WO2017186073A1

WO2017186073A1 - Preparation method of sustained release microparticulates, sustained release microparticulates thereby and use thereof

Info

Publication number: WO2017186073A1
Application number: PCT/CN2017/081636
Authority: WO
Inventors: 刘锋; 赖树挺; 郑阳; 曹付春; 连远发
Original assignee: 广州帝奇医药技术有限公司
Priority date: 2016-04-26
Filing date: 2017-04-24
Publication date: 2017-11-02
Also published as: CN105878191A; CN105878191B

Abstract

A preparation method of sustained release microparticulates, comprising the following steps: 1) preparing a solid dispersion of water-soluble drug and biodegradable and biocompatible water-insoluble polymer; 2) dissolving the prepared solid dispersion in an organic solvent to obtain a solid dispersing emulsion; 3) injecting the obtained solid dispersing emulsion into an oil solution which contains surfactant to form a uniform emulsion; and 4) solidifying microparticulates in the emulsion by means of solvent volatilization or solvent extraction, collecting and washing the solidified microparticulates, and drying the microparticulates to obtain the sustained release microparticulates. The sustained release microparticulates prepared by the preparation method and use thereof in an implanted sustained release pharmaceutical composition. The whole process of the preparation method of the sustained release microparticulates is carried out at normal temperature or low temperature; the prepared sustained release microparticulates have a sustained-released effect close to zero order; the drug concentration is stable in the sustained release period.

Description

Preparation method of sustained-release microparticles, prepared sustained-release microparticles and application thereof

Technical field

The invention belongs to the field of water-soluble medicines, in particular to a preparation method of sustained-release microparticles, a preparation of sustained-release microparticles and sustained-release microparticles in an implantable sustained-release pharmaceutical composition.

Background technique

In recent years, a large number of biologically active substances such as oligopeptides, polypeptides, and proteins have received much attention as drug candidates, and they play an important role in the treatment of severe conditions (cancer, anemia, multiple sclerosis, hepatitis, etc.). However, these macromolecular active ingredients are relatively fragile because of their poor stability in the gastrointestinal tract (easily degraded at low pH or proteolysis), short circulatory half-lives, and poor permeability through the intestinal wall, leading to organisms. The degree of utilization is very low, so it is difficult to orally administer. Administration by injection or parenteral route is still the preferred route of administration for active ingredients such as polypeptides and proteins. Many formulations of peptides and proteins that can be injected by intravenous, intramuscular or subcutaneous routes have been marketed or under development, such as leuprolide slow release microparticles, goserelin sustained release implants, triptorelin sustained release microparticles, etc. .

For many peptide agents, particularly hormones, which require long-term continuous administration at a controlled rate, the systemic concentrations required to produce the desired effect on the target tissue or organ are high and require frequent injections of high doses. The concentration required in the therapeutic utility window often results in systemic toxicity that is detrimental to the patient. At the same time, the administration of the injection is painful, and therefore, the patient's compliance is low, the curative effect is poor, and the side effects are large. These problems can be solved by a polymer-based active ingredient, the Drug Delivery System (DDS), which is made into microcapsules by encapsulating the active ingredient in a biodegradable and biocompatible polymer matrix. In the form of microparticles or a transplantable rod, the active ingredient is released stably for a long period of time, thereby achieving the purpose of sustained release and controlled release.

There have been various methods for preparing microparticles, such as solvent evaporation, coacervation, spray drying, and spray freeze drying. Among them, the solvent is used in the aqueous phase, which can be subdivided into a single emulsion method (Oil/Water, O/W; Water/Oil, W/O) and a double emulsion method (Water/Oil/Water, W). /O/W; Water/Oil/Oil, W/O/O).

An improved double emulsification method (such as CN102245210 A, CN1826170 B) directly suspends the polypeptide powder in an organic phase to form an oil-encapsulated (S/O) suspension, and then disperses the suspension into the aqueous phase. In the middle, a complex emulsion of S/O/W is formed. Since the polypeptide powder is generally insoluble in the intermediate organic phase solvent, this method can avoid the diffusion of the internal aqueous phase to the outer aqueous phase in the Wl/O/W2 double emulsification method, thereby increasing the encapsulation efficiency.

In the S/O/W method, the size of the pre-prepared active material particles is very important. When the diameter of the solid protein particles is increased from 5 micrometers to 20 micrometers, not only the initial release rate is doubled, but also the microencapsulation rate. From 80% to 20%. The protein or polypeptide lyophilized powder generally obtained has an average particle diameter of 10 to 1000 μm. For example, a typical protein and polypeptide freeze-dried powder has an average particle diameter of about 10 to 500 μm. If the S/O suspension is directly suspended in an organic solvent with such a large particle size powder, and then the S/O/W double emulsification method is used to prepare the microparticles, the drug will not be well encapsulated, resulting in an encapsulation efficiency. Low or slow release results are unsatisfactory (previous drug release is large, but late drug release is insufficient), or the prepared microparticles are too large in particle size to be administered by injection; at the same time, the shape of the drug particles also affects the microparticles. shape. Therefore, it is generally necessary to preliminarily reduce the average particle diameter of the drug powder to 1-10 μm, and then use this powder for S/O/W double emulsification to prepare sustained-release particles (USP 6270700; Takada S, et al. Journal of Controlled Release. 2003, 88(2): 229-42).

However, the preparation of small particle size drugs is often complicated by processes such as grinding, spray drying, ultrasonic pulverization, jet pulverization, crystallization, etc. (such as CN1494900 B), and easily leads to inactivation of the active ingredient. Grinding is limited by protein denaturation caused by dust, heavy metal contamination, and heat of grinding; spray drying may provide small enough protein particles, but high shear forces near the nozzle and interfacial tension between liquid and air can denature proteins. In addition, surfactants must be used in spray drying or spray lyophilization, which in turn cause the protein to interact with the solvent in the next formulation procedure. At the same time, if the complexity of the particle preparation process is reduced and the particle size is sacrificed to ensure the activity of the drug, the particle size prepared is slightly smaller than the particle size (ie, the particle radius is much larger than the thickness of the polymer layer). There is a case where each particle only wraps one or a few active substance particles, resulting in very little drug release in the early stage, and the drug release is released quickly, and the sustained release effect is not achieved.

An improved double emulsification method (CN102233129 B, CN 102871969 A, CN102266294 B, etc.), preparing a small particle size of an active substance with one or more additives (such as dextran, polyethylene glycol, sodium alginate, etc.) The particles are then washed away with some or all of the additives with an organic solvent to obtain small particles of porous, semi-emptied or hollowed active material, which are then prepared by an S/O/hO process. This method adds a more complicated step in the preparation of small particles and requires removal of the additives therein with an organic solvent. Moreover, most of these additives are water-soluble substances, and if not completely removed, it is easy to affect the release effect, accelerate the release of active substances, or form a gel (such as high molecular weight dextran, sodium alginate), which may lead to particle breakage, drugs Delay in release or incomplete release.

A further optimized preparation method (US5556642), the water-soluble active substance and the polymer are dissolved in a co-solvent, and then the organic solvent is removed by evaporation to obtain a solid dispersion, and then the solid dispersion is dissolved in an organic solvent, and passed through an O/W method. Microparticles were prepared. This method overcomes the shortcomings of the traditional S/O/W method without drug release in the early stage, and the rapid release of drug release in the later stage. However, the process of preparing a solid by volatile organic solvent is not conducive to temperature-sensitive active materials, and it is easy to cause denaturation; if the organic solvent is volatilized at a lower temperature, the active substance is aggregated and precipitated when solidified due to slow evaporation of the solvent, drying. The active material in the subsequent solid dispersion also exists in a large volume, such as a block, a ribbon, or a filament, which causes difficulty or waste to the subsequent process of preparing the microparticles, and also causes unstable release.

Therefore, the object of the present invention is to provide a sustained-release composition prepared by an emulsification-solvent volatilization method without preparing a small-diameter drug powder in advance, which is simple in process and can ensure the biological activity of the active substance and an ideal encapsulation efficiency. And excellent method of sustained release effect.

Summary of the invention

On the one hand, in order to solve the problems existing in the prior art, the present invention provides a preparation method of sustained-release microparticles, which is prepared at a normal temperature or a low temperature throughout the whole process, and is very advantageous for a high-temperature sensitive drug, and can maintain the active material to the greatest extent. Biological activity.

The technical scheme adopted by the invention is: a preparation method of sustained-release particles, comprising the following steps:

1) preparing a solid dispersion of a water-soluble drug and a biodegradable and biocompatible water-insoluble polymer;

2) Dissolving the solid dispersion prepared in the step 1) in the organic solvent C to form a solid dispersion emulsion (internal oil phase) which is incapable of dissolving the water-soluble drug but can dissolve the water poorly soluble a polymer, an organic solvent having a boiling point lower than water and insoluble or poorly soluble in water;

3) injecting the solid dispersion emulsion obtained in the step 2) into a surfactant-containing oil solution (outer oil phase) to form a uniform emulsion;

4) The particles in the emulsion are solidified by solvent evaporation or solvent extraction, the particles are collected, washed several times with the organic solvent D, and then washed several times with ultrapure water to remove the surfactant attached to the surface of the particles, and then dried. Obtaining the sustained-release microparticles; wherein the organic solvent D is incapable of dissolving a water-soluble drug and a poorly water-soluble polymer, which is miscible with the oil solution, and has good solubility to the surfactant;

The water-soluble drug is at least one of a basic substance, a substance containing a basic group, and a salt thereof.

The water-soluble drug includes a polypeptide, a protein, a nucleic acid, an antibody, an antigen, an antibiotic, and the like. Preferably, the water-soluble drug is at least one of a protein drug, a peptide drug, and a nucleic acid drug. Preferably, the water soluble drug has a molecular weight greater than about 3350 Da.

The protein includes a natural, synthetic, semi-synthetic or recombinant compound or protein, or a basic constituent structure comprising an alpha amino acid covalently linked by a peptide bond, or is functionally related. Specifically, including but not limited to globular proteins (such as albumin, globulin, histone), fibrin (such as collagen, elastin, keratin), compound proteins (which may contain one or more non-peptide components, such as sugar) Proteins, nuclear proteins, mucins, lipoproteins, metalloproteins), therapeutic proteins, fusion proteins, receptors, antigens (such as synthetic or recombinant antigens), viral surface proteins, hormones and hormone analogues, antibodies (such as At least one of a clone or polyclonal antibody, an enzyme, a Fab fragment, an interleukin and a derivative thereof, an interferon and a derivative thereof.

The nucleic acid refers to a naturally occurring, synthetic, semi-synthetic, or at least partially recombinant compound formed from two or more identical or different nucleotides, and may be single-stranded or double-stranded. Non-limiting examples of nucleic acids include oligonucleotides, antisense oligonucleotides, aptamers, polynucleotides, deoxyribonucleic acids, siRNAs, nucleotide constructs, single or double stranded segments, and precursors and Derivatives (such as glycosylation, hyperglycosylation, PEGylation, FITC labeling, nucleosides, and salts thereof). Specifically, the nucleic acid includes, but is not limited to, Mipomersen, Alicaforsen, Nusinersen, Volanesorsen, Custirsen, Apatorsen, Plazomicin, RG-012, RG-101, ATL1102, ATL1103, IONIS-HBV _Rx , IONIS-HBV-L _Rx , IONIS- GCGR _Rx , IONIS-GCCR _Rx , IONIS-HTT _Rx , IONIS-TTR _Rx , IONIS-PKK _Rx , IONIS-FXI _Rx , IONIS-APO(a)-L _Rx , IONIS-ANGPTL3-L _Rx , IONIS-AR-2.5 _{At least one of Rx} , IONIS-DMPK-2.5 _Rx , IONIS-STAT3-2.5 _Rx , IONIS-SOD1 _Rx , IONIS-GSK4-L _Rx , IONIS-PTP1B _Rx , IONIS-FGFR4 _Rx , IONIS-DGAT2 _Rx . The above noun is the name or code of the nucleic acid drug.

The water-soluble drug preferably contains at least one basic group of water-soluble substances (such as peptide drugs), including but not limited to adrenocorticotropic hormone (ACTH) and its derivatives, epidermal growth factor (EGF), platelet-derived growth Factor (TOGF), gonadotropin releasing hormone (LHRH) and its derivatives or analogues, calcitonin, insulin-like growth factor (IGF-I, IGF-II), cell growth factors (eg EGF, TGF-α, TGF-β, PDGF, FGF, basic FGF, etc.), glucagon-like peptides (such as GLP-1, GLP-2) and their derivatives or analogues, neurotrophic factors (such as NT-3, NT- 4. CNTF, GDNF, BDNF, etc.), colony stimulating factors (e.g., CSF, GCSF, GMCSF, MCSF, etc.), and at least one of their synthetic analogs, modifications, and pharmaceutically active fragments. Derivatives or analogs of GLP-1 include, but are not limited to, exendin-3 and exendin-4.

The water-soluble drug containing at least one basic group is preferably at least one of a peptide substance, a derivative thereof, and the like, and the peptide substance includes, but is not limited to, glucagon (29 peptide), shemorie Lin (29 peptide), adiformil (28 peptide), secretin (27 peptide), ziconotide (25 peptide), ticocarp (24 peptide), bivalirudin (20 peptide), Somatostatin (14 peptide), terlipressin (12 peptide), goserelin (10 peptide), leuprolide (10 peptide), triptorelin (10 peptide), nafarelin ( 10 peptide), gonarelin (10 peptide), cetrorelix (10 peptide), degarelix (10 peptide), antipeptide (10 peptide), angiotensin (6-10 peptide), Allah Relin (9 peptide), buserelin (9 peptide), desherrin (9 peptide), octreotide (8 peptide), lanreotide (8 peptide), Bremer wave (7 peptide), erti Non-peptapeptide (7 peptide), sarsarelin (6 peptide), spleen pentapeptide (5 peptide), thymopentin (5 peptide), calcitonin (31 peptide), somaglutide (31 peptide) , glucagon-like peptide-1 (31 peptide), liraglutide (34 peptide), teriparatide (34 peptide), pramlintide (37 peptide), enfuvirtide (38 peptide), AI Senamin (39 peptide), adrenocorticotropic hormone (39 peptide), adrenal gland Hormone releasing hormone (41 peptide), temoline (44 peptide), lixisenatide (44 peptide), follicle stimulating hormone (118 peptide), duraglutide (274 peptide), abiprolide ( At least one of 645 peptides).

The peptide substance preferably has a polypeptide of not less than 30 amino acid residues. The derivative or analog of the peptide substance means that at least one of a polypeptide having not less than 30 amino acid residues and a variant or the like thereof is modified with a water-soluble or poorly water-soluble group or substance. A product that has higher biological and pharmacological activity, stability, or has new functions or properties.

The peptide drug derivatives, analogs include at least one of a glucagon-like peptide (such as GLP-1, GLP-2) and derivatives, analogs thereof, including but not limited to exendin-3, exendin -4 and at least one of their variants and derivatives.

The variant, the analog refers to a peptide in which one or more amino acid residues of the amino acid sequence are substituted (or substituted), deleted, inserted, fused, truncated or any combination thereof, and the variant polypeptide may be fully functional. Sexual or may lack one or more functions. For example, glucagon peptide-1 (GLP-1), exendin-4, is the second position of glycine, while GLP-1 is the second position of alanine, and exendin-4 can interact with GLP-1 receptor. Binding and producing a cascade of cellular signaling.

The water-soluble or poorly water-soluble group or substance is selected from the group consisting of polyethylene glycol and derivatives thereof, cyclodextrin, hyaluronic acid, short peptide, albumin, amino acid sequence, nucleic acid, gene, antibody, phosphoric acid, sulfonate Acid, fluorescent dye, KLH, OVA, PVP, PEO, PVA, alkane, aromatic hydrocarbon, biotin, immunoglobulin, albumin, polyamino acid, gelatin, succinylated gelatin, acrylamide derivative, fatty acid, polysaccharide, lipid amino acid At least one of chitosan and dextran. Preferred are polyethylene glycol and/or a derivative thereof, the polyethylene glycol and derivatives thereof The structure can be branched, linear, bifurcated or dumbbell shaped. Derivatives of the polyethylene glycol include, but are not limited to, monomethoxypolyethylene glycol, methoxypolyethylene glycol propionate. The polyethylene glycols and their derivatives are either commercially available or can be prepared by themselves by techniques well known to those skilled in the art.

The water-soluble or poorly water-soluble substance is modified to be a modifying agent with an activating group, and is coupled to the peptide substance derivative, and the activating group is selected from the group consisting of maleimide, halogen, and vinyl. At least one of a sulfone, a disulfide bond, a thiol group, an aldehyde group, a carbonyl group, an O-substituted hydroxylamine, an active ester, an alkenyl group, an alkynyl group, an azide group, and other groups having a high chemical reactivity; preferably, The activating group is selected from at least one of a maleimide, a halogen, a vinyl sulfone, and a disulfide bond; more preferably a maleimide and/or a disulfide bond. The number of activating groups carried on the polymer is one or more, and when the number of activating groups is more than one, the activating groups may be the same or different.

The one or more of the water-soluble or poorly water-soluble substances have a molecular weight of from 1 to 60 kDa, preferably from 2 to 50 kDa, more preferably from 5 to 40 kDa.

The modifying agent having an activating group can be coupled to the peptide or a variant thereof or the like by an amino group, a carboxyl group, a hydroxyl group or a thiol group on the amino acid sequence. Such groups are typically located at amino acid residues such as Lys (lysine), Asp (aspartic acid), Glu (glutamic acid), Cys (cysteine), His (histidine), 4-mercapto Any one of valine, Trp (tryptophan), Arg (arginine), Ala (alanine), Gly (glycine), Ser (serine) or Thr (threonine) or their derivatives The N-terminus, C-terminus, side chain or any site of the object is preferably a site containing a thiol group. For example, in Exendin-4 and its analogs, any cysteine residue site or other amino acid residue at 2, 14, 21, 25, 28, 35, 38 or any position is replaced with a cysteine. The site of the residue.

Modifications of the peptides and variants, analogs thereof are random modifications, localization modifications (specific modifications), single-site modifications or multi-point modifications, preferably single-site localization modifications.

The peptide and its variants and analogs are prepared by conventional polypeptide synthesis methods, including solid phase polypeptide synthesis methods, liquid phase polypeptide synthesis methods, solid phase-liquid phase polypeptide synthesis methods, and recombinant methods; peptides and variants thereof, The reaction of the analog with the modifying agent is carried out in an aqueous solution or a buffered salt solution, and the pH of the reaction system is appropriately controlled, and the modified product is monitored by HPLC, GPC, etc., and separated and purified by ion exchange, gel chromatography, etc., concentrated and frozen. Dry to obtain the target product.

The water-soluble drug mentioned above may be in the form of a free form or a pharmaceutically acceptable salt, and the salt-forming acid may be selected from a mineral acid or an organic acid. The inorganic acid includes hydrochloric acid, sulfuric acid, phosphoric acid, and the organic acid includes acetic acid, formic acid, propionic acid, lactic acid, trifluoroacetic acid, citric acid, fumaric acid, malonic acid, maleic acid, tartaric acid, aspartic acid, Benzoic acid, methanesulfonic acid, benzenesulfonic acid, citric acid, malic acid, oxalic acid, succinic acid, carbonic acid; preferably hydrochloric acid, acetic acid, fumaric acid, maleic acid; more preferably acetic acid.

The biodegradable and biocompatible water poorly soluble polymers in step 1) include polyesters, polycarbonates, polyacetals, polyanhydrides, polyhydroxy fatty acids, and copolymers or blends thereof. In detail, the biodegradable and biocompatible polymers are polylactide (PLA), polyglycolide (PGA), lactide-glycolide copolymer (PLGA) and their polycaprolactone (PCL) or polyethylene glycol (PEG) copolymer (such as PLA-PEG, PLGA-PEG, PLGA-PEG-PLGA, PLA-PEG-PLA, PEG-PCL, PCL-PLA-PCL, PCL-PLGA- PCL, PEG-PLA-PEG, PEG-PLGA-PEG), polycaprolactone and its copolymer with polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, polydioxanone (PPDO) , chitosan, alginic acid and its salts, polycyanoacrylates, fibrin, polyanhydrides, polyorthoesters, polyamides, polyphosphazenes, polyphosphates and copolymers or mixtures thereof; preferably PLA, PLGA And their copolymers with PCL or PEG, and mixtures thereof; more preferred are PLA, PLGA or mixtures thereof.

The PLA, PLGA and their copolymers with PCL or PEG have a weight average molecular weight of from 2,000 to 30,000 Da, preferably a molecular weight of from 25,000 to 110,000 Da, more preferably a molecular weight of from 3,000 to 100,000 Da. The weight average molecular weight used in the present specification is a value obtained by gel permeation chromatography (GPC) measurement.

The viscosity of the PLA, PLGA and their copolymers with PCL or PEG (test conditions of -0.5% (w/v), CHCl3, 25 ° C) is from 0.18 to 1.0 dL/g, preferably from 0.22 to 0.9 dL/g. More preferably, it is 0.27-0.85dL/g.

The molecular chains of the poorly water-soluble polymer may carry anionic or cationic groups or may not carry these groups. Preferred, poly The compound has a terminal carboxyl group or a terminal ester group, and more preferably a polymer having a terminal carboxyl group.

The PLA, PLGA and their copolymers with PCL or PEG, wherein the ratio of lactide to glycolide is from 100:0 to 50:50, preferably from about 90:10 to 50:50, more preferably 85: 15 to 50:50.

The polymer for preparing sustained-release fine particles of the present invention may be a single polymer or a mixture of a plurality of polymers, such as a combination of a ratio of a lactide to a glycolide and a PLGA having the same molecular weight but a different carrying group. The combination, molecular weight, and carrier group of PLGA, the ratio of lactide to glycolide, and the combination of PLGA, molecular weight, and carrier group having the same carrier group but different molecular weights but different ratios of lactide to glycolide And a combination of PLGA having different ratios of lactide to glycolide, a combination of PLGA and PLA, and the like.

The organic solvent C does not dissolve the water-soluble drug, but dissolves the biodegradable and biocompatible water-insoluble polymer, has a boiling point lower than water and is insoluble or poorly soluble in water. The organic solvent C may be a single organic solvent or a miscible two or more organic solvents. The organic solvent C is selected from aliphatic hydrocarbons (molecular structure is linear, branched or cyclic, such as n-hexane, n-heptane, n-pentane, cyclohexane, petroleum ether, etc.), halogenated hydrocarbons (such as dichloro Methane, chloroform, ethyl chloride, tetrachloroethylene, trichloroethylene, dichloroethane, trichloroethane, carbon tetrachloride, fluorocarbons, chlorobenzene (mono, di, trisubstituted), trichlorofluoromethane Et,) fatty acid esters (such as ethyl acetate, butyl acetate, etc.), aromatic hydrocarbons (such as benzene, toluene, xylene, etc.), ethers (such as diethyl ether, diisopropyl ether, methyl isobutyl) At least one of ether, methyl tert-butyl ether, methoxylated ether, alkyl ether, dihaloether, trihaloether, cyclic ether, crown ether, etc., preferably a halogenated aliphatic hydrocarbon solvent, More preferably, at least one of dichloromethane and chloroform. The type and proportion of the organic solvent C in the internal oil phase are different according to different drugs and polymers, and are formulated according to actual conditions.

The concentration of the poorly water-soluble polymer in the organic solvent C varies depending on the type of the polymer, the weight average molecular weight, and the type of the organic solvent; generally, the mass concentration (polymer mass / organic solvent C mass * 100%) is about 1 -18% (w/w), preferably about 2-15% (w/w), more preferably about 3-12% (w/w).

The organic solvent D does not dissolve the water-soluble drug and the biodegradable and biocompatible water-insoluble polymer at the same time, but is miscible with the oil solution while having good solubility to the surfactant. The organic solvent D may be a single organic solvent or a miscible two or more organic solvents. The organic solvent D is at least one selected from the group consisting of anhydrous diethyl ether, cyclohexane, n-hexane, n-heptane, and petroleum ether, preferably at least one of n-hexane, cyclohexane, and n-heptane. The type and proportion of the solvent D are different depending on the surfactant and the oil solution, and are formulated according to actual conditions.

The organic solvent having a boiling point lower than water and insoluble or poorly soluble in water means an organic solvent which is only miscible with water in a volume ratio of <5% by volume, and has a lower boiling point (less than or much less than 100 ° C) so that It is easily removed by, for example, lyophilization, evaporation, or blasting.

The emulsion is at a low temperature, which is understood to be 20 ° C or below, preferably 15 ° C or below, more preferably 6 ° C or below.

The surfactant-containing oil solution (also referred to as the outer oil phase) is a low temperature, which is understood to be 18 ° C or below, preferably 12 ° C or below, more preferably 8 ° C.

The oil base of the surfactant-containing oil solution is at least one of any pharmaceutically acceptable polyol, vegetable oil, mineral oil, and other oils in the pharmaceutical arts. The oil base may be a single component or a mixture of two or more components. The vegetable oil includes, but is not limited to, soybean oil, cottonseed oil, rapeseed oil, peanut oil, safflower oil, sesame oil, rice bran oil, corn germ oil, sunflower oil, poppy oil, olive oil, corn oil, cottonseed oil, coconut oil, flax. At least one of a seed oil, castor oil, and palm oil, and at least one of soybean oil, peanut oil, and castor oil is preferably used in the vegetable oil, more preferably peanut oil; and the mineral oil includes, but not limited to, silicone oil, liquid paraffin; other oil The oil obtained by partial hydrogenation of vegetable oil (such as hydrogenated castor oil) and at least one of liquid saturated fatty acids (such as caproic acid, octanoic acid, etc.) are included; the polyol includes glycerin, polyethylene glycol. The oil base is preferably a vegetable oil and/or a mineral oil, more preferably a vegetable oil.

The surfactant can increase the wetting property of the organic phase, improve the stability and shape of the small liquid bead during the emulsification process, avoid re-polymerization of the small liquid bead, and reduce the number of unencapsulated or partially encapsulated small spherical particles, thereby avoiding The initial burst of the drug during release.

The surfactant is a compound such as an anionic surfactant, a zwitterionic surfactant, a nonionic surfactant or a surface active biomolecule, preferably an anionic surfactant, a nonionic surfactant, more preferably an anionic surfactant. Agent.

The nonionic surfactant includes, but is not limited to, at least one of sorbitan ester (Span), glyceryl monostearate, cetyl alcohol, octadecyl alcohol, stearyl alcohol .

The anionic surfactants include, but are not limited to, phospholipids and derivatives thereof, glycerides, fatty acid esters, fatty alcohols, and other bile acids (eg, bile acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycine deoxygenation). At least one of cholic acid).

The anionic surfactant is preferably a phospholipid and a derivative thereof, including but not limited to phosphatidylcholine (lecithin), phosphatidylethanolamine (cephalin), phosphatidylserine, phosphatidylinositol, Phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), glycerophosphatidic acid, lysophospholipid, soybean phospholipid, dipalmitoyl-phosphatidylcholine, dioleoylphosphatidyl-ethanolamine, dioleoylphosphatidylcholine and two flesh Myristoyl-phosphatidylglycerol, and mixtures thereof. The phospholipids may be salified or non-salted, hydrogenated or partially hydrogenated, natural, semi-synthetic or fully synthetic. The phospholipid and its derivative are preferably phosphatidylcholine, soybean phospholipid, phosphatidylglycerol, more preferably soybean phospholipid.

The mass percentage of the surfactant (or stabilizer) in the oil base is generally from 0.05 to 10%, preferably from 0.25 to 8%, more preferably from 0.5 to 5%.

The amount of the external oil phase used is usually about 50 times or more by volume of the inner oil phase, preferably about 70 times by volume and particularly preferably about 100 times by volume or more.

The method of forming a uniform emulsion is the same as the well-known emulsification method, using a device that generates high shear force (such as a magnetic stirrer, a mechanical stirrer, a high speed homogenizer, an ultrasound machine, a membrane emulsifier, a rotor-stator mixer). , static mixer, high pressure homogenizer, etc.) The inner oil phase is mixed with the outer oil to form a uniform emulsion.

The following method can be applied to remove the organic solvent in the step 4):

(1) removing the organic solvent by heating, decompression (or combined heating);

(2) the gas stream blows the surface of the liquid, and controls the contact area of the liquid phase with the gas phase, the rate of emulsion agitation and circulation (such as JP-A-9-221418) to accelerate the volatilization of the organic solvent, preferably the dry gas;

(3) The organic solvent (for example, W00183594) is quickly removed by a hollow fiber membrane, and the hollow fiber membrane is preferably a silicone rubber pervaporation film (particularly a pervaporation film prepared from polydimethylsiloxane).

The microparticles obtained in the step 4) are separated by centrifugation, sieving or filtration.

The temperature of the ultrapure water used for washing the microparticles in the step 4) is a low temperature, which is understood to be 12 ° C or less, preferably 9 ° C or less, more preferably 6 ° C or less.

The ultrapure water used for washing in the step 4) may further contain an inorganic salt (such as a zinc salt) to reduce the infiltration of the water-soluble active substance into the aqueous phase during the washing process, thereby improving the encapsulation efficiency of the drug, and the mechanism is to improve the external phase. The osmotic pressure or the solubility of the active substance in the external phase. For active substances such as peptides, proteins, nucleic acids, antibodies, antigens, antibiotics, etc., zinc ion-containing compounds are ideal, including but not limited to zinc acetate, zinc chloride, zinc sulfate, zinc hydrogen sulfate, zinc nitrate, gluconic acid. Zinc, zinc carbonate or any mixture thereof. The mass concentration of the inorganic salt in the ultrapure water is from 0.01 to 3%, preferably from 0.01 to 1.5%, more preferably from 0.01 to 1%.

Preferably, the step 1) is carried out by the following steps:

1) completely dissolving the biodegradable and biocompatible water-insoluble polymer and the water-soluble drug in the organic solvent A to form a mixed solution of the drug and the polymer;

2) Injecting the mixed solution into the organic solvent B or injecting the organic solution B into the mixed solution to produce a uniform, fine precipitate, collecting the precipitate, and washing it with the organic solvent B several times to remove the organic solvent B, and obtaining A solid dispersion of a water-soluble drug and a poorly water-soluble polymer; wherein the organic solvent B is incapable of dissolving the water-insoluble polymer and the water-soluble drug.

The organic solvent A can simultaneously dissolve a water-soluble drug and a biodegradable, biocompatible water-insoluble polymer. The organic solvent A may be a single organic solvent or a miscible two or more organic solvents. The organic solvent A is at least one selected from the group consisting of glacial acetic acid, acetonitrile, trifluoroacetic acid, and dimethyl sulfoxide, preferably glacial acetic acid and/or acetonitrile, more preferably glacial acetic acid. The kind and proportion of the organic solvent in the mixture vary according to different drugs and polymers, and can be formulated according to actual conditions.

The organic solvent B does not dissolve the water-soluble drug and the biodegradable, biocompatible water-insoluble polymer at the same time. The organic solvent B may be a single organic solvent or a miscible two or more organic solvents. The organic solvent B is selected from at least one of anhydrous diethyl ether, hexane (including cyclohexane, n-hexane), and n-heptane, and preferably at least one of anhydrous diethyl ether and hexane (including cyclohexane, n-hexane). One, more preferably anhydrous diethyl ether. The kind and proportion of the organic solvent in the mixture vary according to different drugs and polymers, and can be formulated according to actual conditions.

The organic solvent A is controlled to be below normal temperature or low temperature, and the normal temperature is generally understood to be 20 ° C, preferably 10-15 ° C; the low temperature is generally understood to be 10 ° C or lower, preferably 4-6 ° C or below; The organic solvent B is controlled to a low temperature, which is generally understood to be 15 ° C or lower, preferably 10 ° C or lower, more preferably 6 ° C or lower; the organic solvent A is 0 to 10 ° C higher than the temperature of the organic solvent B, preferably 3 8 ° C.

In the solid dispersion, the mass ratio of the water-soluble drug to the biodegradable and biocompatible water-insoluble polymer is from 1:1 to 1:99, preferably from 2:3 to 3:97, more preferably 7:13. ~1:19.

The concentration of the water-insoluble polymer in the organic solvent A varies depending on the type of the polymer, the weight average molecular weight, and the type of the organic solvent. Usually, the mass concentration (polymer mass / organic solvent A mass * 100%) is 1-18% (w/w), preferably 2-15% (w/w), more preferably 3-12% (w) /w).

The step of removing the organic solvent B does not include a temperature raising procedure, which is carried out below normal temperature or at a low temperature, which is generally understood to be 20-30 ° C, preferably 20-25 ° C; the low temperature is generally understood to be 15 ° C. Hereinafter, it is preferably 10 ° C or less. Methods of removing organic solvents include, but are not limited to, vacuum drying, freeze drying, and fluidized drying.

Further, the sustained release microparticles of the present invention may contain one or more auxiliary agents. The preparation method of the sustained-release fine particles of the present invention further comprises the step of adding an auxiliary agent, which is added during the step of preparing the solid dispersion in the step 1), or added during the preparation of the solid dispersion emulsion in the step 2); It is added at the time of preparing the solid dispersion emulsion in the step 2). The adjuvant is dissolved in the internal phase or suspended in the internal oil phase. When the auxiliary agent is added, it may be a very fine powder having a particle diameter of less than 0.5 μm, preferably a particle diameter of less than 0.1 μm, more preferably a particle diameter of less than 0.05 μm.

The adjuvant may impart additional characteristics to the active drug or microparticles, such as increasing the stability of the microparticles, active drug or polymer, promoting controlled release of the active drug from the microparticles, or modulating the biological tissue permeability of the active drug. The auxiliary agent is 0.01 to 10%, preferably 0.1 to 8%, more preferably 0.5 to 8% by mass of the sum of the mass of the water-soluble drug and the poorly water-soluble polymer.

The adjuvant includes, but is not limited to, at least one of a saccharide, an amino acid, a fatty acid, an alcohol, an antioxidant, and a buffer.

The buffering agents include, but are not limited to, mineral or organic acid salts such as salts of carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid. Specifically, including but not limited to calcium carbonate, calcium hydroxide, calcium myristate, calcium oleate, calcium palmitate, calcium stearate, calcium phosphate, calcium acetate, magnesium acetate, magnesium carbonate, magnesium hydroxide, phosphoric acid Magnesium, magnesium myristate, magnesium oleate, magnesium palmitate, magnesium stearate, zinc carbonate, zinc hydroxide, zinc oxide, zinc myristate, zinc oleate, zinc acetate, zinc chloride, zinc sulfate, Zinc hydrogen sulfate, zinc nitrate, zinc gluconate, zinc palmitate, zinc stearate, zinc phosphate, sodium carbonate, sodium hydrogencarbonate, sodium hydrogen sulfite, sodium thiosulfate, acetic acid-sodium acetate buffer, and their random combination. A zinc salt of an inorganic acid or an organic acid is preferred, and zinc chloride is more preferred. The buffering agent is 0-5%, preferably 0.01-3%, more preferably 0.01-2%, of the sum of the mass of the water-soluble drug and the poorly water-soluble polymer.

The antioxidants include, but are not limited to, tert-butyl-p-hydroxyanisole, dibutylphenol, tocopherol, isopropyl myristate, tocopheryl daacetate, ascorbic acid, ascorbyl palmitate, butylated hydroxybenzoic acid Ether, butylated hydroxy hydrazine, hydroxy coumarin, butylated hydroxy Base toluene, decanoic acid fatty acid ester (such as ethyl ester, propyl ester, octyl ester, lauryl ester), propyl hydroxybenzoate, hydroxybutanone, vitamin E, vitamin E-TPGS, ρ-hydroxybenzoic acid At least one of an ester such as a methyl ester, an ethyl ester, a propyl ester, or a butyl ester. The antioxidant can effectively remove free radicals or peroxides in the sustained release microparticles. The antioxidant is 0-1%, preferably 0-0.05%, more preferably 0-0.01%, of the sum of the mass of the water-soluble drug and the poorly water-soluble polymer.

Such saccharides include, but are not limited to, monosaccharides, oligosaccharides, and polysaccharides, as well as derivatives thereof. Specifically, including but not limited to trehalose, glucose, sucrose, glycerol, erythritol, arabitol, xylitol, sorbitol, mannitol, glucuronic acid, iduronic acid, nervous amino acid, Galacturonic acid, gluconic acid, mannuronic acid, hyaluronic acid and its salts, chondroitin sulfate and its salts, heparin, inulin, chitin and its derivatives, dextrin, dextran and alginic acid At least one of its salts. At least one of sucrose, mannitol, and xylitol is preferred. The saccharide is 0.1 to 10%, preferably 0.5 to 8%, more preferably 1 to 6%, based on the sum of the mass of the water-soluble drug and the water-insoluble polymer.

The amino acids include, but are not limited to, glycine, alanine, serine, aspartic acid, glutamic acid, threonine, tryptophan, lysine, hydroxylysine, histidine, arginine, cyst At least one of amino acid, cysteine, methionine, phenylalanine, leucine, isoleucine, and derivatives thereof; preferably a basic amino acid, including but not limited to arginine, At least one of histidine and lysine. The amino acid is 0-4%, preferably 0-2%, more preferably 0.01-1%, of the sum of the mass of the water-soluble drug and the poorly water-soluble polymer.

The fatty acid includes 12-24 alkanoic acid and its derivatives including, but not limited to, oleic acid, stearic acid, lauric acid, myristic acid, palmitic acid, arachidic acid, behenic acid, lignin acid, preferably stearic acid. At least one of behenic acid and palmitic acid. The fatty acid is 0 to 5%, preferably 0.01 to 4%, more preferably 0.05 to 3%, of the sum of the mass of the water-soluble drug and the water-insoluble polymer.

The alcohols include, but are not limited to, polyethylene glycol. The polyethylene glycol has a molecular weight of from 400 to 6000 Da, preferably from 400 to 4000 Da, more preferably from 400 to 2000 Da. The alcohol is 0 to 5%, preferably 0.01 to 4%, more preferably 0.05 to 3%, of the sum of the mass of the water-soluble drug and the water-insoluble polymer.

Formulations for injection require sterility, and specific sterilization methods are within the ordinary knowledge and skill of those skilled in the art, such as aseptic processing, hot pressing, ethylene oxide or gamma radiation to assure sterility of the formulation. The preparation of the sustained-release microparticles of the present invention is preferably aseptic operation, such as filtering the outer phase aqueous solution with a cellulose acetate membrane, filtering the PLGA acetic acid solution with a polyethersulfone membrane, filtering the dichloromethane with a polytetrafluoroethylene membrane, and all the equipment is easily sealed. It is equipped with an organic solvent recovery unit to prevent bacterial contamination and the diffusion of organic solvents into the air.

In another aspect, the present invention also provides sustained release microparticles prepared according to the method for preparing sustained release microparticles.

Because the particles are used for injection, the particle size is too large to cause needle sticking. The larger needle must be used, and the patient's pain is stronger. If the particle size is too small, the copolymer will not be able to wrap the drug well. Not a good sustained release effect. The sustained release microparticles prepared by the present invention preferably have an average geometric particle size of less than 200 μm. The sustained-release fine particles have a particle diameter of 10 to 200 μm, preferably 10 to 150 μm, more preferably 20 to 150 μm. The particle size of the sustained release particles is measured by a dynamic light scattering method (for example, laser diffraction method) or a microscopic technique (such as scanning electron microscopy).

The sustained-release microparticles of the present invention may encapsulate a large amount of active ingredients, and the dosage may depend on the type and content of the active ingredient, the dosage form, the duration of release, the subject to be administered, the route of administration, the purpose of administration, the target disease and symptoms, and the like. Choose as appropriate. However, the dosage can be considered satisfactory as long as the active ingredient can be maintained in the active concentration of the drug for the desired duration in vivo.

In the sustained-release fine particles of the present invention, the water-soluble drug has a mass content percentage of about 1 to 40%, preferably 3 to 35%, more preferably 5 to 30%.

When a range is recited herein, it is meant to include any range or combination of ranges.

In still another aspect, the present invention also provides a suspension formulation comprising the sustained release microparticles and a dispersion medium.

When the microparticles are administered as a suspension, they can be formulated as a suspension with a suitable dispersion medium.

The dispersion medium includes a nonionic surfactant, a polyoxyethylene castor oil derivative, a cellulose thickener, sodium alginate, and a hyaluronic acid. At least one of an acid, a dextrin, and a starch. Alternatively, it may be combined with other components such as isotonic agents (such as sodium chloride, mannitol, glycerol, sorbitol, lactose, xylitol, maltose, galactose, sucrose, glucose, etc.), pH adjusters ( For example, carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid or salts of these acids, such as sodium carbonate, sodium hydrogencarbonate, etc., preservatives (for example, parabens, propylparaben, benzyl alcohol, At least one of chlorobutanol, sorbic acid, boric acid, etc. is combined to form an aqueous solution, or subsequently cured by freeze drying, drying under reduced pressure, spray drying, etc., and the cured product is dissolved in water for injection before use. A dispersion medium that disperses the particles.

Further, the sustained release injection can also be obtained by dispersing the sustained release microparticles in a vegetable oil such as sesame oil and corn oil or a vegetable oil to which a phospholipid such as lecithin is added, or dispersed in a medium chain triglyceride, Obtain an oily suspension.

In still another aspect, the present invention also provides the use of the solid dispersion, sustained release microparticles in an implantable sustained release pharmaceutical composition.

The water-soluble drug sustained-release pharmaceutical composition prepared by the present invention, particularly the sustained-release pharmaceutical composition of the protein, the nucleic acid and the peptide drug, may also be a stick or a sheet. Further, the present invention also provides an implantable type. A method for preparing a sustained release pharmaceutical composition, comprising the steps of:

1 preparing a solid dispersion of a water-soluble drug and a biodegradable and biocompatible water-insoluble polymer;

2 The solid dispersion prepared in the step 1 is heated and then molded by a molding method, and the implanted sustained-release pharmaceutical composition is prepared by cooling.

The molding method is not limited herein, and molding methods well known to those skilled in the art can be used, such as compression molding, extrusion molding, and the sustained-release composition can be formed into a rod shape or a sheet shape.

The sustained-release pharmaceutical composition for preparing a water-soluble drug, particularly a protein or a peptide drug, of the present invention is a rod-shaped or sheet-like implant.

Further, a method for preparing an implantable sustained release pharmaceutical composition comprises the following steps:

1′ according to the preparation method of the sustained release microparticles to prepare sustained release microparticles;

2' The sustained release microparticles obtained in step 1' are prepared into an implantable sustained release pharmaceutical composition by a molding method well known to those skilled in the art. The sustained release pharmaceutical composition can be formed into a rod shape or a sheet shape.

The sustained-release microparticles obtained by the present invention can be used in the form of granules, suspensions, implants, injections, adhesive preparations, and the like, and can be administered orally or parenterally (intramuscular injection, subcutaneous injection). , transdermal administration, mucosal administration (intracrine, intravaginal, rectal, etc.)).

The implant of the invention is based on a biodegradable and biocompatible material, and has a thin rod shape, a round rod shape or a sheet shape (disc shape), and can be implanted into the body by injection or surgery, and the drug is completely released. No need to remove it by surgery. The advantage of the implant is that it is easy to obtain high encapsulation rate and drug loading rate, low burst release rate, and can continuously release the therapeutic dose of the active drug at a stable rate for one month to several months, greatly reducing medical treatment. Cost to improve patient compliance.

The beneficial effects of the present invention are as follows: in the present invention, the preparation of the sustained-release microparticles is normal or low temperature throughout the whole process, and is highly advantageous for the preparation of the polymer matrix for drugs sensitive to high temperatures, particularly proteins, nucleic acids and peptide drugs. The technology can maintain the biological activity of the active substance to the greatest extent in the whole process; at the same time, the prepared sustained-release particles have an excellent sustained release effect close to zero order, and the drug concentration is stable during the release, which solves the traditional advance The S/O/W process for preparing the fine particles of the drug has no drug release in the early stage, and has the disadvantage of rapid release of the drug in the later stage; further, the sustained release particles have a higher drug loading rate and drug encapsulation efficiency.

After the administration of the sustained-release microparticles of the present invention, active substances such as proteins, peptides, nucleic acids, and alkaloids can be continuously transported in the body for a period of time, and the release period is as long as several weeks or several months.

DRAWINGS

Figure 1 is a graph showing the mean HbA _1c value-time curve of diabetic model mice administered with Exendin extended-release microparticles or liraglutide sustained-release microparticles prepared in Examples 6-11.

detailed description

The present invention will be further described with reference to specific embodiments in order to better illustrate the objects, aspects and advantages of the invention. The "microparticles" in the following examples are also "sustained release microparticles"; the "sustained release implant" is also an implantable sustained release pharmaceutical composition.

Example 1 Preparation of Abreglutide/PLGA Microparticles

(I) Preparation of solid dispersion

0.90 g of PLGA (molecular weight 25 kDa, monomer ratio 65/35, terminal carboxyl group) was dissolved in about 6.00 mL of glacial acetic acid, then 0.10 g of albendide acetate was added, dissolved under vortex, and then slowly infused without stirring. A white precipitate was obtained in water diethyl ether (6 ° C). The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 6.00 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 230 mL of 0.05% (w/w) lecithin/peanut oil which had been previously thermostated to about 4 ° C. The S/O/O emulsion (rotor speed about 3000 rpm, 5 min) was prepared in solution and using a high speed homogenizer. The S/O/O emulsion was mechanically stirred for about 3 hours (400 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The obtained microparticles were found to have an aspirin content of 9.12% and a particle diameter of 19 to 90 μm.

Example 2 Preparation of Durarupeptide/PLGA Microparticles

(I) Preparation of solid dispersion

0.95 g of PLGA (molecular weight 30 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 7.92 mL of acetonitrile, then 0.05 g of duraglutide acetate was added, dissolved under vortex, and then slowly injected into the stirred ring. In the alkane (6 ° C), a white precipitate was obtained, and a white precipitate was collected and extracted with cyclohexane for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 7.92 g of chloroform to obtain an internal oil phase, and then the inner oil phase was injected into 420 mL of a 0.1% (w/w) lecithin/liquid paraffin solution which had been previously thermostated to about 5 °C. The S/O/O emulsion (rotor speed about 3000 rpm, 5 min) was prepared using a high speed homogenizer. The S/O/O emulsion was mechanically stirred for about 3 hours (400 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the microparticles were washed with anhydrous diethyl ether for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain microparticles. The content of duraglutide in the obtained microparticles was measured to be 4.60%, and the particle diameter of the microparticles was 20 to 95 μm.

Example 3 Preparation of follicle-stimulating hormone/PLA microparticles

(I) Preparation of solid dispersion

0.97 g of PLA (molecular weight 20 kDa, terminal ester group) was dissolved in about 5.39 mL of dimethyl sulfoxide, then 0.03 g of follicle stimulating hormone, 0.05 g of xylitol and 0.03 g of zinc chloride were added and dissolved under vortexing. Then, the mixture was slowly poured into n-hexane (8 ° C) under stirring to produce a white precipitate. The white precipitate was collected and extracted with n-hexane for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in a mixture of about 5.39 g of dichloromethane and chloroform to obtain an internal oil phase, and then the inner oil phase was injected into 410 mL of 0.25% which had been previously thermostated to about 6 ° C (w/w). The lecithin/soybean oil solution was emulsified using a wheeled uniform mixer to prepare an S/O/O emulsion (running speed of about 5500 rpm, 5 min). The S/O/O emulsion was transferred to a sealed glass flask and mechanical stirring was continued for about 3 hours (400 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 2500 rpm, 5 min) using a centrifuge. After the particles were washed with cyclohexane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of follicle-stimulating hormone in the obtained microparticles was measured to be 2.73%, and the particle diameter of the microparticles was 30-82 μm.

Example 4 Preparation of Lixila/PLGA Microparticles

(I) Preparation of solid dispersion

0.99 g PLGA (molecular weight 22 kDa, monomer ratio 90/10, terminal carboxyl group) was dissolved in about 5.50 mL of trichloroacetic acid, then 0.01 g of lixisenatide acetate, 0.05 g of xylitol and 0.03 g of zinc carbonate were added, and vortexed. Dissolve, then slowly inject n-heptane (6 ° C) under stirring to produce a white precipitate. The white precipitate was collected and extracted with n-heptane about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h ( 10 ° C), a solid dispersion was obtained.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 5.50 g of tetrachloroethylene to obtain an internal oil phase, and then the inner oil phase was injected into 330 mL of 0.5% (w/w) lecithin/corn which had been previously thermostated to about 5 °C. An S/O/O emulsion (film pore size 30-80 μm, cycle 3 times) was prepared in an oil solution using a SPG membrane emulsifier. The S/O/O emulsion was mechanically stirred for about 3.5 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-hexane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of lixisenatide in the obtained fine particles was 0.93%, and the particle diameter was 34-98 μm.

Example 5 Preparation of Corticotropin-releasing Hormone/PLGA Microparticles

(I) Preparation of solid dispersion

0.85 g of PLGA (molecular weight 25 kDa, monomer ratio 85/15, terminal carboxyl group) was dissolved in a mixture of about 8.50 mL of glacial acetic acid and acetonitrile, then 0.15 g of corticotropin-releasing hormone was added, dissolved under vortex, and then Slowly inject a mixture of anhydrous diethyl ether and cyclohexane (6 ° C) under stirring to give a white precipitate. The white precipitate was collected and extracted with a mixture of anhydrous diethyl ether and cyclohexane for about 5 times. After collection, it was dried in a vacuum oven for 24 h (10 ° C) to obtain a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 8.50 g of n-heptane to obtain an internal oil phase, and then the inner oil phase was injected into 580 mL of 0.75% (w/w) lecithin/蓖 which had been previously thermostated to about 6 ° C. The S/O/O emulsion was prepared in a sesame oil solution using a static mixer (rotation speed 5000 rpm, 3 cycles). The S/O/O emulsion was transferred to a sealed glass flask and mechanical stirring was continued for about 3.5 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with petroleum ether for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of somatostatin in the obtained microparticles was measured to be 13.77%, and the particle diameter of the microparticles was 31 to 95 μm.

Example 6 Preparation of Exenatide/PLGA Microparticles

(I) Preparation of solid dispersion

0.95 g of PLGA (molecular weight 35 kDa, monomer ratio 75/25, terminal carboxyl group) was dissolved in about 6.33 mL of glacial acetic acid, then 0.05 g of exenatide acetate and 0.08 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 6.33 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 430 mL of 1% (w/w) lecithin/peanut oil which had been previously thermostated to about 5 °C. The S/O/O emulsion (1000 rpm, 5 min) was prepared in solution and by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 4 hours (400 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. The microparticles were washed about 5 times with a mixture of n-heptane and petroleum ether, and then again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of exenatide in the obtained microparticles was measured to be 4.65%, and the particle diameter of the microparticles was 24-93 μm.

Example 7 Preparation of Liraglutide/PLGA Microparticles

(I) Preparation of solid dispersion

0.93 g of PLGA (molecular weight 40 kDa, monomer ratio 65/35, terminal carboxyl group) was dissolved in about 7.75 mL of glacial acetic acid, then 0.07 g of liraglutide acetate and 0.06 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I1 was uniformly dispersed in about 7.75 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 470 mL of 1.25% (w/w) lecithin/peanut oil which had been previously thermostated to about 7 ° C. The S/O/O emulsion (1000 rpm, 5 min) was prepared in solution and by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 4 hours (400 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of liraglutide in the obtained microparticles was measured to be 6.50%, and the particle diameter of the microparticles was 30 to 102 μm.

Example 8 Preparation of Exenatide/PLGA Microparticles

(I) Preparation of solid dispersion

0.90 g of PLGA (molecular weight 45 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 9.00 mL of glacial acetic acid, then 0.10 g of exenatide acetate and 0.04 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 9.00 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 680 mL of 1.5% (w/w) lecithin/peanut oil which had been previously thermostated to about 9 °C. The S/O/O emulsion (1500 rpm, 7 min) was prepared in solution and mechanically stirred. The S/O/O emulsion was mechanically stirred for about 4 hours (700 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of exenatide in the obtained microparticles was measured to be 9.23%, and the particle diameter of the microparticles was 25 to 92 μm.

Example 9 Preparation of Liraglutide/PLGA Microparticles

(I) Preparation of solid dispersion

0.86 g of PLGA (molecular weight 50 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 10.75 mL of glacial acetic acid, then 0.14 g of liraglutide acetate and 0.02 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 10.75 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 970 mL of 2% (w/w) lecithin/peanut oil which had been previously thermostated to about 10 °C. The S/O/O emulsion (1800 rpm, 5 min) was prepared in solution and mechanically agitated. The S/O/O emulsion was mechanically stirred for about 4 hours (800 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of liraglutide in the obtained microparticles was 12.81%, and the particle diameter was 22-89 μm.

Example 10 Preparation of Exenatide/PLGA Microparticles

(I) Preparation of solid dispersion

0.82 g of PLGA (molecular weight 55 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 11.71 mL of glacial acetic acid, then 0.18 g of exenatide acetate and 0.01 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 11.71 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 700 mL of 2% (w/w) lecithin/peanut oil which had been previously thermostated to about 8 °C. The S/O/O emulsion (1500 rpm, 5 min) was prepared in solution and by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 5 hours (600 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of exenatide in the obtained microparticles was measured to be 17.00%, and the particle diameter was 22-90 μm.

Example 11 Preparation of Liraglutide/PLGA Microparticles

(I) Preparation of solid dispersion

0.80 g of PLGA (molecular weight 60 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 13.33 mL of glacial acetic acid, then 0.20 g of liraglutide acetate was added, dissolved under vortex, and then slowly infused without stirring. A white precipitate was obtained in water diethyl ether (6 ° C). The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 13.33 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 900 mL of 3% (w/w) lecithin/peanut oil which had been previously thermostated to about 11 ° C. The S/O/O emulsion (1600 rpm, 5 min) was prepared in solution and mechanically stirred. The S/O/O emulsion was mechanically stirred for about 5 hours (700 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of liraglutide in the obtained microparticles was measured to be 18.83%, and the particle diameter of the microparticles was 25 to 107 μm.

Example 12 Preparation of Enfuvirtide/PLGA Microparticles

(I) Preparation of solid dispersion

0.75 g of PLGA (molecular weight 65 kDa, monomer ratio 65/35, terminal carboxyl group) was dissolved in about 15.00 mL of glacial acetic acid, then 0.25 g of enfuviryl acetate, 0.03 g of sucrose and 0.01 g of stearic acid were added, and vortexed. Dissolved, and then slowly poured into anhydrous diethyl ether (6 ° C) under stirring to give a white precipitate. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10) °C), a solid dispersion was obtained.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 15.00 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 1.1 L of 4% (w/w) lecithin which had been previously thermostated to about 13 ° C/ The S/O/O emulsion (2000 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 5 hours (850 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The obtained microparticles had an enfuvirtide content of 22.97% and a particle diameter of 18-93 μm.

Example 13 Preparation of pramlintide/PLGA microparticles

(I) Preparation of solid dispersion

0.70 g of PLGA (molecular weight 70 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 17.50 mL of glacial acetic acid, then 0.30 g of pramlintide acetate and 0.02 g of mannitol were added, dissolved under vortex, and then slowly injected. Under agitation of anhydrous diethyl ether (6 ° C), a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to obtain a solid dispersion. body.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 17.50 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 1.3 L of 5% (w/w) lecithin which had been previously thermostated to about 20 ° C/ S/O/O emulsion (2200 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 5 hours (800 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of pramlintide in the obtained microparticles was measured to be 27.49%, and the particle diameter was 27-98 μm.

Example 14 Preparation of teriparatide/PLGA microparticles

(I) Preparation of solid dispersion

0.65 g of PLGA (molecular weight 85 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 21.67 mL of glacial acetic acid, then 0.35 g of teriparatide acetate, 0.03 g of mannitol and 0.03 g of PEG-400 were added and dissolved under vortexing. Then, it was slowly poured into anhydrous diethyl ether (6 ° C) under stirring to give a white precipitate. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) ), a solid dispersion is obtained.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 21.67 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 1.6 L of 6% (w/w) lecithin which had been previously thermostated to about 15 ° C. The S/O/O emulsion (2400 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 5 hours (900 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of teriparatide in the obtained microparticles was measured to be 32.16%, and the particle diameter was 20-92 μm.

Example 15 Preparation of Liraglutide /PLGA Microparticles

(I) Preparation of solid dispersion

0.60 g of PLGA (molecular weight 100 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 30.00 mL of glacial acetic acid, then 0.40 g of liraglutide acetate and 0.005 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 30.00 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 2 L of 7% (w/w) lecithin/glycerol which had been previously thermostated to about 15 ° C. The S/O/O emulsion (2000 rpm, 5 min) was prepared in solution and by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 5 hours (700 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of liraglutide in the obtained microparticles was measured to be 37.18%, and the particle diameter of the microparticles was 23 to 90 μm.

Example 16 Preparation of Somatoglutide/PLGA Microparticles

(I) Preparation of solid dispersion

0.50 g of PLGA (molecular weight 110 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 50.00 mL of glacial acetic acid, then 0.50 g of somatoglutide acetate and 0.001 g of xylitol were added, dissolved under vortex, and then slowly Slowly injecting anhydrous diethyl ether (6 ° C) under stirring, a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C). Solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in step I was uniformly dispersed in about 50.00 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 2.6 L of 8% (w/w) lecithin which had been previously thermostated to about 15 ° C/ The S/O/O emulsion (film pore size 20-50 μm, cycle 3 times) was prepared in a peanut oil solution using a SPG membrane emulsifier. The S/O/O emulsion was mechanically stirred for about 5 hours (600 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The obtained microparticles had a content of somaglutide of 45.04% and a particle diameter of 23-87 μm.

Example 17 Preparation of glucagon-like peptide-1/PLGA microparticles

(I) Preparation of solid dispersion

0.50 g of PLGA (molecular weight 130 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 50.00 mL of glacial acetic acid, then 0.50 g of glucagon-like peptide-1 was added, dissolved under vortex, and then slowly injected. Under agitation of anhydrous diethyl ether (6 ° C), a white precipitate was obtained. The white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to obtain a solid dispersion. body.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 50.00 g of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 3 L of 10% (w/w) lecithin/peanut oil which had been previously thermostated to about 20 ° C. The S/O/O emulsion was prepared by emulsification in a solution using a wheel-type homomixer (running speed of about 7000 rpm, 5 min). The S/O/O emulsion was transferred to a sealed glass flask and mechanical stirring was continued for about 5 hours (800 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of glucagon-like peptide-1 in the obtained microparticles was measured to be 46.21%, and the particle diameter was 19-85 μm.

Example 18 Preparation of Exendin-4 Derivative/PLGA Microparticles

(I) Preparation of solid dispersion

The solid dispersion contains the following components in mass percent: water-soluble drug: Exendin-4 derivative 20%, poorly water-soluble polymer: PLGA 79.5%, adjuvant: xylitol 0.5%; wherein the molecular weight of the PLGA It is 50 kDa in which the ratio of lactide to glycolide is 50/50, and the PLGA has a terminal carboxyl group.

(1) Preparation of Exendin-4 derivative: Preparation of 10kDa PEG-NHS ester, then reacted with asparagine at position 28 in Exendin-4 in PBS buffer, purified by ion exchange, gel chromatography, concentrated and freeze-dried Obtained the Exendin-4 derivative.

(2) completely dissolving the poorly water-soluble polymer in glacial acetic acid, and then adding a water-soluble drug and an auxiliary agent to completely dissolve; wherein the poorly water-soluble polymer is 6.5% by mass of glacial acetic acid; and then injecting anhydrous diethyl ether ( 6 ° C) resulted in a white precipitate, which was collected and extracted 5 times with anhydrous diethyl ether. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 12 times of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 970 mL of 2% (w/w) lecithin which had been previously thermostated to about 5 °C. The S/O/O emulsion (1400 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 4 hours (500 rpm) to solidify the microparticles, and then centrifuged (about 3500 rpm, using a centrifuge). 5 min) Collect the particles. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of the Exendin-4 derivative in the obtained fine particles was measured to be 18.40%, and the particle diameter was 27-109 μm.

Example 19 Preparation of Exendin-4 Derivative/PLGA Microparticles

(I) Preparation of solid dispersion

The solid dispersion contains the following components by mass: water-soluble drug: Exendin-4 derivative 15%, poorly water-soluble polymer: PLGA 84%, adjuvant: xylitol 1%; wherein the PLGA has a molecular weight of 50 kDa Wherein the ratio of lactide to glycolide is 50/50 and the PLGA has a terminal carboxyl group.

(1) Preparation of Exendin-4 derivative: Exendin-4 variant in which asparagine at position 28 in Exendin-4 was replaced with cysteine by solid phase peptide synthesis method, and then in 10 kbD with PBS buffer The monomethoxypolyethylene glycol-maleimide reaction was purified by ion exchange, gel chromatography, concentrated, and lyophilized to obtain an Exendin-4 derivative.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 13 times of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 970 mL of 1.75% (w/w) lecithin which had been previously thermostated to about 5 °C. S/O/O emulsion (1300 rpm, 5 min) was prepared in a peanut oil solution and mechanically agitated. The S/O/O emulsion was mechanically stirred for about 4 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of the Exendin-4 derivative in the obtained fine particles was 13.25%, and the particle diameter was 30-113 μm.

Example 20 Preparation of Exendin-4 Derivative/PLGA Microparticles

(I) Preparation of solid dispersion

The solid dispersion contains the following components in mass percent: water-soluble drug: Exendin-4 derivative 20%, poorly water-soluble polymer: PLGA 78%, adjuvant: sorbitol 2%; wherein the PLGA has a molecular weight of 55 kDa, Wherein the ratio of lactide to glycolide is 50/50, and the PLGA has a terminal carboxyl group.

(1) Preparation of Exendin-4 derivative: Exendin-4 variant in which arginine at position 20 in Exendin-4 was replaced with cysteine by solid phase peptide synthesis method, and then 5kDa in PBS buffer The oxypolyethylene glycol-maleimide reaction is purified by ion exchange, gel chromatography, concentrated, and lyophilized to obtain an Exendin-4 derivative.

(2) completely dissolving the poorly water-soluble polymer in glacial acetic acid, and then adding a water-soluble drug and an auxiliary agent to completely dissolve; wherein the poorly water-soluble polymer is 6% by mass of glacial acetic acid; and then injecting anhydrous diethyl ether ( 6 ° C) resulted in a white precipitate, which was collected and extracted 5 times with anhydrous diethyl ether. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 14 times of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 1 L of 1.5% (w/w) lecithin which had been previously thermostated to about 5 ° C / S/O/O emulsion (1500 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 4 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of the Exendin-4 derivative in the obtained fine particles was measured to be 18.31%, and the particle diameter was 32-126 μm.

Example 21 Preparation of Exendin-4 Derivative/PLGA Microparticles

(I) Preparation of solid dispersion

The solid dispersion contains the following components by mass: water-soluble drug: Exendin-4 derivative 16%, poorly water-soluble polymer: PLGA 81%, adjuvant: xylitol 3%; wherein the PLGA has a molecular weight of 45 kDa Wherein the ratio of lactide to glycolide is 50/50 and the PLGA has a terminal carboxyl group.

(1) Preparation of Exendin-4 derivative: Exendin-4 variant in which methionine at position 14 in Exendin-4 was replaced with cysteine was prepared by solid phase peptide synthesis method, and then singly with 20 kDa in PBS buffer. The methoxypolyethylene glycol-maleimide reaction was purified by ion exchange, gel chromatography, concentrated, and lyophilized to obtain an Exendin-4 derivative.

(2) completely dissolving the poorly water-soluble polymer in glacial acetic acid, and then adding a water-soluble drug and an auxiliary agent to completely dissolve; wherein the poorly water-soluble polymer is 7% by mass of glacial acetic acid; and then injecting anhydrous ether ( 6 ° C) resulted in a white precipitate, which was collected and extracted 5 times with anhydrous diethyl ether. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in Step I was uniformly dispersed in about 11 times of dichloromethane to obtain an internal oil phase, and then the internal oil phase was injected into 970 mL of 1.25% (w/w) lecithin which had been previously thermostated to about 5 °C. The S/O/O emulsion (1400 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 4 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of the Exendin-4 derivative in the obtained fine particles was measured to be 13.74%, and the particle diameter was 32-128 μm.

Example 22 Preparation of Exendin-4 Derivative/PLGA Microparticles

The solid dispersion contains the following components by mass: water-soluble drug: Exendin-4 derivative 12%, poorly water-soluble polymer: PLGA 84%, adjuvant: xylitol 4%; wherein the PLGA has a molecular weight of 40 kDa Wherein the ratio of lactide to glycolide is 50/50 and the PLGA has a terminal carboxyl group.

(1) Preparation of Exendin-4 derivative: Exendin-4 variant in which the glycine at position 2 of Exendin-4 was replaced with cysteine by solid phase peptide synthesis method, and then 40 kDa monomethoxy group in PBS buffer The polyethylene glycol-maleimide reaction was purified by ion exchange, gel chromatography, concentrated, and lyophilized to obtain an Exendin-4 derivative.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 10 times of dichloromethane to obtain an internal oil phase, and then the inner oil phase was injected into 970 mL of 1% (w/w) lecithin which had been previously thermostated to about 5 ° C / The S/O/O emulsion (1400 rpm, 5 min) was prepared in a peanut oil solution by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 4 hours (600 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with n-heptane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of the Exendin-4 derivative in the obtained fine particles was 10.68%, and the particle diameter was 35-132 μm.

Example 23 Preparation of Mipomersen/PLGA Microparticles

(I) Preparation of solid dispersion

0.80 g of PLGA (molecular weight 30 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in a mixture of about 6.53 mL of glacial acetic acid and acetonitrile, and then 0.20 g of Mipomersen sodium and 0.01 g of xylitol were added and dissolved under vortexing. Then slowly inject the anhydrous ether under stirring (6 ° C), a white precipitate was obtained, and a white precipitate was collected and extracted with n-hexane for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to obtain a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in about 6.53 g of tetrachloroethylene to obtain an internal oil phase, and then the inner oil phase was injected with 500 mL of 1% (w/w) lecithin/peanut oil which had been previously thermostated to about 6 ° C. The S/O/O emulsion (1000 rpm, 5 min) was prepared in solution and by mechanical stirring. The S/O/O emulsion was mechanically stirred for about 3.5 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the particles were washed with cyclohexane for about 5 times, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of Mipomersen in the obtained fine particles was measured to be 18.10%, and the particle diameter was 32-109 μm.

Example 24 Preparation of Interleukin/PLGA Microparticles

(I) Preparation of solid dispersion

0.82 g of PLGA (molecular weight 35 kDa, monomer ratio 50/50, terminal carboxyl group) was dissolved in about 6.12 mL of glacial acetic acid, then 0.18 g of interleukin and 0.02 g of xylitol were added, dissolved under vortex, and then slowly poured under stirring. A white precipitate was obtained in anhydrous diethyl ether (6 ° C). A white precipitate was collected and extracted with anhydrous diethyl ether for about 5 times. The precipitate was collected and dried in a vacuum oven for 24 h (10 ° C) to give a solid dispersion.

(II) Preparation of microparticles

The solid dispersion obtained in the step I was uniformly dispersed in a mixture of about 6.12 g of dichloromethane and chloroform to obtain an internal oil phase, and then the inner oil phase was injected into 500 mL of 0.5% (w/w which had been previously thermostated to about 5 ° C). In a lecithin/soybean oil solution, an S/O/O emulsion (1000 rpm, 5 min) was prepared by mechanical stirring. The S/O/O emulsion was transferred to a sealed glass flask and mechanical stirring was continued for about 4 hours (500 rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500 rpm, 5 min) using a centrifuge. After the microparticles were washed about 5 times with a mixture of n-heptane and n-hexane, they were again dispersed in ultrapure water (5 ° C) for about 2 times, then collected by centrifugation, and lyophilized in a freeze dryer to obtain fine particles. The content of interleukin in the obtained fine particles was 16.36%, and the particle diameter was 31-114 μm.

Example 25 Preparation of Exenatide/PLGA Sustained Release Implant

The dried solid dispersion prepared in the step I of Example 10 was filled in a mold of 1 mm * 10 mm (the inner cavity was cylindrical, the diameter of the round bottom was 1 mm, and the depth was about 10 mm), and the temperature was raised to about 43 ° C, and then compression molding was carried out. A columnar (1 mm * 5.31 mm) exenatide sustained release implant was obtained. The content of liraglutide in the obtained implant was measured to be 17.24%.

Example 26 Preparation of Exenatide/PLGA Sustained Release Implant

The microparticles obtained in the step II of Example 10 were fed into a hot melt extruder, hot melt extruded into strips having a diameter of about 1 mm, and after cooling, cut into exenatide sustained release implants having a length of about 5 mm. Agent. The content of liraglutide in the obtained implant was measured to be 17.03%.

The method for analyzing the drug loading rate and the drug encapsulation efficiency of the microparticles or implants in the above embodiments is as follows: taking 5 mg of microparticles or an implant, dissolving in 50 mL of acetonitrile (ACN), and then adding 0.1 μ of TFA 500 μL, and after thoroughly mixing, The supernatant was centrifuged, and the concentration of the drug was analyzed by high performance liquid chromatography. The ratio of the total mass of the drug encapsulated in the microparticle (or implant) to the dose is the encapsulation efficiency of the drug, the mass of the drug encapsulated in the microparticle (or implant) and the mass of the microparticle (or implant). The ratio is the drug loading rate of the drug. All experiments were repeated more than 3 times.

The particle size analysis method of the fine particles in the above examples was as follows: about 10 mg of the fine particles were dispersed in liquid paraffin, ultrasonically dispersed for about 30 seconds, and measured using a Beckman Coulter laser particle size analyzer.

Example 27 Determination of burst and in vitro release profiles of microparticles and implants

The sustained-release microparticles and the implant prepared in the above examples were subjected to the burst release and in vitro release curves, and the measurement method was as follows: accurately weigh the drug-containing microparticles or the implant 20 mg into a 15 mL centrifuge tube, and the pH was 7.4. The buffer (containing 0.02% sodium azide as a bacteriostatic agent) is a release medium, placed in a constant temperature air bath shaker, and the particles and implants are carried out under the conditions of an oscillation speed of 100 rpm and a temperature of 37 ° C ± 0.5 ° C. In vitro release assay. All release media were taken at l, 2, 7, 14, 21, 28, 40, 50, and 60 days, and the same amount of new release medium was added. The drug release was determined by high performance liquid chromatography. The measurement method is:

Liquid chromatograph: Agilent 1260;

Column: Proteonavi 4.6 × 250mm;

Mobile phase: water-acetonitrile (containing 0.1% trifluoroacetic acid), gradient elution;

Flow rate: 1 mL/min;

Detection wavelength: 280 nm.

The test results are shown in Table 1.

Table 1 Results of cumulative release of sustained release microparticles and implants in vitro

样品sample	1天1 day	2天2 days	7天7 days	14天14 days	21天21 days	28天28 days	40天40 days	50天50 days	60天60 days
实施例1Example 1	0.79％0.79%	1.88％1.88%	5.37％5.37%	12.86％12.86%	23.19％23.19%	42.35％42.35%	67.81％67.81%	89.61％89.61%	100.00％100.00%
实施例2Example 2	0.95％0.95%	2.24％2.24%	13.90％13.90%	28.34％28.34%	43.64％43.64%	68.82％68.82%	95.56％95.56%	99.96％99.96%	99.95％99.95%
实施例3Example 3	0.90％0.90%	2.07％2.07%	5.88％5.88%	13.09％13.09%	25.28％25.28%	38.15％38.15%	56.01％56.01%	66.85％66.85%	83.45％83.45%
实施例4Example 4	1.12％1.12%	2.08％2.08%	4.39％4.39%	9.73％9.73%	21.19％21.19%	32.39％32.39%	49.63％49.63%	62.74％62.74%	80.64％80.64%
实施例5Example 5	0.96％0.96%	1.81％1.81%	4.06％4.06%	8.76％8.76%	19.28％19.28%	32.61％32.61%	51.82％51.82%	70.52％70.52%	85.20％85.20%
实施例6Example 6	1.05％1.05%	2.12％2.12%	4.31％4.31%	10.79％10.79%	22.16％22.16%	35.27％35.27%	58.57％58.57%	76.13％76.13%	90.34％90.34%
实施例7Example 7	0.91％0.91%	1.88％1.88%	7.41％7.41%	16.83％16.83%	29.08％29.08%	45.46％45.46%	69.94％69.94%	83.91％83.91%	94.92％94.92%
实施例8Example 8	1.98％1.98%	4.16％4.16%	8.36％8.36%	16.25％16.25%	27.24％27.24%	41.61％41.61%	78.25％78.25%	93.75％93.75%	99.96％99.96%
实施例9Example 9	1.02％1.02%	2.12％2.12%	8.46％8.46%	19.78％19.78%	34.21％34.21%	52.57％52.57%	76.98％76.98%	90.03％90.03%	100.00％100.00%
实施例10Example 10	1.14％1.14%	2.23％2.23%	9.77％9.77%	24.31％24.31%	45.81％45.81%	60.64％60.64%	82.72％82.72%	94.20％94.20%	100.00％100.00%
实施例11Example 11	1.69％1.69%	2.98％2.98%	15.81％15.81%	31.25％31.25%	50.53％50.53%	70.36％70.36%	88.64％88.64%	98.57％98.57%	100.00％100.00%
实施例12Example 12	1.53％1.53%	3.45％3.45%	7.45％7.45%	14.81％14.81%	24.25％24.25%	38.57％38.57%	51.69％51.69%	71.40％71.40%	90.08％90.08%
实施例13Example 13	1.48％1.48%	2.52％2.52%	13.88％13.88%	28.95％28.95%	47.35％47.35%	66.36％66.36%	82.60％82.60%	93.00％93.00%	100.00％100.00%
实施例14Example 14	1.21％1.21%	2.38％2.38%	12.59％12.59%	26.33％26.33%	42.54％42.54%	61.77％61.77%	79.32％79.32%	89.05％89.05%	99.40％99.40%
实施例15Example 15	0.95％0.95%	2.24％2.24%	13.90％13.90%	28.34％28.34%	43.64％43.64%	68.82％68.82%	85.56％85.56%	99.96％99.96%	99.95％99.95%
实施例16Example 16	1.69％1.69%	3.61％3.61%	8.37％8.37%	15.82％15.82%	26.13％26.13%	39.49％39.49%	63.24％63.24%	83.36％83.36%	95.80％95.80%
实施例17Example 17	1.81％1.81%	3.89％3.89%	8.42％8.42%	17.87％17.87%	28.02％28.02%	42.37％42.37%	72.80％72.80%	93.35％93.35%	100.00％100.00%
实施例18Example 18	0.80％0.80%	1.52％1.52%	7.12％7.12%	16.03％16.03%	28.05％28.05%	40.50％40.50%	60.78％60.78%	78.42％78.42%	89.95％89.95%
实施例19Example 19	0.92％0.92%	1.64％1.64%	7.46％7.46%	15.78％15.78%	28.21％28.21%	41.17％41.17%	61.48％61.48%	80.03％80.03%	91.20％91.20%
实施例20Example 20	1.37％1.37%	2.15％2.15%	13.70％13.70%	26.25％26.25%	44.83％44.83%	58.66％58.66%	71.50％71.50%	85.00％85.00%	98.10％98.10%
实施例21Example 21	0.94％0.94%	1.83％1.83%	8.59％8.59%	16.91％16.91%	28.81％28.81%	44.67％44.67%	62.72％62.72%	80.10％80.10%	90.50％90.50%
实施例22Example 22	0.85％0.85%	1.94％1.94%	9.10％9.10%	17.36％17.36%	30.64％30.64%	48.80％48.80%	65.56％65.56%	83.37％83.37%	95.95％95.95%
实施例23Example 23	1.14％1.14%	2.15％2.15%	11.34％11.34%	24.68％24.68%	38.00％38.00%	42.67％42.67%	64.34％64.34%	79.21％79.21%	91.05％91.05%
实施例24Example 24	0.98％0.98%	1.97％1.97%	10.05％10.05%	22.80％22.80%	31.95％31.95%	40.34％40.34%	65.76％65.76%	81.50％81.50%	93.14％93.14%
实施例25Example 25	0.84％0.84%	1.73％1.73%	8.77％8.77%	22.81％22.81%	43.81％43.81%	59.64％59.64%	77.22％77.22%	94.00％94.00%	100.00％100.00%
实施例26Example 26	1.19％1.19%	2.28％2.28%	14.81％14.81%	29.75％29.75%	45.00％45.00%	61.26％61.26%	78.14％78.14%	94.26％94.26%	100.00％100.00%

It can be seen from the in vitro release results of Table 1 that the sustained release microparticles prepared by using the solid dispersion of the present invention and the prepared implant have no burst phenomenon or obvious delayed release, and the whole release tendency is close to zero. Level release. Among them, some samples have a long release period in vitro. For 40-50 days, some samples have an in vitro release period of 50-60 days, and some samples have an in vitro release period of more than 60 days, which has an excellent sustained release effect.

Example 28 microparticle needle test

Approximately 20 mg of the microparticle sample was suspended in 2 mL of diluent (3% carboxymethylcellulose, 0.9% NaCl), and then inhaled into a syringe and injected into a commercially available 1 kg weight of the hind leg (muscle) through a 24-30 G injection needle. )in. The injection was observed for 20 seconds or less per injection, and the results are shown in Table 2.

Table 2 results of microscopic needle test

Note: ++ push needle smoothness is very good, + push needle smoothness is general, - push needle has a sense of block, -- plugging.

The general results of Table 2 indicate that the particle suspensions of different particle sizes prepared by the present invention have the ability to inhale the syringe through the 30 gauge needle and completely inject the contents of the syringe into the pork without any blockage or The phenomenon of needle plugging indicates that the microparticles of the present invention can be administered by subcutaneous or intramuscular injection.

Example 29 Determination of residual amount of organic solvent

The residual amount of the organic solvent A, the organic solvent B, the organic solvent C, and the organic solvent D in the solid dispersion and the sustained-release fine particles prepared in Examples 1 to 24 of the present invention is measured, and the measurement method is a well-known measurement method. The measurement results are shown in Table 3.

Table 3 Determination of residual amount of organic solvent

Note: - indicates no detection or content below the detection limit.

It can be seen from the results of the residual amount of the organic solvent in Table 3 that in the solid dispersion and the sustained-release fine particles prepared by the present invention, the residual amount of the organic solvent is low, or is not detected, or the residual amount is lower than the detectable range, and the drug is administered. After the patient has no side effects caused by organic solvents, it is also beneficial to maintain the stability of the particles and extend the shelf life.

Example 30 Animal Test

56 diabetic mice were selected, weighing 20±5g, male and female, randomly divided into the drug-administered group (6 groups) and the blank group (1 group). The mice in the drug-administered group were injected subcutaneously into the neck and back of the skin. 11 exenatide microparticles or liraglutide microparticles, the microparticles were suspended with a diluent containing 3% carboxymethylcellulose and 0.9% NaCl, and each mouse in the administration group was injected with exenatide 2 mg/kg or Liraglutide 10 mg / kg, the blank group was injected subcutaneously with the same volume of normal saline. Blood samples were taken from the tail vein at the same time on the 0th, 0.5d, 1d, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, 70d doses, and the average HbA _{1c was prepared.} A plot of the values (% of glycated hemoglobin as a percentage of total hemoglobin, %) and time (d) is shown in Figure 1.

As can be seen from the graph of Fig. 1, the Exenatide sustained-release microparticles or the liraglutide sustained-release microparticles prepared in Examples 6-11 of the present invention can well control the HbA _1c value within 70 days after administration, and The HbA _1c value was between 5 and 7 in the 7-70 days after administration, which was significantly lower than that of the blank group, indicating that the exenatide sustained-release microparticles or liraglutide sustained-release microparticles of the present invention can be long after administration. Time to release the active drug, and achieve the desired therapeutic effect, can reduce the frequency of administration, and help to improve patient compliance.

It should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention, although the present invention will be described in detail with reference to the preferred embodiments, The technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims

A method for preparing sustained-release microparticles, comprising the steps of:

1) preparing a solid dispersion of a water-soluble drug and a biodegradable and biocompatible water-insoluble polymer;

2) Dissolving the solid dispersion prepared in the step 1) in an organic solvent C to form a solid dispersion emulsion which is incapable of dissolving the water-soluble drug but capable of dissolving the poorly water-soluble polymer and having a low boiling point An organic solvent that is insoluble in water or poorly soluble in water;

3) injecting the solid dispersion emulsion obtained in the step 2) into the oil solution containing the surfactant to form a uniform emulsion;

4) The particles in the emulsion are solidified by solvent evaporation or solvent extraction, the particles are collected, washed several times with the organic solvent D, and then washed several times with ultrapure water to remove the surfactant attached to the surface of the particles, and then dried. Obtaining the sustained-release microparticles; wherein the organic solvent D is incapable of dissolving a water-soluble drug and a poorly water-soluble polymer, which is miscible with the oil solution, and has good solubility to the surfactant;

The water-soluble drug is at least one of a protein drug, a peptide drug, and a nucleic acid drug.
The method for producing sustained-release microparticles according to claim 1, wherein the water-soluble drug is at least one of a polypeptide having not less than 30 amino acid residues, a derivative thereof, and the like.
The method for producing sustained release microparticles according to claim 2, wherein the polypeptide derivative or the analog is at least one of a polypeptide having not less than 30 amino acid residues, a variant thereof, and the like. A product modified with a water-soluble or poorly water-soluble group or substance.
The method for producing sustained-release fine particles according to claim 3, wherein the water-soluble or poorly water-soluble group or substance is polyethylene glycol and/or a derivative thereof.
The method for preparing sustained-release microparticles according to claim 1, wherein the step 1) is carried out by the following steps:

1) completely dissolving the biodegradable and biocompatible water-insoluble polymer and the water-soluble drug in the organic solvent A to form a mixed solution of the drug and the polymer;

2) Injecting the mixed solution into the organic solvent B or injecting the organic solution B into the mixed solution to produce a uniform, fine precipitate, collecting the precipitate, and washing it with the organic solvent B several times to remove the organic solvent B, and obtaining A solid dispersion of a water-soluble drug and a poorly water-soluble polymer; wherein the organic solvent B is incapable of dissolving the water-insoluble polymer and the water-soluble drug.
The method for preparing sustained-release fine particles according to claim 5, wherein the organic solvent A is at least one selected from the group consisting of glacial acetic acid, acetonitrile, trifluoroacetic acid, and dimethyl sulfoxide;

The organic solvent B is selected from at least one of anhydrous diethyl ether, hexane, and n-heptane.
The method for producing sustained-release fine particles according to claim 1, wherein a mass ratio of the water-soluble drug to the poorly water-soluble polymer in the solid dispersion is from 1:1 to 1:99.
The method for preparing sustained-release fine particles according to claim 1, wherein the organic solvent C is at least one selected from the group consisting of aliphatic hydrocarbons, halogenated hydrocarbons, fatty acid esters, aromatic hydrocarbons, and ethers;

The organic solvent D is at least one selected from the group consisting of anhydrous diethyl ether, cyclohexane, n-hexane, n-heptane, and petroleum ether.
The method for preparing sustained-release microparticles according to claim 1, wherein the poorly water-soluble polymer in the step 1) is a polylactide, a polyglycolide, a lactide-glycolide copolymer, and They are copolymers with polycaprolactone or polyethylene glycol, polycaprolactone and copolymers thereof with polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, polydioxanone, chitosan At least one of sugar, alginic acid and salts thereof, polycyanoacrylate, fibrin, polyanhydride, polyorthoester, polyamide, polyphosphazene, polyphosphate, and copolymers and/or mixtures thereof .
The method for preparing sustained-release microparticles according to claim 1, wherein the method further comprises the step of adding an auxiliary agent, the auxiliary agent is added during the step of preparing the solid dispersion in the step 1), or Step 2) adding a solid dispersion emulsion; The adjuvant is 0.01-10% of the sum of the mass of the water-soluble drug and the poorly water-soluble polymer.
The method for producing sustained-release fine particles according to claim 10, wherein the auxiliary agent is at least one of an amino acid, an antioxidant, a buffer, a saccharide, a fatty acid, and an alcohol.
The sustained-release fine particles obtained by the method for producing sustained-release fine particles according to any one of claims 1 to 11.