CN105878191B - Preparation method of sustained-release microparticles, prepared sustained-release microparticles and application thereof - Google Patents

Abstract

The invention discloses a preparation method of sustained-release particles, which comprises the following steps: 1) preparing a solid dispersion of a water-soluble drug and a biodegradable and biocompatible poorly water-soluble polymer; 2) dissolving the prepared solid dispersion in an organic solvent C to form a solid dispersion emulsion; 3) injecting the obtained solid dispersion emulsion into an oil solution containing a surfactant to form a uniform emulsion; 4) solidifying the particles in the emulsion by solvent volatilization or solvent extraction, collecting the particles, washing and drying to obtain the slow-release particles; the invention also discloses the sustained-release particles prepared by the preparation method of the sustained-release particles and the application of the sustained-release particles in an implanted sustained-release pharmaceutical composition. The preparation method of the sustained-release particles is completely normal temperature or low temperature, and is very beneficial to preparing polymer matrix compositions from high-temperature sensitive medicines; meanwhile, the prepared sustained-release particles have an excellent sustained-release effect close to zero order, and the drug concentration is stable in a 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, and particularly relates to a preparation method of sustained-release particles, the prepared sustained-release particles and application of the sustained-release particles in an implanted sustained-release medicine composition.

Background

In recent years, a large number of bioactive substances such as oligopeptides, polypeptides and proteins have gained a great deal of attention as drug candidates, which play important roles in treating severe disease states (cancer, anemia, multiple sclerosis, hepatitis, etc.). However, these macromolecular active ingredients are fragile because of their poor stability in the gastrointestinal tract (susceptible to degradation at low pH or proteolysis), short circulatory half-life, and their poor permeability across the intestinal wall, resulting in very low bioavailability and hence difficult oral administration. Injection or parenteral administration remains 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 are already on the market or under development, such as leuprorelin sustained 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 of these active ingredients required to produce the desired effect on the target tissue or organ are high, thus requiring frequent injections of high doses to achieve the required concentrations in the therapeutic utility window, often resulting in systemic toxicity which is detrimental to the patient. Meanwhile, the injection administration is painful, so that the compliance of patients is low, the treatment effect is poor, and the side effect is great. These problems are solved by a Drug Delivery System (DDS) based on polymeric active ingredients by encapsulating the active ingredient in a biodegradable and biocompatible polymer matrix in the form of microcapsules, microparticles or implantable rods, allowing a long-term stable release of the active ingredient, thereby achieving controlled release.

There are various methods for preparing microparticles, such as solvent evaporation (solvent evaporation), coacervation (coarvation), spray drying, and spray freeze drying, which have been reported. The most used method is the solvent evaporation method in the Water phase, which can be divided into single emulsion method (Oil/Water, O/W; Water/Oil, W/O) and multiple emulsion method (Water/Oil/Water, W/O/W; Water/Oil, W/O/O).

An improved double emulsion method (such as CN102245210A, CN1826170B) comprises directly suspending polypeptide powder in organic phase to form solid-in-oil (S/O) suspension, and dispersing the suspension in water phase to form S/O/W double emulsion. Because the polypeptide powder is generally insoluble in an intermediate organic phase solvent, the method can avoid the diffusion of the internal water phase to the external water phase in the Wl/O/W2 double-emulsification method, thereby improving the encapsulation efficiency.

In the S/O/W process, the size of the pre-prepared active particles is important, and as the diameter of the solid protein particles increases from 5 microns to 20 microns, not only does the initial release rate double, its microencapsulation efficiency decreases from 80% to 20%. The average particle size of the lyophilized powder of protein and polypeptide is usually 10-1000 μm, for example, the average particle size of the lyophilized powder of protein and polypeptide is typically about 10-500. mu.m. If the powder with large particle size is directly suspended in an organic solvent to form S/O suspension, and then the S/O/W multiple emulsion method is used for preparing the particles, the medicament cannot be well encapsulated, so that the encapsulation efficiency is low, or the release result is unsatisfactory (the burst release of the medicament at the early stage is large, and the release amount of the medicament at the later stage is insufficient), or the prepared particles have too large particle size and are difficult to inject and administer; at the same time, the shape of the drug particles also affects the shape of the microparticles. Therefore, the mean particle size of the drug powder is generally reduced to 1 to 10 μm in advance by a method and then the powder is used in an S/O/W double emulsion process to prepare release fine 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 grinding, spray drying, ultrasonic pulverization, jet pulverization, crystallization, and the like (e.g., CN1494900B), and tends to result in inactivation of the active ingredient. The grinding process is limited by the contamination of dust, heavy metals and protein denaturation caused by grinding heat; spray drying may provide sufficiently small protein particles, but high shear forces near the nozzle and interfacial tension between the liquid and air denature the protein, and in addition, spray drying or spray freeze drying requires the use of surfactants which cause the protein to interact with the solvent in the next formulation procedure. Meanwhile, if the complexity of the particle preparation process is reduced, the particle size is sacrificed to ensure the activity of the medicament, and the particle size of the prepared particles is slightly smaller than that of the particles (namely, the particle radius is much larger than the thickness of the polymer layer), the situation that each particle only wraps one or a few active substance particles occurs, so that few medicaments are released at the early stage, and the medicaments are released quickly at the later stage, and the slow-release effect cannot be achieved.

An improved multiple emulsion method (CN 102233129B, CN 102871969A, CN 102266294B, etc.) comprises preparing active substance and one or more additives (such as dextran, polyethylene glycol, sodium alginate, etc.) into small-particle-diameter particles, washing off part or all of the additives with organic solvent to obtain porous, semi-hollow or hollow active substance small particles, and preparing microparticles by S/O/hO process. This method adds a relatively complicated step of preparing small particles and requires the use of an organic solvent to remove the additives. Moreover, most of these additives are water-soluble substances, and if they are not completely removed, they may easily affect the release effect, accelerate the release of the active substance, or form a gel (such as high molecular weight dextran, sodium alginate), which may cause the particles to burst, delay the release of the drug, or cause incomplete release.

A further optimized preparation process (US5556642) provides for the preparation of microparticles by the O/W process by dissolving the water-soluble active substance and the polymer in a cosolvent, then removing the organic solvent by evaporation to give a solid dispersion, and then dissolving the solid dispersion in the organic solvent. The method overcomes the defects that the traditional S/O/W method has no drug release at the early stage and the drug release is rapidly released at the later stage. However, the process of preparing a solid by volatilizing an organic solvent is not favorable for the active substance sensitive to temperature, and the active substance is easy to denature; if the organic solvent is volatilized at a lower temperature, the active substance is aggregated and separated during solidification due to slow volatilization of the solvent, and the active substance in the dried solid dispersion exists in a larger volume, such as a block shape, a belt shape or a thread shape, so that the subsequent particle preparation process is difficult or wasted, and the release is unstable.

Accordingly, it is an object of the present invention to provide a method for preparing -release composition by emulsion-solvent evaporation method without preparing small-particle size drug powder in advance, which is relatively simple in process and can ensure the bioactivity of active substance, desirable encapsulation efficiency and excellent -release effect.

Disclosure of Invention

On one hand, in order to solve the problems in the prior art, the invention provides a preparation method of sustained-release particles, and the preparation method is completely at normal temperature or low temperature, is very favorable for high-temperature sensitive medicines, and can keep the biological activity of active substances to the maximum extent.

The technical scheme adopted by the invention is as follows: 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 poorly water-soluble polymer;

2) dissolving the solid dispersion prepared in the step 1) in an organic solvent C which is an organic solvent which can not dissolve the water-soluble drug but can dissolve the water-insoluble polymer, has a boiling point lower than that of water, and is insoluble or hardly soluble in water to form a solid dispersion emulsion (internal oil phase);

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

4) solidifying the particles in the emulsion by solvent volatilization or solvent extraction, collecting the particles, washing the particles for a plurality of times by using an organic solvent D, washing the particles for a plurality of times by using ultrapure water to remove the surfactant attached to the surfaces of the particles, and drying the particles to obtain the slow-release particles; wherein the organic solvent D is not capable of dissolving a water-soluble drug and a poorly water-soluble polymer, is miscible with the oil solution, and has good solubility for 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 drugs include polypeptides, proteins, nucleic acids, antibodies, antigens, antibiotics, 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 proteins include natural, synthetic, semi-synthetic or recombinant compounds or proteins, or basic building blocks containing alpha amino acids covalently linked by peptide bonds, or functionally related. Specifically, it includes, but is not limited to, at least one of globular proteins (e.g., albumin, globulin, histone), fibrin (e.g., collagen, elastin, keratin), compound proteins (which may contain one or more non-peptide components, such as glycoprotein, nucleoprotein, mucin, lipoprotein, metalloprotein), therapeutic proteins, fusion proteins, receptors, antigens (e.g., synthetic or recombinant antigens), viral surface proteins, hormones and hormone analogs, antibodies (e.g., monoclonal or polyclonal antibodies), enzymes, Fab fragments, interleukins and derivatives thereof, interferons and derivatives thereof.

The nucleic acid refers to a compound that is natural, synthetic, semi-synthetic, or at least partially recombinant formed from two or more nucleotides that are the same or different, and may be single-stranded or double-stranded. Non-limiting examples of nucleic acids include oligonucleotides, antisense oligonucleotides, aptamers, polynucleotides, deoxyribonucleic acids, siRNA, nucleotide constructs, single-or double-stranded fragments and precursors and derivatives thereof (e.g., glycosylation, hyperglycosylation, PEGylation, FITC labels, nucleosides, andsalts thereof). Specifically, the nucleic acid includes, but is not limited to, Miporensen, Alicafensen, Nusines, Volanesorsen, Custirsen, Apatorsen, Plazomib, 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_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_RxAt least one of (1). The term refers to the name or code number of the nucleic acid drug.

The water-soluble drug is preferably a water-soluble substance (e.g., peptide drug) containing at least one basic group, 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 analogs, calcitonin, insulin-like growth factor (IGF-I, IGF-II), cell growth factors (e.g., EGF, TGF- α, TGF- β, PDGF, FGF hydrochloride, basic FGF, etc.), glucagon-like peptides (e.g., GLP-1, GLP-2) and derivatives or analogs thereof, neurotrophic factors (e.g., NT-3, NT-4, CNTF, GDNF, BDNF, etc.), colony stimulating factors (e.g., CSF, GCSF, GMCSF, MCSF, etc.), and synthetic analogs, modifications, and pharmaceutically active fragments thereof. The derivative or analog of GLP-1 includes, but is not limited to exendin-3 and exendin-4.

The water-soluble drug containing at least one basic group is preferably at least one of peptide substances and derivatives and analogues thereof, wherein the peptide substances include but are not limited to glucagon (29 peptide), sertraline (29 peptide), aviptadil (28 peptide), secretin (27 peptide), ziconotide (25 peptide), teicoplanin (24 peptide), bivalirudin (20 peptide), somatostatin (14 peptide), terlipressin (12 peptide), goserelin (10 peptide), leuprorelin (10 peptide), triptorelin (10 peptide), nafarelin (10 peptide), gonadorelin (10 peptide), cetrorelix (10 peptide), degarelix (10 peptide), antipeptide (10 peptide), angiotensin (6-10 peptide), alarelin (9 peptide), buserelin (9 peptide), deserelin (9 peptide), octreotide (8 peptide), lanreotide (8 peptide), and derivatives and analogues thereof, At least one of bremerrandan (7 peptide), eptifibatide (7 peptide), halaprelin (6 peptide), spleen pentapeptide (5 peptide), thymic pentapeptide (5 peptide), elcalcitonin (31 peptide), somaglutide (31 peptide), glucagon-like peptide-1 (31 peptide), liraglutide (34 peptide), teriparatide (34 peptide), pramlintide (37 peptide), enfuvirtide (38 peptide), exenatide (39 peptide), adrenocorticotropin-releasing hormone (41 peptide), temorelin (44 peptide), lixisenatide (44 peptide), follitropin (118 peptide), dolapride (274 peptide), and albiglutide (645 peptide).

The peptide substance is preferably a polypeptide having not less than 30 amino acid residues. The derivatives and analogs of the peptide substances refer to products modified by at least one of water-soluble or water-insoluble groups or substances in polypeptides with no less than 30 amino acid residues and variants and analogs thereof, and have higher biological and pharmacological activity and stability or new functions or attributes.

The derivatives and analogs of the peptide drugs comprise at least one of glucagon-like peptides (such as GLP-1 and GLP-2) and derivatives and analogs thereof, including but not limited to exendin-3 and exendin-4 and at least one of variants and derivatives thereof.

By variant, analog is meant a peptide that differs by the substitution (or substitution), deletion, insertion, fusion, truncation, or any combination thereof of one or more amino acid residues of the amino acid sequence, and the variant polypeptide may be fully functional or may lack one or more functions. The analog exendin-4, such as glucagon-like peptide-1 (GLP-1), has a glycine at position 2 and alanine at position 2 of GLP-1, and exendin-4 binds to the GLP-1 receptor and produces cellular signaling cascades.

The water-soluble or water-insoluble group or substance is selected from at least one of polyethylene glycol and derivatives thereof, cyclodextrin, hyaluronic acid, short peptide, albumin, amino acid sequence, nucleic acid, gene, antibody, phosphoric acid, sulfonic acid, fluorescent dye, KLH, OVA, PVP, PEO, PVA, alkane, aromatic hydrocarbon, biotin, immunoglobulin, albumin, polyamino acid, gelatin, succinylated gelatin, acrylamide derivatives, fatty acid, polysaccharide, lipoamino acid, chitosan, and dextran. Polyethylene glycol and/or derivatives thereof are preferred, which may be branched, straight-chained, branched or dumbbell-shaped in structure. Derivatives of the polyethylene glycol include, but are not limited to, monomethoxypolyethylene glycol, methoxypolyethylene glycol propionate. The polyethylene glycols and derivatives thereof are either commercially available or prepared themselves by techniques well known to those skilled in the art.

The water-soluble or water-insoluble substance is modified into a modifier with an activated group and then coupled with the peptide substance derivative, wherein the activated group is selected from at least one of maleimide, halogen, vinyl sulfone, disulfide bond, sulfydryl, aldehyde group, carbonyl, O-substituted hydroxylamine, active ester, alkenyl, alkynyl, azido and other groups with high chemical reactivity; preferably, the activating group is selected from at least one of maleimide, halogen, vinyl sulfone, and disulfide bond; more preferably maleimide and/or disulfide bonds. The number of the activating groups carried on the polymer is one or more, and when the number of the activating groups is more than one, the activating groups may be the same or different.

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

The modifying agent having an activating group may be coupled to the peptide or a variant or the like thereof via an amino group, a carboxyl group, a hydroxyl group, a thiol group or the like in the amino acid sequence. Such a group is usually located at the N-terminus, C-terminus, side chain or any site of any one of amino acid residues such as Lys (lysine), Asp (aspartic acid), Glu (glutamic acid), Cys (cysteine), His (histidine), 4-mercaptoproline, Trp (tryptophan), Arg (arginine), Ala (alanine), Gly (glycine), Ser (serine) or Thr (threonine), preferably at a site containing a thiol group. Such as Exendin-4 and its analogues, at any one of the cysteine residues atpositions 2, 14, 21, 25, 28, 35, 38 or at any position or at any other position where the amino acid residue is replaced with a cysteine residue.

The modification of the peptide and the variant and the analogue thereof is random modification, location modification (specific modification), single-point modification or multi-point modification, and the single-point location modification is preferred.

The peptide and the variant and analogue thereof are prepared by a conventional polypeptide synthesis method, and comprise a solid-phase polypeptide synthesis method, a liquid-phase polypeptide synthesis method, a solid-liquid-phase polypeptide synthesis method and a recombination method; the reaction of the peptide and its variant, analog with the modifying agent is carried out in an aqueous solution or a buffered salt solution, the pH value of the reaction system is appropriately controlled, the modified product is monitored by HPLC, GPC or the like, and is separated and purified by ion exchange, gel chromatography or the like, concentrated and freeze-dried to obtain the target product.

The above-mentioned water-soluble drugs may be in free form or in the form of pharmaceutically acceptable salts, and the acids for forming the salts thereof may be inorganic acids or organic acids. The inorganic acid comprises hydrochloric acid, sulfuric acid and phosphoric acid, and the organic acid comprises 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 and carbonic acid; preferably hydrochloric acid, acetic acid, fumaric acid, maleic acid; acetic acid is more preferred.

The biodegradable and biocompatible water-insoluble polymer in step 1) comprises polyester, polycarbonate, polyacetal, polyanhydride, polyhydroxyfatty acid, and copolymer or blend thereof. In detail, the biodegradable and biocompatible polymer is Polylactide (PLA), Polyglycolide (PGA), poly (lactide-co-glycolide) (PLGA) and their copolymers with Polycaprolactone (PCL) or polyethylene glycol (PEG) (e.g., PLA-PEG, PLGA-PEG-PLGA, PLA-PEG-PLA, PEG-PCL, PCL-PLA-PCL, PCL-PLGA-PCL, PEG-PLA-PEG, PEG-PLGA-PEG), polycaprolactone and its copolymers with polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, polydioxanone (o), chitosan, ppd alginic acid and its salts, polycyanoacrylate, fibrin, polyanhydride, polyorthoester, polyamide, polyphosphazene, polyphosphate and their copolymers or mixtures; preferably PLA, PLGA and their copolymers with PCL or PEG, and mixtures thereof; more preferably PLA, PLGA or mixtures thereof.

The weight average molecular weight of the PLA, the PLGA and the copolymer of the PLA, the PLGA and the PCL or PEG is 20000-130000Da, preferably 25000-110000Da, and more preferably 30000-100000 Da. The weight average molecular weight used in the present specification is a value obtained by Gel Permeation Chromatography (GPC) measurement.

The viscosities of the PLA, PLGA and their copolymers with PCL or PEG (test conditions 0.5% (w/v), CHCl3, 25 ℃) are 0.18-1.0dL/g, preferably 0.22-0.9dL/g, more preferably 0.27-0.85 dL/g.

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

The PLA, PLGA and copolymers thereof 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 microparticles of the present invention may be a single polymer, or a mixture of a plurality of polymers, such as a combination of PLGAs having the same lactide/glycolide ratio and different carrying groups, a combination of PLGAs having the same molecular weight and carrying groups and different lactide/glycolide ratios, a combination of PLGAs having different molecular weights and carrying groups and different lactide/glycolide ratios, a combination of PLGAs having different molecular weights and carrying groups and different PLGAs/glycolide ratios, and a combination of PLGAs and PLA.

The organic solvent C can not dissolve water-soluble drugs, but can dissolve biodegradable and biocompatible water-insoluble polymers, has a boiling point lower than that of water, and is insoluble or poorly soluble in water. The organic solvent C can be a single organic solvent, or can be two or more miscible organic solvents. The organic solvent C is at least one 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 dichloromethane, chloroform, ethyl chloride, tetrachloroethylene, trichloroethylene, dichloroethane, trichloroethane, carbon tetrachloride, fluorocarbons, chlorobenzene (mono-, di-, tri-substituted), trichlorofluoromethane, etc.), 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 ether, methyl tert-butyl ether, methoxylated ether, alkyl ether, dihalogenated ether, trihalo ether, cyclic ether, crown ether, etc.), preferably halogenated aliphatic hydrocarbon solvents, more preferably at least one selected from dichloromethane and chloroform. The type and proportion of the organic solvent C in the inner oil phase are different according to different medicines and polymers, and are prepared according to actual conditions.

The concentration of the sparingly 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, its 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, while being incapable of dissolving both a water-soluble drug and a biodegradable and biocompatible water-insoluble polymer, is miscible with the oil solution while having good solubility for the surfactant. The organic solvent D may be a single organic solvent, or may be two or more organic solvents that are miscible. The organic solvent D is selected from at least one of anhydrous ether, cyclohexane, normal hexane, normal heptane and petroleum ether, preferably at least one of normal hexane, cyclohexane and normal heptane, the type and proportion of the organic solvent D are different according to different surfactants and oil solutions, and the organic solvent D is prepared according to actual conditions.

The organic solvent having a boiling point lower than that of water and insoluble or poorly soluble in water means an organic solvent which is miscible with water only at < 5% by volume and has a lower boiling point (less than or much less than 100 ℃) so as to be easily removed by, for example, lyophilization, evaporation or air blowing.

The emulsion is at a low temperature, which may be understood as 20 ℃ or less, preferably 15 ℃ or less, more preferably 6 ℃ or less.

The surfactant-containing oil solution (also referred to as the external oil phase) is at a low temperature, which may be understood as 18 ℃ or less, preferably 12 ℃ or less, more preferably 8 ℃.

The oil matrix of the oil solution containing the surfactant is at least one of any pharmaceutically acceptable polyol, vegetable oil, mineral oil and other oil in the field of pharmaceutical technology. The oil matrix may be a single component or two or more components that are miscible. The vegetable oil includes but is not limited to at least one of soybean oil, cotton seed oil, rapeseed oil, peanut oil, safflower oil, sesame oil, rice bran oil, corn germ oil, sunflower oil, poppy oil, olive oil, corn oil, cotton seed oil, coconut oil, linseed oil, castor oil and palm oil, and among these vegetable oils, at least one of soybean oil, peanut oil and castor oil is preferably used, and peanut oil is more preferably used; the mineral oil includes but is not limited to silicone oil, liquid paraffin; other oils include at least one of oils obtained by partial hydrogenation of vegetable oils (e.g., hydrogenated castor oil) and liquid saturated fatty acids (e.g., caproic acid, caprylic acid, etc.); the polyalcohol comprises glycerol and 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 globules during emulsification, avoid repolymerization of the globules, reduce the number of unencapsulated or partially encapsulated globule particles, and thus avoid the initial burst of 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 or a nonionic surfactant, more preferably an anionic surfactant.

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

The anionic surfactant includes, but is not limited to, at least one of phospholipids and derivatives thereof, glycerol esters, fatty acid esters, fatty alcohols, and other bile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid).

The anionic surfactant is preferably a phospholipid and derivatives thereof including, but not limited to, phosphatidylcholine (lecithin), phosphatidylethanolamine (cephalin), phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), glycerophosphatidic acid, lysophospholipids, soybean phospholipids, dipalmitoyl-phosphatidylcholine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and dimyristoyl-phosphatidylglycerol, and mixtures thereof. The phospholipids may be salted or unsalted, hydrogenated or partially hydrogenated, natural, semi-synthetic or fully synthetic. The phospholipid and its derivatives are preferably phosphatidylcholine, soybean phospholipid, and phosphatidylglycerol, more preferably soybean phospholipid.

The concentration of the surfactant (or stabilizer) in the oil matrix is generally between 0.05 and 10% by mass, preferably between 0.25 and 8% by mass, more preferably between 0.5 and 5% by mass.

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

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

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

(1) removing the organic solvent by heating, reducing pressure (or heating in combination);

(2) blowing ｃA gas stream, preferably dry nitrogen, over the liquid surface and controlling the contact areｃA of the liquid phase with the gas phase, the rate of stirring and circulation of the emulsion (e.g. JP-A-9-221418) to accelerate the volatilization of the organic solvent;

(3) organic solvents (e.g., W00183594) are rapidly removed with hollow fiber membranes, preferably silicone rubber pervaporation membranes (especially made from polydimethylsiloxane).

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

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

The ultrapure water used for washing in the step 4) can also contain inorganic salts (such as zinc salts) to reduce the permeation of water-soluble active substances into the water phase in the washing process and improve the drug encapsulation efficiency, wherein the mechanism is to improve the osmotic pressure of the external phase or reduce the solubility of the active substances in the external phase. For active substances such as polypeptides, proteins, nucleic acids, antibodies, antigens, antibiotics, etc., compounds containing zinc ions are desirable choices, including but not limited to zinc acetate, zinc chloride, zinc sulfate, zinc bisulfate, zinc nitrate, zinc gluconate, zinc carbonate, or any mixture thereof. The mass concentration of the inorganic salt in the ultrapure water is 0.01 to 3%, preferably 0.01 to 1.5%, more preferably 0.01 to 1%.

Preferably, the step 1) is performed by:

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

12) injecting the mixed solution into an organic solvent B or injecting the organic solution B into the mixed solution to generate uniform and fine precipitates, collecting the precipitates, washing the precipitates for a plurality of times by using the organic solvent B, and removing the organic solvent B to obtain a solid dispersion of the water-soluble drug and the water-insoluble polymer; wherein the organic solvent B is incapable of dissolving the poorly water-soluble polymer and the water-soluble drug.

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

The organic solvent B can not dissolve water-soluble drugs and biodegradable and biocompatible water-insoluble polymers at the same time. The organic solvent B may be a single organic solvent, or may be two or more organic solvents that are miscible with each other. The organic solvent B is at least one selected from anhydrous ether, hexane (including cyclohexane and n-hexane) and n-heptane, preferably at least one selected from anhydrous ether and hexane (including cyclohexane and n-hexane), and more preferably anhydrous ether. The types and the proportion of the organic solvents in the mixture are different according to different medicines and polymers, and can be prepared according to actual conditions.

The organic solvent A is controlled to be below the normal temperature or at a low temperature, wherein the normal temperature can be generally understood as 20 ℃, and is preferably 10-15 ℃; the low temperature is generally understood to be 10 ℃ or less, preferably 4 to 6 ℃ or less; the organic solvent B is controlled to a low temperature, which is generally understood to be 15 ℃ or less, preferably 10 ℃ or less, more preferably 6 ℃ or less; the temperature of the organic solvent A is 0 to 10 ℃ higher than that of the organic solvent B, preferably 3 to 8 ℃.

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

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

The step of removing the organic solvent B does not comprise a temperature raising procedure, and is carried out below normal temperature or at low temperature, wherein the normal temperature can be generally understood as 20-30 ℃, and is preferably 20-25 ℃; the low temperature is generally understood to be 15 ℃ or lower, preferably 10 ℃ or lower. Methods for removing the organic solvent include, but are not limited to, vacuum drying, freeze drying, fluidized drying.

Further, one or more auxiliary agents may be included in the sustained-release microparticles of the present invention. The preparation method of the sustained-release particles further comprises the step of adding an auxiliary agent, wherein the auxiliary agent is added in the process of preparing the solid dispersion in the step 1) or is added in the process of preparing the solid dispersion emulsion in the step 2); preferably, the solid dispersion is added when the solid dispersion emulsion is prepared in the step 2). The adjuvant is dissolved in the internal phase or suspended in the internal oil phase. The auxiliaries, when added, may be very fine powders having a particle size of less than 0.5. mu.m, preferably less than 0.1. mu.m, more preferably less than 0.05. mu.m.

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

The auxiliary agent includes but is not limited to at least one of saccharides, amino acids, fatty acids, alcohols, antioxidants and buffering agents.

The buffer includes, but is not limited to, salts of inorganic or organic acids, 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 croquette fringed pink, calcium oleate, calcium palmitate, calcium stearate, calcium phosphate, calcium acetate, magnesium carbonate, magnesium hydroxide, magnesium phosphate, magnesium myristate, magnesium oleate, magnesium palmitate, magnesium stearate, zinc carbonate, zinc hydroxide, zinc oxide, zinc croquette fringed pink, zinc oleate, zinc acetate, zinc chloride, zinc sulfate, zinc bisulfate, zinc nitrate, zinc gluconate, zinc palmitate, zinc stearate, zinc phosphate, sodium carbonate, sodium bicarbonate, sodium bisulfite, sodium thiosulfate, acetic acid-sodium acetate buffer salt, and any combination thereof. Preferred are zinc salts of inorganic or organic acids, more preferred is zinc chloride. The buffer is 0 to 5%, preferably 0.01 to 3%, more preferably 0.01 to 2% of the sum of the mass of the water-soluble drug and the mass of the poorly water-soluble polymer.

The antioxidants include, but are not limited to, at least one of t-butyl p-hydroxyanisole, dibutylphenol, tocopherol, isopropyl phaseolus vulgaris fringed pink, d-a tocopheryl acetate, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, hydroxycoumarins, butylated hydroxytoluene, dehydrofatty acid esters (e.g., ethyl ester, propyl ester, octyl ester, lauryl ester), propylhydroxybenzoate, trihydroxybutanone, vitamin E-TPGS, rho-hydroxybenzoate (e.g., methyl ester, ethyl ester, propyl ester, butyl ester). The antioxidant can effectively remove free radicals or peroxides in the sustained-release microparticles. The antioxidant is 0 to 1%, preferably 0 to 0.05%, more preferably 0 to 0.01% of the sum of the mass of the water-soluble drug and the mass of the poorly water-soluble polymer.

The saccharides include, but are not limited to, monosaccharides, oligosaccharides, and polysaccharides, and derivatives thereof. Specifically, the polysaccharide derivative includes, but is not limited to, at least one of trehalose, glucose, sucrose, glycerol, erythritol, arabitol, xylitol, sorbitol, mannitol, glucuronic acid, iduronic acid, neuraminic acid, galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid and salts thereof, chondroitin sulfate and salts thereof, heparin, inulin, chitin and derivatives thereof, dextrin, dextran, and alginic acid and salts thereof. Preferably at least one of sucrose, mannitol, and xylitol. The saccharide is 0.1-10%, preferably 0.5-8%, more preferably 1-6% of the sum of the mass of the water-soluble drug and the mass of the water-insoluble polymer.

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

The fatty acid includes 12-24 alkanoic acid and derivatives thereof, including but not limited to oleic acid, stearic acid, lauric acid, myristic acid, palmitic acid, arachidic acid, behenic acid, and ligninic acid, preferably at least one of stearic acid, 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 mass of the poorly water-soluble polymer.

The alcohols include, but are not limited to, polyethylene glycol. The molecular weight of the polyethylene glycol is 400-6000Da, preferably 400-4000Da, and more preferably 400-2000 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 mass of the water-insoluble polymer.

The preparation for injection requires sterility, and the specific sterilization method belongs to the general knowledge and technology of the technicians in this field, such as sterile operation, hot pressing, ethylene oxide or gamma ray to ensure the sterility of the preparation. The preparation of sustained-release fine particles according to the present invention is preferably performed aseptically such as filtration of an external phase aqueous solution with a cellulose acetate membrane, filtration of an acetic acid solution of PLGA with a polyether sulfone membrane, and filtration of methylene chloride with a polytetrafluoroethylene membrane, and all the equipment is easily closed and equipped with an organic solvent recovery device to prevent contamination of bacteria and diffusion of organic solvent into the air.

In another aspect, the invention also provides sustained-release microparticles prepared according to the preparation method of the sustained-release microparticles.

When the particles are used for injection administration, the particle size is too large, so that the needle is easily blocked, a larger injection needle head is needed, the pain of a patient is stronger, and the particle size is too small, so that the copolymer cannot well wrap the medicine, and a good slow release effect cannot be achieved. The sustained-release microparticles prepared in the present invention preferably have an average geometric particle size of less than 200 μm. The particle size of the sustained-release microparticles is 10-200 μm, preferably 10-150 μm, and more preferably 20-150 μm. The sustained-release microparticle particle size is measured by dynamic light scattering methods (e.g., laser diffraction methods), or microscopic techniques (e.g., scanning electron microscopy).

The sustained-release fine particles of the present invention can encapsulate a large amount of active ingredient, and the dose can be appropriately selected depending on the type and content of the active ingredient, the dosage form, the release duration, the administration subject, the administration route, the administration purpose, the target disease and symptom, and the like. However, the dosage may be considered satisfactory as long as the active ingredient can be maintained in vivo at a pharmaceutically effective concentration for the desired duration.

In the sustained-release microparticle of the present invention, the water-soluble drug is contained in an amount of about 1 to 40% by mass, preferably 3 to 35% by mass, and more preferably 5 to 30% by mass.

When ranges are stated herein, it is meant to encompass any range or combination of ranges therein.

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

When the microparticles are administered in the form of a suspension, they may be formulated with a suitable dispersing medium into a suspension formulation.

The dispersion medium comprises at least one of nonionic surfactant, polyoxyethylene castor oil derivative, cellulose thickener, sodium alginate, hyaluronic acid, dextrin and starch. Or alternatively, it can be combined with other components such as isotonic agent (such as sodium chloride, mannitol, glycerol, sorbitol, lactose, xylitol, maltose, galactose, sucrose, glucose, etc.), pH regulator (such as carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid or their salts, such as sodium carbonate, sodium bicarbonate, etc.), antiseptic (such as p-hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol, chlorobutanol, sorbic acid, boric acid, etc.) to make into aqueous solution, or solidified by freeze drying, drying under reduced pressure, spray drying, etc., and dissolved in water for injection to obtain dispersion medium for dispersing microparticles before use.

In addition, the sustained-release injection can also be obtained by the following method: the sustained-release microparticles are dispersed in a vegetable oil (such as sesame oil and corn oil) or a vegetable oil added with a phospholipid (such as lecithin), or in a medium-chain triglyceride to obtain an oily suspension.

In another aspect, the invention also provides the application of the solid dispersion and the sustained-release microparticles in an implantable sustained-release pharmaceutical composition.

The water-soluble drug sustained-release pharmaceutical composition prepared by the invention, in particular to a sustained-release pharmaceutical composition of protein, nucleic acid and peptide drugs, which can also be a rod-shaped object and a sheet-shaped object, and further, the invention also provides a preparation method of the implantable sustained-release pharmaceutical composition, which comprises the following steps:

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

secondly, the solid dispersion prepared in the step I is heated and then molded by a molding method, and the implanted medicine-releasing composition is prepared after cooling.

The molding method is not limited herein, and molding methods known to those skilled in the art can be used, such as compression molding, extrusion molding, and molding of -releasing composition into stick or tablet.

The slow released medicine composition for preparing water soluble medicine, especially protein and peptide medicine, is rod-shaped and sheet-shaped implanting agent.

Further, a preparation method of the implantable sustained-release pharmaceutical composition comprises the following steps:

preparing sustained-release particles according to the preparation method of the sustained-release particles;

secondly, the slow release particles prepared in the step I are prepared into the implanted medicine-releasing composition by a forming method which is well known to a person skilled in the art. the drug delivery composition may be in the form of stick or tablet.

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

The implant of the invention takes biodegradable and biocompatible materials as a substrate, has the appearance of thin rod shape, round rod shape or sheet shape (disc shape), can be implanted into the body by injection or operation, and does not need to be taken out by operation after the drug is completely released. The implant has the advantages of easily obtaining high encapsulation rate and drug loading rate, low burst release rate, and being capable of continuously releasing the active drug with the dosage required by treatment for one to several months at a stable speed, greatly reducing the medical cost and improving the compliance of patients.

The invention has the beneficial effects that: in the invention, the preparation of the sustained-release particles is carried out at normal temperature or low temperature in the whole process, which is very beneficial to high-temperature sensitive medicines, especially to the composition of polymer matrix prepared from protein, nucleic acid and peptide medicines, and compared with the disclosed technology, the preparation method can furthest keep the biological activity of active substances in the whole process; meanwhile, the prepared sustained-release particles have an excellent sustained-release effect close to zero level, the drug concentration is stable in the release period, and the defects that no drug is released at the early stage and the drug is released rapidly at the later stage of the particles obtained by the traditional S/O/W process for preparing the drug particles in advance are overcome; moreover, the sustained-release particles have higher drug loading rate and drug encapsulation rate.

After the sustained-release particles are administrated, active substances such as proteins, peptides, nucleic acids, alkaloids and the like can be continuously delivered in vivo for a period of time, and the release period is up to several weeks or months.

Drawings

FIG. 1 is the mean HbA of diabetic model mice to which the exenatide sustained release microparticles or the liraglutide sustained release microparticles prepared in examples 6 to 11 were administered_1cValue versus time graph.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. "microparticles" in the following examples, i.e., "sustained release microparticles"; "sustained release implants" are also implantable sustained release pharmaceutical compositions.

Example 1Preparation of Abeliglutide/PLGA microparticles

(I) Preparation of solid dispersions

0.90g of PLGA (molecular weight 25kDa, monomer proportion 65/35, carboxyl end group) was dissolved in about 6.00mL of glacial acetic acid, then 0.10g of albiglutide acetate was added, dissolved under vortexing, and then slowly poured into stirred anhydrous ether (6 ℃) to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24h (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 2Preparation of dolacilin/PLGA microparticles

(I) Preparation of solid dispersions

0.95g PLGA (molecular weight 30kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 7.92mL acetonitrile, then 0.05g dolabrin acetate was added, dissolved under vortex, and then slowly poured into stirred cyclohexane (6 ℃) to generate white precipitate, the white precipitate was collected and extracted with cyclohexane for about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24h (10 ℃) to obtain solid dispersion.

(II) preparation of microparticles

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

Example 3Preparation of follicle stimulating hormone/PLA microparticles

(I) Preparation of solid dispersions

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

(II) preparation of microparticles

The solid dispersion obtained in step I was uniformly dispersed in a mixture of about 5.39g of methylene chloride and chloroform to obtain an internal oil phase, and then the internal oil phase was injected into 410mL of a 0.25% (w/w) lecithin/soybean oil solution which had been previously thermostated to about 6 ℃ and emulsified using a wheel-type homomixer to prepare an S/O/O emulsion (lubrication speed about 5500rpm, 5 min). The S/O/O emulsion was transferred to a sealed glass flask and mechanical stirring was continued for about 3 hours (400rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 2500rpm, 5min) using a centrifuge. After the fine particles were washed with cyclohexane about 5 times, they were dispersed again in ultrapure water (5 ℃ C.) and washed about 2 times, and then collected by centrifugation and freeze-dried with a freeze-dryer to obtain fine particles. The resulting microparticles were found to have an FSP content of 2.73% and a particle size of 30-82 μm.

Example 4Preparation of lixisenatide/PLGA microparticles

(I) Preparation of solid dispersions

0.99g of PLGA (molecular weight 22kDa, monomer proportion 90/10, carboxyl end group) was dissolved in about 5.50mL of trichloroacetic acid, then 0.01g of lixisenatide acetate, 0.05g of xylitol and 0.03g of zinc carbonate were added, dissolved with swirling, then slowly poured into n-heptane (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with n-heptane about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to give a solid dispersion.

(II) preparation of microparticles

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

Example 5Preparation of corticotropin releasing hormone/PLGA microparticles

(I) Preparation of solid dispersions

0.85g of PLGA (molecular weight 25kDa, monomer proportion 85/15, carboxyl end group) was dissolved in about 8.50mL of a mixture of glacial acetic acid and acetonitrile, 0.15g of adrenocorticotropic hormone releasing acetate was added and dissolved by vortexing, and then the mixture was slowly poured into a stirred mixture of anhydrous ether and cyclohexane (6 ℃) to produce a white precipitate, which was collected and extracted with the mixture of anhydrous ether and cyclohexane for about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 6Preparation of Exenatide/PLGA microparticles

(I) Preparation of solid dispersions

0.95g of PLGA (molecular weight 35kDa,monomer proportion 75/25, carboxyl end group) was dissolved in about 6.33mL of glacial acetic acid, then 0.05g of exenatide acetate and 0.08g of xylitol were added, dissolved by vortexing, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 7Preparation of liraglutide/PLGA microparticles

(I) Preparation of solid dispersions

0.93g of PLGA (molecular weight 40kDa, monomer proportion 65/35, carboxyl end group) was dissolved in about 7.75mL of glacial acetic acid, then 0.07g of liraglutide acetate and 0.06g of xylitol were added, dissolved with vortex, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 8Preparation of Exenatide/PLGA microparticles

(I) Preparation of solid dispersions

0.90g of PLGA (molecular weight 45kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 9.00mL of glacial acetic acid, then 0.10g of exenatide acetate and 0.04g of xylitol were added, dissolved by vortexing, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 9Preparation of liraglutide/PLGA microparticles

(I) Preparation of solid dispersions

0.86g of PLGA (molecular weight 50kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 10.75mL of glacial acetic acid, then 0.14g of liraglutide acetate and 0.02g of xylitol were added, dissolved with vortex, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24h (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 10Preparation of Exenatide/PLGA microparticles

(I) Preparation of solid dispersions

0.82g of PLGA (molecular weight 55kDa, monomer proportion 50/50, terminal carboxyl group) was dissolved in about 11.71mL of glacial acetic acid, then 0.18g of exenatide acetate and 0.01g of xylitol were added, dissolved under vortex, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 11Preparation of liraglutide/PLGA microparticles

(I) Preparation of solid dispersions

0.80g of PLGA (molecular weight 60kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 13.33mL of glacial acetic acid, then 0.20g of liraglutide acetate was added, dissolved under vortex, and then slowly poured into stirred anhydrous ether (6 ℃) to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24h (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 12Preparation of Enfuvirdine/PLGA microparticles

(I) Preparation of solid dispersions

0.75g of PLGA (molecular weight 65kDa, monomer proportion 65/35, carboxyl end group) was dissolved in about 15.00mL of glacial acetic acid, then 0.25g of enfuvirdine acetate, 0.03g of sucrose and 0.01g of stearic acid were added, dissolved with swirling, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 13Preparation of pramlintide/PLGA microparticles

(I) Preparation of solid dispersions

0.70g of PLGA (molecular weight 70kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 17.50mL of glacial acetic acid, then 0.30g of pramlintide acetate and 0.02g of mannitol were added, dissolved with vortex, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 14Preparation of teriparatide/PLGA microparticles

(I) Preparation of solid dispersions

0.65g of PLGA (molecular weight 85kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 21.67mL of glacial acetic acid, then 0.35g of teriparatide acetate, 0.03g of mannitol and 0.03g of PEG-400 were added, dissolved with vortex, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 15 liraglutidePreparation of PLGA microparticles

(I) Preparation of solid dispersions

0.60g of PLGA (molecular weight 100kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 30.00mL of glacial acetic acid, then 0.40g of liraglutide acetate and 0.005g of xylitol were added, dissolved with vortex, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 16Preparation of somaglutide/PLGA microparticles

(I) Preparation of solid dispersions

0.50g of PLGA (molecular weight 110kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 50.00mL of glacial acetic acid, then 0.50g of somaglutide and 0.001g of xylitol were added, dissolved with vortexing, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24h (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

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

(I) Preparation of solid dispersions

0.50g of PLGA (molecular weight 130kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 50.00mL of glacial acetic acid, 0.50g of glucagon-like peptide-1 acetate was added, dissolved under vortex, and then slowly poured into stirred anhydrous ether (6 ℃) to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

The solid dispersion obtained in step I was dispersed homogeneously in about 50.00g of methylene chloride to obtain an internal oil phase, which was then injected into 3L of a 10% (w/w) lecithin/peanut oil solution which had been previously thermostated to about 20 ℃ and emulsified using a wheel homogenizer to prepare an S/O/O emulsion (lubrication speed of about 7000rpm, 5 min). The S/O/O emulsion was transferred to a sealed glass flask and mechanical stirring was continued for about 5 hours (800rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 4000rpm, 5min) using a centrifuge. After the fine particles were washed with n-heptane about 5 times, they were again dispersed in ultrapure water (5 ℃ C.) and washed about 2 times, and then collected by centrifugation and freeze-dried with a freeze-dryer to obtain fine particles. The resulting microparticles were found to have a glucagon-like peptide-1 content of 46.21% and a particle size of 19-85 μm.

Example 18Preparation of Exendin-4 derivative/PLGA microparticles

(I) Preparation of solid dispersions

The solid dispersion comprises the following components in percentage by mass: water-soluble drugs: 20% of Exendin-4 derivative, water-insoluble polymer: PLGA 79.5%, adjuvant: 0.5 percent of xylitol; wherein the PLGA has a molecular weight of 50kDa, wherein the ratio of lactide to glycolide is 50/50, and the PLGA has terminal carboxyl groups.

(1) Preparation of Exendin-4 derivatives: preparing 10kDa PEG-NHS ester, then reacting with asparagine atposition 28 in Exendin-4 in PBS buffer solution, separating and purifying by ion exchange and gel chromatography, concentrating and freeze-drying to obtain the Exendin-4 derivative.

(2) Completely dissolving the water-insoluble polymer in glacial acetic acid, and then adding the water-soluble medicine and the auxiliary agent until the water-soluble medicine and the auxiliary agent are completely dissolved; wherein the water-insoluble polymer accounts for 6.5 percent of the mass of the glacial acetic acid; then, anhydrous ether (6 ℃ C.) was poured in to cause a white precipitate to be generated, the precipitate was collected and extracted 5 times with anhydrous ether, and the precipitate was collected and dried in a vacuum oven for 24 hours (10 ℃ C.) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 19Preparation of Exendin-4 derivative/PLGA microparticles

(I) Preparation of solid dispersions

The solid dispersion comprises the following components in percentage by mass: water-soluble drugs: 15% of Exendin-4 derivative, water-insoluble polymer: PLGA 84%, adjuvant: 1% of xylitol; wherein the PLGA has a molecular weight of 50kDa, wherein the ratio of lactide to glycolide is 50/50, and the PLGA has terminal carboxyl groups.

(1) Preparation of Exendin-4 derivatives: preparing an Exendin-4 variant with the asparagine at the 28 th position in the Exendin-4 replaced by cysteine by a solid phase polypeptide synthesis method, then reacting with 10kDa Y-type monomethoxypolyethylene glycol-maleimide in a PBS buffer solution, separating and purifying by ion exchange and gel chromatography, concentrating and freeze-drying to obtain the Exendin-4 derivative.

(2) Completely dissolving the water-insoluble polymer in glacial acetic acid, and then adding the water-soluble medicine and the auxiliary agent until the water-soluble medicine and the auxiliary agent are completely dissolved; wherein the water-insoluble polymer accounts for 6.5 percent of the mass of the glacial acetic acid; then, anhydrous ether (6 ℃ C.) was poured in to cause a white precipitate to be generated, the precipitate was collected and extracted 5 times with anhydrous ether, and the precipitate was collected and dried in a vacuum oven for 24 hours (10 ℃ C.) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 20Preparation of Exendin-4 derivative/PLGA microparticles

(I) Preparation of solid dispersions

The solid dispersion comprises the following components in percentage by mass: water-soluble drugs: 20% of Exendin-4 derivative, water-insoluble polymer: PLGA 78%, adjuvant: 2% of sorbitol; wherein the PLGA has a molecular weight of 55kDa, wherein the ratio of lactide to glycolide is 50/50, and the PLGA has terminal carboxyl groups.

(1) Preparation of Exendin-4 derivatives: preparing an Exendin-4 variant with cysteine substituted for arginine at position 20 in Exendin-4 by a solid phase polypeptide synthesis method, then reacting with 5kDa monomethoxy polyethylene glycol-maleimide in a PBS buffer solution, separating and purifying by ion exchange and gel chromatography, concentrating and freeze-drying to obtain the Exendin-4 derivative.

(2) Completely dissolving the water-insoluble polymer in glacial acetic acid, and then adding the water-soluble medicine and the auxiliary agent until the water-soluble medicine and the auxiliary agent are completely dissolved; wherein the water-insoluble polymer accounts for 6% of the mass of the glacial acetic acid; then, anhydrous ether (6 ℃ C.) was poured in to cause a white precipitate to be generated, the precipitate was collected and extracted 5 times with anhydrous ether, and the precipitate was collected and dried in a vacuum oven for 24 hours (10 ℃ C.) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 21Preparation of Exendin-4 derivative/PLGA microparticles

(I) Preparation of solid dispersions

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

(1) Preparation of Exendin-4 derivatives: an Exendin-4 variant with the methionine at the 14 th position replaced by cysteine in the Exendin-4 is prepared by a solid phase polypeptide synthesis method, then the Exendin-4 variant reacts with 20kDa monomethoxy polyethylene glycol-maleimide in a PBS buffer solution, and the Exendin-4 derivative is obtained by ion exchange, gel chromatography separation and purification, concentration and freeze drying.

(2) Completely dissolving the water-insoluble polymer in glacial acetic acid, and then adding the water-soluble medicine and the auxiliary agent until the water-soluble medicine and the auxiliary agent are completely dissolved; wherein the water-insoluble polymer accounts for 7% of the mass of the glacial acetic acid; then, anhydrous ether (6 ℃ C.) was poured in to cause a white precipitate to be generated, the precipitate was collected and extracted 5 times with anhydrous ether, and the precipitate was collected and dried in a vacuum oven for 24 hours (10 ℃ C.) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 22Preparation of Exendin-4 derivative/PLGA microparticles

The solid dispersion comprises the following components in percentage by mass: water-soluble drugs: 12% of Exendin-4 derivative, and water-insoluble polymer: PLGA 84%, adjuvant: 4% of xylitol; wherein the PLGA has a molecular weight of 40kDa, wherein the ratio of lactide to glycolide is 50/50, and the PLGA has terminal carboxyl groups.

(1) Preparation of Exendin-4 derivatives: preparing an Exendin-4 variant with cysteine substituted for glycine at position 2 in Exendin-4 by a solid-phase polypeptide synthesis method, then reacting with 40kDa monomethoxy polyethylene glycol-maleimide in a PBS buffer solution, separating and purifying by ion exchange and gel chromatography, concentrating and freeze-drying to obtain the Exendin-4 derivative.

(2) Completely dissolving the water-insoluble polymer in glacial acetic acid, and then adding the water-soluble medicine and the auxiliary agent until the water-soluble medicine and the auxiliary agent are completely dissolved; wherein the water-insoluble polymer accounts for 6.5 percent of the mass of the glacial acetic acid; then, anhydrous ether (6 ℃ C.) was poured in to cause a white precipitate to be generated, the precipitate was collected and extracted 5 times with anhydrous ether, and the precipitate was collected and dried in a vacuum oven for 24 hours (10 ℃ C.) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 23Preparation of Mipomersen/PLGA microparticles

(I) Preparation of solid dispersions

0.80g of PLGA (molecular weight 30kDa, monomer proportion 50/50, carboxyl end group) was dissolved in about 6.53mL of a mixture of glacial acetic acid and acetonitrile, then 0.20g of Miporesen sodium and 0.01g of xylitol were added, dissolved with vortexing, and then slowly poured into anhydrous ether (6 ℃) under stirring to produce a white precipitate, which was collected and extracted with n-hexane about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

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

Example 24Preparation of interleukin/PLGA microparticles

(I) Preparation of solid dispersions

0.82g of PLGA (molecular weight 35kDa, monomer proportion 50/50, terminal carboxyl group) was dissolved in about 6.12mL of glacial acetic acid, then 0.18g of interleukin and 0.02g of xylitol were added, dissolved with vortexing, and then slowly poured into anhydrous ether (6 ℃) with stirring to produce a white precipitate, which was collected and extracted with anhydrous ether about 5 times, and the precipitate was collected and dried in a vacuum drying oven for 24 hours (10 ℃) to obtain a solid dispersion.

(II) preparation of microparticles

The solid dispersion obtained in step I was dispersed homogeneously in a mixture of about 6.12g of methylene chloride and chloroform to obtain an internal oil phase, and then the internal oil phase was injected into 500mL of a 0.5% (w/w) lecithin/soybean oil solution which had been previously thermostated to about 5 ℃ and an S/O/O emulsion (1000rpm, 5min) 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 (500rpm) to solidify the microparticles, and then the microparticles were collected by centrifugation (about 3500rpm, 5min) using a centrifuge. The microparticles were washed with a mixed solution of n-heptane and n-hexane for about 5 times, then dispersed again in ultrapure water (5 ℃) and washed for about 2 times, and then collected by centrifugation and freeze-dried with a freeze-dryer to obtain microparticles. The content of interleukin in the obtained fine particles was found to be 16.36%, and the particle size of the fine particles was found to be 31 to 114 μm.

Example 25Preparation of exenatide/PLGA sustained-release implant

The dried solid dispersion prepared in step I of example 10 was filled in a 1mm x 10mm mold (inner cavity is cylindrical, round bottom diameter is 1mm, depth is about 10mm), and after warming to about 43 ℃, compression molding was performed to obtain a cylindrical (1mm x 5.31mm) exenatide sustained release implant. The liraglutide content in the resulting implants was determined to be 17.24%.

Example 26Preparation of exenatide/PLGA sustained-release implant

The microparticles obtained in step II of example 10 were fed into a hot-melt extruder, hot-melt extruded into a stick having a diameter of about 1mm, cooled and cut to give an exenatide sustained release implant having a length of about 5 mm. The liraglutide content in the resulting implants was determined to be 17.03%.

The method for analyzing the drug loading rate and the drug encapsulation rate of the microparticles or the implants in the above embodiments comprises: 5mg of microparticles or implants were dissolved in 50mL of Acetonitrile (ACN), 500. mu.L of 0.1% TFA was added thereto, and after thorough mixing, the supernatant was centrifuged to analyze the drug concentration by high performance liquid chromatography. The ratio of the total mass of the encapsulated medicine in the particles (or the implant) to the dosage is the encapsulation rate of the medicine, and the ratio of the mass of the encapsulated medicine in the particles (or the implant) to the mass of the particles (or the implant) is the loading rate of the medicine. All experiments were repeated 3 more times.

The particle size analysis method of the microparticles in the above examples was: about 10mg of microparticles were dispersed in liquid paraffin, dispersed by sonication for about 30s, and measured using a laser particle size analyzer from Beckman Coulter.

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

The sustained release microparticles and the implant prepared in the above examples are subjected to burst release and in vitro release curve measurement, and the measurement method comprises the following steps: 20mg of drug-containing particles or implants are precisely weighed and placed in a 15mL centrifuge tube, phosphate buffer solution with the pH value of 7.4 (containing 0.02 percent of sodium azide as bacteriostatic agent) is used as release medium and placed in a constant temperature gas bath shaking table, and the in-vitro release degree of the particles and implants is measured under the conditions of oscillation speed of 100rpm and temperature of 37 +/-0.5 ℃. Taking out all release media and supplementing new release media with the same amount at l day, 2 days, 7 days, 14 days, 21 days, 28 days, 40 days, 50 days and 60 days respectively, and determining the drug release amount by high performance liquid chromatography, wherein the determination method comprises the following steps:

liquid chromatograph: agilent 1260;

a chromatographic column: proteonavi 4.6X 250 mm;

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 cumulative in vitro Release Rate results for Slow Release microparticles and implants

As can be seen from the in vitro release results in table 1, the sustained release microparticles prepared by using the solid dispersion of the present invention and the implant prepared therefrom have no burst release phenomenon or significant delayed release phenomenon, and the whole release trend is close to zero-order release. Wherein, the in vitro release period of some samples is as long as 40-50 days, the in vitro release period of some samples is as long as 50-60 days, and the in vitro release period of some samples exceeds 60 days, so that the sustained-release effect is excellent.

Example 28Fine particle penetration test

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

TABLE 2 fine particle penetration test results

Note: the + push pin has good smoothness, the + push pin has general smoothness, -the-push pin has blocking feeling, -the-needle is blocked.

The needle penetration results in table 2 show that the fine particle suspensions of different particle sizes prepared by the present invention are all fine particles sucked into the syringe through the 30-gauge needle and the contents of the syringe are completely injected into pork without blocking or clogging the needle, indicating that the fine particles of the present invention can be administered by subcutaneous or intramuscular injection.

Example 29 organic solvent residual amount measurement test

The residual amounts of the organic solvent a, the organic solvent B, the organic solvent C and the organic solvent D in the solid dispersions and sustained-release fine particles prepared in examples 1 to 24 of the present invention were measured by a known measurement method. The measurement results are shown in Table 3.

TABLE 3 measurement results of the residual amount of organic solvent

Note: -means no detection or a content below the detection limit.

As can be seen from the results of the residual amounts of organic solvents in Table 3, the solid dispersion and sustained-release microparticles prepared according to the present invention have very low, or no detectable, or residual amounts below the detectable range, and thus have no side effects on patients after administration due to organic solvents, and are also advantageous in maintaining the stability of the microparticles and prolonging the shelf life.

Example 30Animal testing

56 diabetes model mice with weight of 20 +/-5 g and half of male and female were selected and divided into an administration group (6 group) and a blank group (1 group) at random, each 8 mice in the administration group were injected subcutaneously into the neck and the back of the neck of each mouse with exenatide or liraglutide of examples 6 to 11, the microparticles were suspended with a diluent containing 3% carboxymethylcellulose and 0.9% NaCl, each mouse in the administration group was injected with 2mg/kg of exenatide or 10mg/kg of liraglutide, and the blank group was injected subcutaneously with the same volume of physiological saline. Blood glucose was measured by collecting blood from the tail vein at the same time of 0d, 0.5d, 1d, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, and 70d of administration, and mean HbA was prepared_1cThe results of the value (percentage of glycated hemoglobin in total hemoglobin,%) and time (d) are plotted in FIG. 1.

As can be seen from the graph of FIG. 1, the Exenatide sustained-release microparticles or liraglutide sustained-release microparticles prepared in examples 6 to 11 of the present invention can well control HbA within 70 days after administration_1cValue, and HbA within 7-70 days after administration_1cThe value is between 5 and 7 and is obviously lower than that of the blank group, which shows that the exenatide sustained release particles or the liraglutide sustained release particles can release the active drug for a long time after being administrated, achieve ideal treatment effect, reduce administration frequency and be beneficial to improving the compliance of patients.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. A method for preparing sustained-release microparticles, which is characterized in that: the method comprises the following steps:

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

2) dissolving the solid dispersion prepared in the step 1) in an organic solvent C to form a solid dispersion emulsion, wherein the organic solvent C is an organic solvent which can not dissolve the water-soluble drug but can dissolve the water-insoluble polymer and has a boiling point lower than that of water and can not dissolve or hardly dissolve water;

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

4) solidifying the particles in the emulsion by solvent volatilization or solvent extraction, collecting the particles, washing the particles for a plurality of times by using an organic solvent D, washing the particles for a plurality of times by using ultrapure water to remove the surfactant attached to the surfaces of the particles, and drying the particles to obtain the slow-release particles; wherein the organic solvent D is not capable of dissolving a water-soluble drug and a poorly water-soluble polymer, is miscible with the oil solution, and has good solubility for the surfactant;

the water-soluble medicine is at least one of protein medicine, peptide medicine and nucleic acid medicine;

the step 1) is implemented by the following steps:

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

12) injecting the mixed solution into an organic solvent B or injecting the organic solvent B into the mixed solution to generate uniform and fine precipitates, collecting the precipitates, washing the precipitates for a plurality of times by using the organic solvent B, and removing the organic solvent B to obtain a solid dispersion of the water-soluble drug and the water-insoluble polymer; wherein the organic solvent B is incapable of dissolving the poorly water-soluble polymer and the water-soluble drug;

the organic solvent A is selected from at least one of glacial acetic acid, acetonitrile, trifluoroacetic acid and dimethyl sulfoxide;

the organic solvent B is at least one of anhydrous ether, hexane and n-heptane;

in the solid dispersion, the mass ratio of the water-soluble drug to the water-insoluble polymer is 1: 1-1: 99;

the organic solvent C is at least one selected from aliphatic hydrocarbon, halogenated hydrocarbon, fatty acid ester, aromatic hydrocarbon and ether;

the organic solvent D is at least one selected from anhydrous ether, cyclohexane, n-hexane, n-heptane and petroleum ether;

the surfactant is lecithin;

the oil solution is at least one selected from polyalcohol, vegetable oil and mineral oil.

2. The method for producing sustained-release microparticles according to claim 1, wherein: the water-soluble drug is polypeptide with no less than 30 amino acid residues and at least one product modified by water-soluble or water-insoluble groups or substances.

3. The method for producing sustained-release microparticles according to claim 2, wherein: the water-soluble or water-insoluble group or substance is polyethylene glycol.

4. The method for producing sustained-release microparticles according to claim 1, wherein: the water-insoluble polymer in the step 1) is at least one of polylactide, polyglycolide, lactide-glycolide copolymer and copolymer of the polylactide, the polyglycolide or the polyethylene glycol, polycaprolactone and copolymer of the polycaprolactone and the polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, polydioxanone, chitosan, alginic acid and salt thereof, polycyanoacrylate, fibrin, polyanhydride, polyorthoester, polyamide, polyphosphazene, polyphosphoester, copolymer and/or mixture of the polycaprolactone and the polyethylene glycol.

5. The method for producing sustained-release microparticles according to claim 1, wherein: the method also comprises a step of adding an auxiliary agent, wherein the auxiliary agent is added in the process of preparing the solid dispersion in the step 1) or is added in the process of preparing the solid dispersion emulsion in the step 2); the auxiliary agent is 0.01-10% of the sum of the mass of the water-soluble drug and the mass of the water-insoluble polymer.

6. The method for producing sustained-release microparticles according to claim 5, wherein: the auxiliary agent is at least one of amino acid, antioxidant, buffering agent, saccharide, fatty acid and alcohol.

7. The sustained-release microparticle produced by the method for producing sustained-release microparticles according to any one of claims 1 to 6.

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