 1.2%.
  • the Exenatide microsphere formulation comprises the following components by weight: 3%-8% exenatide or exenatide salt, 90% -96% polylactic acid-glycolic acid copolymer (PLGA), 0% to 2% of a protective agent.
  • the Exenatide microsphere formulation comprises the following components by weight: 5%-8% exenatide or exenatide salt, 92% -95% polylactic acid-glycolic acid copolymer (PLGA), 0% to 2% of a protective agent.
  • 5%-8% exenatide or exenatide salt 92% -95% polylactic acid-glycolic acid copolymer (PLGA)
  • PLGA polylactic acid-glycolic acid copolymer
  • the Exenatide microsphere formulation comprises the following components by weight: 5%-6% exenatide or exenatide salt, 93% -94% polylactic acid-glycolic acid copolymer (PLGA), 0% to 2% of a protective agent.
  • 5%-6% exenatide or exenatide salt 93% -94% polylactic acid-glycolic acid copolymer (PLGA)
  • PLGA polylactic acid-glycolic acid copolymer
  • the Exenatide microsphere formulation comprises the following components by weight: 5%-6% exenatide or exenatide salt, 93% - 94% polylactic acid-glycolic acid copolymer (PLGA), 1% - 1.2% of a protective agent.
  • the polylactic acid-glycolic acid copolymer is a mixture of the above low viscosity PLGA and the above high viscosity PLGA, and the low viscosity PLGA and high viscosity PLGA weight ratio is 1. From 1 to 9:1, preferably from 1.1:1 to 8:1, more preferably from 1.2:1 to 6:1, further preferably from 1.5:1 to 4:1.
  • the protective agent is preferably a polyhydroxy sugar, and the polyhydroxy sugar substance easily forms a hydrogen bond with a side chain group such as a hydroxyl group or an amino group in the exenatide molecule, and can improve exenatide in the PLGA microsphere.
  • the conformation is more stable and not easily destroyed, thereby making the exenatide microspheres more stable during long-term storage.
  • the above polyhydroxy sugars are sucrose and/or mannitol.
  • the Exenatide microsphere formulation is comprised of the following weight percent components: 5.5% exenatide, 56% 5050 DLG 2A PLGA, 37.3% 5050DLG 4.5A PLGA, 1.2% sucrose.
  • the exenatide microsphere formulation is loaded in an amount of from 1% to 10%, preferably from 2% to 9.8%, more preferably 2% by weight. -8%.
  • the present invention also provides a preparation method of the exenatide microsphere preparation, wherein the preparation method adopts an O/W emulsification volatilization method, wherein the organic solvent in the oil phase is methanol and dichloromethane. a mixture, or a mixture of dimethyl sulfoxide and dichloromethane, or methanol, A mixture of dimethyl sulfoxide and dichloromethane.
  • O/W emulsion evaporation method is a common preparation method of PLGA microspheres, also known as one-step emulsion solvent evaporation method.
  • the O phase (oil phase) is dispersed in the W phase (aqueous phase) by ultrasonic or mechanical shearing to form an O/W emulsion, and the solvent in the emulsion droplet is slowly extracted and volatilized by the W phase to complete solidification, collection, washing and drying. That is, the final microspheres are obtained.
  • the one-step emulsion solvent evaporation method requires that both the drug and the PLGA can be dissolved in the O phase, and the single solvent as the O phase is generally difficult to achieve the purpose of simultaneously dissolving the drug and PLGA.
  • a preferred method for preparing an Exenatide microsphere formulation comprising the steps of:
  • a weigh exenatide or exenatide salt and PLGA, the two are dissolved in an organic solvent to prepare an oil phase, preferably the stabilizer is dissolved in water to prepare an aqueous phase;
  • the oil phase is injected into the water phase at a constant rate, and sheared at high speed to prepare an O/W emulsion, which is stirred and solidified at 20-30 ° C;
  • the organic solvent is a mixture of methanol and dichloromethane, or a mixture of dimethyl sulfoxide and dichloromethane, or a mixture of methanol, dimethyl sulfoxide and dichloromethane.
  • the main role of methanol is to dissolve exenatide and accelerate the surface curing speed at the initial stage of emulsion droplet formation, which is beneficial to improve the encapsulation efficiency;
  • the role of methylene chloride is to dissolve PLGA and maintain the globing ability of the mixed solvent during emulsification and curing.
  • the main function of dimethyl sulfoxide is to accelerate the initial solidification of the emulsion droplets, so that the surface structure of the microspheres after curing is more compact and the microspheres are released.
  • one of the three mixed solvents is used as the organic solvent in the oil phase, and the inventors have found that such a mixed solvent has a remarkable effect on solving the burst effect of the exenatide microsphere preparation, and can reduce the microsphere preparation.
  • the burst effect it is particularly important that the volume of methylene chloride is more than 50% in the three mixed solvents, so that when the exenatide and the PLGA are dissolved, the solvent is formed by mixing the solvent at the time of curing.
  • the droplets can maintain a good spherical shape, so that the emulsion droplets can complete the curing process in a good spherical shape, and the surface properties of the microspheres formed by the curing are favorable for reducing the burst release and making the dispersion of exenatide in the sphere more uniform.
  • the volume ratio of methanol to dichloromethane is 1/3 to 2/3; in a mixture of dimethyl sulfoxide and dichloromethane, dimethyl sulfoxide and dichloro
  • the volume ratio of methane is 2/3 to 9/11; in a mixture of methanol, dimethyl sulfoxide and dichloromethane, the volume ratio of methanol to dichloromethane is 1/3 to 2/3, wherein dimethyl sulfoxide is methanol. And 0.5% to 15% of the total volume of dichloromethane.
  • the PLGA concentration in the oil phase is 0.04 g/ml to 0.2 g/ml, more preferably 0.054 g/ml, 0.06 g/ml, 0.08 g/ml, or 0.1 g/ml.
  • the aqueous phase contains from 0.1% to 1.5% by weight of stabilizer, further preferably from 0.5%, 0.75%, and 1% by weight.
  • the stabilizer may be one or more of polyvinyl alcohol, sodium carboxymethyl cellulose, trehalose, and Tween.
  • the stabilizer comprises at least polyvinyl alcohol.
  • Polyvinyl alcohol can make oil phase emulsion droplets formed at the beginning of preparation more stable.
  • the volume of the aqueous phase is from 26 to 66 times the volume of the oil phase, further preferably 33 times.
  • the organic solvent described in step a is a mixed solvent of dimethyl sulfoxide and dichloromethane 9:11; the PLGA concentration in the oil phase is 0.054 gml. -1 ; the aqueous phase contains 0.5% by weight of polyvinyl alcohol; the volume of the aqueous phase is 33 times that of the oil phase.
  • the invention also provides the use of the Exenatide microsphere formulation in the manufacture of a medicament for the treatment of Type II diabetes.
  • the inventors have studied and studied the release process of the prior art exenatide microsphere preparation, and found that, generally, in the initial stage of release of the PLGA microsphere, water molecules diffuse to the surface of the microsphere and gradually enter the interior of the microsphere, and the surface of the microsphere is dense.
  • the combination of degree, porosity, physical state of the PLGA molecule, and solubility of the polypeptide results in a difference in the diffusion rate of the polypeptide, which in turn results in different degrees of burst release.
  • the PLGA near the surface of the microspheres will gradually expand, resulting in the closure of the diffusion channels, and the interaction of exenatide with PLGA increases with the entry of water molecules.
  • Exenatide is difficult to diffuse into the medium, and the first stage of drug release lag occurs; after the water molecules continue to diffuse into the microspheres, the PLGA molecular fragment increases activity, and exenatide is released under the guidance of a large amount of free water. The rate is accelerated, and the first phase of rapid release is formed, and the first Cmax is formed. As the concentration of the polypeptide inside the microsphere gradually decreases, and the PLGA does not undergo significant dissolution, the diffusion potential of the polypeptide decreases, resulting in the microsphere entering the second stage.
  • the inventors hope to obtain a formulation with a good release behavior by mixing PLGA materials. Although such a method improves the release lag period of the microsphere preparation, the obtained microsphere release behavior is obtained. Still not ideal.
  • the inventors have found that the preparation of microspheres by O/W emulsification volatilization of the oil phase using a specially selected mixed solvent can obtain microspheres with more uniform distribution of exenatide, thereby significantly reducing Cmax and burst release.
  • the present invention provides an exenatide microsphere preparation in which a mixture of a low viscosity PLGA and a high viscosity PLGA is used as a carrier material to significantly shorten the release lag phase.
  • the invention also provides an improved O/W preparation method, which uses a mixed solvent as an oil phase, dissolves exenatide and a carrier material PLGA, and makes the distribution of exenatide more uniform, and the micro after curing
  • the ball surface structure is more dense, significantly reducing drug burst and C max /C ave .
  • the exenatide microsphere preparation prepared by the invention has small burst release, shortened release lag phase and reduced C max /C ave , which is beneficial to reducing the risk caused by large fluctuation of blood concentration during the treatment of type II diabetes. Has a good clinical application prospects.
  • microsphere preparation of the present invention is usually prepared as a suspension for injection or in the form of a sterile powder.
  • the microsphere preparation of the present invention can be administered by suspension with a solvent.
  • a preferred solvent is an aqueous vehicle containing a pharmaceutical excipient for ensuring isotonicity and improving wettability and sedimentation properties of the microparticles, for example, including Viscosity enhancer (such as sodium carboxymethyl cellulose), wetting agent (such as nonionic surfactants including poloxamer, polysorbate or sorbitan fatty acid ester), osmotic pressure regulator
  • aqueous solvent such as (sorbitol, sodium chloride, mannitol, glucose).
  • the Exenatide microsphere preparation of the present invention is for treating diabetes, including Type I and Type II diabetes; can be used in the treatment of diseases which benefit from an agent which lowers plasma glucose levels; for preventing hyperglycemia; Prevention of high blood pressure; and treatment for diseases that benefit from delaying and/or delaying gastric emptying; can also be used to reduce food intake Inhibition of appetite and treatment of obesity; the exenatide microsphere preparation of the present invention can also be used for the treatment of congestive heart failure.
  • "obesity" is generally defined as having a body mass index of more than 30, for the purposes of the present disclosure, any subject in need or desire to lose weight, including those having a body mass index of less than 30, is included within the scope of "obesity.”
  • exenatide microsphere formulations of the invention can generally be administered by methods generally known in the art. May be administered by injection, implantation (eg subcutaneous, intramuscular, intraperitoneal, intracranial and intradermal), mucosal (eg intranasal, intravaginal, intrapulmonary or by suppository), orally, by needle-free injection or in situ
  • the sustained release microsphere formulation of the present invention is delivered (e.g., by an enema or an aerosol) to a patient (e.g., a human in need of the formulation) or other animal.
  • the microsphere formulation can be administered at any administration time that achieves the desired therapeutic level within the desired time period. For example, a microsphere formulation can be administered and the patient monitored until the level of drug delivered is returned to baseline. After returning to the baseline, the microsphere preparation can be administered again. Alternatively, the microsphere formulation can be administered to a patient at a baseline level prior to reaching the baseline level.
  • the microsphere preparation of the present invention can also be administered together with a corticosteroid.
  • the administration of the microsphere preparation of the present invention together with a corticosteroid further enhances the bioavailability of the biologically active polypeptide in the microsphere preparation.
  • Corticosteroids as defined herein are steroid anti-inflammatory agents, also known as glucocorticoids.
  • patient refers to a human, such as a human in need of the agent or treatment method, prophylactic method or diagnostic method.
  • Figure 1 is a graph showing the in vitro cumulative release rate of Example 1, Comparative Example 1, Comparative Example 3, and marketed Exendin microspheres (Bydureon) at 45 ° C;
  • Figure 2 is a graph showing the blood concentration of the enzymes of Examples 1, 4 and the marketed exenatide microspheres (Bydureon);
  • Figure 3 is a graph showing the in vitro cumulative release rate of Examples 1, 2, 3 and the marketed Exendin microspheres (Bydureon) at 45 ° C;
  • Figure 4 is an electron micrograph of Example 4 and the marketed Exendin microspheres (Bydureon);
  • Figure 5 is a graph showing the in vitro cumulative release rate of Example 4, Comparative Example 2, Comparative Example 4, and marketed Exendin microspheres (Bydureon) at 45 ° C;
  • Figure 6 is a graph showing the in vitro cumulative release rate of Examples 4, 5, 6 and the marketed Exendin microspheres (Bydureon) at 45 ° C;
  • Figure 7 is a graph showing the in vitro cumulative release rate at 45 °C of Example 4 and Comparative Examples 5, 6.
  • Figure 8 is a graph showing the in vitro cumulative release rate at 45 °C for Examples 1, 4 and Comparative Example 7.
  • the raw materials and equipment used in the specific embodiments of the present invention are known products and are obtained by purchasing commercially available products.
  • PLGA is an abbreviation for Poly(lactic-co-glycolic acid), which is a polylactic acid-glycolic acid copolymer.
  • the ring-opening polymer which is a cyclic dimer of lactide and glycolide catalyzed by a nucleophilic initiator is a biodegradable polymer material.
  • the PLGA terminal group may be a hydroxyl group, a carboxyl group, an ester group or the like, and the PLGA in the present invention is preferably a carboxyl group terminal.
  • the intrinsic viscosity can be determined by a conventional method for measuring the effluent time. Unless otherwise stated, the intrinsic viscosity of the present invention is determined by dissolving the PLGA polymer in chloroform at a concentration of 0.5% (w/v) at 30 ° C, according to the United Kingdom. Determination of the method (capillary method) described in the Pharmacopoeia 2013 Edition Appendix VH. Viscosity Measurement Method II.
  • the PLGA nomenclature composition in the examples of the present invention is the polymerization ratio of lactide and glycolide + optical rotation + logarithmic viscosity + polymer end type.
  • the specific meaning of 5050DLG 1A PLGA means that PLGA is a ring-opening polymer catalyzed by a nucleophilic initiator from a cyclic dipolymer of lactide or glycolide.
  • the polymerization ratio is 1:1, which is a foreign product.
  • the PLGA has a logarithmic viscosity of about 1, and the PLGA terminal group is a carboxyl group.
  • the blood samples in vivo were determined by cell method to determine the concentration of exenatide, and the standard curve was fitted by the reaction rate of the cells at different concentrations of exenatide, with a linear range of 0.2 ng to 4 ng.
  • the drug loading method is to first destroy the microspheres with acetic acid and acetonitrile solution, release the exenatide into the solution, and then precipitate the PLGA with water, centrifuge, and determine the concentration of exenatide in the supernatant.
  • Drug loading (%) actually measured exenatide content / microsphere weight * 100%
  • the particle size of the microspheres was measured using a Maserizer 3000 laser particle size analyzer.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the cumulative release profile of the microsphere preparation at 45 ° C is shown in Figure 1, and the plasma concentration curve in the rat is shown in Figure 2.
  • aqueous phase 3.3 g of polyvinyl alcohol (molecular weight 13,000-23000) was dissolved in a 1000 ml beaker containing 660 ml of deionized water to prepare an aqueous phase.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • microspheres After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 3.
  • aqueous phase 3.3 g of polyvinyl alcohol (molecular weight 13,000-23000) was dissolved in a 1000 ml beaker containing 660 ml of deionized water to prepare an aqueous phase.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • microspheres After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 3.
  • aqueous phase 3.3 g of polyvinyl alcohol (molecular weight 13,000-23000) was dissolved in a 1000 ml beaker containing 660 ml of deionized water to prepare an aqueous phase.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • microspheres After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the plasma concentration curve of the microsphere preparation in rats is shown in Fig. 2, the electron microscope image is shown in Fig. 4, and the cumulative release curve at 45 °C is shown in Fig. 5.
  • microspheres After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved through a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray and lyophilized to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 6.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was 10 ml/min with a 30 ml syringe at room temperature of 25 °C.
  • the velocity was injected into the aqueous phase at a constant rate. After the complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 6.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • aqueous phase 3.3 g of polyvinyl alcohol (molecular weight 13,000-23000) was dissolved in a 1000 ml beaker containing 660 ml of deionized water to prepare an aqueous phase.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • microspheres After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • aqueous phase 3.3 g of polyvinyl alcohol (molecular weight 13,000-23000) was dissolved in a 1000 ml beaker containing 660 ml of deionized water to prepare an aqueous phase.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • microspheres After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 1.
  • the oil phase was homogenized at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 °C. Injected into the aqueous phase, after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size. After stirring and solidification at 25 ° C for 3 h, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 5.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 1.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 5.
  • the colostrum was injected into a 3 L 0.5% aqueous solution of polyvinyl alcohol at 1300 rpm and emulsified for 3 min. After stirring and solidification at room temperature for 25 hours at 25 ° C, the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray for lyophilization to obtain a microsphere preparation. . 45 of the microsphere preparation.
  • the cumulative release curve for °C is shown in Figure 7.
  • the cutting head was placed below the liquid level and the shear switch was turned on to adjust the speed to 1300 rpm.
  • the oil phase was uniformly injected into the aqueous phase at a rate of 10 ml/min with a 30 ml syringe at room temperature of 25 ° C, and after complete injection, shearing was maintained for 2 min to form incompletely cured microspheres of appropriate particle size.
  • the microspheres were sieved by a 25-125 ⁇ m metal sieve, and the microspheres were rinsed with 3 volumes of deionized water in water phase, and then transferred to a freeze-dried tray to freeze-dry, to obtain a microsphere preparation.
  • the 45 ° C cumulative release profile of the microsphere formulation is shown in Figure 8.
  • Table 1 summarizes the properties of the Amylin-listed one-week preparation Bydureon and the exenatide microspheres, preparation method, yield, drug loading amount and particle size referred to in the above examples, in order to facilitate an understanding of the beneficial effects of the present invention.
  • the in vivo blood drug concentration data of the microsphere formulations of Examples 1 and 4 and Bydureon are listed below in tabular form, as shown in Table 2. It should be noted that the numerical values of the blood drug concentrations in Table 2 were obtained based on conventional means in the art.
  • the blood concentration of the commercially available Bydureon reached 800 pg/ml on day 0, which was significantly higher than 450 pg/ml of Example 1 and 200 pg/ml of Example 2, therefore, the examples The burst release of the microspheres prepared in Example 1 and Example 4 was significantly lower than that of the commercially available Bydureon, and in addition, the C max /C ave of Example 1 and Example 4 was significantly lower than that of the commercially available Bydureon, particularly the examples.
  • the C max /C ave of 4 is as low as 2.1, which is much smaller than about 3 of Bydureon. ( Cmax refers to the peak plasma concentration and Cave refers to the mean plasma concentration).
  • microspheres are prepared by using the mixed PLGA material in the embodiment of the present invention, and there is no obvious release lag period in the release behavior of the microspheres, and the proper release period can be maintained, about 20 days.
  • the microspheres of Comparative Example 3 had a significant release lag period, and the release was slow in the first 16 days, and the cumulative release was only 14.79%.
  • Example 1, Comparative Example 1, 3 is a microsphere prepared by an O/W method using methanol and dichloromethane as the O phase. Based on this preparation method, Comparative Example 1 prepared microspheres using low-viscosity PLGA. Figure 1 shows that the microspheres obtained in Comparative Example 1 have a fast drug release rate, no drug release lag phase, and 12% release on the first day, and the release cycle is only 6 days; Comparative Example 3 Preparation of microspheres by high viscosity PLGA, the obtained microspheres had obvious release lag period, the release rate was slow in the first 16 days, and the cumulative release was only 14.79%; Example 1 was prepared by using the mixed PLGA material of the present invention.
  • the ball in the microsphere release behavior, has no obvious release lag period, and can maintain an appropriate release cycle, about 20 days.
  • the in vitro cumulative release profile of the microsphere preparation of Example 1, Comparative Example 1, Comparative Example 3 and the marketed exenatide microsphere preparation (Bydureon) at 45 ° C is shown in Figure 1.
  • Example 2 and Example 3 employ a preparation method for introducing dimethyl sulfoxide in the O phase to reduce the burst release of the microsphere preparation.
  • the in vitro release rate measurement showed that the release on day 1 of Example 1 was 12%, which was greater than 1% of the listed exenatide microsphere preparation (Bydureon); and after the introduction of dimethyl sulfoxide in the O phase, and the examples 1 Comparison, the cumulative release of the first day of Example 2 and Example 3 was 5% and 4.5%, respectively, with a significant decrease, no significant release lag phase, and the release period was comparable to that of Bydureon.
  • the in vitro cumulative release profile of the microsphere preparations of Examples 1, 2, and 3 and the Bydureon microspheres at 45 ° C is shown in FIG.
  • Comparative Example 2 is a microsphere prepared by using 5050 2A PLGA (low viscosity PLGA) alone. The main problem is that there is a release platform period of about 3 days, and the drug release is too fast during the rapid release period, resulting in C max /C ave is greater than 3, and the release fluctuation is large.
  • Comparative Example 4 is a microsphere prepared by using 5050 4.5A PLGA (high viscosity PLGA) alone, and the main problem is that there is a significant release plateau period (1-8 days). The release behavior of the microspheres prepared in Comparative Example 1 (5050 2A PLGA) and Comparative Example 2 (5050 4.5A PLGA) at 45 ° C is shown in Figure 5.
  • the preferred prescription example 4 was obtained , and the cumulative release curve of the microspheres at 45 ° C is shown in Fig. 5, and the blood concentration curve in the body is shown in Fig. 2.
  • the burst release is low, from 0.8 ng/ml to 0.2 ng/ml; the release lag period is short, from about 2 weeks to about Within 1 week; C max /C ave is low, decreasing from about 3 to about 2 (C max refers to peak blood concentration, and Cave refers to mean blood concentration).
  • FIG. 4 is an electron micrograph of the microsphere preparation of Example 4 and the marketed exenatide microsphere (Bydureon).
  • Figure 4 shows that the surface of the microsphere prepared in Example 4 of the present invention is smoother and denser than Bydureon, and the risk of occurrence of burst release can be reduced.
  • the optimal formulation of the present invention uses 5050 2A PLGA (low viscosity PLGA) and 5050 4.5A PLGA (high viscosity PLGA) to prepare microspheres in a specific ratio of mixed materials, and the obtained microspheres have better surface morphology, and Compared with Bydureon, it can significantly reduce burst release, shorten drug release lag phase, and reduce C max /C ave .
  • Example 5 and Example 6 are prescriptions in which the theoretical drug loading is approximately 3% and 10%. Compared with Example 4, the drug loading increased from 3% to 10% has little effect on the release behavior of the microspheres. See Figure 6. The drug loading increased, and the release rate of the microspheres increased slightly, but did not affect the overall drug release trend.
  • Comparative Example 5 Comparative Example 6 Microspheres were prepared using S/O/O and W/O/W, respectively.
  • Figure 7 compares the difference between the two preparation methods and the release behavior of the microspheres of Example 4 of the present invention. The results showed that the main problem with S/O/O and W/O/W was the large burst release, which was as high as 63.77% and 16.8% on the first day.
  • Comparative Example 7 The O/W of the present invention was used to prepare microspheres, but the mixing ratio of the low viscosity PLGA to the high viscosity PLGA was 1:3, which was not in the range of 1:1 to 9:1 of the present invention, as can be seen from FIG.
  • the high-viscosity PLGA content is higher than 50%, it has a significant release platform period, and only 10.36% is released in the previous week.

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Abstract

一种艾塞那肽微球制剂，包含下述重量百分比的组分：3％-10％艾塞那肽或艾塞那肽盐，88％-97％PLGA，其中，PLGA为低粘度PLGA和高粘度PLGA的混合物，所述低粘度PLGA和高粘度PLGA重量比为1∶1～9∶1。所述制剂采用O/W乳化挥发法制备。所得艾塞那肽微球制剂突释小、释药迟滞期短、C max/C ave低，有利于降低II型糖尿病接受治疗时由于血药浓度大幅度波动带来的风险，具有较好的临床应用前景。

Description

一种艾塞那肽微球制剂及其制备方法 技术领域
本发明涉及一种艾塞那肽微球制剂及其制备方法，涉及医药领域。
背景技术
艾塞那肽是人工合成的北美毒晰外泌肽，由39个氨基酸残基组成，为胰高血糖素-1(GLP-1)受体激动剂。艾塞那肽已知的药理作用包括：(1)增加葡萄糖依赖性促胰岛素分泌；(2)抑制II型糖尿病患者胰高血糖素的分泌；(3)抑制餐后胃肠动力及分泌功能，延迟胃排空，从而有利于餐后血糖的控制；(4)降低食欲，减少食物的摄入；(5)刺激β细胞凋亡，从而增加β细胞的数量；(6)改善II型糖尿病患者的空腹及餐后血糖水平。与传统的治疗糖尿病药物相比，艾塞那肽具有不可比拟的优点。美国食品药品监督管理局(FDA)于2005年4月批准了艾塞那肽在美国上市。然而由于半衰期短，该产品为每日2次注射剂，频繁的注射使患者顺应性较差。
为降低给药频率、提高患者顺应性，Amylin公司开发了一周注射一次的艾塞那肽微球制剂Bydureon，但该微球制剂在体内存在的初期突释明显、释药迟滞期长、Cmax(血药浓度峰值)偏高等问题(参考专利CN 101065116A)，易造成病人血糖波动大。Amylin公司开发一月注射一次的艾塞那肽微球制剂，在II期临床试验中因峰谷浓度的波动，导致毒副反应严重，暂未能开展III期临床研究。
因此，目前亟需开发一种新的艾塞那肽微球制剂和制备工艺，在达到长效缓释目的的同时，解决同类产品存在的突释明显、释药迟滞期长、Cmax/Cave过高的问题，以期得到释药行为更好的艾塞那肽微球制剂，降低由于血药浓度大幅度波动带来的治疗风险。
发明内容
本发明的目的在于提供一种艾塞那肽微球制剂及其制备方法和用途，以解决现有同类产品存在的突释明显、释药迟滞期长、Cmax/Cave过高的问题。
第一方面，本发明提供了一种艾塞那肽微球制剂，包含下述重量 百分比的组分：3％-10％艾塞那肽或艾塞那肽盐，88％-97％PLGA，其中，PLGA为低粘度PLGA和高粘度PLGA的混合物，所述低粘度PLGA和高粘度PLGA重量比为1∶1至9∶1。
其中，在上述艾塞那肽微球制剂中，所述的艾塞那肽或艾塞那肽盐重量百分比优选为3％-8％，进一步优选为5％-8％，更优选为5％-6％，最优选为5.4％、5.5％、5.6％、5.7％、5.8％。
其中，在上述艾塞那肽微球制剂中，所述的PLGA重量百分比优选为90％-96％，进一步优选为92％-95％，更优选为93％-94％，最优选为93％、93.1％、93.2％、93.3％、93.4％、94％、94.4％、94.8％、95％。
上述艾塞那肽盐可以为任何可以药用的艾塞那肽盐，包括艾塞那肽有机酸盐和艾塞那肽无机酸盐，例如目前已经药用的为艾塞那肽醋酸盐，还包括艾塞那肽盐酸盐、艾塞那肽乳酸盐、艾塞那肽三氟乙酸盐、艾塞那肽枸橼酸盐、艾塞那肽富马酸盐、艾塞那肽丙二酸盐、艾塞那肽马来酸盐、艾塞那肽酒石酸盐、艾塞那肽门冬氨酸盐、艾塞那肽苯甲酸盐、艾塞那肽琥珀酸盐或艾塞那肽扑酸盐，可将上述艾塞那肽盐作为优选的艾塞那肽盐原料用于制备艾塞那肽微球制剂。
进一步的，所述低粘度PLGA的特性粘度为0.05dl/g至0.25dl/g。
进一步的，所述高粘度PLGA的特性粘度为0.40dl/g至0.75dl/g。
进一步的，所述低粘度PLGA的分子量为3000-40000道尔顿。
进一步的，所述高粘度PLGA的分子量为30000-100000道尔顿。
所述的特性粘度是指PLGA的比浓对数粘度，即PLGA溶液相对黏度的自然对数与其重量浓度之比。
优选的，所述的低粘度PLGA为5050DLG 1A PLGA、5050DLG 2A PLGA中的一种或两种。
优选的，所述的高粘度PLGA为5050DLG 4A PLGA、5050DLG4.5A PLGA、5050DLG 7A PLGA中的一种或多种。
在本发明所述的PLGA混合物中，高粘度PLGA的重量百分比不大于50％，可以显著改进本发明微球制剂在释药平台期的表现。
更进一步的，所述低粘度PLGA和高粘度PLGA重量比为1.1∶1至8∶1、优选为1.2∶1至6∶1、更优选为1.5∶1至4∶1。
其中，所述的艾塞那肽微球制剂还包含重量百分比为0％-2％的保护剂。
其中，上述保护剂的重量百分比优选为0.8％-1.5％，进一步优选为1％-1.5％，更优选为1％-1.2％，最优选为1％、1.2％。
在一个优选的艾塞那肽微球制剂中，所述的艾塞那肽微球制剂包含下述重量百分比的组分：3％-8％艾塞那肽或艾塞那肽盐、90％-96％聚乳酸-羟基乙酸共聚物(PLGA)、0％-2％的保护剂。
在一个优选的艾塞那肽微球制剂中，所述的艾塞那肽微球制剂包含下述重量百分比的组分：5％-8％艾塞那肽或艾塞那肽盐、92％-95％聚乳酸-羟基乙酸共聚物(PLGA)、0％-2％的保护剂。
在一个优选的艾塞那肽微球制剂中，所述的艾塞那肽微球制剂包含下述重量百分比的组分：5％-6％艾塞那肽或艾塞那肽盐、93％-94％聚乳酸-羟基乙酸共聚物(PLGA)、0％-2％的保护剂。
在一个优选的艾塞那肽微球制剂中，所述的艾塞那肽微球制剂包含下述重量百分比的组分：5％-6％艾塞那肽或艾塞那肽盐、93％-94％聚乳酸-羟基乙酸共聚物(PLGA)、1％-1.2％的保护剂。
上述优选的任一艾塞那肽微球制剂中，聚乳酸-羟基乙酸共聚物(PLGA)为上述低粘度PLGA和上述高粘度PLGA的混合物，所述低粘度PLGA和高粘度PLGA重量比为1∶1至9∶1，优选为1.1∶1至8∶1、更优选为1.2∶1至6∶1、进一步优选为1.5∶1至4∶1。
进一步的，所述保护剂优选为多羟基糖类，多羟基糖类物质易与艾塞那肽分子中的羟基、氨基等侧链基团形成氢键，可以提高艾塞那肽在PLGA微球中的胶化程度，构象更稳定不易被破坏，进而使艾塞那肽微球在长期保存过程中更稳定。
进一步优选的，上述多羟基糖类为蔗糖和/或甘露醇。
在一个更优选的艾塞那肽微球制剂中，所述的艾塞那肽微球制剂是由下述重量百分比的组分组成：5.5％艾塞那肽、56％5050DLG 2A PLGA、37.3％5050DLG 4.5A PLGA、1.2％蔗糖。
经检测，该更优选的艾塞那肽微球制剂的Cmax/Cave在2.5以内。
在一个优选的艾塞那肽微球制剂中，所述的艾塞那肽微球制剂的载药量按重量百分比计为1％-10％，优选为2％-9.8％，更优选2％-8％。
第二方面，本发明还提供了一种所述艾塞那肽微球制剂的制备方法，所述制备方法采用O/W乳化挥发法，制备方法中油相中的有机溶剂为甲醇和二氯甲烷混合物，或二甲亚砜和二氯甲烷混合物，或甲醇、 二甲亚砜和二氯甲烷混合物。
在微球制剂的制备领域中，O/W乳化挥发法是一种常见的PLGA微球制备方法，又称一步乳化溶剂挥发法。通过超声或机械剪切将O相(油相)分散在W相(水相)中，形成O/W型乳剂，乳滴中的溶剂再被W相缓慢萃取挥发完成固化，收集、清洗和干燥即获得最终的微球。
一步乳化溶剂挥发法要求药物和PLGA均能溶解在O相中，而单一溶剂作为O相通常难以达到同时溶解药物和PLGA的目的。
一种优选的艾塞那肽微球制剂的制备方法，它包括如下步骤：
a、按比例称取艾塞那肽或艾塞那肽盐和PLGA，将两者溶解于有机溶剂中制备成油相，优选地将稳定剂溶解于水中制备成水相；
b、在20-30℃下，将油相匀速注入到水相中，并高速剪切，制备成O/W乳液，20-30℃条件下搅拌固化；
c、用水冲洗微球，过筛收集，干燥，例如冻干；
所述有机溶剂为甲醇和二氯甲烷混合物，或二甲亚砜和二氯甲烷混合物，或甲醇、二甲亚砜和二氯甲烷混合物。
其中，甲醇的主要作用是溶解艾塞那肽和加快乳滴形成初期的表面固化速度，有益于提高包封率；二氯甲烷的作用是溶解PLGA和维持混合溶剂乳化和固化时的成球能力；二甲亚砜主要作用是加快乳滴形成初期的固化，使固化后的微球表面结构更致密，降低微球的突释。
上述制备方法中采用三种混合溶剂中一种作为油相中有机溶剂，发明人发现，这样的混合溶剂对于解决艾塞那肽微球制剂的突释效应效果显著，能减小微球制剂的突释效应，特别重要的是，在三种混合溶剂中，二氯甲烷的体积量都在50％以上，以使得在溶解艾塞那肽和PLGA的同时，在固化时，混合溶剂形成的乳滴可以维持良好的球形，从而能保证乳滴以良好的球形完成固化过程，固化形成的微球表面性质有利于降低突释，且能使艾塞那肽在球内的分散更加均匀。
作为一种优选，在甲醇和二氯甲烷混合物中，甲醇与二氯甲烷的体积比为1/3至2/3；在二甲亚砜和二氯甲烷混合物中，二甲亚砜与二氯甲烷体积比为2/3至9/11；在甲醇、二甲亚砜和二氯甲烷混合物中，甲醇与二氯甲烷的体积比为1/3至2/3，其中二甲亚砜是甲醇和二氯甲烷总体积的0.5％至15％。
优选的，所述的油相中PLGA浓度是0.04g/ml-0.2g/ml，进一步优选为0.054g/ml、0.06g/ml、0.08g/ml、0.1g/ml。
优选的，所述的水相中含重量百分比为0.1％-1.5％的稳定剂，进一步优选为重量百分比0.5％、0.75％、1％。
优选的，所述的稳定剂可以为聚乙烯醇、羧甲基纤维素钠、海藻糖、吐温中的一种或多种。
进一步优选的，所述稳定剂至少包括聚乙烯醇。聚乙烯醇能使制备初期形成的油相乳滴更加稳定。
优选的，水相体积是油相体积的26倍至66倍，进一步优选为33倍。
在一个更优选的艾塞那肽微球制剂制备方法中，a步骤所述的有机溶剂为体积比为二甲亚砜和二氯甲烷9∶11的混合溶剂；油相中PLGA浓度是0.054gml-1；水相中含重量百分比为0.5％的聚乙烯醇；水相体积是油相的33倍。
本发明还提供了所述艾塞那肽微球制剂在制备用于治疗II型糖尿病的药物中的用途。
发明人对现有技术的艾塞那肽微球制剂的释放过程进行研究分析发现，通常，在PLGA微球释药初期，水分子扩散到达微球表面并逐渐进入微球内部，微球表面致密程度、孔隙度、PLGA分子的物理状态和多肽溶解度等众多因素的共同作用导致多肽的扩散速率差异，进而形成不同程度的突释。一般情况下，在水分子进入微球内部的同时，微球近表面的PLGA会逐渐发生膨胀，导致扩散孔道的关闭，且随着水分子的进入，艾塞那肽与PLGA的相互作用增强，艾塞那肽难扩散进入介质中，出现第一阶段的释药迟滞期；水分子继续扩散进入微球后，PLGA分子片段活动性增加，艾塞那肽在大量自由水的介导下，释放速率加快，进入第一阶段的快速释药期，形成第一个Cmax；随着微球内部多肽浓度逐渐降低，且PLGA未发生明显溶蚀，多肽扩散势能的降低，导致微球进入第二阶段的释药迟滞期，在此之后，PLGA开始逐渐降解，溶蚀作用明显，扩散孔道增加，微球内部局部结构坍塌，微球进入第二阶段的快速释药期，形成第二个Cmax。因此，现有技术的艾塞那肽PLGA微球在注射后通常容易出现突释大、释药迟滞期长和Cmax(血药浓度峰值)过高等问题，导致艾塞那肽血药浓度出现较 大的波动。当血药浓度不在治疗窗内时，PLGA缓释微球易出现疗效不理想或毒副作用明显的风险。
在开发制备方法的过程中，发明人通过混合PLGA材料的方式，期望获得释药行为良好的制剂处方，这样的方式虽然对于微球制剂的释药迟滞期有改善，但是得到的微球释放行为仍不理想。
发明人发现，采用特定选择的混合溶剂为油相的O/W乳化挥发法制备微球，可以获得艾塞那肽分布更为均匀的微球，从而显著降低Cmax和突释。
在此基础上，通过特定比例混合PLGA材料，可以有效减少或者消除艾塞那肽微球制剂的释药迟滞期，从而降低在连续多次给药时由于释药模式缺陷导致的血药浓度波动带来的毒副作用。
综上所述，本发明提供了一种艾塞那肽微球制剂，在处方中采用低粘度PLGA和高粘度PLGA的混合物作为载体材料，使释药迟滞期明显缩短。本发明还提供了一种改进的O/W制备方法，使用混合溶剂为油相，在溶解艾塞那肽和载体材料PLGA的同时，使艾塞那肽分布更为均匀，且固化后的微球表面结构更致密，显著降低了药物突释和Cmax/Cave。
本发明制备得到的艾塞那肽微球制剂突释小、释药迟滞期缩短、Cmax/Cave降低，有利于降低II型糖尿病接受治疗时由于血药浓度大幅度波动带来的风险，具有较好的临床应用前景。
本发明所述的微球制剂，通常制备成注射用的混悬剂，或以无菌粉末的形式存在。
本发明所述的微球制剂，可以采用溶媒混悬后给药，优选的溶媒是含有为了确保等张性和改善微粒的润湿性和沉降性质的药物赋形剂的水性溶媒，例如包括含有粘度增强剂(如羧甲基纤维素钠)、润湿剂(如包括泊洛沙姆、聚山梨酯或失水山梨酯脂肪酸酯等在内的非离子表面活性剂)、渗透压调节剂(山梨醇、氯化钠、甘露醇、葡萄糖)等的水性溶媒。
本发明的艾塞那肽微球制剂用于治疗糖尿病，包括I型和II型糖尿病；可以用于通过降低血浆葡萄糖水平的药剂而获益的疾病的治疗中；用于预防高血糖；用于预防高血压；和用于治疗会通过给予延迟和/或延缓胃排空的药剂而获益的疾病的治疗；还可以用于减少食物摄 入、抑制食欲并治疗肥胖症；本发明的艾塞那肽微球制剂还可以用于治疗充血性心力衰竭。尽管“肥胖症”通常被定义为体重指数超过30，出于本发明公开的目的，需要或希望减轻体重的任何对象，包括体重指数小于30的那些，都被包括在“肥胖”的范围内。
本发明艾塞那肽微球制剂通常可按照本领域通常所知方法给药。可通过注射、植入(例如皮下、肌内、腹膜内、颅内和皮内)、粘膜给药(例如鼻内、阴道内、肺内或借助栓剂)、口服、通过无针注射或原位递送(例如通过灌肠剂或气雾剂)将本发明的缓释微球制剂给予患者(例如需要该制剂的人类)或其它动物。
可用在所需时间内达到所需治疗水平的任何给药时间给予该微球制剂。举例而言，可给予微球制剂，监测患者直到所递送的药物水平回到基线。回到基线后，可再次给予微球制剂。或者，可在患者体内达到基线水平前序惯给予微球制剂。
本发明的微球制剂还可与皮质类固醇一起给予。本发明的微球制剂与皮质类固醇一起给予可进一步提高微球制剂中生物活性多肽的生物利用度。本发明所定义的皮质类固醇指类固醇类抗炎性剂，也称为糖皮质激素。
本发明中所用术语患者是指人类，例如需要该药剂或治疗方法、预防方法或诊断方法的人类。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实施例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为实施例1、对比例1、对比例3和上市的艾塞那肽微球(Bydureon)45℃体外累积释放率曲线图；
图2为实施例1、4和上市的艾塞那肽微球(Bydureon)大鼠体内血药浓度曲线图；
图3为实施例1、2、3和上市的艾塞那肽微球(Bydureon)45℃体外累积释放率曲线图；
图4为实施例4和上市的艾塞那肽微球(Bydureon)电镜图；
图5为实施例4、对比例2、对比例4和上市的艾塞那肽微球(Bydureon)45℃体外累积释放率曲线图；
图6为实施例4、5、6和上市的艾塞那肽微球(Bydureon)45℃体外累积释放率曲线图；
图7为实施例4和对比例5、6的45℃体外累积释放率曲线图。
图8为实施例1、4和对比例7的45℃体外累积释放率曲线图。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品，通过购买市售产品获得。
PLGA是Poly(lactic-co-glycolic acid)的缩写，即聚乳酸-羟基乙酸共聚物，
为丙交酯、乙交酯的环状二聚合物在亲核引发剂催化作用下的开环聚合物，是一种生物可降解高分子材料。PLGA末端基团可以是羟基、羧基、酯基等，在本发明中的PLGA优选羧基基团末端。
特性粘度可以通过测定流出时间的常规方法测定，除非另有说明，本发明的特性粘度测定是将PLGA高分子以0.5％(W/V)的浓度溶于氯仿中，于30℃下，按照英国药典2013版Appendix VH.粘度测定方法II所述方法(毛细管法)测定。
本发明实施例中的PLGA命名组成是丙交酯和乙交酯聚合比例+旋光性+比浓对数粘度+高分子末端类型。例如，5050DLG 1A PLGA的具体含义，是指PLGA是由丙交酯、乙交酯的环状二聚合物在亲核引发剂催化作用下的开环聚合物，聚合比例1∶1，是外消旋物质，PLGA比浓对数粘度在1左右，PLGA末端基团是羧基。
方法：
体内血药浓度测定：
体内血样采用细胞法测定艾塞那肽浓度，通过不同艾塞那肽浓度下细胞对应的反应率拟合标准曲线，线性范围0.2ng-4ng。
45℃体外释放率测定或计算：
45℃体外评价在pH9.4的tris缓冲液中进行。具体方法为在50mlTris缓冲盐中，加入50mg微球，静置，在不同时间点采样，采样方法为每次取出1.5ml Tris缓冲盐，过滤留样，并在释放瓶中补充新鲜的Tris缓冲盐1.5ml。HPLC测量取样的艾塞那肽浓度，并折算释放至缓冲液中的多肽量，累积释放率计算方法为已释放出的艾塞那肽量/称量的50mg微球中艾塞那肽总量。
收率计算：
收率(％)＝实际收集微球重量/(艾塞那肽或其盐投料量+PLGA投料量)*100％
载药量测定或计算：
载药量测定方法是先用乙酸和乙腈溶液破坏微球，使艾塞那肽释放至溶液中，再用水将PLGA析出，离心，测定上清液中的艾塞那肽浓度。
载药量(％)＝实际测得的艾塞那肽含量/微球重量*100％
粒径测定或计算：
采用Maserizer 3000激光粒度仪测得微球粒径。
实施例1  本发明微球制剂的制备I
采用甲醇和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 1A PLGA(特性粘度0.14dL/g、分子量12000道尔顿)、0.72g 5050 7A PLGA(特性粘度0.65dL/g、分子量102000道尔顿)、107mg艾塞那肽于25ml西林瓶中，用13ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂45℃累积释放曲线见图1，大鼠体内血药浓度曲线见图2。
实施例2  本发明微球制剂的制备II
采用甲醇、二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 1A PLGA(特性粘度0.14dL/g、分子量12000道尔顿)、0.72g 5050 7A PLGA(特性粘度0.65dL/g、分子量102000道尔顿)、107mg艾塞那肽和20mg蔗糖于25ml西林瓶中，用13ml甲醇、二甲亚砜和二氯甲烷(4∶3∶6，v/v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图3。
实施例3  本发明微球制剂的制备III
采用二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 1A PLGA(特性粘度0.14dL/g、分子量12000道尔顿)、0.72g 5050 7A PLGA(特性粘度0.65dL/g、分子量102000道尔顿)、111mg艾塞那肽和23mg蔗糖于25ml西林瓶中，用20ml二甲亚砜和二氯甲烷(9∶11，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图3。
实施例4  本发明微球制剂的制备IV
采用二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.72g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道 尔顿)、107mg艾塞那肽和23mg蔗糖于25ml西林瓶中，用20ml二甲亚砜和二氯甲烷(9∶11，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的大鼠体内血药浓度曲线见图2，电镜图片见图4，45℃累积释放曲线见图5。
实施例5  本发明微球制剂的制备V
采用二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.11g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.74g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)、60mg艾塞那肽和38mg蔗糖于25ml西林瓶中，用20ml二甲亚砜和二氯甲烷(9∶11，v/v)混合溶剂溶解，制备成油相。将9.9g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干得到微球制剂。该微球制剂的45℃累积释放曲线见图6。
实施例6  本发明微球制剂的制备VI
采用二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.03g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.68g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)、192mg艾塞那肽和23mg蔗糖，用40ml二甲亚砜和二氯甲烷(9∶11，v/v)混合溶剂溶解，制备成油相。将9.9g聚乙烯醇(分子量13000-23000)溶解于含1320ml去离子水的2000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min 的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图6。
实施例7
采用甲醇和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 1A PLGA(特性粘度0.14dL/g、分子量12000道尔顿)、0.72g 5050 7A PLGA(特性粘度0.65dL/g、分子量102000道尔顿)、117mg醋酸艾塞那肽于25ml西林瓶中，用13ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。
实施例8
采用二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.72g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)、117mg醋酸艾塞那肽和23mg蔗糖于25ml西林瓶中，用20ml二甲亚砜和二氯甲烷(9∶11，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。
实施例9
采用二甲亚砜和二氯甲烷为O相的O/W法制备混合PLGA微球
称量1.08g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.72g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)、120mg酒石酸艾塞那肽和23mg蔗糖于25ml西林瓶中，用20ml二甲亚砜和二氯甲烷(9∶11，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。
以下通过对比实验证明本发明的有益效果。
对比例1  采用甲醇和二氯甲烷为O相的O/W法制备低粘度PLGA微球I
称量1.8g 5050 1A PLGA(特性粘度0.14dL/g、分子量12000道尔顿)、112mg艾塞那肽于30ml西林瓶中，用13ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图1。
对比例2  采用甲醇和二氯甲烷为O相的O/W法制备低粘度PLGA微球II
称量1.8g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、110mg艾塞那肽于25ml西林瓶中，用15ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速 注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图5。
对比例3  采用甲醇和二氯甲烷为O相的O/W法制备高粘度PLGA微球I
称量1.8g 5050 7A PLGA(特性粘度0.65dL/g、分子量102000道尔顿)、112mg艾塞那肽于25ml西林瓶中，用20ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图1。
对比例4  采用甲醇和二氯甲烷为O相的O/W法制备高粘度PLGA微球II
称量1.8g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)、120mg艾塞那肽于25ml西林瓶中，用13ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图5。
对比例5  采用S/O/O法制备混合PLGA微球
称量1.08g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.72g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)，并用10ml二氯甲烷溶解，得到PLGA溶液；将107mg艾塞 那肽和23mg蔗糖溶解于5ml冰醋酸中，得到艾塞那肽溶液；将艾塞那肽溶液滴加至PLGA溶液中搅拌，形成S/O混悬液。在16000rpm剪切的条件下，加入9ml硅油，然后转移至0℃的硅油乙醇溶液中，固化1.5h后，正庚烷清洗3次，浸泡1h。真空干燥条件下，25℃维持24h，34℃维持24h，38℃维持24h，过109μm筛，收集干燥后微球。该微球的45℃累积释放曲线见图7。
对比例6  采用W/O/W制备混合PLGA微球
称量107mg艾塞那肽和23mg蔗糖溶解于1ml去离子水中制备成水相，称量1.08g 5050 2A PLGA(特性粘度0.20dL/g、分子量18000道尔顿)、0.72g 5050 4.5A PLGA(特性粘度0.43dL/g、分子量64000道尔顿)，用30ml二氯甲烷溶解，制备成油相。在12000rpm条件下，将水相滴加入油相中制备成初乳。在1300rpm条件下，将初乳注入到3L 0.5％聚乙烯醇水溶液中，乳化3min。在25℃的室温条件下搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45。℃累积释放曲线见图7。
对比例7  采用甲醇和二氯甲烷为O相的O/W法制备低粘度PLGA和高粘度PLGA混合微球I
称量0.45g 5050 1A PLGA(特性粘度0.14dL/g、分子量12000道尔顿)和1.35g 50507A PLGA(特性粘度0.65dL/g、分子量102000道尔顿)、112mg艾塞那肽于25ml西林瓶中，用20ml甲醇和二氯甲烷(2∶3，v/v)混合溶剂溶解，制备成油相。将3.3g聚乙烯醇(分子量13000-23000)溶解于含660ml去离子水的1000ml烧杯中，制备成水相。将剪切头至于水相液面以下，并打开剪切开关，调节转速为1300rpm。在25℃的室温条件下，用30ml注射器将油相以10ml/min的速度匀速注入到水相中，完全注入后，维持剪切2min，形成适当粒径的未完全固化微球。在25℃搅拌固化3h后，25-125μm金属筛筛分微球，并用水相3倍体积的去离子水冲洗微球，然后转移至冻干盘中冻干，得到微球制剂。该微球制剂的45℃累积释放曲线见图8。
表1汇总了Amylin公司上市的1周制剂Bydureon和上述实施例涉及的艾塞那肽微球、制备方法、收率、载药量和粒径等特性，以便于对本发明有益效果的理解。
表1艾塞那肽微球特性
Figure PCTCN2016111215-appb-000001
为了便于比较各实施例和对比例微球的释药行为，以下以表格的形式列出实施例1、4的微球制剂和Bydureon的体内血药浓度数据，如表2所示。应予说明，表2中的血药浓度的数值基于本领域的常规手段而获得。
表2  实施例1、4的微球制剂和上市的Bydureon在大鼠体内血药浓度(pg/ml)
Figure PCTCN2016111215-appb-000002
从表2和图2可以看出，第0天时，市售的Bydureon体内血药浓度达到800pg/ml，显著高于实施例1的450pg/ml和实施例2的200pg/ml，因此，实施例1和实施例4制备的微球的突释显著低于市售的Bydureon，此外，与市售的Bydureon相比，实施例1和实施例4的Cmax/Cave明显降低，特别是实施例4的Cmax/Cave低至2.1，远小于Bydureon的3左右。(Cmax指峰值血药浓度，Cave指平均血药浓度)。
为了便于比较各实施例和对比例微球的释药行为，以下以表格的形式列出各实施例微球的45℃体外释放率数据，如表3-表11所示。
表3  本发明实施例1～8微球的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000003
从表3可以看出，本发明实施例采用混合PLGA材料制备微球，在微球释药行为上，没有明显的释药迟滞期，又能维持适当的释药周期，约20天左右。
表4  上市艾塞那肽微球制剂Bydureon的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000004
从表4可以看出，市售Bydureon的释药迟滞期比较长，并且表2表明Bydureon的Cmax/Cave较高，血药浓度波动较大。
表5  对比例1微球的45℃体外释放率(％)
时间(天) 0 1 2 3 4 7
对比例1 0 12.09 22.46 58.19 86.6 103.45
从表5可以看出，对比例1的微球释药速度过快，第1天释放12％，突释明显，释药周期短，仅6天。
表6  对比例2微球的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000005
从表6可以看出，对比例2的微球具有3天左右的释药平台期，而在快速释药期内药物又释放太快，导致Cmax/Cave大于3，释药波动较大。
表7  对比例3微球的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000006
从表7可以看出，对比例3的微球具有明显的释药迟滞期，前16天释药缓慢，累积释放仅14.79％。
表8  对比例4微球的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000007
从表8可以看出，对比例4的微球具有明显的释药平台期(1-8天)。
表9  对比例5微球的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000008
从表9可以看出，对比例5的微球突释较大，第1天释放高达63.77％。
表10  对比例6微球的45℃体外释放率(％)
时间(天) 1 2 3 5 6 8 11
对比例6 16.8 17.9 18.8 24.35 30.17 35 48
时间(天) 13 15 17 18 19 20 23
对比例6 64 76 80 88 96 98.15 98.23
从表10可以看出，对比例6的微球突释较大，第1天释放高达16.8％。
表11  对比例7微球的45℃体外释放率(％)
Figure PCTCN2016111215-appb-000009
从表11可以看出，对比例7的微球存在明显释药平台期，前1周释放仅10.36％。
其中，表中的“-”代表未取样
实验结果分析：
1、实施例1、对比例1、3是采用甲醇和二氯甲烷为O相的O/W法制备微球。在此制备方法基础上，对比例1采用低粘度PLGA制备微球，图1显示：对比例1所得微球释药速度快，无释药迟滞期，第1天释放12％，释药周期仅6天；对比例3采用高粘度PLGA制备微球，所得微球有明显的释药迟滞期，前16天释药缓慢，累积释放仅14.79％；实施例1是采用本发明混合PLGA材料制备微球，在微球释药行为上，没有明显的释药迟滞期，又能维持适当的释药周期，约20天左右。实施例1、对比例1、对比例3的微球制剂和上市的艾塞那肽微球制剂(Bydureon)的45℃体外累积释放曲线见图1。
以上实验结果表明：使用本发明混合PLGA材料制备的微球，表 现出理想的释药行为，其他不管是单独使用低粘度PLGA还是单独使用高粘度PLGA，均不能得到释药行为良好的微球。
2、实施例2和实施例3采用在O相中引入二甲亚砜的制备方法，来降低微球制剂的突释。体外释放率测定结果表明：实施例1的第1天释放为12％，大于上市艾塞那肽微球制剂(Bydureon)的1％；而在O相中引入二甲亚砜后，与实施例1比较，实施例2和实施例3第1天累积释药分别为5％和4.5％，有了明显降低，无明显释药迟滞期，且释药周期与Bydureon相当。实施例1、2、3的微球制剂和Bydureon微球的45℃体外累积释放曲线见图3。
以上实验结果表明：在本发明制剂处方和制备工艺的基础上，O相中引入二甲亚砜后可显著降低微球的初期突释。
3、对比例2是单独使用5050 2A PLGA(低粘度PLGA)制备的微球，存在主要问题是有3天左右的释药平台期，而在快速释药期内药物释放太快，导致Cmax/Cave大于3，释药波动较大。对比例4是单独使用5050 4.5A PLGA(高粘度PLGA)制备的微球，存在主要问题是有明显的释药平台期(1-8天)。对比例1(5050 2A PLGA)和对比例2(5050 4.5A PLGA)制备的微球45℃释药行为见图5。将前述两种PLGA材料按一定比例混合后，获得了优选处方实施例4，其微球45℃累积释药曲线见图5，体内血药浓度曲线见图2。
测定结果表明：本发明的优选处方实施例4与Bydureon相比，具有如下优点：突释较低，从0.8ng/ml降低至0.2ng/ml；释药迟滞期短，从2周左右降低至1周以内；Cmax/Cave低，从3左右降低至2左右(Cmax指峰值血药浓度，Cave指平均血药浓度)。
微球在体内的突释是注射后的迅速释药，主要取决于微球表面的致密程度和摄水能力，通常制备方法起决定性作用。图4为实施例4的微球制剂和上市的艾塞那肽微球(Bydureon)电镜图。图4表明：本发明实施例4制备的微球表面较Bydureon光滑致密，能降低发生突释的风险。
以上实验结果表明：本发明最优处方使用5050 2A PLGA(低粘度PLGA)和5050 4.5A PLGA(高粘度PLGA)在特定配比下的混合材料制备微球，所得微球表面形态更佳，与Bydureon相比，可明显降低突释、缩短释药迟滞期、降低Cmax/Cave。
4、实施例5和实施例6是理论载药近似为3％和10％的处方，与实施例4比较，载药量从3％升高到10％对微球释药行为影响不大，见图6。载药量升高，微球释药速率略有变快，但不影响整体释药趋势。
5、对比例5、对比例6分别采用S/O/O和W/O/W制备微球，图7比较了这两种制备方法与本发明实施例4微球释药行为的差异。结果显示：采用S/O/O和W/O/W存在的主要问题是突释较大，第1天释放高达63.77％和16.8％。
6、对比例7采用本发明的O/W制备微球，但低粘度PLGA与高粘度PLGA混合比例是1∶3，不在本发明的1∶1-9∶1范围内，从图8可以看出，当高粘度PLGA含量高于50％时，其存在明显释药平台期，前1周释放仅10.36％。

Claims (13)

  1. 一种艾塞那肽微球制剂，其特征是：包含下述重量百分比的组分：3％-10％、优选为3％-8％的艾塞那肽或艾塞那肽盐，88％-97％、优选为90％-96％的聚乳酸-羟基乙酸共聚物(PLGA)，其中，PLGA为低粘度PLGA和高粘度PLGA的混合物，所述低粘度PLGA和高粘度PLGA重量比为1∶1至9∶1。
  2. 如权利要求1所述的艾塞那肽微球制剂，其特征是：所述艾塞那肽盐为艾塞那肽醋酸盐、艾塞那肽盐酸盐、艾塞那肽乳酸盐、艾塞那肽三氟乙酸盐、艾塞那肽枸橼酸盐、艾塞那肽富马酸盐、艾塞那肽丙二酸盐、艾塞那肽马来酸盐、艾塞那肽酒石酸盐、艾塞那肽门冬氨酸盐、艾塞那肽苯甲酸盐、艾塞那肽琥珀酸盐或艾塞那肽扑酸盐。
  3. 如权利要求1或2所述的艾塞那肽微球制剂，其特征是：所述低粘度PLGA的特性粘度为0.05dl/g至0.25dl/g；所述高粘度PLGA的特性粘度为0.40dl/g至0.75dl/g。
  4. 如权利要求3所述的艾塞那肽微球制剂，其特征是：所述低粘度PLGA为5050 DLG 1A PLGA、5050 DLG 2A PLGA中的一种或两种；所述的高粘度PLGA为5050 DLG 4A PLGA、5050 DLG 4.5A PLGA、5050 DLG 7A PLGA中的一种或多种。
  5. 如权利要求1-4任意一项所述的艾塞那肽微球制剂，其特征是：所述低粘度PLGA和高粘度PLGA重量比为1.1∶1～8∶1、优选为1.2∶1～6∶1、更优选为1.5∶1～4∶1。
  6. 如权利要求1-5任意一项所述的艾塞那肽微球制剂，其特征是：还包含重量百分比为0％-2％的保护剂，所述保护剂优选为多羟基糖类，进一步优选为蔗糖和/或甘露醇。
  7. 一种权利要求1-6任意一项所述艾塞那肽微球制剂的制备方法，其特征是：所述制备方法采用O/W乳化挥发法，制备方法中油相中的有机溶剂为甲醇和二氯甲烷混合物，或二甲亚砜和二氯甲烷混合物，或甲醇、二甲亚砜和二氯甲烷混合物。
  8. 如权利要求7所述的制备方法，其特征是：包括如下步骤：
    a、称取艾塞那肽或艾塞那肽盐和PLGA，将两者溶解于有机溶剂中制备成油相，优选地将稳定剂溶解于水中制备成水相；
    b、在20-30℃下，将油相匀速注入到水相中，并高速剪切，制备成O/W乳液，20-30℃条件下搅拌固化；和
    c、用水冲洗微球，过筛收集，干燥。
  9. 如权利要求8所述的制备方法，其特征是：所述的油相中PLGA浓度是0.04g/ml-0.2g/ml。
  10. 如权利要求7-9任意一项所述的制备方法，其特征是：所述甲醇和二氯甲烷混合物中，甲醇与二氯甲烷体积比为1/3至2/3；所述的二甲亚砜和二氯甲烷混合物中，二甲亚砜与二氯甲烷体积比为2/3至9/11；所述的甲醇、二甲亚砜和二氯甲烷混合物中，甲醇与二氯甲烷体积比为1/3至2/3，二甲亚砜是甲醇和二氯甲烷总体积的0.5％至15％。
  11. 权利要求1-6任意一项所述艾塞那肽微球制剂在制备用于治疗II型糖尿病的药物中的用途。
  12. 权利要求1-6任意一项所述的艾塞那肽微球制剂用于治疗II型糖尿病的用途。
  13. 一种治疗患有II型糖尿病的患者的方法，该方法包括给予患者治疗有效量的权利要求1-6任意一项所述的艾塞那肽微球制剂。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112791066A (zh) * 2019-11-13 2021-05-14 鲁南制药集团股份有限公司 一种注射用西罗莫司缓释微球及其制备方法
CN113207799A (zh) * 2021-03-19 2021-08-06 中山大学 一种二型糖尿病小鼠快速心衰模型的构建方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114903859A (zh) * 2022-06-07 2022-08-16 烟台大学 一种来曲唑微球制剂及其制备方法
CN115779144B (zh) * 2022-09-27 2024-07-19 成都奇璞生物科技有限公司 可降解微球及其制备方法、应用以及用于治疗压力性尿失禁的填充剂

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008117927A1 (en) * 2007-03-27 2008-10-02 Peptron Co., Ltd Composition and microsphere for controlled-release of exendin, and method of preparing the same
CN102198103A (zh) * 2011-05-30 2011-09-28 深圳翰宇药业股份有限公司 一种稳定的艾塞那肽缓释微球制剂及制备方法
CN102370624A (zh) * 2010-08-17 2012-03-14 东莞太力生物工程有限公司 Exendin-4缓释微球及其注射剂和该缓释微球的制备方法
CN102488619A (zh) * 2011-12-05 2012-06-13 上海交通大学 连续生产艾塞那肽微球的装置及控制微球释放速度的方法
CN103585114A (zh) * 2013-11-25 2014-02-19 深圳翰宇药业股份有限公司 一种改进的制备艾塞那肽缓释微球的方法
CN103990114A (zh) * 2014-05-06 2014-08-20 浙江圣兆医药科技有限公司 一种艾塞那肽缓释微球组合物

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103142475B (zh) * 2012-11-28 2014-08-06 深圳市健元医药科技有限公司 一种醋酸艾塞那肽缓释微球制剂及其制备方法
CN103417957B (zh) * 2013-06-26 2015-06-10 深圳翰宇药业股份有限公司 一种艾塞那肽缓释微球及其制备方法和制剂
CN104382861A (zh) * 2014-10-30 2015-03-04 浙江美华鼎昌医药科技有限公司 一种改进的艾塞那肽缓释微球制剂及其制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008117927A1 (en) * 2007-03-27 2008-10-02 Peptron Co., Ltd Composition and microsphere for controlled-release of exendin, and method of preparing the same
CN102370624A (zh) * 2010-08-17 2012-03-14 东莞太力生物工程有限公司 Exendin-4缓释微球及其注射剂和该缓释微球的制备方法
CN102198103A (zh) * 2011-05-30 2011-09-28 深圳翰宇药业股份有限公司 一种稳定的艾塞那肽缓释微球制剂及制备方法
CN102488619A (zh) * 2011-12-05 2012-06-13 上海交通大学 连续生产艾塞那肽微球的装置及控制微球释放速度的方法
CN103585114A (zh) * 2013-11-25 2014-02-19 深圳翰宇药业股份有限公司 一种改进的制备艾塞那肽缓释微球的方法
CN103990114A (zh) * 2014-05-06 2014-08-20 浙江圣兆医药科技有限公司 一种艾塞那肽缓释微球组合物

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112791066A (zh) * 2019-11-13 2021-05-14 鲁南制药集团股份有限公司 一种注射用西罗莫司缓释微球及其制备方法
CN112791066B (zh) * 2019-11-13 2024-04-02 鲁南制药集团股份有限公司 一种注射用西罗莫司缓释微球及其制备方法
CN113207799A (zh) * 2021-03-19 2021-08-06 中山大学 一种二型糖尿病小鼠快速心衰模型的构建方法
CN113207799B (zh) * 2021-03-19 2022-03-15 中山大学 一种二型糖尿病小鼠快速心衰模型的构建方法

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