CN105878191A - Sustained-release microgranules, method for preparing same and application of sustained-release microgranules - Google Patents

Abstract

The invention discloses a method for preparing sustained-release microgranules. The method includes steps of 1), preparing solid dispersion of biodegradable and biocompatible water-insoluble polymer and water-soluble drug; 2), dissolving the prepared solid dispersion in an organic solvent C to form solid dispersion emulsion; 3), injecting the obtained solid dispersion emulsion into oil solution with surfactants to form uniform emulsion; 4), curing microgranules in the emulsion by means of solvent evaporation or solvent extraction, and collecting, washing and drying the microgranules to obtain the sustained-release microgranules. The invention further discloses the sustained-release microgranules prepared by the aid of the method and application of the sustained-release microgranules to implantable sustained-release pharmaceutical compositions. The method, the sustained-release microgranules and the application have the advantages that full procedures for preparing the sustained-release microgranules by the aid of the method are carried out at the normal temperature or the low temperatures, and accordingly the method is quite favorable for preparing polymer matrix compositions from high-temperature-sensitive drugs; excellent similarly zero-level sustained-release effects can be realized by the prepared sustained-release microgranules, and the drugs are stable in concentration in sustained-release period.

Description

The preparation method of sustained-release microparticle, prepared sustained-release microparticle and application thereof

Technical field

The invention belongs to water soluble drug field, particularly relate to the preparation method of sustained-release microparticle, prepared sustained-release microparticle and slow release micro- Grain application in implanted sustained release pharmaceutical composition.

Background technology

In recent years, substantial amounts of biological activity class material such as oligopeptide, polypeptide and albumen obtain a large amount of concern as drug candidate, its The condition of illness (cancer, anemia, multiple sclerosis, hepatitis etc.) that treatment is serious plays an important role.But, these Large molecule active comparison of ingredients is fragile, because they poor stabilities in the gastrointestinal tract (easily decline at low pH or proteolysis Solve), circulating half-life is shorter, and they are poor through the permeability of intestinal wall, thus causes bioavailability the lowest, therefore difficult With oral administration.Injection or Parenteral are still polypeptide and the preferred route of administering of albumen isoreactivity composition.Can be by quiet A lot of preparations of arteries and veins, muscle or the peptide of subcutaneous route injection and protein have listed or in the middle of research and development, such as leuprorelin slow release Microgranule, goserelin slow-release implant, triptorelin sustained-release microparticle etc..

For many peptide reagents, particularly hormone, needing with the controlled long-term successive administration of speed, these active component are in target group Systemic concentrations needed for knitting or producing intended effect on organ is the highest, so that obtain treatment by injection high dose continually Concentration required in effectiveness window, this often results in general toxicity prejudicial to patient.Meanwhile, drug administration by injection is pain, because of This, complying with of patient is low, weak curative effect, and side effect is big.These problems can be by the long-acting biography of active component based on polymer Delivery system (Drug Delivery System, DDS) is addressed, and this system is by being encapsulated into biodegradable by active component With in biocompatible polymeric matrix, make microcapsule, microparticle or the form of transplantation bar, make active component steady in a long-term Discharge, thus reach to release the purpose of controlled release.

It has been reported that microgranule preparation method have multiple, such as solvent evaporation method (solvent evaporation), coacervation (coacervation), spray drying method and atomizing freeze drying method etc..Wherein using most is solvent evaporation method in aqueous phase, It can be subdivided into again single emulsion process (Oil/Water, O/W；Water/Oil, W/O) and double emulsion method (Water/Oil/Water, W/O/W；Water/Oil/Oil, W/O/O).

A kind of double emulsion method (such as CN102245210A, CN1826170B) of improvement, has directly been suspended in polypeptide powder Machine mutually in, formed oil Bao Gu (S/O) suspension, then this suspension is distributed in aqueous phase, formed S/O/W emulsion Liquid.Due to polypeptide powder typically insoluble in middle organic phase solvent, the method can avoid aqueous phase in the double emulsion method of Wl/O/W2 The outwards diffusion of aqueous phase, and then improve envelop rate.

In S/O/W method, the size of pre-prepared active material particle is particularly significant, when solid protein granule diameter from 5 microns when increasing to 20 microns, not only the rate of release at initial stage is double, and its microencapsulation rate drops to 20% from 80%.Generally The protein that obtains, polypeptide lyophilized powder end mean diameter are in 10-1000 μm, such as, and typical protein, polypeptide lyophilization Powder average particle size is about in 10-500 μm.S/O is formed in organic solvent suspended if directly suspended with the powder of the biggest particle diameter Liquid, then the application double emulsion method of S/O/W prepares microgranule, and medicine will can not get well encapsulating, and cause envelop rate low, or Release result unsatisfactory (early stage burst drug release is relatively big, and later stage release amount of medicine is not enough), or the diameter of particle of preparation is too Greatly, it is difficult to drug administration by injection；Meanwhile, the shape of drug particles also affects the shape of microgranule.Therefore, typically to use someway In advance drug powder mean diameter is reduced to 1-10 μm, then this powder is used for the preparation of S/O/W double emulsion method and releases microgranule (USP 6270700；Takada S,et al.Journal of Controlled Release.2003,88(2):229-42).

But, the medicine of preparation small particle often to pass through grinding, spray drying, Ultrasonic Pulverization, comminution by gas stream, crystallization process etc. Method (such as CN1494900B) complex process, and it is easily caused active component inactivation.Polishing be limited to cause dust, Heavy metal pollution and grind thermally-induced protein denaturation；Spray drying method perhaps can provide sufficiently small protein body, but It is that the interfacial tension between the high shear force near nozzle and liquid-to-air can make albuminous degeneration, additionally, be spray-dried or atomizing Lyophilization must use surfactant, and surfactant causes protein mutual with solvent in next step preparation program Effect.Meanwhile, if the complexity of granule technique is prepared in reduction, sacrifice size to ensure the activity of medicine, preparation Grain particle diameter relatively small particle smaller (i.e. particle radius is more much bigger than polymer layer of thickness), then there will be each microgranule only wrap up one or The situation of little several active material particle, thus cause the few drug release of early stage, and quickly release released by later stage medicine, reaches not To slow release effect.

The double emulsion method of a kind of improvement (CN 102233129 B, CN 102871969 A, CN 102266294 B etc.), by activity Material and one or more additives (such as glucosan, Polyethylene Glycol, sodium alginate etc.) prepare the granule of small particle, then use Organic solvent washes away all or part of additive, it is thus achieved that the little granule of active substance of porous, half hollow out or hollow out, uses the most again S/O/hO technique prepares microgranule.Such a process increases and prepare the step that this step of little granule is more complicated, and need with organic molten Agent removes additive therein.Further, these additives are mostly water-soluble substances, without removing completely, easily affect Releasing effect, accelerates active substance release, or forms gel (such as glucosan, the sodium alginate of high molecular), may cause Microgranule rises brokenly, drug release postpones or release is the most thorough.

The preparation method (US5556642) further optimized, is dissolved in cosolvent by water-soluble actives and polymer, then Obtain solid dispersion by evaporative removal organic solvent, then solid dispersion is dissolved in organic solvent, by O/W legal system Standby microgranule.This method overcomes traditional S/O/W method early stage does not has drug release, and the shortcoming drastically discharged released by later stage medicine. But, volatile organic solvent is prepared the technique of solid and is unfavorable for thermally sensitive active substance, easily causes its degeneration；If Volatile organic solvent under lower temperature, owing to solvent volatilization then can cause active substance segregation when solidification relatively slowly, is dried After solid dispersion in active substance also can exist with bigger volume, such as bulk, banding, thread, to follow-up prepare micro- Grain technique causes difficulty or causes waste, also results in release instability.

Therefore, it is an object of the invention to, it is provided that a kind of without previously prepared small particle drug powder, volatilized by emulsifying-solvent Compositions is released in method preparation, and technics comparing simply and can guarantee that again the biological activity of active substance, preferable envelop rate and excellence The method releasing effect.

Summary of the invention

On the one hand, for solving problems of the prior art, the invention provides the preparation method of a kind of sustained-release microparticle, this side The preparation whole process room temperature of method or low temperature, the medicine for sensitive is highly beneficial, it is possible to keep active substance to the full extent Biological activity.

The technical solution used in the present invention is: the preparation method of a kind of sustained-release microparticle, comprises the following steps:

1) solid dispersion of water soluble drug and biodegradable and biocompatible slightly water-soluble polymer is prepared；

2) by step 1) solid dispersion prepared is dissolved in organic solvent C, forms solid dispersion emulsion (interior oil phase), Described organic solvent C for can not dissolve described water soluble drug but can dissolve described slightly water-soluble polymer, boiling point less than water and Do not dissolve in or be insoluble in the organic solvent of water；

3) by step 2) the solid dispersion emulsion that obtains injects in the oil solution (outer oil phase) containing surfactant and formed all Even emulsion；

4) volatilized by solvent or solvent extraction make the microgranule in emulsion solidify, collect microgranule, wash for several times with organic solvent D, Again with milli-Q water for several times, to remove the surfactant being attached to microparticle surfaces, then it is dried, it is thus achieved that described slow release Microgranule；Wherein, described organic solvent D can not dissolve water soluble drug and slightly water-soluble polymer, and it can mix with described oil solution Molten, described surfactant there is is good dissolubility simultaneously；

Described water soluble drug is alkaline matter, at least one in material containing basic group and their salt.

Described water soluble drug includes polypeptide, albumen, nucleic acid, antibody, antigen, antibiotic etc..Preferably, described water solublity Medicine is at least one in protein medicaments, peptide medicament and nucleic acid drug.Preferably, the molecule of described water soluble drug Amount greater than about 3350Da.

Described albumen includes natural, synthesis, the semisynthetic or compound of restructuring or protein, or containing by peptide bond altogether The alpha amino acid that valency connects substantially form structure, or be functionally correlated with.Concrete, include but not limited to that globular preteins is (as in vain Albumen, globulin, histone), fibrin (such as collagen, elastin laminin, keratin), compound protein matter (can contain One or more non-peptide compositions, such as glycoprotein, nucleoprotein, mucin, lipoprotein, metalloprotein), human cytokines, merges Albumen, receptor, antigen (such as synthesize or the antigen of restructuring), virus surface proteins, hormone and hormone analogs, antibody (as Monoclonal or polyclonal antibody), enzyme, Fab fragment, interleukin and derivant thereof, at least one in interferon and derivant thereof.

Described nucleic acid refers to natural, synthesis, semisynthetic, or is formed by two or more identical or different nucleotide The compound of at least partly restructuring, and can be strand or double-strand.The non-limitative example of nucleic acid includes oligonucleotide, instead MODN, fit, polynucleotide, DNA (deoxyribonucleic acid), siRNA, constructs, strand or double-strand section thing And precursor and derivant (such as glycosylation, super glycosylation, PEGization, FITC labelling, nucleoside, and their salt) thereof. Concrete, described nucleic acid include but not limited to Mipomersen, Alicaforsen, Nusinersen, Volanesorsen, Custirsen, Apatorsen、Plazomicin、RG-012、RG-101、ATL1102、ATL1103、IONIS-HBV_Rx、IONIS-HBV-L_Rx、 IONIS-GCGR_Rx、IONIS-GCCR_Rx、IONIS-HTT_Rx、IONIS-TTR_Rx、IONIS-PKK_Rx、IONIS-FXI_Rx、 IONIS-APO(a)-L_Rx、IONIS-ANGPTL3-L_Rx、IONIS-AR-2.5_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_RxIn at least one.Above-mentioned noun is title or the code name of nucleic acid drug.

Described water soluble drug preferably comprises the water-soluble substances (such as peptide medicament) of at least one basic group, including and be not limited to Thyroliberin (ACTH) and derivant thereof, epidermal growth factor (EGF), platelet derived growth factor (TOGF), Gonadotropin-releasing hormone (LHRH) and derivant or the like thereof, calcitonin, insulin like growth factor (IGF-I, IGF-II), Cell growth factor (such as EGF, TGF-α, TGF-β, PDGF, hydrochloric acid FGF, basic FGF etc.), glucagon Sample peptide (such as GLP-1, GLP-2) and derivant or the like thereof, neurotrophic factor (such as NT-3, NT-4, CNTF, GDNF, BDNF etc.), colony stimulating factor (such as CSF, GCSF, GMCSF, MCSF etc.), and they synthesis be similar to At least one in thing, trim and pharmaceutically active fragment.Derivant of described GLP-1 or the like includes but not limited to Exendin-3 and exendin-4.

It is described containing at least one at least one preferred peptide matters of basic group water soluble drug and derivant, analog, Described peptide matters includes and is not limited to glucagon (29 peptide), Sermorelin (29 peptide), aviptadil (28 peptide), pancreas Secretin (27 peptide), ziconotide (25 peptide), tetracosactide (24 peptide), Angiomax (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), Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 (10 peptide), Antide Acetate (10 peptide), blood Angiotensin (6-10 peptide), alarelin (9 peptide), buserelin (9 peptide), De She Rayleigh (9 peptide), octreotide (8 peptide), Lanreotide (8 peptide), Bremelanotide (7 peptide), Eptifibatide (7 peptide), Hexarelin (6 peptide), spleen pentapeptide (5 Peptide), Thymopentin (5 peptide), elcatonin (31 peptide), Suo Malu peptide (31 peptide), glucagon-like-peptide-1 (31 peptide), Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (34 peptide), teriparatide (34 peptide), Pramlintide (37 peptide), enfuirtide (38 peptide), Exenatide (39 Peptide), thyroliberin (39 peptide), corticotropin releasing hormone (41 peptide), Tesamorelin (44 peptide), In lixisenatide (44 peptide), FSH (118 peptide), Du Lalu peptide (274 peptide), albiglutide (645 peptide) At least one.

Described peptide matters preferably has the polypeptide no less than 30 amino acid residues.The derivant of described peptide matters, analog Refer to have no less than at least one in the polypeptide of 30 amino acid residues and variant, analog through water solublity or slightly water-soluble Group or material modify product, it has higher biology and pharmacologically active, stability, or has new function or attribute.

The derivant of described peptide medicament, analog include glucagon-like peptide (such as GLP-1, GLP-2) and derivant thereof, At least one in analog, includes but not limited at least in exendin-3, exendin-4 and their variant, derivant Kind.

Described variant, analog refer to that one or more amino acid residues of aminoacid sequence are replaced (or replace), lack, insert Enter, merge, truncate or its combination in any and different peptides, variant polypeptide can be fully functional or can lack one or Several functions.The 2nd of analog exendin-4 such as glucagon-like peptide-1 (GLP-1) is glycine, and the of GLP-1 2 is alanine, and exendin-4 can be combined and produce cell signalling cascade conduction with GLP-1 receptor.

Described water solublity or the group of slightly water-soluble or material are selected from Polyethylene Glycol and derivant thereof, cyclodextrin, hyaluronic acid, short Peptide, albumin, aminoacid sequence, nucleic acid, gene, antibody, phosphoric acid, sulfonic acid, fluorescent dye, KLH, OVA, PVP, PEO, PVA, alkane, aromatic hydrocarbons, biotin, immunoglobulin, albumin, polyamino acid, gelatin, succinyl gelatin, At least one in acrylamide derivative, fatty acid, polysaccharide, lipidic amino acid, chitosan and glucosan.The most poly-second two Alcohol and/or its derivant, the structure of described Polyethylene Glycol and derivant thereof can be side chain, straight chain, bifurcated or dumbbell shaped 's.The derivant of described Polyethylene Glycol includes but not limited to mono methoxy polyethylene glycol, propanoic acid methoxy poly (ethylene glycol).Described poly- Ethylene glycol and derivant thereof are commercially available, or prepared voluntarily by technology well known to those skilled in the art.

Described water solublity or slightly water-soluble material modified be after the dressing agent with activated group with described peptide matters derivant phase Coupling, described activated group takes selected from maleimide, halogen, vinyl sulfone, disulfide bond, sulfydryl, aldehyde radical, carbonyl, O- For oxyammonia, active ester, thiazolinyl, alkynyl, azido and other there is at least one in the group of high chemical reactivity；Excellent Selection of land, at least one in maleimide, halogen, vinyl sulfone and disulfide bond of described activated group；More preferably horse Come acid imide and/or disulfide bond.On polymer with activated group number be one or more, and when activated group number big In one time, described activated group can be identical or different.

The molecular weight of one or more described water solublity or slightly water-soluble material is 1-60kDa, preferably 2-50kDa, more preferably 5-40kDa。

Dressing agent with activated group can be by amino, carboxyl, hydroxyl or the sulfydryl etc. on aminoacid sequence and peptide or its change Body, analog phase coupling.Such group is usually located at amino acid residue such as Lys (lysine), Asp (aspartic acid), Glu (glutamic acid), Cys (cysteine), His (histidine), 4-mercaptoproline, Trp (tryptophan), Arg (arginine), Any one aminoacid in Ala (alanine), Gly (glycine), Ser (serine) or Thr (threonine) or they The N end of derivant, C end, side chain or any site, be preferably the site containing sulfydryl.Such as Exendin-4 and the like In, any one the cysteine residues site or other aminoacid that are positioned at 2,14,21,25,28,35,38 or any positions are residual Base is replaced by the site of cysteine residues.

Described peptide and variant thereof, analog be modified to modify at random, locator qualification thing (specificity modification), single-point are modified or many Point is modified, and preferably One-Point Location is modified.

The polypeptide synthesis method of described peptide and variant, analog routine is prepared, including solid-phase peptide synthesis, liquid phase Polypeptide synthesis method, solid-liquid polypeptide synthesis method and recombination method；Peptide and the reaction of variant, analog and dressing agent thereof Aqueous solution or buffer salt solution are carried out, suitably controls the pH value of reaction system, with HPLC, GPC etc. to modified outcome It is monitored, and isolated and purified by ion exchange, gel chromatography etc., concentrate and lyophilization obtains target product.

Water soluble drug mentioned above can be to be free form or the form of pharmaceutically acceptable salt, and the acid of its one-tenth salt is optional With mineral acid or organic acid.Described mineral acid includes hydrochloric acid, sulphuric acid, phosphoric acid, and organic acid includes acetic acid, formic acid, propanoic acid, breast Acid, trifluoroacetic acid, citric acid, fumaric acid, malonic acid, maleic acid, tartaric acid, Aspartic Acid, benzoic acid, methanesulfonic acid, Benzenesulfonic acid, citric acid, malic acid, oxalic acid, succinic acid, carbonic acid；Preferably hydrochloric acid, acetic acid, fumaric acid, maleic acid；More excellent Select acetic acid.

Described step 1) in biodegradable and biocompatible slightly water-soluble polymer include polyester, Merlon, bunching Aldehyde, polyanhydride, poly-hydroxy fatty acid, and their copolymer or blend.Detailed, described biodegradable and biofacies Hold polymer be polylactide (PLA), PGA (PGA), PLGA (PLGA) and they With the copolymer of polycaprolactone (PCL) or Polyethylene Glycol (PEG) (as PLA-PEG, PLGA-PEG, PLGA-PEG-PLGA, PLA-PEG-PLA、PEG-PCL、PCL-PLA-PCL、PCL-PLGA-PCL、PEG-PLA-PEG、 PEG-PLGA-PEG), polycaprolactone and with the copolymer of Polyethylene Glycol, poly butyric, poly-hydroxypentanoic acid, poly-to dioxy Ketohexamethylene (PPDO), chitosan, alginic acid and salt thereof, polybutylcyanoacrylate, fibrin, condensing model, poe, Polyamide, polyphosphazene, poly phosphate and their copolymer or mixture；Preferably PLA, PLGA and they and PCL Or the copolymer of PEG, and they mixture；More preferably PLA, PLGA or their mixture.

The weight average molecular weight of described PLA, PLGA and their copolymers with PCL or PEG is 20000-130000Da, Preferred molecular weight be 25000-110000Da, more preferably molecular weight be 30000-100000Da.Weight average used in this explanation Molecular weight is to measure, by gel permeation chromatography (GPC), the value obtained.

The viscosity of described PLA, PLGA and their copolymers with PCL or PEG (test condition is～0.5% (w/v), CHCl3,25 DEG C) it is 0.18-1.0dL/g, preferably 0.22-0.9dL/g, more preferably 0.27-0.85dL/g.

The strand of described slightly water-soluble polymer can carry anion or cation group, or does not carry these groups. Preferably, polymer has end carboxyl or end ester group, the preferred polymer with end carboxyl.

Described PLA, PLGA and their copolymers with PCL or PEG, wherein third hand over the fat ratio with glycolide from 100:0 To 50:50, preferably from about 90:10 to 50:50, more preferably 85:15 to 50:50.

The polymer preparing sustained-release microparticle of the present invention, can be single polymer, it is also possible to for the mixture of multiple polymers, As third hands over fat identical from the ratio of glycolide and molecular weight but carries the combination of the different PLGA of group, third hands over fat and glycolide Ratio and carry that group is identical but combination, the molecular weight of PLGA that molecular weight is different and to carry group identical but third hand over fat to hand over second The different combination of PLGA of the ratio of fat, molecular weight, carry group and third and hand over the fat PLGA the most different from the ratio of glycolide Combination, the combination etc. of PLGA Yu PLA.

Described organic solvent C, it is impossible to dissolve water soluble drug, but biodegradable and biocompatible slightly water-soluble can be dissolved Polymer, boiling point is less than water and does not dissolves in or is insoluble in water.Described organic solvent C can be single organic solvent, it is also possible to for Two kinds miscible and above organic solvent.Described organic solvent C selected from aliphatic hydrocarbon (molecular structure is straight chain, side chain or ring-type, Such as normal hexane, normal heptane, pentane, hexamethylene, petroleum ether etc.), halogenated hydrocarbons (as dichloromethane, chloroform, ethyl chloride, Tetrachloroethylene, trichloro ethylene, dichloroethanes, trichloroethane, carbon tetrachloride, fluorohydrocarbon, chlorobenzene (single, double, triple replacement), Arcton 11 etc.), fatty acid ester (such as ethyl acetate, butyl acetate etc.), aromatic hydrocarbon (such as benzene,toluene,xylene etc.), Ether is (such as Anaesthetie Ether, Di Iso Propyl Ether, methyl-isobutyl ether, methyl tertiary butyl ether(MTBE), methoxylation ether, alkyl ether, dihalo- For ether, trihalogen ether, cyclic ethers, crown ether etc.) at least one, preferably halogenated aliphatic varsol, more preferably dichloromethane With at least one in chloroform.In described interior oil phase, the kind of organic solvent C and ratio are according to different pharmaceutical and polymer the most not With, allocate according to practical situation.

The slightly water-soluble polymer concentration foundation type of polymer, weight average molecular weight and organic solvent in organic solvent C Type and change；Generally, its mass concentration (polymer quality/organic solvent C mass * 100%) is about 1-18% (w/w), excellent Elect about 2-15% (w/w), even more preferably about 3-12% (w/w) as.

Described organic solvent D, can not dissolve water soluble drug and biodegradable and biocompatible slightly water-soluble polymer simultaneously, But can be miscible with described oil solution, described surfactant there is is good dissolubility simultaneously.Described organic solvent D can be single Organic solvent, it is also possible to for two kinds and above organic solvent that can be miscible.Described organic solvent D selected from absolute ether, In at least one in hexamethylene, normal hexane, normal heptane, petroleum ether, preferably normal hexane, hexamethylene, normal heptane at least one Kind, kind and the ratio of described organic solvent D are different according to different surfaces activating agent, oil solution, adjust according to practical situation Join.

Described boiling point less than water and insoluble in or be insoluble in the organic solvent of water and refer to be merely able to water so that < 5% volume ratio is miscible Organic solvent, and there is relatively low boiling (being less than or much smaller than 100 DEG C), in order to easily pass through such as lyophilizing, evaporate or drum Wind removes.

Described emulsion is low temperature, and described low temperature can be understood as 20 DEG C or less, preferably 15 DEG C or less, more preferably 6 DEG C or with Under.

The described oil solution (being also called outer oil phase) containing surfactant is low temperature, described low temperature can be understood as 18 DEG C or with Under, preferably 12 DEG C or less, more preferably 8 DEG C.

The oil matrix of the described oil solution containing surfactant is any pharmaceutically acceptable polyhydric alcohol in pharmaceutical technology field, plants At least one in thing oil, mineral oil and other oil.This oil matrix can be one-component, it is also possible to for miscible two kinds and more than Component.Described vegetable oil includes but not limited to Oleum Glycines, Oleum Gossypii semen, Oleum Brassicae campestris, Oleum Arachidis hypogaeae semen, safflower oil, Oleum sesami, Testa oryzae Oil, Fructus Maydis oil, sunflower oil, poppy seed oil, olive oil, Semen Maydis oil, Oleum Gossypii semen, Oleum Cocois, Semen Lini oil, Oleum Ricini, At least one in Petiolus Trachycarpi oil, is preferably used at least one in Oleum Glycines, Oleum Arachidis hypogaeae semen, Oleum Ricini, more preferably in these vegetable oil Oleum Arachidis hypogaeae semen；Described mineral oil includes but not limited to silicone oil, liquid paraffin；Other oil include being hydrogenated by the part of vegetable oil obtaining Oil (such as castor oil hydrogenated) and at least one of satisfied fatty acid (such as caproic acid, octanoic acid etc.) of liquid；Described polyhydric alcohol bag Include glycerol, Polyethylene Glycol.Described oil matrix preferably vegetable oil and/or mineral oil, more preferably vegetable oil.

Described surfactant can increase the moistening character of organic facies, the stability improving emulsion process medium and small liquid pearl and shape, Avoid little liquid pearl to regroup, reduce the quantity of non-encapsulated or partly encapsulating small spherical particles, thus avoid medicine discharging Initial burst in journey.

Described surfactant is anion surfactant, zwitterionic surfactant, nonionic surfactant or table The such compound of face active biomolecule, preferred anionic surfactant, nonionic surfactant, more preferably cloudy from Sub-surface activating agent.

Described nonionic surfactant include but not limited to sorbitan esters (span), glyceryl monostearate, 16 At least one in alkanol, 18 hexadecanol, octadecanol.

Described anion surfactant include but not limited to phospholipid and derivant thereof, glyceride, fatty acid ester, fatty alcohol and its At least one in its bile acid (such as cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodesoxycholic acid).

The preferred phospholipid of described anion surfactant and derivant thereof, described phospholipid and derivant thereof include but not limited to phosphatidyl Choline (lecithin), PHOSPHATIDYL ETHANOLAMINE (cephalin), Phosphatidylserine, phosphatidylinositols, phosphatidyl glycerol, double phosphorus Phosphatidyl glycerol (cuorin), phosphoglyceride acid, lysophosphatide, soybean phospholipid, two palmityls-phosphatidylcholine, dioleoyl phosphorus Acyl-ethanolamine, DOPC and two myristoyls-phosphatidyl glycerol, and mixture.Described phospholipid can be Salinization or non-salinization, hydrogenation or partially hydrogenated, natural, semisynthetic or complete synthesis.Described phospholipid and spreading out Biological preferably phosphatidylcholine, soybean phospholipid, phosphatidyl glycerol, more preferably soybean phospholipid.

The described surfactant (or stabilizer) mass percent concentration in oil matrix, typically at 0.05-10%, is preferably 0.25-8%, more preferably 0.5-5%.

The usage amount of outer oil phase, usually in oil phase about 50 times of volumes more than, preferably from about 70 times volumes and particularly preferably More than about 100 times of volumes.

The method of the uniform emulsion of described formation is identical with well-known emulsification method, use produce high shear force device (as Magnetic stirring apparatus, mechanical agitator, high-shear homogenizer, Ultrasound Instrument, membrane emulsifier, Rotor-stator mixers, static mixer, High pressure homogenizer etc.) interior oil phase is mixed with outer oil phase, to form homogeneous latex emulsion.

Described step 4) in remove organic solvent and can apply following method:

(1) organic solvent is removed by heating, decompression (or combined heat)；

(2) air-flow advertises liquid surface, and control liquid phase and the contact area of gas phase, emulsion stirring and circulation speed (as JP-A-9-221418) volatilization of organic solvent, the nitrogen that described air-flow is preferably dried are accelerated；

(3) rapidly removing organic solvent (such as W00183594) with hollow fiber membrane, the preferred silicone rubber of hollow fiber membrane is complete Thin evaporated film (the pervaporation thin film particularly prepared by polydimethylsiloxane).

Described step 4) obtained in microgranule separated by the way of being centrifuged, sieve or filtering.

Described step 4) in washing ultra-pure water temperature used by microgranule be low temperature, described low temperature can be understood as 12 DEG C or less, Preferably 9 DEG C or less, more preferably 6 DEG C or less.

Described step 4) in washing used by ultra-pure water in also can contain inorganic salt (such as zinc salt), water-soluble to reduce in washing process Property active substance is molten oozes to aqueous phase, improve entrapment efficiency, its mechanism is to improve the osmotic pressure of foreign minister or reduce active substance outside Dissolubility in mutually.For polypeptide, albumen, nucleic acid, antibody, antigen, antibiotic isoreactivity material, the chemical combination containing zinc ion Thing is more satisfactory selection, including and be not limited to zinc acetate, zinc chloride, zinc sulfate, zinc hydrogen sulfate, zinc nitrate, glucose Acid zinc, zinc carbonate or their any mixture.In ultra-pure water, the mass concentration of inorganic salt is 0.01-3%, preferably 0.01-1.5%, More preferably 0.01-1%.

Preferably, described step 1) implemented by following steps:

11) biodegradable and biocompatible slightly water-soluble polymer and water soluble drug are dissolved completely in organic solvent A, Form medicine and the mixed solution of polymer；

12) described mixed solution is injected in organic solvent B or organic solution B is injected in described mixed solution, produce uniformly, Trickle precipitate, collects precipitate, and washs for several times by organic solvent B, remove organic solvent B, it is thus achieved that water soluble drug Solid dispersion with slightly water-soluble polymer；Wherein, organic solvent B can not dissolve described slightly water-soluble polymer and described water Soluble drug.

Described organic solvent A, can dissolve water soluble drug and slightly water-soluble polymer biodegradable, biocompatible simultaneously. Described organic solvent A can be single organic solvent, it is also possible to for miscible two kinds and above organic solvent.Described organic molten At least one in glacial acetic acid, acetonitrile, trifluoroacetic acid, dimethyl sulfoxide of agent A, preferably glacial acetic acid and/or acetonitrile, more excellent Select glacial acetic acid.In described mixture, kind and the ratio of organic solvent are different according to different pharmaceutical and polymer, can be according to reality Border situation allotment.

Described organic solvent B, can not dissolve water soluble drug and slightly water-soluble polymer biodegradable, biocompatible simultaneously. Described organic solvent B can be single organic solvent, it is also possible to for two kinds and above organic solvent that can be miscible.Described have At least one in absolute ether, hexane (including hexamethylene, normal hexane), normal heptane of machine solvent B, preferably absolute ether With at least one in hexane (including hexamethylene, normal hexane), more preferably absolute ether.The kind of organic solvent in described mixture Class and ratio are different according to different pharmaceutical and polymer, can allocate according to practical situation.

Described organic solvent A controls to be below room temperature or low temperature, and described room temperature it is generally understood that 20 DEG C, preferably 10-15 DEG C； Described low temperature it is generally understood that less than 10 DEG C, preferably 4-6 DEG C or less；Described organic solvent B controls as low temperature, institute State low temperature and it is generally understood that less than 15 DEG C, preferably 10 DEG C or less, more preferably less than 6 DEG C；Organic solvent A ratio has Temperature height 0-10 DEG C of machine solvent B, preferably 3-8 DEG C.

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

Described insoluble polymer concentration in organic solvent A is according to the type of polymer, weight average molecular weight and organic molten The type of agent and change.Generally, its mass concentration (polymer quality/organic solvent A quality * 100%) is 1-18% (w/w), It is preferably 2-15% (w/w), more preferably 3-12% (w/w).

The step of described removal organic solvent B does not contains heating schedule, and this step is carried out below room temperature or under low temperature, described room temperature It is generally understood that 20-30 DEG C, preferably 20-25 DEG C；Described low temperature it is generally understood that less than 15 DEG C, preferably 10 DEG C And below.The method removing organic solvent includes but not limited to vacuum drying, lyophilization, fluidized drying.

Further, the sustained-release microparticle of the present invention can comprise one or more auxiliary agents.The preparation side of the sustained-release microparticle of the present invention Method also includes the step adding auxiliary agent, and auxiliary agent is in described step 1) prepare addition during solid dispersion, or in described step Rapid 2) add when preparing solid dispersion emulsion；Preferably in described step 2) add when preparing solid dispersion emulsion.Described help Agent is dissolved in internal phase or is suspended in interior oil phase.It is imperceptible powder that described auxiliary agent adds fashionable, and its particle diameter is less than 0.5 μm, It is preferably particle diameter less than 0.1 μm, more preferably particle diameter less than 0.05 μm.

Described auxiliary agent can give active medicine or microgranule other feature, such as, increase the steady of microgranule, active medicine or polymer The controllable release qualitative, promotion active medicine is from microgranule or biological organization's permeability of regulation active medicine.Described auxiliary agent is The 0.01-10% of the quality sum of described water soluble drug and slightly water-soluble polymer, preferably 0.1-8%, more preferably 0.5-8%.

Described auxiliary agent includes but not limited at least one in saccharide, aminoacid, fatty acid, alcohols, antioxidant, buffer agent.

Described buffer agent includes but not limited to the salt of mineral acid or organic acid, as carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, The salt of hydrochloric acid.Concrete, include but not limited to calcium carbonate, calcium hydroxide, lima bean fringed pink acid calcium, calcium oleate, calcium palmitate, hard Fat acid calcium, calcium phosphate, calcium acetate, magnesium acetate, magnesium carbonate, magnesium hydroxide, magnesium phosphate, magnesium myristate, magnesium oleate, palm fibre Palmitic acid acid magnesium, magnesium stearate, zinc carbonate, zinc hydroxide, zinc oxide, lima bean fringed pink acid zinc, zinc oleate, zinc acetate, zinc chloride, Zinc sulfate, zinc hydrogen sulfate, zinc nitrate, zinc gluconate, Hexadecanoic acid, zinc salt, zinc stearate, zinc phosphate, sodium carbonate, bicarbonate Sodium, sodium sulfite, sodium thiosulfate, Acetic acid-sodium acetate buffer salt, and their combination in any.Preferably mineral acid or organic The zinc salt of acid, more preferably zinc chloride.Described buffer agent is the quality sum of described water soluble drug and described slightly water-soluble polymer 0-5%, preferably 0.01-3%, more preferably 0.01-2%.

Described antioxidant includes but not limited to butylated hydroxyarisol, dibutylphenol, tocopherol, lima bean fringed pink acid isopropyl Ester, d-a tocopherol acetate, ascorbic acid, ascorbic palmitate, butylated hydroxyanisol, butylatedhydroxyquinone, hydroxyl Butylcoumariii, Yoshinox BHT, amass wealth by heavy taxation acid fatty acid ester (such as ethyl ester, propyl ester, monooctyl ester, lauryl), the third hydroxy benzoic acid Ester, trihydroxybutyrophenone, vitamin E, D-ALPHA-tocopheryl polyethylene glycol 1000 succinate, ρ-hydroxybenzoate are (such as methyl ester, ethyl ester, propyl ester, fourth Ester) at least one.Antioxidant can remove the free radical in sustained-release microparticle or peroxide effectively.Described antioxidation Agent is 0-1%, the preferably 0-0.05% of described water soluble drug and the quality sum of described slightly water-soluble polymer, more preferably 0-0.01%.

Described saccharide includes but not limited to monosaccharide, oligosaccharide and polysaccharide, and their derivant.Concrete, include but not limited to Trehalose, glucose, sucrose, glycerol, erithritol, arabitol, xylitol, sorbitol, mannitol, glucuronic acid, Iduronic acid, neuraminic acid, galacturonic acid, glucose keto acid, mannuronic acid, hyaluronic acid and salt, sulphuric acid In chrondroitin and salt, heparin, inulin, chitin and derivant thereof, dextrin, glucosan and alginic acid and salt thereof at least one Kind.At least one in preferably sucrose, mannitol, xylitol.Described saccharide is that described water soluble drug is molten with described shipwreck 0.1-10%, the preferably 0.5-8% of the quality sum of property polymer, more preferably 1-6%.

Described aminoacid includes but not limited to glycine, alanine, serine, aspartic acid, glutamic acid, threonine, color ammonia Acid, lysine, hydroxylysine, histidine, arginine, cystine, cysteine, methionine, phenylalanine, bright ammonia At least one in acid, isoleucine and their derivant；Preferably basic amino acid, includes but not limited to arginine, group At least one in propylhomoserin, lysine.Described amino acids is the quality of described water soluble drug and described slightly water-soluble polymer The 0-4% of sum, preferably 0-2%, more preferably 0.01-1%.

Described fatty acid includes 12～24 alkanoic acid and derivants thereof, includes but not limited to oleic acid, stearic acid, lauric acid, Semen Myristicae At least one in acid, Palmic acid, arachidic acid, mountain Yu acid, lignin acid, preferably stearic acid, mountain Yu acid, Palmic acid.Institute State 0-5%, preferably 0.01-4% that fatty acid is described water soluble drug and the quality sum of described slightly water-soluble polymer, more excellent Select 0.05-3%.

Described alcohols includes but not limited to Polyethylene Glycol.The molecular weight of described Polyethylene Glycol is 400-6000Da, is preferably 400-4000Da, more preferably 400-2000Da.Described alcohols is the matter of described water soluble drug and described slightly water-soluble polymer The 0-5% of amount sum, preferably 0.01-4%, more preferably 0.05-3%.

The formulation requirements of injection is aseptic, and concrete sterilizing methods belongs to general knowledge and the technology of those skilled in the art, as adopted Ensure that preparation is aseptic with sterile working, hot pressing, oxirane or gamma ray.The present invention prepares the preferred aseptic behaviour of sustained-release microparticle Make, as with cellulose acetate membrane filtration foreign minister's aqueous solution, with poly (ether sulfone) film filter PLGA acetic acid solution, use politef Membrane filtration dichloromethane, and armamentarium is the most airtight and is equipped with organic solvent recovery device, with prevent the pollution of antibacterial with And organic solvent is diffused in air.

On the other hand, present invention also offers the sustained-release microparticle that the preparation method according to described sustained-release microparticle prepares.

Because microgranule is when drug administration by injection, particle diameter is easily caused the most greatly stifled pin, it is necessary to use the injection needle of more large size, patient's Pain is higher, and particle diameter the least will cause copolymer cannot good packaging medicine, do not reach good slow release effect.This The sustained-release microparticle of bright preparation preferably has less than the average geometric granular size of 200 μm.The particle diameter of described sustained-release microparticle is 10-200 μm, preferably 10-150 μm, more preferably 20-150 μm.Sustained-release microparticle size passes through dynamic light scattering method (example Such as laser diffractometry) or microtechnique (such as scanning electron microscope method) measure.

The sustained-release microparticle of the present invention can encapsulate substantial amounts of active component, dosage can according to the type of active component and content, dosage form, Release duration, it is administered experimenter, route of administration, administration purpose, targeted condition and symptom etc. and properly selects.But, As long as active component can reach the intended persistent period in vivo maintaining medicine effective concentration, then this dosage is regarded as making us full Meaning.

In the sustained-release microparticle of the present invention, the mass content percentage ratio of described water soluble drug is about 1-40%, preferably 3-35%, More preferably 5-30%.

In this article when stated ranges, it is meant that contain any scope therein or the combination of scope.

Another aspect, present invention also offers a kind of suspension formulations, and it includes described sustained-release microparticle and disperse medium.

When microgranule is administered with suspended form, it can make suspension formulations with suitable disperse medium.

Described disperse medium includes nonionic surfactant, castor oil derivatives, cellulose thickener, alginic acid At least one in sodium, hyaluronic acid, dextrin, starch.Or selectable, it is also possible to other components such as isotonic agent (such as chlorine Change sodium, mannitol, glycerol, sorbitol, lactose, xylitol, maltose, galactose, sucrose, glucose etc.), pH adjusts Joint agent (the such as salt of carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid or these acid, such as sodium carbonate, sodium bicarbonate Deng), preservative (such as p-Hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol, chlorobutanol, sorbic acid, boric acid Deng) at least one combination make aqueous solution, or subsequently through methods such as lyophilization, drying under reduced pressure, spray drying Solidification, is dissolved in water for injection the disperse medium obtaining disperse particles again by solidfied material before using.

Additionally, slow releasing injection also can be obtained by following method: sustained-release microparticle to be scattered in vegetable oil (such as Oleum sesami and jade Miyou) or be added with in the vegetable oil of phospholipid (such as lecithin), or be scattered in medium chain triglyceride, to obtain oiliness Suspension.

Another aspect, present invention also offers described solid dispersion, sustained-release microparticle in implanted sustained release pharmaceutical composition Application.

Water-soluble drug sustained-release pharmaceutical composition prepared by the present invention, the particularly slow releasing pharmaceutical of albumen, nucleic acid and peptide medicament Compositions can also be club, tablet, further, present invention also offers a kind of implanted sustained release pharmaceutical composition Preparation method, comprises the following steps:

1. the solid dispersion of water soluble drug and biodegradable and biocompatible slightly water-soluble polymer is prepared；

2. by step 1. in prepare solid dispersion heating after use forming method molding, cooling i.e. prepare implanted release thing Compositions.

This is not restricted for forming method, and the forming method known to professional and technical personnel all can use, such as compression molding, extrusion Molding, releases compositions and may be molded to bar-shaped, lamellar.

The present invention prepares water soluble drug, and particularly albumen, the sustained release pharmaceutical composition of peptide medicament is planting of bar-shaped, lamellar Enter agent.

Further, the preparation method of a kind of implanted sustained release pharmaceutical composition, comprise the following steps:

1. ' prepare sustained-release microparticle according to the preparation method of described sustained-release microparticle；

2. ' by step 1. ' prepare sustained-release microparticle prepare implanted release thing by the forming method known to professional and technical personnel Compositions.Release compositions may be molded to bar-shaped, lamellar.

The present invention obtain sustained-release microparticle can be used for granular form, suspending agent form, the preparation of heeling-in form, injection form, Adhesion preparation form etc., it is possible to (intramuscular injection, subcutaneous injection, percutaneous dosing, mucosa are given for oral or parenteral administration Medicine (in cheek, intravaginal, internal rectum etc.)).

The implant of the present invention is with biodegradable, biocompatible materials as substrate, and outward appearance is in the most bar-shaped, pole shape or lamellar (discoid), can be implanted to internal by injection or modus operandi, takes out without operation after drug release is complete.This implant Advantage is to be readily available high encapsulation rate and carrying drug ratio, and dashing forward, it is low to release rate, and can be with stable speed sustained-release therapeutic required dosage Active medicine reach one month to the several months long, be substantially reduced medical treatment cost, improve the compliance of sufferer.

The invention have the benefit that in the present invention, the preparation whole process room temperature of sustained-release microparticle or low temperature, for the medicine of sensitive The compositions that thing, particularly albumen, nucleic acid and peptide medicament prepare polymeric matrix is highly beneficial, compares published technology, The biological activity of active substance can be kept to the full extent in whole technical process；Meanwhile, prepared sustained-release microparticle has Close to the superior sustained-release effect of zero level, drug level is stable at deenergized period, solves and traditional wants small of previously prepared medicine The microgranule early stage that the S/O/W technique of grain obtains does not has drug release, and the shortcoming quickly discharged released by later stage medicine；Furthermore, slow release Microgranule has higher carrying drug ratio and entrapment efficiency.

Upon administration, albumen, peptide, nucleic acid, alkaloid isoreactivity material can the most persistently carry the sustained-release microparticle of the present invention A period of time, deenergized period is up to a few weeks or months.

Accompanying drawing explanation

The diabetic mice that Fig. 1 is the Exenatide release microgranule prepared of embodiment 6-11 or Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sustained-release microparticle is administered Average HbA_1cValue-time plot.

Detailed description of the invention

For better illustrating the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment, the present invention is made into one Step explanation." microgranule " namely " sustained-release microparticle " in following example；" sustained-release implant " namely implanted sustained release pharmaceutical composition.

Embodiment 1The preparation of albiglutide/PLGA microgranule

(I) preparation of solid dispersion

0.90g PLGA (molecular weight 25kDa, monomer ratio 65/35, end carboxyl) is dissolved in about 6.00mL glacial acetic acid, then adds Enter 0.10g acetic acid albiglutide, dissolve under vortex, the most slowly inject in the absolute ether (6 DEG C) under stirring, produce white Precipitate, collects white depositions and extracts about 5 times with absolute ether, is dried 24h after being collected by precipitate in vacuum drying oven (10 DEG C), obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 6.00g dichloromethane obtain in oil phase, then interior oil phase is injected In 0.05% (w/w) lecithin/peanut oil solution of 230mL constant temperature the most in advance to about 4 DEG C, and high-shear homogenizer is used to prepare S/O/O emulsion (spinner velocity about 3000rpm, 5min).S/O/O emulsion is continued mechanical agitation about 3 hours (400rpm) Solidification microgranule, then uses centrifuge to collect microgranule by centrifugal (about 3500rpm, 5min).With normal heptane, microgranule is washed After about 5 times, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, Obtain microgranule.Recording the content of albiglutide in gained microgranule is 9.12%, and diameter of particle is 19-90 μm.

Embodiment 2The preparation of Du Lalu peptide/PLGA microgranule

(I) preparation of solid dispersion

0.95g PLGA (molecular weight 30kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 7.92mL acetonitrile, is subsequently adding 0.05g acetic acid Du Lalu peptide, dissolves under vortex, the most slowly injects in the hexamethylene (6 DEG C) under stirring, produces white precipitate Thing, collects white depositions and extracts about 5 times with hexamethylene, is dried 24h (10 DEG C) after being collected by precipitate in vacuum drying oven, Obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 7.92g chloroform obtain in oil phase, then by interior oil phase injection 420mL The most in advance in 0.1% (w/w) lecithin/liquid paraffin solution of constant temperature to about 5 DEG C, and high-shear homogenizer is used to prepare S/O/O breast Liquid (spinner velocity about 3000rpm, 5min).S/O/O emulsion is continued mechanical agitation about 3 hours (400rpm) solidification microgranule, Then centrifuge is used to collect microgranule by centrifugal (about 3500rpm, 5min).After microgranule being washed about 5 times with absolute ether, Again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule. Recording the content of Du Lalu peptide in gained microgranule is 4.60%, and diameter of particle is 20-95 μm.

Embodiment 3The preparation of FSH/PLA microgranule

(I) preparation of solid dispersion

0.97g PLA (molecular weight 20kDa holds ester group) is dissolved in about 5.39mL dimethyl sulfoxide, is subsequently adding 0.03g acetic acid FSH, 0.05g xylitol and 0.03g zinc chloride, dissolve under vortex, the most slowly injects the normal hexane (8 DEG C) under stirring In, produce white depositions, collect white depositions and with n-hexane extraction about 5 times, in vacuum drying after being collected by precipitate Case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate obtain in the mixed liquor of about 5.39g dichloromethane and chloroform in oil phase, then Interior oil phase is injected in 0.25% (w/w) lecithin/soybean oil solution of 410mL constant temperature the most in advance to about 6 DEG C, and use profit wheeled S/O/O emulsion (profit rotary speed about 5500rpm, 5min) is prepared in homo-mixer emulsifying.It is transferred to S/O/O emulsion seal glass Glass flask continues mechanical agitation about 3 hours (400rpm) solidification microgranule, then use centrifuge by centrifugal (about 2500rpm, 5min) collect microgranule.After microgranule being washed about 5 times with hexamethylene, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), It is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Recording the content of FSH in gained microgranule is 2.73%, diameter of particle is 30-82 μm.

Embodiment 4The preparation of lixisenatide/PLGA microgranule

(I) preparation of solid dispersion

0.99g PLGA (molecular weight 22kDa, monomer ratio 90/10, end carboxyl) is dissolved in about 5.50mL trichloroacetic acid, then Add 0.01g acetic acid lixisenatide, 0.05g xylitol and 0.03g zinc carbonate, dissolve under vortex, the most slowly inject under stirring Normal heptane (6 DEG C) in, produce white depositions, collect white depositions and also extract about 5 times with normal heptane, precipitate is received In vacuum drying oven, it is dried 24h (10 DEG C) after collecting, obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 5.50g tetrachloroethylene obtain in oil phase, then interior oil phase is injected In 0.5% (w/w) lecithin/corn oil of 330mL constant temperature the most in advance to about 5 DEG C, and SPG membrane emulsifier is used to prepare S/O/O emulsion (membrane aperture 30-80 μm circulates 3 times).S/O/O emulsion is continued mechanical agitation about 3.5 hours (500rpm) Solidification microgranule, then uses centrifuge to collect microgranule by centrifugal (about 3500rpm, 5min).With normal hexane, microgranule is washed After about 5 times, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, Obtain microgranule.Recording the content of lixisenatide in gained microgranule is 0.93%, and diameter of particle is 34-98 μm.

Embodiment 5The preparation of corticotropin releasing hormone/PLGA microgranule

(I) preparation of solid dispersion

0.85g PLGA (molecular weight 25kDa, monomer ratio 85/15, end carboxyl) is dissolved in the mixed of about 8.50mL glacial acetic acid and acetonitrile Close in liquid, be subsequently adding 0.15g acetic acid corticotropin releasing hormone, dissolve under vortex, the most slowly inject stirring Under absolute ether and hexamethylene mixed liquor (6 DEG C) in, produce white depositions, collect white depositions and also use absolute ether Extract about 5 times with the mixed liquor of hexamethylene, in vacuum drying oven, be dried 24h (10 DEG C) after being collected by precipitate, obtain solid and divide A prose style free from parallelism.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 8.50g normal heptane obtain in oil phase, then interior oil phase is injected In 0.75% (w/w) lecithin/castor oil solution of 580mL constant temperature the most in advance to about 6 DEG C, and static mixer is used to prepare S/O/O emulsion (rotating speed 5000rpm circulates 3 times).It is transferred to S/O/O emulsion in seal glass flask continue mechanical agitation About 3.5 hours (500rpm) solidifies microgranule, then uses centrifuge to collect microgranule by centrifugal (about 3500rpm, 5min). After microgranule being washed about 5 times with petroleum ether, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with Freezer dryer lyophilization, it is thus achieved that microgranule.Recording the content of somatostatin in gained microgranule is 13.77%, and diameter of particle is 31-95μm。

Embodiment 6The preparation of Exenatide/PLGA microgranule

(I) preparation of solid dispersion

0.95g PLGA (molecular weight 35kDa, monomer ratio 75/25, end carboxyl) is dissolved in about 6.33mL glacial acetic acid, then adds Enter 0.05g Exendin-4 and 0.08g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 6.33g dichloromethane obtain in oil phase, then interior oil phase is injected In 1% (w/w) lecithin/peanut oil solution of 430mL constant temperature the most in advance to about 5 DEG C, and prepare S/O/O breast by mechanical agitation Liquid (1000rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (400rpm) solidification microgranule, then uses Centrifuge collects microgranule by centrifugal (about 3500rpm, 5min).With the mixed liquor of normal heptane and petroleum ether, microgranule is washed about After 5 times, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, Obtain microgranule.Recording the content of Exenatide in gained microgranule is 4.65%, and diameter of particle is 24-93 μm.

Embodiment 7The preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]/PLGA microgranule

(I) preparation of solid dispersion

0.93g PLGA (molecular weight 40kDa, monomer ratio 65/35, end carboxyl) is dissolved in about 7.75mL glacial acetic acid, then adds Enter 0.07g acetic acid Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and 0.06g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I1 gained is divided uniformly dissipate in about 7.75g dichloromethane obtain in oil phase, then interior oil phase is injected In 1.25% (w/w) lecithin/peanut oil solution of 470mL constant temperature the most in advance to about 7 DEG C, and prepare S/O/O by mechanical agitation Emulsion (1000rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (400rpm) solidification microgranule, then makes Microgranule is collected by centrifugal (about 3500rpm, 5min) with centrifuge.After microgranule being washed about 5 times with normal heptane, again divide Dissipate washing about 2 times in ultra-pure water (5 DEG C), be then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record In gained microgranule, the content of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is 6.50%, and diameter of particle is 30-102 μm.

Embodiment 8The preparation of Exenatide/PLGA microgranule

(I) preparation of solid dispersion

0.90g PLGA (molecular weight 45kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 9.00mL glacial acetic acid, then adds Enter 0.10g Exendin-4 and 0.04g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 9.00g dichloromethane obtain in oil phase, then interior oil phase is injected In 1.5% (w/w) lecithin/peanut oil solution of 680mL constant temperature the most in advance to about 9 DEG C, and prepare S/O/O by mechanical agitation Emulsion (1500rpm, 7min).S/O/O emulsion is continued mechanical agitation about 4 hours (700rpm) solidification microgranule, then makes Microgranule is collected by centrifugal (about 3500rpm, 5min) with centrifuge.After microgranule being washed about 5 times with normal heptane, again divide Dissipate washing about 2 times in ultra-pure water (5 DEG C), be then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record In gained microgranule, the content of Exenatide is 9.23%, and diameter of particle is 25-92 μm.

Embodiment 9The preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]/PLGA microgranule

(I) preparation of solid dispersion

0.86g PLGA (molecular weight 50kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 10.75mL glacial acetic acid, then Add 0.14g acetic acid Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and 0.02g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 10.75g dichloromethane obtain in oil phase, then interior oil phase is injected In 2% (w/w) lecithin/peanut oil solution of 970mL constant temperature the most in advance to about 10 DEG C, and prepare S/O/O by mechanical agitation Emulsion (1800rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (800rpm) solidification microgranule, then makes Microgranule is collected by centrifugal (about 3500rpm, 5min) with centrifuge.After microgranule being washed about 5 times with normal heptane, again divide Dissipate washing about 2 times in ultra-pure water (5 DEG C), be then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record In gained microgranule, the content of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is 12.81%, and diameter of particle is 22-89 μm.

Embodiment 10The preparation of Exenatide/PLGA microgranule

(I) preparation of solid dispersion

0.82g PLGA (molecular weight 55kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 11.71mL glacial acetic acid, then Add 0.18g Exendin-4 and 0.01g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 11.71g dichloromethane obtain in oil phase, then interior oil phase is injected In 2% (w/w) lecithin/peanut oil solution of 700mL constant temperature the most in advance to about 8 DEG C, and prepare S/O/O breast by mechanical agitation Liquid (1500rpm, 5min).S/O/O emulsion is continued mechanical agitation about 5 hours (600rpm) solidification microgranule, then uses Centrifuge collects microgranule by centrifugal (about 3500rpm, 5min).After microgranule being washed about 5 times with normal heptane, again disperse Washing about 2 times in ultra-pure water (5 DEG C), are then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record institute Obtaining the content of Exenatide in microgranule is 17.00%, and diameter of particle is 22-90 μm.

Embodiment 11The preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]/PLGA microgranule

(I) preparation of solid dispersion

0.80g PLGA (molecular weight 60kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 13.33mL glacial acetic acid, then Add 0.20g acetic acid Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], dissolve under vortex, the most slowly inject in the absolute ether (6 DEG C) under stirring, produce white Color precipitate, collects white depositions and extracts about 5 times with absolute ether, being dried after being collected by precipitate in vacuum drying oven 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 13.33g dichloromethane obtain in oil phase, then interior oil phase is injected In 3% (w/w) lecithin/peanut oil solution of 900mL constant temperature the most in advance to about 11 DEG C, and prepare S/O/O by mechanical agitation Emulsion (1600rpm, 5min).S/O/O emulsion is continued mechanical agitation about 5 hours (700rpm) solidification microgranule, then makes Microgranule is collected by centrifugal (about 4000rpm, 5min) with centrifuge.After microgranule being washed about 5 times with normal heptane, again divide Dissipate washing about 2 times in ultra-pure water (5 DEG C), be then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record In gained microgranule, the content of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is 18.83%, and diameter of particle is 25-107 μm.

Embodiment 12The preparation of enfuirtide/PLGA microgranule

(I) preparation of solid dispersion

0.75g PLGA (molecular weight 65kDa, monomer ratio 65/35, end carboxyl) is dissolved in about 15.00mL glacial acetic acid, then Add 0.25g acetic acid enfuirtide, 0.03g sucrose and 0.01g stearic acid, dissolve under vortex, the most slowly inject under stirring In absolute ether (6 DEG C), produce white depositions, collect white depositions and extract about 5 times, by precipitate with absolute ether In vacuum drying oven, it is dried 24h (10 DEG C) after collection, obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 15.00g dichloromethane obtain in oil phase, then interior oil phase is injected In 4% (w/w) lecithin/peanut oil solution of 1.1L constant temperature the most in advance to about 13 DEG C, and prepare S/O/O breast by mechanical agitation Liquid (2000rpm, 5min).S/O/O emulsion is continued mechanical agitation about 5 hours (850rpm) solidification microgranule, then uses Centrifuge collects microgranule by centrifugal (about 4000rpm, 5min).After microgranule being washed about 5 times with normal heptane, again disperse Washing about 2 times in ultra-pure water (5 DEG C), are then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record institute Obtaining the content of enfuirtide in microgranule is 22.97%, and diameter of particle is 18-93 μm.

Embodiment 13The preparation of Pramlintide/PLGA microgranule

(I) preparation of solid dispersion

0.70g PLGA (molecular weight 70kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 17.50mL glacial acetic acid, then Add 0.30g pramlintide acetate and 0.02g mannitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 17.50g dichloromethane obtain in oil phase, then interior oil phase is injected In 5% (w/w) lecithin/peanut oil solution of 1.3L constant temperature the most in advance to about 20 DEG C, and prepare S/O/O breast by mechanical agitation Liquid (2200rpm, 5min).S/O/O emulsion is continued mechanical agitation about 5 hours (800rpm) solidification microgranule, then uses Centrifuge collects microgranule by centrifugal (about 4000rpm, 5min).After microgranule being washed about 5 times with normal heptane, again disperse Washing about 2 times in ultra-pure water (5 DEG C), are then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record institute Obtaining the content of Pramlintide in microgranule is 27.49%, and diameter of particle is 27-98 μm.

Embodiment 14The preparation of teriparatide/PLGA microgranule

(I) preparation of solid dispersion

0.65g PLGA (molecular weight 85kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 21.67mL glacial acetic acid, then Add 0.35g teriparatide acetate, 0.03g mannitol and 0.03gPEG-400, dissolve under vortex, the most slowly inject under stirring Absolute ether (6 DEG C) in, produce white depositions, collect white depositions and also extract about 5 times with absolute ether, will precipitation Thing is dried 24h (10 DEG C) after collecting in vacuum drying oven, obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 21.67g dichloromethane obtain in oil phase, then interior oil phase is injected In 6% (w/w) lecithin/peanut oil solution of 1.6L constant temperature the most in advance to about 15 DEG C, and prepare S/O/O breast by mechanical agitation Liquid (2400rpm, 5min).S/O/O emulsion is continued mechanical agitation about 5 hours (900rpm) solidification microgranule, then uses Centrifuge collects microgranule by centrifugal (about 4000rpm, 5min).After microgranule being washed about 5 times with normal heptane, again disperse Washing about 2 times in ultra-pure water (5 DEG C), are then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record institute Obtaining the content of teriparatide in microgranule is 32.16%, and diameter of particle is 20-92 μm.

Embodiment 15 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]The preparation of/PLGA microgranule

(I) preparation of solid dispersion

0.60g PLGA (molecular weight 100kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 30.00mL glacial acetic acid, then Add 0.40g acetic acid Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and 0.005g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 30.00g dichloromethane obtain in oil phase, then interior oil phase is injected In 7% (w/w) lecithin/glycerite of 2L constant temperature the most in advance to about 15 DEG C, and prepare S/O/O emulsion by mechanical agitation (2000rpm, 5min).By S/O/O emulsion continue mechanical agitation about 5 hours (700rpm) solidification microgranule, then use from Scheming collects microgranule by centrifugal (about 4000rpm, 5min).After microgranule being washed about 5 times with normal heptane, again it is scattered in Washing about 2 times in ultra-pure water (5 DEG C), are then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record gained In microgranule, the content of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is 37.18%, and diameter of particle is 23-90 μm.

Embodiment 16The preparation of Suo Malu peptide/PLGA microgranule

(I) preparation of solid dispersion

0.50g PLGA (molecular weight 110kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 50.00mL glacial acetic acid, then Add 0.50g acetic acid Suo Malu peptide and 0.001g xylitol, dissolve under vortex, the most slowly inject the absolute ether (6 DEG C) under stirring In, produce white depositions, collect white depositions and extract about 5 times with absolute ether, doing in vacuum after precipitate is collected Dry case is dried 24h (10 DEG C), obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 50.00g dichloromethane obtain in oil phase, then interior oil phase is injected In 8% (w/w) lecithin/peanut oil solution of 2.6L constant temperature the most in advance to about 15 DEG C, and SPG membrane emulsifier is used to prepare S/O/O Emulsion (membrane aperture 20-50 μm circulates 3 times).S/O/O emulsion is continued mechanical agitation about 5 hours (600rpm) and solidifies micro- Grain, then uses centrifuge to collect microgranule by centrifugal (about 4000rpm, 5min).With normal heptane, microgranule is washed about 5 times After, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that Microgranule.Recording the content of Suo Malu peptide in gained microgranule is 45.04%, and diameter of particle is 23-87 μm.

Embodiment 17The preparation of glucagon-like-peptide-1/PLGA microgranule

(I) preparation of solid dispersion

0.50g PLGA (molecular weight 130kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 50.00mL glacial acetic acid, then Add 0.50g acetic acid glucagon-like-peptide-1, dissolve under vortex, the most slowly inject in the absolute ether (6 DEG C) under stirring, Produce white depositions, collect white depositions and extract about 5 times with absolute ether, in vacuum drying oven after precipitate is collected In be dried 24h (10 DEG C), obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 50.00g dichloromethane obtain in oil phase, then interior oil phase is injected In 10% (w/w) lecithin/peanut oil solution of 3L constant temperature the most in advance to about 20 DEG C, and use the wheeled homo-mixer emulsifying system of profit Standby S/O/O emulsion (profit rotary speed about 7000rpm, 5min).It is transferred to S/O/O emulsion in seal glass flask continue machinery Stir about 5 hours (800rpm) solidification microgranule, then uses centrifuge to collect micro-by centrifugal (about 4000rpm, 5min) Grain.After microgranule being washed about 5 times with normal heptane, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, With freezer dryer lyophilization, it is thus achieved that microgranule.Recording the content of glucagon-like-peptide-1 in gained microgranule is 46.21%, micro- Grain particle diameter is 19-85 μm.

Embodiment 18The preparation of Exendin-4 derivant/PLGA microgranule

(I) preparation of solid dispersion

Solid dispersion contains the composition of following weight/mass percentage composition: water soluble drug: Exendin-4 derivant 20%, shipwreck Soluble polymer: PLGA79.5%, auxiliary agent: xylitol 0.5%；The molecular weight of wherein said PLGA is 50kDa, Qi Zhongbing The ratio of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 50/50, and described PLGA has end carboxyl.

(1) prepare Exendin-4 derivant: preparation 10kDa PEG-NHS ester, then in PBS with Exendin-4 In 28 agedoite reaction, by ion exchange, gel chromatography separation purification, concentrate and lyophilization obtain Exendin-4 Derivant.

(2) slightly water-soluble polymer is dissolved completely in glacial acetic acid, then adds water soluble drug and auxiliary agent to the most molten Solve；Wherein slightly water-soluble polymer be glacial acetic acid quality 6.5%；It is then injected into absolute ether (6 DEG C) to make to produce white Precipitate, collects precipitate, and extracts 5 times with absolute ether, is dried 24h after being collected by precipitate in vacuum drying oven (10 DEG C), obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in the dichloromethane of about 12 times obtain in oil phase, then by interior oil Inject mutually in 2% (w/w) lecithin/peanut oil solution of 970mL constant temperature the most in advance to about 5 DEG C, and prepared by mechanical agitation S/O/O emulsion (1400rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (500rpm) solidification microgranule, Then centrifuge is used to collect microgranule by centrifugal (about 3500rpm, 5min).After microgranule being washed about 5 times with normal heptane, Again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule. Recording the content of Exendin-4 derivant in gained microgranule is 18.40%, and diameter of particle is 27-109 μm.

Embodiment 19The preparation of Exendin-4 derivant/PLGA microgranule

(I) preparation of solid dispersion

Solid dispersion contains the composition of following weight/mass percentage composition: water soluble drug: Exendin-4 derivant 15%, shipwreck Soluble polymer: PLGA84%, auxiliary agent: xylitol 1%；The molecular weight of wherein said PLGA is 50kDa, wherein third hands over The ratio of ester and Acetic acid, hydroxy-, bimol. cyclic ester is 50/50, and described PLGA has end carboxyl.

(1) Exendin-4 derivant is prepared: prepared the Radix Asparagi acyl of in Exendin-4 28 by solid-phase peptide synthesis Amine replaces with the Exendin-4 variant of cysteine, then in PBS with 10kDa Y type mono methoxy polyethylene glycol- Maleimide reacts, and by ion exchange, gel chromatography separation purification, concentrates and lyophilization obtains Exendin-4 and derives Thing.

(2) slightly water-soluble polymer is dissolved completely in glacial acetic acid, then adds water soluble drug and auxiliary agent to the most molten Solve；Wherein slightly water-soluble polymer be glacial acetic acid quality 6.5%；It is then injected into absolute ether (6 DEG C) to make to produce white Precipitate, collects precipitate, and extracts 5 times with absolute ether, is dried 24h after being collected by precipitate in vacuum drying oven (10 DEG C), obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in the dichloromethane of about 13 times obtain in oil phase, then by interior oil Inject mutually in 1.75% (w/w) lecithin/peanut oil solution of 970mL constant temperature the most in advance to about 5 DEG C, and prepared by mechanical agitation S/O/O emulsion (1300rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (500rpm) solidification microgranule, Then centrifuge is used to collect microgranule by centrifugal (about 3500rpm, 5min).After microgranule being washed about 5 times with normal heptane, Again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule. Recording the content of Exendin-4 derivant in gained microgranule is 13.25%, and diameter of particle is 30-113 μm.

Embodiment 20The preparation of Exendin-4 derivant/PLGA microgranule

(I) preparation of solid dispersion

Solid dispersion contains the composition of following weight/mass percentage composition: water soluble drug: Exendin-4 derivant 20%, shipwreck Soluble polymer: PLGA78%, auxiliary agent: sorbitol 2%；The molecular weight of wherein said PLGA is 55kDa, wherein third hands over The ratio of ester and Acetic acid, hydroxy-, bimol. cyclic ester is 50/50, and described PLGA has end carboxyl.

(1) Exendin-4 derivant is prepared: prepare the arginine of 20 in Exendin-4 by solid-phase peptide synthesis Replace with the Exendin-4 variant of cysteine, then in PBS with 5kDa mono methoxy polyethylene glycol-maleoyl Imine reaction, by ion exchange, gel chromatography separation purification, concentrates and lyophilization obtains Exendin-4 derivant.

(2) slightly water-soluble polymer is dissolved completely in glacial acetic acid, then adds water soluble drug and auxiliary agent to the most molten Solve；Wherein slightly water-soluble polymer be glacial acetic acid quality 6%；It is then injected into absolute ether (6 DEG C) to make to produce white sinking Shallow lake thing, collects precipitate, and extracts 5 times with absolute ether, is dried 24h (10 DEG C) after being collected by precipitate in vacuum drying oven, Obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in the dichloromethane of about 14 times obtain in oil phase, then by interior oil Inject mutually in 1.5% (w/w) lecithin/peanut oil solution of 1L constant temperature the most in advance to about 5 DEG C, and prepare S/O/O by mechanical agitation Emulsion (1500rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (500rpm) solidification microgranule, then makes Microgranule is collected by centrifugal (about 3500rpm, 5min) with centrifuge.After microgranule being washed about 5 times with normal heptane, again divide Dissipate washing about 2 times in ultra-pure water (5 DEG C), be then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record In gained microgranule, the content of Exendin-4 derivant is 18.31%, and diameter of particle is 32-126 μm.

Embodiment 21The preparation of Exendin-4 derivant/PLGA microgranule

(I) preparation of solid dispersion

Solid dispersion contains the composition of following weight/mass percentage composition: water soluble drug: Exendin-4 derivant 16%, shipwreck are molten Property polymer: PLGA81%, auxiliary agent: xylitol 3%；The molecular weight of wherein said PLGA is 45kDa, wherein lactide It is 50/50 with the ratio of Acetic acid, hydroxy-, bimol. cyclic ester, and described PLGA has end carboxyl.

(1) Exendin-4 derivant is prepared: prepared the first sulfur ammonia of in Exendin-4 14 by solid-phase peptide synthesis Acid replaces with the Exendin-4 variant of cysteine, then in PBS with 20kDa mono methoxy polyethylene glycol-Malaysia Imide reaction, by ion exchange, gel chromatography separation purification, concentrates and lyophilization obtains Exendin-4 derivant.

(2) slightly water-soluble polymer is dissolved completely in glacial acetic acid, then adds water soluble drug and auxiliary agent to the most molten Solve；Wherein slightly water-soluble polymer be glacial acetic acid quality 7%；It is then injected into absolute ether (6 DEG C) to make to produce white sinking Shallow lake thing, collects precipitate, and extracts 5 times with absolute ether, is dried 24h (10 DEG C) after being collected by precipitate in vacuum drying oven, Obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in the dichloromethane of about 11 times obtain in oil phase, then by interior oil phase Inject in 1.25% (w/w) lecithin/peanut oil solution of 970mL constant temperature the most in advance to about 5 DEG C, and prepared by mechanical agitation S/O/O emulsion (1400rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (500rpm) solidification microgranule, Then centrifuge is used to collect microgranule by centrifugal (about 3500rpm, 5min).After microgranule being washed about 5 times with normal heptane, Again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule. Recording the content of Exendin-4 derivant in gained microgranule is 13.74%, and diameter of particle is 32-128 μm.

Embodiment 22The preparation of Exendin-4 derivant/PLGA microgranule

Solid dispersion contains the composition of following weight/mass percentage composition: water soluble drug: Exendin-4 derivant 12%, shipwreck Soluble polymer: PLGA84%, auxiliary agent: xylitol 4%；The molecular weight of wherein said PLGA is 40kDa, wherein third hands over The ratio of ester and Acetic acid, hydroxy-, bimol. cyclic ester is 50/50, and described PLGA has end carboxyl.

(1) Exendin-4 derivant is prepared: prepare the glycine of 2 in Exendin-4 by solid-phase peptide synthesis and replace Be changed to the Exendin-4 variant of cysteine, then in PBS with 40kDa mono methoxy polyethylene glycol-maleimide Amine reacts, and by ion exchange, gel chromatography separation purification, concentrates and lyophilization obtains Exendin-4 derivant.

(2) slightly water-soluble polymer is dissolved completely in glacial acetic acid, then adds water soluble drug and auxiliary agent to the most molten Solve；Wherein slightly water-soluble polymer be glacial acetic acid quality 6.5%；It is then injected into absolute ether (6 DEG C) to make to produce white Precipitate, collects precipitate, and extracts 5 times with absolute ether, is dried 24h after being collected by precipitate in vacuum drying oven (10 DEG C), obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in the dichloromethane of about 10 times obtain in oil phase, then by interior oil Inject mutually in 1% (w/w) lecithin/peanut oil solution of 970mL constant temperature the most in advance to about 5 DEG C, and prepared by mechanical agitation S/O/O emulsion (1400rpm, 5min).S/O/O emulsion is continued mechanical agitation about 4 hours (600rpm) solidification microgranule, Then centrifuge is used to collect microgranule by centrifugal (about 3500rpm, 5min).After microgranule being washed about 5 times with normal heptane, Again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), is then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule. Recording the content of Exendin-4 derivant in gained microgranule is 10.68%, and diameter of particle is 35-132 μm.

Embodiment 23The preparation of Mipomersen/PLGA microgranule

(I) preparation of solid dispersion

0.80g PLGA (molecular weight 30kDa, monomer ratio 50/50, end carboxyl) is dissolved in the mixed of about 6.53mL glacial acetic acid and acetonitrile Close in liquid, be subsequently adding 0.20g Mipomersen sodium and 0.01g xylitol, dissolve under vortex, the most slowly inject and stir In absolute ether (6 DEG C) under mixing, produce white depositions, collect white depositions and with n-hexane extraction about 5 times, will be heavy Shallow lake thing is dried 24h (10 DEG C) after collecting in vacuum drying oven, obtains solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate in about 6.53g tetrachloroethylene obtain in oil phase, then interior oil phase is noted In 1% (w/w) lecithin/peanut oil solution of 500mL constant temperature the most in advance to about 6 DEG C, and prepare S/O/O breast by mechanical agitation Liquid (1000rpm, 5min).S/O/O emulsion is continued mechanical agitation about 3.5 hours (500rpm) solidification microgranule, then makes Microgranule is collected by centrifugal (about 3500rpm, 5min) with centrifuge.After microgranule being washed about 5 times with hexamethylene, again divide Dissipate washing about 2 times in ultra-pure water (5 DEG C), be then centrifuged for collecting, with freezer dryer lyophilization, it is thus achieved that microgranule.Record In gained microgranule, the content of Mipomersen is 18.10%, and diameter of particle is 32-109 μm.

Embodiment 24The preparation of interleukin/PLGA microgranule

(I) preparation of solid dispersion

0.82g PLGA (molecular weight 35kDa, monomer ratio 50/50, end carboxyl) is dissolved in about 6.12mL glacial acetic acid, then adds Enter 0.18g interleukin and 0.02g xylitol, dissolve under vortex, the most slowly inject in the absolute ether (6 DEG C) under stirring, Produce white depositions, collect white depositions and extract about 5 times with absolute ether, in vacuum drying oven after precipitate is collected In be dried 24h (10 DEG C), obtain solid dispersion.

(II) preparation of microgranule

The solid dispersion of step I gained is divided uniformly dissipate obtain in the mixed liquor of about 6.12g dichloromethane and chloroform in oil phase, then Interior oil phase is injected in 0.5% (w/w) lecithin/soybean oil solution of 500mL constant temperature the most in advance to about 5 DEG C, and stirred by machinery Mixing is for S/O/O emulsion (1000rpm, 5min).It is transferred to S/O/O emulsion in seal glass flask continue mechanical agitation about 4 hours (500rpm) solidifies microgranule, then uses centrifuge to collect microgranule by centrifugal (about 3500rpm, 5min).With After microgranule is washed about 5 times by the mixed liquor of normal heptane and normal hexane, again it is scattered in washing about 2 times in ultra-pure water (5 DEG C), so Rear centrifugal collection, with freezer dryer lyophilization, it is thus achieved that microgranule.Recording the content of interleukin in gained microgranule is 16.36%, Diameter of particle is 31-114 μm.

Embodiment 25The preparation of Exenatide/PLGA sustained-release implant

Prepared by step I of embodiment 10 be dried solid dispersion insert 1mm*10mm mould (inner chamber is cylindric, The a diameter of 1mm of round bottom, the degree of depth is about 10mm) in, after being warming up to about 43 DEG C, compression molding, obtain column (1mm*5.31mm) Exenatide release implant.Recording the content of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] in gained implant is 17.24%.

Embodiment 26The preparation of Exenatide/PLGA sustained-release implant

In in step II of embodiment 10, gained microgranule is fed to hot-melt extruded machine, hot-melt extruded becomes the strip of diameter about 1mm Thing, after cooling, cutting obtains the Exenatide release implant that length is about 5mm.Record containing of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] in gained implant Amount is 17.03%.

In above-described embodiment, microgranule or the carrying drug ratio of implant and entrapment efficiency analysis method are: take 5mg microgranule or implant, It is dissolved in 50mL acetonitrile (ACN), adds 0.1%TFA 500 μ L, after being sufficiently mixed, centrifuging and taking supernatant, use high-efficient liquid Analysis of hplc drug level therein.In microgranule (or implant), the medicine gross mass of encapsulating is medicine with the ratio of dosage The envelop rate of thing, in microgranule (or implant), the drug quality of encapsulating is medicine with the ratio of microgranule (or implant) quality Carrying drug ratio.All of experiment is all repeated 3 times above.

In above-described embodiment, the Particle size analysis methods of microgranule is: be dispersed in liquid paraffin by about 10mg microgranule, ultrasonic about 30s Dispersion, uses the laser particle size analyzer of Beckman Coulter to measure.

Embodiment 27Microgranule and the prominent mensuration released with In-vitro release curves of implant

The sustained-release microparticle prepared by above-described embodiment and implant carry out dashing forward and release the mensuration with In-vitro release curves, and assay method is: Precision weighs pastille microgranule or implant 20mg puts in 15mL centrifuge tube, with phosphate buffer that pH is 7.4 (containing 0.02% Hydrazoic acid,sodium salt is as antibacterial) it is release medium, it is placed in constant temperature gas bath shaking table, in hunting speed 100rpm, temperature The vitro release carrying out microgranule and implant under the conditions of 37 DEG C ± 0.5 DEG C measures.Respectively l days, 2 days, 7 days, 14 days, 21 My god, within 28 days, 40 days, 50 days and 60 days, take out whole release medium and supplement equivalent new release medium, high performance liquid chromatography Method measures release amount of medicine, and assay method is:

Chromatograph of liquid: Agilent 1260；

Chromatographic column: Proteonavi 4.6 × 250mm；

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

Flow velocity: 1mL/min；

Detection wavelength: 280nm.

Test result is as shown in table 1.

Table 1 sustained-release microparticle and implant release in vitro accumulative releasing degree result

By the release in vitro result of table 1 it can be seen that use the sustained-release microparticle and institute that solid dispersion of the present invention prepares The implant prepared, does not has phenomenon of burst release or obvious slowbreak phenomenon, and whole releasing trend is close to Zero order release.Wherein, have The sample release in vitro cycle is up to 40-50 days, and some sample release in vitro cycles are up to 50-60 days, some sample release in vitro Cycle, more than 60 days, has the slow release effect of excellence.

Embodiment 28Microgranule syringeability is tested

About 20mg particulate samples is suspended in 2mL diluent (3% carboxymethyl cellulose, 0.9%NaCl), then sucks Syringe, is injected by the injection needle of 24-30G in the pig back leg (muscle) of commercially available 1kg weight respectively.Per injection is carried out 20 seconds or less, observing syringeability, result is as shown in table 2.

Table 2 microgranule syringeability experimental result

Note: ++ smoothness of pushing pin is fine, and+smoothness of pushing pin is general ,-retardance sense of having pushed pin,--stifled pin.

The syringeability result of table 2 shows, the equal microgranule of microparticle suspending liquid of different-grain diameter prepared by the present invention is sucked by No. 30 syringe needles , there is not retardance or the phenomenon of stifled pin, says in the ability in syringe and the inclusions in syringe being expelled to completely in Carnis Sus domestica The microgranule of the bright present invention can be administered by the way of subcutaneously or intramuscularly injecting.

Embodiment 29 Determination of Residual Organic Solvents determination test

Organic solvent A, organic solvent B, organic solvent in solid dispersion prepared by embodiment of the present invention 1-24 and sustained-release microparticle C and the determination of residual amount of organic solvent D, assay method is well-known assay method.Measurement result is as shown in table 3.

Table 3 Determination of Residual Organic Solvents measurement result

Note :-expression is not detected by or content is less than detection limit.

By the Determination of Residual Organic Solvents result of table 3 it can be seen that in the solid dispersion prepared of the present invention and sustained-release microparticle, organic The residual quantity of solvent is the lowest, or is not detected by, or residual quantity less than can detection range, to patient without because of organic molten after administration The side effect that agent produces, is also beneficial to keep the stability of microgranule, Shelf-life.

Embodiment 30Animal experiment

Choose diabetic mice 56, body weight 20 ± 5g, male and female half and half, the most every 8 be divided into administration group (6 groups) and Blank group (1 group), the Exenatide microgranule of administration group mouse carotid dorsal sc injection embodiment 6-11 or Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] microgranule, micro- Grain is with containing 3% carboxymethyl cellulose, the diluent suspendible of 0.9%NaCl, every injected in mice Exenatide 2mg/kg of administration group Or Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] 10mg/kg, the normal saline of blank group subcutaneous injection same volume.Be administered 0d, 0.5d, 1d, 3d, The same time of 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, 70d carries out blood glucose survey from tail venous blood sampling Fixed, then make average HbA_1cValue (glycolated hemoglobin accounts for the percentage ratio of total hemoglobin, %) and the curve chart of time (d), Result is as shown in Figure 1.

From Fig. 1 curve chart, Exenatide release microgranule prepared by embodiments of the invention 6-11 or Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sustained-release microparticle HbA can be controlled well in 70 days upon administration_1cValue, and the upon administration be HbA in 7-70 days_1cValue is in 5-7 Between, hence it is evident that lower than blank group, can grow after the Exenatide release microgranule of the present invention or the administration of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sustained-release microparticle be describeds Time discharges active medicine therein, and reaches preferable therapeutic effect, it is possible to reduce administration frequency, is conducive to improving patient's Compliance.

Last institute is it should be noted that, above example is only in order to illustrate technical scheme rather than to scope Restriction, although the present invention being explained in detail with reference to preferred embodiment, it will be understood by those within the art that, can Technical scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention.

Claims (12)

1. the preparation method of a sustained-release microparticle, it is characterised in that: comprise the following steps:

1) solid dispersion of water soluble drug and biodegradable and biocompatible slightly water-soluble polymer is prepared；

2) by step 1) solid dispersion prepared is dissolved in organic solvent C, forms solid dispersion emulsion, described organic molten Agent C for can not dissolve described water soluble drug but can dissolve described slightly water-soluble polymer, boiling point less than water and insoluble in or difficult It is dissolved in the organic solvent of water；

3) by step 2) the solid dispersion emulsion that obtains injects in the oil solution containing surfactant and forms uniform emulsion；

4) volatilized by solvent or solvent extraction make the microgranule in emulsion solidify, collect microgranule, wash for several times with organic solvent D, Again with milli-Q water for several times, to remove the surfactant being attached to microparticle surfaces, then it is dried, it is thus achieved that described slow release Microgranule；Wherein, described organic solvent D can not dissolve water soluble drug and slightly water-soluble polymer, and it can mix with described oil solution Molten, described surfactant there is is good dissolubility simultaneously；

Described water soluble drug is at least one in protein medicaments, peptide medicament and nucleic acid drug.

The preparation method of sustained-release microparticle the most according to claim 1, it is characterised in that: described water soluble drug is for having not Less than at least one in the polypeptide of 30 amino acid residues and derivant, analog.

The preparation method of sustained-release microparticle the most according to claim 2, it is characterised in that: described polypeptide derivative, analog For having no less than at least one in the polypeptide of 30 amino acid residues and variant, analog through water solublity or slightly water-soluble The product that group or material are modified.

The preparation method of sustained-release microparticle the most according to claim 3, it is characterised in that: described water solublity or slightly water-soluble Group or material be Polyethylene Glycol and/or its derivant.

The preparation method of sustained-release microparticle the most according to claim 1, it is characterised in that: described step 1) by following step Rapid enforcement:

11) biodegradable and biocompatible slightly water-soluble polymer and water soluble drug are dissolved completely in organic solvent A, Form medicine and the mixed solution of polymer；

12) described mixed solution is injected in organic solvent B or organic solution B is injected in described mixed solution, produce uniformly, Trickle precipitate, collects precipitate, and washs for several times by organic solvent B, remove organic solvent B, it is thus achieved that water soluble drug Solid dispersion with slightly water-soluble polymer；Wherein, organic solvent B can not dissolve described slightly water-soluble polymer and described water Soluble drug.

The preparation method of sustained-release microparticle the most according to claim 5, it is characterised in that: described organic solvent A is selected from ice At least one in acetic acid, acetonitrile, trifluoroacetic acid, dimethyl sulfoxide；

At least one in absolute ether, hexane, normal heptane of described organic solvent B.

The preparation method of sustained-release microparticle the most according to claim 1, it is characterised in that: in described solid dispersion, water-soluble Property medicine and slightly water-soluble polymer mass ratio be 1:1～1:99.

The preparation method of sustained-release microparticle the most according to claim 1, it is characterised in that: described organic solvent C is selected from fat At least one in fat hydrocarbon, halogenated hydrocarbons, fatty acid ester, aromatic hydrocarbon, ether；

At least one in absolute ether, hexamethylene, normal hexane, normal heptane, petroleum ether of described organic solvent D.

The preparation method of sustained-release microparticle the most according to claim 1, it is characterised in that: described step 1) in shipwreck molten Property polymer be polylactide, PGA, PLGA and they are with polycaprolactone or the copolymerization of Polyethylene Glycol Thing, polycaprolactone and with the copolymer of Polyethylene Glycol, poly butyric, poly-hydroxypentanoic acid, PPDO, shell gather Sugar, alginic acid and salt thereof, polybutylcyanoacrylate, fibrin, condensing model, poe, polyamide, polyphosphazene, poly- At least one in phosphate ester and their copolymer and/or mixture.

The preparation method of sustained-release microparticle the most according to claim 1, it is characterised in that: described method also includes that addition helps The step of agent, auxiliary agent is in described step 1) prepare solid dispersion during add, or in described step 2) prepare solid and divide Add during prose style free from parallelism emulsion；Described auxiliary agent is the 0.01-10% of described water soluble drug and the quality sum of described slightly water-soluble polymer.

The preparation method of 11. sustained-release microparticles according to claim 10, it is characterised in that: described auxiliary agent is aminoacid, resists Oxidant, buffer agent, saccharide, fatty acid, alcohol apoplexy due to endogenous wind at least one.

12. sustained-release microparticles prepared according to the preparation method of the sustained-release microparticle according to any one of claim 1～11.