CN1241139A - Method of producing sustained-release preparation - Google Patents
Method of producing sustained-release preparation Download PDFInfo
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- CN1241139A CN1241139A CN 97180846 CN97180846A CN1241139A CN 1241139 A CN1241139 A CN 1241139A CN 97180846 CN97180846 CN 97180846 CN 97180846 A CN97180846 A CN 97180846A CN 1241139 A CN1241139 A CN 1241139A
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Abstract
The invention relates to a method of producing sustained-release microcapsules which comprises dispersing a physiologically active polypeptide into a solution of a biodegradable polymer and zinc oxide in an organic solvent, followed by removing the organic solvent; which provides a sustained-release preparation showing a high entrapment ratio of the physiologically active polypeptide and its constant high blood concentration levels over a long period of time.
Description
Technical field
The present invention relates to produce the method for the slow releasing preparation that contains physiological active polypeptide.
Background technology
We know that the physiological active polypeptide or derivatives thereof can show various pharmacologically actives in vivo.Genetic engineering and cytologic technology that the utilization immediate development is got up, by using escherichia coli, yeast, zooblast or host animal such as goat and hamster, some physiological active polypeptide mass production comes out and is used for medical industry.But, because generally the biological half-life of physiological active polypeptide is very short, so must frequent drug administration.And the injection meeting brings very heavy body burden to patient repeatedly.
For example, growth hormone (hereinafter claiming GH sometimes) is that a kind of initial generation justacrine is in prehypophyseal typical hormone, it is a kind of physiological active polypeptide, has propagation and the differentiation of extensive and various physiologically active as metabolism, proteinic anabolism and the cell of promotion physical growth, glucose and lipid.The utilization gene recombination technology by utilizing the escherichia coli GH that can be mass-produced, and is used for clinical medicinal career in worldwide.But, because the biological half-life of GH is very short, so in order to keep effective blood concentration, GH must frequent drug administration.Especially when the treatment pituitary dwarfism, to ill baby and young patient, carry out subcutaneous injection administration (time is not waited by at least ten years from some months) long-term every day is the method for using at present.
In order to address this problem, people have repaid and have tried several different methods and invent the slow releasing preparation that contains physiological active polypeptide.
JP-A 3055/1996 (EP-A 633020) discloses a kind of method of producing slow releasing preparation, be included in the aqueous solution water soluble polypeptide infiltration is contained in the biodegradable substrate of biodegradable polymer and fatty acid metal salts, also disclose the slow-release microcapsule (hereinafter being sometimes referred to as MC) of preparation in this way simultaneously.
JP-A 217691/1996 (WO 96/07399) discloses and has prepared water insoluble or be slightly soluble in the method for the multivalent metal salt of water, promptly utilize the aqueous solution of the water-soluble peptide type of biological active substances and zinc chloride etc. to prepare, also disclose the method that preparation contains the slow releasing preparation of this salt and biodegradable polymer simultaneously.
WO 94/12158 discloses the method for preparing the slow releasing preparation that contains people GH and biodegradable polymer, polymer erodes rate adaptation agent soon such as zinc hydroxide join in the polymer solution with the amount of 0.1%-30% (weight ratio), and wherein said percentage by weight is for the amount of polymer.This open file also further discloses the method for preparing MC such as porous particle, is about to people GH and the solution of polymer in organic solvent and sprays into liquid nitrogen, keeps its biological activity simultaneously.
WO 92/17200 and Nature Medicine, the 2nd volume, p.795 (1996) zinc salt of disclosing personnel selection GH prepares the method for slow releasing preparation.
WO 95/29664 discloses the method for preparing MC, comprising following step: slaine such as zinc carbonate are distributed in the polymer solution with solid-state form, add biological active substances such as hormone, then biological active substances and metal cation component are distributed in the biodegradable polymer respectively.
Yet just as mentioned before, though people have repaid the still retainable slow releasing preparation of physiologically active that has tried the whole bag of tricks generation in next life reason active polypeptide, but still fail to produce gratifying clinically preparation, its reason is that there are many problems in some physiological active polypeptides, for example the embedding ratio of physiological active polypeptide in preparation is low, excessive release of initial stage after the administration, can not be for a long time in constant release, keep the blood drug level of being satisfied with in can not be for a long time.In addition, its manufacture method is not suitable for the mass industrialized production under the present condition in many aspects.
Of the present invention open
In order to address the above problem, the application's inventors have done the very research of effort, lactic acid/ethanol copolymer (hereinafter being sometimes referred to as PLGA) that they find to be used as MC substrate coexists in organic solvent with zinc oxide the effect of dissolved oxygen zinc can unexpectedly be provided, and zinc oxide is undissolvable in organic solvent originally, simultaneously can produce high-load PLGA-zinc oxide complex effectively, they also find with physiological active polypeptide directly be dispersed in PLGA-zinc oxide complex in the solution of organic solvent and one-tenth mould effect subsequently can produce slow releasing preparation with premium properties, for example can improve the embedding ratio of physiological active polypeptide, the excessive release of initial period after the minimizing administration, and keep good stable release.In addition, present inventors find that also this production method has reduced many steps, are a kind of industrialized production methods that is well suited for.After further research, inventors have been developed the present invention.
In other words, the present invention relates to:
(1) method of production slow releasing preparation comprises physiological active polypeptide is distributed in biodegradable polymer and the solution of zinc oxide in organic solvent, removes organic solvent then;
(2) according to the method for above-mentioned (1), wherein said physiological active polypeptide is a growth hormone;
(3) according to the method for above-mentioned (1), wherein said biodegradable polymer is lactic acid/ethanol copolymer;
(4) according to the method for above-mentioned (3), wherein in lactic acid/ethanol copolymer, the molecular composition ratio of lactic acid/glycolic is about 85/15-about 50/50;
(5) according to the method for above-mentioned (3), wherein the weight average molecular weight of lactic acid/ethanol copolymer is about 8000-about 20000;
(6) according to the method for above-mentioned (1), wherein in the solution of organic solvent, zinc is about about 2% weight ratio of 0.001%-with respect to the content of degradable polymer;
(7) according to the method for above-mentioned (1), wherein the average particulate diameter of slow releasing preparation is about the about 300 μ m of 0.1-;
(8) according to the method for above-mentioned (1), wherein said slow releasing preparation is an injection;
(9) according to the method for above-mentioned (1), wherein oil/aqueous emulsion to be carried out in the water dryly, said oil/aqueous emulsion contains by growth hormone being distributed to the dispersion liquid for preparing in lactic acid/ethanol copolymer and the zinc oxide solution in organic solvent as oil phase;
(10) according to the method for above-mentioned (1), wherein said preparation is a microcapsule;
(11) lactic acid/ethanol copolymer and the zinc oxide solution in organic solvent;
(12) dissolve in the lactic acid/ethanol copolymer-zinc oxide complex of organic solvent, it obtains by lactic acid/ethanol copolymer and zinc oxide are co-existed in organic solvent;
(13) by physiological active polypeptide being distributed to the dispersion liquid that is prepared in the organic solvent solution of lactic acid/ethanol copolymer and zinc oxide;
(14) according to the dispersion liquid of above-mentioned (13), wherein said physiological active polypeptide is a growth hormone; And
(15) slow releasing preparation for preparing with method according to above-mentioned (1).
Biodegradable polymer preferred examples used in the present invention comprises, synthesize obtain and polymer that contain the free-end carboxyl from one or more alpha-hydroxy carboxylic acid compounds (for example glycolic, lactic acid), hydroxydicarboxylic acid's (for example malic acid), hydroxyl tricarboxylic acids (for example citric acid) etc. by the dehydration polycondensation that does not contain catalyst, its mixture, poly--a-cyanoacrylate, polyamino acid (for example poly--γ-benzyl-L-glutamic acid) and copolymer-maleic anhydride (for example styrene-maleic acid copolymer).
Polymerization can be random, block type or graft type.When above-mentioned alpha-hydroxy carboxylic acid compounds, hydroxydicarboxylic acid and hydroxyl tricarboxylic acids had rotophore in its molecular structure, they can be D-, L-or DL-configuration.
In these polymer, the biodegradable polymer with free-end carboxyl is as from alpha-hydroxy carboxylic acid compounds (for example glycolic, lactic acid) synthetic polymer (for example lactic acid/ethanol copolymer) and poly--a-cyanoacrylate is preferred.Biodegradable polymer is more preferably from the synthetic polymer of alpha-hydroxy carboxylic acid compounds, preferred especially lactic acid/ethanol copolymer.
When the biodegradable polymer of using was lactic acid/ethanol copolymer or homopolymer, it is about 40/60 that its proportion of composing (mol%) is preferably about 100/0-, more preferably about 85/15-about 50/50.
In this manual, lactic acid/ethanol copolymer and homopolymer such as polylactic acid and polyglycolic acid are called lactic acid/glycolic acid polymer sometimes simply.
It is about 25000 that the weight average molecular weight of above-mentioned lactic acid/glycolic acid polymer is preferably about 3000-, more preferably about 5000-about 20000.
It is about 4.0 that the dispersion of lactic acid/glycolic acid polymer (weight-average molecular weight/number-average molecular weight) is preferably about 1.2-, more preferably about 1.5-about 3.5.
As for weight average molecular weight and dispersion, in this manual, the former measures according to polystyrene with gel permeation chromatography (GPC), wherein be respectively 120000,52000,22000,9200,5050,2950,1050,580 and 162 polystyrene as object of reference with 9 kinds of weight average molecular weight, the latter calculates from the former.Said determination is to carry out with GPC post KF804Lx2 (being produced by Japanese Showa Denko) and RI monitor L-3300 (by Japanese Hitachi, Ltd. produces), uses chloroform as mobile phase.
Biodegradable polymer with free-end carboxyl is wherein according to the number-average molecular weight of GPC mensuration and the almost equal each other biodegradable polymer of determining according to end group quantity of number-average molecular weight.
Number-average molecular weight according to end group quantity is calculated with the following method:
About 1-3g biodegradable polymer is dissolved in the mixed solvent of acetone (25ml) and methanol (5ml), under room temperature (20 ℃) stirs,, uses phenolphthalein as the content of indicator with definite carboxyl with the quick titration of potassium hydroxide alcoholic solution of solution with 0.05N; Number-average molecular weight according to end group quantity is calculated with following equation:
Number-average molecular weight=20000 * A/B according to end group quantity
A: the weight of biodegradable polymer (g)
B: when reaching titration end-point, the amount (ml) that the potassium hydroxide alcoholic solution of 0.05 N adds
Because the number-average molecular weight of determining according to end group quantity is an absolute value, the number-average molecular weight of measuring according to GPC is the relative value who changes with different analysis condition (for example the thin layer width of the kind of the kind of mobile phase, post, reference substance, selection, the factors such as baseline of selection); Therefore these two values are difficult to use absolute numeric representation.Yet, from the synthetic polymer of one or more alpha-hydroxy carboxylic acid compounds, the number-average molecular weight of measuring according to GPC is almost consistent with the number-average molecular weight of determining according to end group quantity, for example, the number-average molecular weight of determining according to end group quantity be the number-average molecular weight measured according to GPC 0.5-2 doubly, preferred 0.7-1.5 is doubly.
For example, when polymer have the free-end carboxyl and be by without the dehydration polycondensation of catalyst from one or more alpha-hydroxy carboxylic acid compounds when synthetic, number-average molecular weight of measuring according to GPC and the number-average molecular weight of determining according to end group quantity are almost equal each other.On the contrary, when polymer go up substantially do not contain the free-end carboxyl and be ring-opening polymerization by using catalyst from cyclic dimer when synthetic, the number-average molecular weight high a lot (about more than 2 times or 2 times) that the number-average molecular weight of determining according to end group quantity wants beguine to measure according to GPC.
Can clearly differentiate polymer with free-end carboxyl and the polymer that does not have the free-end carboxyl by this species diversity.
Lactic acid/the glycolic acid polymer that contains the free-end carboxyl can be by method (for example do not use the dehydration polycondensation reaction of catalyst or dehydration polycondensation reaction under the solid inorganic acid catalyst) preparation of known method as describing in JP-A 28512/1986 itself.
According to the difference of ratio of components or weight average molecular weight, the decomposition of lactic acid/glycolic acid polymer/elimination speed can be very different.Because decomposition/elimination effect postpones along with the decline of glycolic ratio usually, thus ratio or increase molecular weight by reducing glycolic, but the persistent period that prolong drug discharges.On the contrary, by ratio or the minimizing molecular weight that increases glycolic, can shorten the persistent period of drug release.The preparation of long-acting in order to obtain (for example 1-4 month) slow release preferably uses proportion of composing and the weight average molecular weight lactic acid/glycolic acid polymer in above-mentioned scope.
Therefore, in the present invention, the composition of used biodegradable polymer is preferably selected according to the kind of required biologically active polypeptide and the release duration of needs.In a specific embodiment, for example when the biologically active polypeptide that uses was GH, lactic acid/ethanol copolymer was preferred the use.In lactic acid/ethanol copolymer, it is about 50/50 that lactic acid/glycolic proportion of composing (mol%) is preferably about 85/15-, more preferably about 75/25-about 50/50.It is about 20000 that the weight average molecular weight of lactic acid/ethanol copolymer is preferably about 8000-, more preferably about 10000-about 20000.In addition, it is about 4.0 that the dispersion of lactic acid/ethanol copolymer (weight-average molecular weight/number-average molecular weight) is preferably about 1.2-, more preferably about 1.5-about 3.5.
Used lactic acid/ethanol copolymer can wait by the method that known method is for example described in above-mentioned publication and prepare.Preferred copolymer is the copolymer by the dehydration polyreaction preparation of not using catalyst.Preferred number-average molecular weight of determining according to end group quantity and the almost equal each other lactic acid/ethanol copolymer of measuring according to GPC (PLGA) of number-average molecular weight of using.
In addition, two kinds of lactic acid/ethanol copolymers different aspect proportion of composing and weight average molecular weight also can use with the form of the mixture of given proportioning.Typical example is the mixture of following two kinds of lactic acid/ethanol copolymers: a kind of proportion of composing of its lactic acid/glycolic (mol%) is about 75/25, and weight average molecular weight is about 10000; The proportion of composing (mol%) of another kind of its lactic acid/glycolic is about 50/50, and weight average molecular weight is about 12000.Preferred weight ratio is about 25/75-about 75/25 in the mixture.
In the present invention, the used zinc oxide of preparation PLGA-zinc oxide complex preparation is the zinc compound that is slightly soluble in water, and itself is insoluble or slight dissolving in organic solvent such as dichloromethane.Make us very unexpectedly, zinc oxide and the PLGA coexistence in organic solvent such as dichloromethane has formed PLGA-zinc oxide complex effectively, and complex can dissolve in organic solvent subsequently.These operations only need to add PLGA in organic solvent and zinc oxide just can be finished simply, do not need to separate the step of PLGA-zinc oxide complex.Physiological active polypeptide is joined in the solution of thus obtained PLGA-zinc oxide complex in organic solvent, can easily prepare the MC that contains physiological active polypeptide.In addition, thus obtained MC can make physiological active polypeptide keep bio-stable, and can provide the initial stage to discharge the slow releasing preparation that is lowered and has good sustained release performance.
The physiological active polypeptide that the present invention uses comprises that molecular weight is preferably about 50000, the physiological active polypeptide of about 5000-about 40000 more preferably of about 1000-.
The typical activity of physiological active polypeptide is a hormonal activity.Physiological active polypeptide can be natural prodcuts, sintetics, semi-synthetic product, with and derivant and analog.The model of action of physiological active polypeptide can be excitement or antagonism.
The physiological active polypeptide that the present invention uses comprises peptide hormone, cytokine, peptide neurotransmitter, the hemopoietic factor, various somatomedin, enzyme, peptide antibiotics and analgesic peptide.
The example of peptide hormone comprises insulin, somatostatin, somatostatin derivant (Sandostatin; Referring to United States Patent (USP) 4087390,4093574,4100117 and 4253998), growth hormone (GH), diuretic hormone sodium, gastrin, prolactin antagonist, thyroliberin (ACTH), ACTH derivant (for example ebiratide), melanotropin (MSH), throtropin releasing hormone (TRH) and its salt and derivant (referring to JP-A 121273/1975 and 116465/1977), thyrotropin (TSH), metakentrin (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), thymosin, motilin, vasopressin, vasopressin derivant (desmopressin, referring to Folia Endocrinologica Japonica, the 54th volume, No.5,676-691 page or leaf (1978)), oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin, Pancreozymin, cholecystokinin, angiotensin, and human placental lactogen.Peptide hormone is preferably insulin and growth hormone.
Cytokine comprises lymphokine and monokine.Lymphokine comprises interferon (α, β and IFN-) and interleukin (IL-2,3,4,5,6,7,8,9,10,11,12).Monokine comprises il-1 (IL-1) and tumor necrosis factor (TNF).Preferred cytokine is a lymphokine, more preferably interferon.Particularly preferred cytokine is an alpha-interferon.
The peptide neurotransmitter comprises P material, 5-hydroxy tryptamine and GABA.
Hemopoietic factor comprise erythropoietin (EPO), colony stimulating factor (G-CSF, GM-CSF, M-CSF), thrombopoietin (TPO), platelet derived growth factor and megalokaryocyte enhancer (popentiator).
Various somatomedin comprise alkalescence and acid fibroblast growth factor (FGF) and its family (for example EGF, TGF-α, TGF-β, PDGF, acid FGF, basic FGF, FGF-9), nerve growth factor (NGF) and its family (for example BDNF, NT-3, NT-4, CNTF, GDNF), insulin like growth factor (for example IGF-1, IGF-2 etc.) and bone morphogenetic protein (BMP) and its family.
Enzyme comprises superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA), asparaginase and kallikrein.
Peptide antibiotics comprises polymyxin B (polymixin B), colistin, Gramicidin and bacitracin.
The analog of peptide comprises enkephalin, enkephalin derivant (referring to United States Patent (USP) 4277394 and EP-A 31567), endorphins and kyotorphin.
In addition, physiological active polypeptide also comprises thymopoietin, dynorphin, bombesin, caerulin, thymostimulin (thymostimulin), thymic humoral factor (THF), blood thymic factor (FTS) and its derivant (referring to United States Patent (USP) 4229438), other thymic factor [Igaku no Ayumi, the 125th volume, No.10,835-843 page or leaf (1983)], neurotensin, Kallidin I and Endothelin-antagonism peptide (referring to EP-A 436189,457195 and 496452, JP-A94692/1991 and 130299/1991).
Particularly preferred biologically active polypeptide comprises growth hormone and insulin.
In the present invention, when biologically active polypeptide contained metal, the content of metal preferably was no more than 0.1%, more preferably no more than 0.01%, was most preferably not exceeding 0.001%.Therefore, the physiological active polypeptide that is substantially devoid of metal be suitable for most of the present invention.For example, crystalline insulin contains a spot of heavy metal such as zinc, nickel, cobalt and cadmium usually.The insulin that contains 0.4% (w/w) zinc exists with stable hexamer form, and is showing very strong inertia aspect the interaction of biodegradable polymer slaine.
If necessary, can in advance the metal that exists in the biologically active polypeptide be removed.Adopt known method of removing metal.For example, with the aqueous hydrochloric acid solution water of insulin or ammonium acetate aqueous solution dialysis, and the amorphous insulin lyophilization that contains the denier metal that dialysis solution is provided.
Growth hormone can derive from any kind, preferably the growth hormone that obtains from philtrum.In addition, though the natural prodcuts of extracting from hypophysis can be used for the present invention, preferred gene recombinant type GH (JP-B 12996/1994,48987/1994).More preferably structure, the recombinant type people GH that at N-end do not have methionine identical with natural type.This GH can slaine form exist, and the GH that is substantially devoid of metal also can use.
The preferred boiling point of organic solvent that the present invention uses is no more than 120 ℃ solvent.These organic solvents comprise halogenated hydrocarbons (for example dichloromethane, chloroform, carbon tetrachloride etc.), alcohol (for example ethanol, methanol, 1,4-butanediol, 1,5-pentanediol etc.), ethyl acetate, acetonitrile etc.These solvents can also use with the form of the mixture of giving certainty ratio.Preferably the organic solvent that uses separately comprises, for example dichloromethane and acetonitrile.
Preferably the organic solvent that uses with form of mixtures comprises, for example the mixture of halogenated hydrocarbons (for example dichloromethane, chloroform) and alcohol (for example ethanol, methanol, 1,4-butanediol, 1,5-pentanediol) or acetonitrile.It is especially extensive that the mixture of dichloromethane and acetonitrile is used.Halogenated hydrocarbons is about 40 with respect to the mixing ratio (volume ratio) of alcohol or acetonitrile: about 1: 1 of 1-, preferred about 20: about 1: 1 of 1-.The independent use of halogenated hydrocarbons such as dichloromethane is especially preferred.
In the present invention, producing the used zinc oxide of slow releasing preparation preferably uses with the state of fine powder.Though the expected response time can shorten along with diminishing of diameter, the problem of operating aspect can appear, because fragility (fryability) has increased simultaneously again.The particle diameter of zinc oxide is about the about 10 μ m of 0.001 μ m-usually, is preferably the about 1 μ m of about 0.005 μ m-, the about 0.1 μ m of more preferably about 0.01 μ m-.
In the organic solvent solution of biodegradable polymer and zinc oxide, the content of zinc (Zn) (with respect to the weight ratio of biodegradable polymer) is preferably about 0.001%-2% (w/w), more preferably about 0.01%-2% (w/w) most preferably is about 0.1%-2% (w/w).Zinc in biodegradable polymer and zinc oxide the content in the solution in organic solvent can measure by known conventional method such as atomic absorption analysis.
In this manual, as long as slow releasing preparation is that it just without limits with the form existence of the fine particle that contains physiological active polypeptide and microcapsule substrate (being biodegradable polymer-zinc oxide complex).The example of fine particle comprises, contains the microcapsule of a medicine nuclear in each granule, contains the multinuclear microcapsule of high amount of drug nuclear in each granule, and medicine is dissolved in molecular forms or is dispersed in microsphere in the microcapsule substrate.
Slow releasing preparation among the present invention can prepare like this, physiological active polypeptide is dispersed in the organic solvent solution of biodegradable polymer and zinc oxide, and then removes organic solvent.In this manual, the product that is generated by biodegradable polymer and zinc oxide, form in biodegradable polymer and zinc oxide are dissolved in settled solution in the organic solvent is called " biodegradable polymer-zinc oxide complex ".
Biodegradable polymer-zinc oxide complex can be the chemical compound that is produced by intermolecular interaction, for example normal salt, complex salt, double salt and organo-metallic compound, or compositions.
The feature that biodegradable polymer-the zinc oxide complex has is, for example is dissolved in organic solvent, makes the slow releasing preparation as final products have good slow-releasing.In addition, when the biodegradable polymer in the complex was PLGA, this complex was called " PLGA-zinc oxide complex ".
In this manual, " dispersion liquid " is meant the homodisperse liquid of biologically active polypeptide in organic solvent.Solution and the suspension of biologically active polypeptide in organic solvent all is included in the dispersion liquid of the present invention.
In the method for the invention, the method for removing organic solvent comprises, for example, and (a) seasoning (oil/water law) in the water, (b) phase separation method (coacervation), (c) spray drying method and its degeneration.
Introduce the method for preparing the microcapsule slow releasing preparation below.
In the method for the invention, biodegradable polymer and zinc oxide are joined prepare the biodegradable polymer-solution of zinc oxide complex in organic solvent in the organic solvent earlier.Though the concentration of biodegradable polymer in solution is different and different according to its molecular weight and organic solvent kind, but it is, about 80% (weight ratio) of for example about 0.1%-is preferably about 70% (weight ratio) of about 1%-, about 60% (weight ratio) of more preferably about 2%-.The amount that adds zinc oxide is according to the difference of organic solvent kind and difference, and be about 5% (weight ratio) of for example about 0.001%-, preferred about 2.5% (weight ratio) of about 0.01%-, about 2.5% (weight ratio) of 0.1%-more preferably from about, wherein wt calculates than the amount that is based on biodegradable polymer.
The order that in organic solvent, adds as for biodegradable polymer and zinc oxide, the zinc oxide that exists with pulverulence or dispersity form can be joined biodegradable polymer in organic solvent in the solution of organic solvent, on the contrary, the solution of biodegradable polymer in organic solvent can be added in the dispersion liquid of zinc oxide in organic solvent.In addition, also can after biodegradable polymer that all exists and zinc oxide mixing, add organic solvent again with pulverulence.
With biodegradable polymer and zinc oxide prepare biodegradable polymer-zinc oxide complex for example the condition of PLGA-zinc oxide complex can be according to the kind of the particle diameter of used biodegradable polymer kind, zinc oxide, organic solvent, these component different and different.For example, when PLGA is used as polymer, PLGA-zinc oxide complex can obtain by following reaction condition, usually at about 0 ℃-Yue 30 ℃, preferred about 2 ℃-Yue 25 ℃, and react about 1-about 168 hours, preferably about 96 hours of about 12-, about 72 hours of 24-more preferably from about.Yet the response time is not limited to above-mentioned scope, and available indicator solution is determined by the perusal reactive state.In the present invention, the preparation of PLGA-zinc oxide complex can confirm by perusal, because the zinc oxide of dispersity is dissolved in the organic solvent to form settled solution adding fashionable meeting.
Though just the coexistence in organic solvent is carried out by PLGA and zinc oxide in this reaction, in order to shorten the response time, by make reaction under agitation or be favourable under the jolting with suitable agitator or jolting device.In addition, it is same preferred reacting under ultrasonic.When reaction temperature uprised some, the response time can shorten.Yet higher reaction temperature can be with the rapid degraded of PLGA.
Thus obtained biodegradable polymer-zinc oxide complex preferably is used for next step with the form of organic solution, if necessary, removes organic solvent and is used for next step with solid form.
Then, with physiological active polypeptide preferably with pulverulence, with about 50% (weight ratio) of about 0.1%-, preferably with about 20% (weight ratio) of about 1%-, more preferably with the amount of about 3%-about 15% (weight ratio), be dissolved in or be dispersed in the organic solution of biodegradable polymer and zinc oxide, with preparation biodegradable polymer, zinc oxide and the dispersion liquid (hereinafter it simply be called physiological active polypeptide dispersion liquid) of biologically active polypeptide in organic solvent.
In the organic solution of biodegradable polymer and zinc oxide, do not dissolve, add affiliation generation muddiness and be difficult to disperse such characteristic with pulverulence, preferred earlier physiological active polypeptide being dispersed in the organic solvent if physiological active polypeptide for example has.The stabilizing agent (for example serum albumin, gelatin, protamine sulfate) that can in organic solution, add physiological active polypeptide.
For physiological active polypeptide is evenly dispersed in the organic solvent, preferably give the external physical energy.These methods comprise, for example, and ultrasonic, turbine type agitator and homogenizer.In this case, the particle diameter of physiological active polypeptide in organic solvent is about the about 200 μ m of 0.01-, preferably is about the about 100 μ m of 0.05-, is more preferably the about 50 μ m of 0.1-.It is about 50% that the concentration of physiological active polypeptide in organic solvent is about 1%-, preferably is about 2%-about 20%.This processing makes the particle diameter of physiological active polypeptide in organic solvent have uniformity, and physiological active polypeptide can be disperseed in the organic solution of biodegradable polymer and zinc oxide equably.
In addition, can physiological active polypeptide be independent of outside biodegradable polymer-zinc oxide complex earlier and be dispersed in the organic solvent individually.In this case, the composition of the composition of the organic solvent of use and the organic solvent that is used to dissolve biodegradable polymer and zinc oxide each other can be identical, also can be different.For example, biodegradable polymer-zinc oxide complex can be dissolved in the dichloromethane, physiological active polypeptide is dispersed in the acetonitrile, then both be mixed.In this case, the ratio (volume ratio) between physiological active polypeptide and biodegradable polymer-zinc oxide complex is, for example, and about 1: about 1: 1 of 1000-, preferred about 1: about 1: 5 of 200-, especially preferred about 1: about 1: 5 of 100-.
(a) seasoning (oil/water law) in the water
In dispersion liquid, add water again to form oil/aqueous emulsion with the physiological active polypeptide of method for preparing.Then the solvent evaporates in the oil phase is gone out to form microcapsule.In this case, can add emulsifying agent to outside aqueous phase.Added emulsifying agent can be any material that stable oil/aqueous emulsion can be provided.The example of these emulsifying agents comprises anionic surfactant, nonionic surfactant, polyethylene glycol oxide-castor oil derivative, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid (hyaruronic acid) etc.Preferred solvent is a polyvinyl alcohol.These emulsifying agents can use separately, or two or more emulsifying agent is united use.The emulsifying agent externally concentration of aqueous phase is about about 20% (weight ratio) of 0.001%-, preferably is about about 10% (weight ratio) of 0.01%-, especially preferably is about about 5% (weight ratio) of 0.05%-.
By centrifugal or filtered and recycled, to remove emulsion breaker etc. attached to the material on the microcapsule, redispersion is in distilled water, and lyophilization with distilled water wash with thus obtained microcapsule.
Then, if necessary, by further adding water and the organic solvent in the heat extraction microcapsule.Heating can be carried out under reduced pressure.As for heating condition, heating and drying are at the glass transition temperature that is not less than biodegradable polymer and not highly carry out to causing under the accumulative temperature of each microcapsule granule.Heating and dry preferred glass transition temperature in biodegradable polymer carry out under than the temperature between the about 30 ℃ temperature of the glass transition temperature height of biodegradable polymer.Glass transition temperature is defined as, when temperature increases with the speed of 10 ℃-20 ℃ of per minutes, and the intermediate glass point that uses differential scanning calorimeter to measure.
(b) phase separation method (coacervation)
When preparing MC, under agitation in the dispersion liquid of above-mentioned physiological active polypeptide, add flocculating agent gradually with precipitation and curing MC with the method.The amount that flocculating agent uses is about the about 1000 times of volumes of 0.01-, preferably is about the about 500 times of volumes of 0.05-, especially preferably is about the about 200 times of volumes of 0.1-.As long as flocculating agent is polymer, mineral oil or vegetable oil, can mix and can not dissolve the chemical compound of used biodegradable polymer with the organic solvent that is used for dissolving biodegradable polymer is molten, it just can use so.Specifically, the example of these flocculating agents comprises silicone oil, Oleum sesami, Oleum Glycines, Semen Maydis oil, Oleum Gossypii semen, Oleum Cocois, Semen Lini oil, mineral oil, normal hexane and normal heptane.Two or more these flocculating agents can be united use.
Thus obtained MC can pass through filtered and recycled, with cyclic washings such as heptane to remove flocculating agent.In addition, the mode that reuse is identical with above-mentioned (a) is washed lyophilization then.
When seasoning in the water or coacervation prepare MC, can in the step of washing MC, add anti-aggregating agent prepared therefrom to prevent agglomeration of particles.The example of anti-aggregating agent prepared therefrom comprises, for example, and water soluble polysaccharide such as mannitol, lactose, glucose, starch (for example corn starch), hyaluronic acid and its alkali metal salt; Aminoacid is glycine and alanine for example; Albumen is fibrin and collagen for example; With inorganic salt for example sodium chloride and sodium hydrogen phosphate, or the like.
(c) spray drying method
When preparing MC, with nozzle the dispersion liquid of physiological active polypeptide is sprayed onto in the drying chamber of spray dryer and generates MC in very short time so that the organic solvent in the fine droplets volatilizees away with the method.The example of nozzle comprises, for example, and second-rate nozzle type, drive nozzle type and rotational circle dish-type.If necessary, above-mentioned anti-aggregating agent prepared therefrom is sprayed to prevent each MC particle aggregation through another nozzle, this also is favourable.
With the mode identical thus obtained MC is washed, if necessary, heat (under reduced pressure, heating if necessary) subsequently and come except that anhydrating and organic solvent with above-mentioned (a).
In the present invention, when using PLGA-zinc oxide complex as microcapsule substrate, the ratio that physiological active polypeptide such as GH embedding are advanced among the MC is preferably at least 50%.
The content of physiological active polypeptide in slow releasing preparation of the present invention is about 30% (weight ratio) of for example about 0.1%-, about 20% (weight ratio) of preferably about 0.2%-, more preferably from about about 10% (weight ratio) of 0.5%-.
As raw material, slow releasing preparation of the present invention can be with for example following dosage form administration with fine particle: fine particle is microcapsule as mentioned above for example, or the multiple dosage form of non-oral formulation (for example intramuscular, subcutaneous or internal organs injection; Can not keep somewhere (indwellable) preparation; Or oral formulations (for example, capsule such as hard capsule and soft capsule etc. the saturating mucosa preparation in intranasal, rectum or uterus etc.); Solid preparation such as granule and powder etc.; Liquid preparation such as suspension etc.).
The preparation of these dosage forms can be with pharmaceutical field known method production commonly used.
Slow releasing preparation of the present invention is preferably made the form of injection.In order to prepare injection as microcapsule by method for preparing with fine particle, can be with fine particle and dispersant (for example, surfactant such as Tween 80, HCO-60; Polysaccharide such as carboxymethyl cellulose, sodium alginate, hyaluronate sodium; Protamine sulfate; PEG400 etc.), antiseptic (for example methyl parahydroxybenzoate, propyl p-hydroxybenzoate etc.), isoosmotic adjusting agent (for example sodium chloride, mannitol, Sorbitol, glucose etc.) and local anesthetic (for example lidocaine hydrochloride, methaform etc.) prepare waterborne suspension together, or disperse or its mixture prepares oily suspensions with phospholipid (for example lecithin) or MCT Oil (for example Miglyol 812) dispersion with vegetable oil (for example Oleum sesami, corn wet goods).
Slow releasing preparation of the present invention especially preferably exists with the form of fine particle.The optional self energy of particle diameter of making the slow releasing preparation of injectable suspensions satisfies the required dispersion of injection and the scope of syringe needle trafficability characteristic.For example, particle diameter is the about 300 μ m of about 0.1-, is preferably the about 150 μ m of about 1-, the about 100 μ m of more preferably about 2-, and wherein said particle diameter is particulate average diameter.
It is the method for carrying out under aseptic condition that the method that above-mentioned fine particle is made sterile preparation includes, but not limited to whole process of preparation, the method for using gamma-rays to sterilize, and the method that adds antibacterial in preparation process.
Slow releasing preparation of the present invention can be used for mammal (for example people, cattle, pig, Canis familiaris L., cat, mice, rat, rabbit etc.) safely with very low toxicity.
The indication of slow releasing preparation of the present invention is according to the difference of used physiological active polypeptide and difference.When insulin was used as physiological active polypeptide, slow releasing preparation can be used for prevention or treatment diabetes; When using alpha-interferon, slow releasing preparation can be used for prevention or treatment viral hepatitis (for example hepatitis C, HBe antigen positive hepatitis) and cancer (for example renal carcinoma, multiple myeloma etc.); When using erythropoietin, slow releasing preparation can be used for prevention or treatment anemia (for example anemia between the kidney dialysis period); When using G-CSF, slow releasing preparation can be used for prevention or treatment neutropenia (for example neutropenia that takes place in cancer treatment procedure) and infect; When using IL-2, slow releasing preparation can be used for prevention or treatment cancer (for example hemangioendothelioma); When using FGF, slow releasing preparation can be used for prevention or treatment fracture, wound (for example decubital ulcer), periodontitis and gastrointestinal ulceration; When using FGF-9, slow releasing preparation can be used for prevention or treatment thrombocytopenia; When using NGF, slow releasing preparation can be used for prevention or treatment alzheimer disease and neuropathy; When using TPA, slow releasing preparation can be used for prevention or treatment thrombosis; When using the tumor necrosis factor period of the day from 11 p.m. to 1 a.m, slow releasing preparation can be used for prevention or treatment cancer.
In addition, based on the growth hormone activity of GH, the slow releasing preparation that contains GH can be used for the treatment of Turner's syndrome, chronic nephropathy, achondroplasia, adult's hypopituitarism and pituitary dwarfism.In addition, it is reported that GH has good curative effect aspect Down syndrome, Silver syndrome, hypochondroplasia and the juvenile chronic arthritis for example treating.
Though the dosage of slow releasing preparation can change according to kind and the disease of content, release time, pretreat, the kind of being treated animal and the different of other factors of biologically active polypeptide, as long as but can keep physiological active polypeptide valid density in vivo, slow releasing preparation can be used with the dosage of any level.
For example, when slow releasing preparation is when being designed to discharge the dosage form in a week, the dosage of biologically active polypeptide is preferably the about 10mg/kg body weight of about 0.0001-/adult, the about 1mg/kg body weight of more preferably about 0.0005-/adult., according to kind and content, dosage form, release duration, the disease of pretreat, the kind of being treated animal and the other factors of physiological active polypeptide, it is 1 inferior that the preferred administration frequency of slow releasing preparation can suitably be elected 1 time weekly, per two weeks as.
When the active component physiological active polypeptide in the slow releasing preparation is for example during insulin, to the about 1mg/kg body weight of the normally about 0.001-of the dosage of the each administration of diabetes patient, the about 0.2mg/kg body weight of preferably about 0.01-.And preferred administration frequency is 1 time weekly.
When the active component physiological active polypeptide in the slow releasing preparation is GH, though dosage changes according to kind and the disease of content, release duration, pretreat, the kind of being treated animal and the different of other factors of GH, as long as but can keep GH valid density in vivo, slow releasing preparation can be used with the dosage of any level.As for to above-mentioned treatment of diseases, when slow releasing preparation is when being designed to discharge the dosage form in two weeks, for each child or adult's safe administration, the dosage of GH can be the about 5mg/kg body weight of about 0.01-, the about 1mg/kg body weight of more preferably about 0.05-.According to content, dosage form, release duration, the disease of pretreat, the kind of being treated animal and the other factors of GH, it is inferior that preferred administration frequency can suitably be elected 1 time weekly, per two 1 time, every month 1 weeks as.
Slow releasing preparation preferably at normal temperatures or shady and cool place store.Slow releasing preparation more preferably stores in the cool." room temperature " and " shady and cool place " has done definition in Japanese Pharmacopoeia.That is, 15 ℃-25 ℃ of " room temperature " expressions, " shady and cool place " expression temperature is no more than 15 ℃.
Implement best mode of the present invention
Describe the present invention in more detail below by preparation example and test example, but should not be construed as limiting the scope of the invention.Preparation example 1
With 1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 10000) and the 6.6mg zinc oxide join in the 1.7ml dichloromethane, 25 ℃ of stirrings (60rpm) 3 days to obtain the lactic acid/ethanol copolymer-settled solution of zinc oxide complex in organic solvent.In this solution, add 53.0mg human growth hormone's lyophilization powder, it is mixed with vortex agitator and small-sized homogenizer.Then, carry out supersound process, to obtain human growth hormone and the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.Pour this organic solution into 400ml in advance at polyvinyl alcohol (PVA) aqueous solution of 18 ℃ of 0.1% (weight/volume) of regulating, then with the emulsifying of turbine type homo-mixer to obtain oil/aqueous emulsion.At room temperature stir this oil/aqueous emulsion so that dichloromethane volatilizees away.The microcapsule that collect to generate by centrifugal (about 1500rpm) with 400ml distilled water wash 2 times, carries out lyophilization with the precipitation that obtains, obtained the Powdered microcapsule that 521mg contains the human growth hormone.Preparation example 2
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 10000) and 13.1mg zinc oxide are dissolved in the 2.3ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.3mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 536mg contains the human growth hormone.Preparation example 3
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 10000) and 21.9mg zinc oxide are dissolved in the 2.8ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.8mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 589mg contains the human growth hormone.Preparation example 4
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 12000) and 5.1mg zinc oxide are dissolved in the 1.9ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 52.9mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 506mg contains the human growth hormone.Preparation example 5
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 12000) and 10.2mg zinc oxide are dissolved in the 2.5ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.2mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 568mg contains the human growth hormone.Preparation example 6
1g lactic acid/ethanol copolymer (lactic acid/glycolic=65/35 (mol%), weight average molecular weight is 12000) and 17.0mg zinc oxide are dissolved in the 3.0ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.5mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 561mg contains the human growth hormone.Preparation example 7
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 15000) and 4.5mg zinc oxide are dissolved in the 2.0ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 52.9mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 540mg contains the human growth hormone.Preparation example 8
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 15000) and 8.9mg zinc oxide are dissolved in the 2.6ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.1mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 559mg contains the human growth hormone.Preparation example 9
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 15000) and 14.9mg zinc oxide are dissolved in the 3.1ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.4mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 464mg contains the human growth hormone.Preparation example 10
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 20000) and 4.0mg zinc oxide are dissolved in the 2.5ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 52.8mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 595mg contains the human growth hormone.Preparation example 11
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 20000) and 7.9mg zinc oxide are dissolved in the 3.6ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.1mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 478mg contains the human growth hormone.Preparation example 12
1g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 20000) and 13.2mg zinc oxide are dissolved in the 5.2ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.3mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 534mg contains the human growth hormone.Preparation example 13
1g lactic acid/ethanol copolymer (lactic acid/glycolic=75/25 (mol%), weight average molecular weight is 10500) and 6.6mg zinc oxide are dissolved in the 3.0ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.0mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 521mg contains the human growth hormone.Preparation example 14
1g lactic acid/ethanol copolymer (lactic acid/glycolic=85/15 (mol%), weight average molecular weight is 12000) and 5.8mg zinc oxide are dissolved in the 2.0ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 53.0mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 503mg contains the human growth hormone.Preparation example 15
1.89g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 10000) and 10mg zinc oxide are dissolved in the 3.4ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.41g contains the human growth hormone.Preparation example 16
1.89g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 12000) and 10mg zinc oxide are dissolved in the 3.5ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.41g contains the human growth hormone.Preparation example 17
1.89g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 14000) and 10mg zinc oxide are dissolved in the 4.0ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.40g contains the human growth hormone.Preparation example 18
1.89g lactic acid/ethanol copolymer (lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 16000) and 10mg zinc oxide are dissolved in the 4.2ml dichloromethane, to obtain the lactic acid/ethanol copolymer-solution of zinc oxide complex in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.34g contains the human growth hormone.Comparative Examples 1
(lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 15000) is dissolved in (950mg/ml) in the dichloromethane with lactic acid/ethanol copolymer, with the solution of preparation lactic acid/ethanol copolymer in organic solvent.This solution of 1ml is mixed with 1ml human growth hormone's the solution (50mg/ml) of lyophilization powder in dichloromethane, use the mode identical to handle, obtained the Powdered microcapsule that 490mg contains the human growth hormone with preparation example 1.Comparative Examples 2
(lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 10000) is dissolved in the 2.6ml dichloromethane with 1.90g lactic acid/ethanol copolymer, with the solution of preparation lactic acid/ethanol copolymer in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.28g contains the human growth hormone.Comparative Examples 3
(lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 12000) is dissolved in the 2.8ml dichloromethane with 1.90g lactic acid/ethanol copolymer, with the solution of preparation lactic acid/ethanol copolymer in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.18g contains the human growth hormone.Comparative Examples 4
(lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 14000) is dissolved in the 3.0ml dichloromethane with 1.90g lactic acid/ethanol copolymer, with the solution of preparation lactic acid/ethanol copolymer in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 0.89g contains the human growth hormone.Comparative Examples 5
(lactic acid/glycolic=50/50 (mol%), weight average molecular weight is 16000) is dissolved in the 3.2ml dichloromethane with 1.90g lactic acid/ethanol copolymer, with the solution of preparation lactic acid/ethanol copolymer in organic solvent.The lyophilization powder that adds the 100mg human growth hormone in this solution uses the mode identical with preparation example 1 to handle, and obtained the Powdered microcapsule that 1.26g contains the human growth hormone.Test example 1
The microcapsule that 308mg is prepared in preparation example 7, contain human growth hormone and PLGA-zinc oxide complex; That 351mg prepares in preparation example 8, as to contain human growth hormone and PLGA-zinc oxide complex microcapsule; That 327mg prepares in preparation example 9, as to contain human growth hormone and PLGA-zinc oxide complex microcapsule; With 229mg microcapsule that prepare, that contain human growth hormone and PLGA in Comparative Examples 1; Be dispersed in respectively in 2.25ml disperse medium (composition of disperse medium: mannitol (5%), carboxymethyl cellulose (0.5%) and polysorbas20 (0.1%) are dissolved in the distilled water, with acetic acid PH are transferred to 6.0 then) (disperse medium that following use is identical), 2.25ml disperse medium, 1.75ml disperse medium and the 1.75ml disperse medium.
With the back of the thus obtained dispersion liquid of 0.5ml (containing 3mg people GH) subcutaneous administration to the rat of using etherization.As time goes on, gather blood and separation of serum through the tail vein.Measure the concentration of people GH in the serum that obtains by radioimmunoassay, RIA (Ab Beads HGH, by Eiken Kagaku, Japan makes).Measurement result is listed in the table 1.
Table 1
| The slow releasing preparation that contains the human growth hormone | Haemoconcentration (ng/ml) | ||||||
| The 1st day | The 2nd day | The 4th day | The 6th day | The 7th day | The 9th day | The 11st day | |
| Preparation example 7 | ??16.7 | ????8.6 | ????8.4 | ????14.8 | ????26.6 | ????13.5 | ????2.8 |
| Preparation example 8 | ??17.9 | ????10.6 | ????14.1 | ????22.5 | ????27.8 | ????15.9 | ????3.5 |
| Preparation example 9 | ??14.0 | ????8.5 | ????15.8 | ????33.9 | ????28.2 | ????17.8 | ????7.7 |
| Comparative Examples 1 | ????4.3 | ????1.7 | ????2.1 | ????3.1 | ????5.0 | ????8.0 | ????2.7 |
Compare with the test group of using the microcapsule administration that contains human growth hormone and PLGA that from comparative example 1, obtains, in the test group of using the microcapsule administration that contains human growth hormone and PLGA-zinc oxide complex that from preparation example 7,8 and 9, obtains, the concentration of people GH shows quite high value, in addition, also show long slow release effect.The method according to this invention can be produced the slow releasing preparation of excellent releasing effect.Test example 2
With 550mg, 556mg, 576mg and 573mg respectively in preparation example 15,16,17 and 18 preparation, the microcapsule that contains human growth hormone and PLGA-zinc oxide complex is dispersed in 3.38ml respectively as in the test example 1 described disperse medium.Simultaneously with 548mg, 548mg, 567mg and 560mg respectively in comparative example 2,3,4 and 5 preparation, the microcapsule that contains human growth hormone and PLGA is dispersed in respectively in the identical disperse medium of 3.38ml.
With the back of the thus obtained dispersion liquid of 0.75ml (containing 6mg people GH) subcutaneous administration to the rat of using etherization.As time goes on, gather blood and separation of serum through the tail vein.Concentration by people GH in the serum that obtains as test example 1 described radioimmunoassay determination.Measurement result is listed among the table 2-5.
Table 2
Table 3
| The slow releasing preparation that contains the human growth hormone | Haemoconcentration (ng/ml) | |||
| The 4th day | The 7th day | The 9th day | The 11st day | |
| Preparation example 15 | ??24.8 | ??24.5 | ????15.2 | ????6.3 |
| Comparative example 2 | ??13.8 | ??8.1 | ????5.1 | ????3.9 |
| The slow releasing preparation that contains the human growth hormone | Haemoconcentration (ng/ml) | |||
| The 4th day | The 7th day | The 9th day | The 11st day | |
| Preparation example 16 | ????24.6 | ????25.3 | ????16.3 | ????8.7 |
| Comparative example 3 | ????9.2 | ????10.1 | ????9.6 | ????6.8 |
Table 4
| The slow releasing preparation that contains the human growth hormone | Haemoconcentration (ng/ml) | |||
| The 4th day | The 7th day | The 9th day | The 11st day | |
| Preparation example 17 | ????12.5 | ????35.4 | ????29.5 | ????9.7 |
| Comparative example 4 | ????7.6 | ????14.8 | ????7.7 | ????6.2 |
Table 5
| The slow releasing preparation that contains the human growth hormone | Haemoconcentration (ng/ml) | |||
| The 4th day | The 7th day | The 9th day | The 11st day | |
| Preparation example 18 | ??11.8 | ??31.8 | ??35.5 | ??11.3 |
| Comparative example 5 | ??4.9 | ??13.8 | ??18.5 | ??6.7 |
Compare with the test group of using the microcapsule administration that contains human growth hormone and PLGA that from comparative example 2,3,4 and 5, obtains, in the test group of using the microcapsule administration that contains human growth hormone and PLGA-zinc oxide complex that obtains from preparation example 15,16,17 and 18, the concentration of people GH shows quite high value.Industrial applicibility
According to the present invention, can provide physiological active polypeptide wherein such as growth hormone to have the slow releasing preparation of high embedding ratio and long-time constant high haemoconcentration level.
Claims (15)
1. produce the method for slow releasing preparation, comprise physiological active polypeptide is distributed in biodegradable polymer and the solution of zinc oxide in organic solvent, remove organic solvent then.
2. according to the process of claim 1 wherein that described physiological active polypeptide is a growth hormone.
3. according to the process of claim 1 wherein that described biodegradable polymer is lactic acid/ethanol copolymer.
4. according to the method for claim 3, wherein in lactic acid/ethanol copolymer, the molecular composition ratio of lactic acid/glycolic is about 85/15-about 50/50.
5. according to the method for claim 3, wherein the weight average molecular weight of lactic acid/ethanol copolymer is about 8000-about 20000.
6. according to the process of claim 1 wherein with respect to biodegradable polymer, the content of zinc in organic solvent solution is about 2% weight ratio of about 0.001%-.
7. according to the process of claim 1 wherein that the average particulate diameter of slow releasing preparation is the about 300 μ m of about 0.1-.
8. according to the process of claim 1 wherein that described slow releasing preparation is an injection.
9. carry out drying in the water according to the process of claim 1 wherein with containing the oil/aqueous emulsion of dispersion liquid as oil phase, described dispersion liquid is to prepare by growth hormone being dispersed in lactic acid/ethanol copolymer and the zinc oxide solution in organic solvent.
10. according to the process of claim 1 wherein that described preparation is a microcapsule.
11. a solution is lactic acid/ethanol copolymer and the zinc oxide solution in organic solvent.
12. lactic acid/ethanol copolymer-zinc oxide complex, wherein said complex dissolves in organic solvent, and prepares by lactic acid/ethanol copolymer and zinc oxide are coexisted in organic solvent.
13. dispersion liquid, it is to prepare by physiological active polypeptide being dispersed in lactic acid/ethanol copolymer and the zinc oxide solution in organic solvent.
14. according to the dispersion liquid of claim 13, wherein said physiological active polypeptide is a growth hormone.
15. use slow releasing preparation according to the method preparation of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97180846 CN1241139A (en) | 1996-12-20 | 1997-12-18 | Method of producing sustained-release preparation |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP342046/96 | 1996-12-20 | ||
| CN 97180846 CN1241139A (en) | 1996-12-20 | 1997-12-18 | Method of producing sustained-release preparation |
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| Publication Number | Publication Date |
|---|---|
| CN1241139A true CN1241139A (en) | 2000-01-12 |
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| CN 97180846 Pending CN1241139A (en) | 1996-12-20 | 1997-12-18 | Method of producing sustained-release preparation |
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| CN (1) | CN1241139A (en) |
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1997
- 1997-12-18 CN CN 97180846 patent/CN1241139A/en active Pending
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