JPH11302156A - Method for micronizing polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing polypeptide fine particles having high recovery and high purity without denaturation. SOLUTION: A mixed aqueous solution of a polypeptide and polyethylene glycol is freeze-dried, and an organic solvent which does not dissolve the polypeptide but dissolves the polyethylene glycol is added to the obtained solid, and the polyethylene glycol in the solid is added. Is dissolved to obtain polypeptide fine particles.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for micronizing a polypeptide.

[0002]

2. Description of the Related Art In recent years, polypeptides having a physiological activity have been widely used for the treatment and prevention of various human or animal diseases.

Heretofore, these polypeptides have been generally administered by daily injection in most cases. However, simpler administration methods have been studied because of problems such as complicated administration methods and burden on patients.

[0004] One example of such a method is pulmonary administration. However, in transpulmonary administration, smaller particles can reach the alveoli, so that the polypeptide must be micronized in order to reliably reach the alveoli.

Further, studies have been made to achieve long-term sustained release by encapsulating a polypeptide in a biodegradable polymer to form microspheres, and to locally administer this into a living body by injection.

As a method for producing such microspheres, first, an aqueous solution of a polypeptide is emulsified in an organic solvent such as methylene chloride in which a biodegradable polymer is dissolved to prepare a W / O emulsion. A method for emulsifying water in water to obtain microspheres is known.

However, in general, many polypeptides have a problem in that when they are dissolved in water and come into contact with an organic solvent, they are denatured and lose their activity.

To avoid this problem, it has been studied to disperse the polypeptide as it is in an organic solvent in which a biodegradable polymer is dissolved to form an oil phase, and to prepare a microsphere using the oil phase. However, in this method, if the particle size of the polypeptide is large, a high coverage cannot be obtained, and a large amount of the polypeptide is released in the initial stage of elution (initial burst phenomenon). I needed to.

[0009] As a method of micronizing a polypeptide,
(1) A method of atomizing a mixed aqueous solution of a water-soluble polymer substance such as gelatin and a water-soluble polypeptide by using a spray dryer (JP-A-4-36233), (2) Water-soluble polypeptide and a water-soluble polymer A method of freeze-drying a mixed aqueous solution with a substance and pulverizing the freeze-dried product using a jet mill (JP-A-8-225454); (3) mixing a surfactant and a polypeptide in water; Rapid drying method (JP-A-9-315997).

However, both of the methods (1) and (2) use a large amount of a water-soluble polymer in order to prevent deactivation of the polypeptide from heat or physical collision. Peptide microparticles cannot be obtained, and the recovery rate is very low. In the method (3), since a surfactant is used, the three-dimensional structure of the polypeptide changes depending on the type of the polypeptide, and the biological activity is reduced. And other problems.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing polypeptide fine particles having a high recovery rate and high purity without denaturation.

[0012]

The present inventors have found that when a mixed aqueous solution of a polypeptide and polyethylene glycol is freeze-dried, phase separation occurs between the two and the polypeptide can be dissolved in the obtained solid. It has been found that by adding an organic solvent that can dissolve polyethylene glycol but not polyethylene glycol and dissolve only polyethylene glycol, high-purity polypeptide microparticles can be obtained as a suspension, thereby completing the present invention.

According to the present method, the denaturation or deactivation of the polypeptide hardly occurs because no heat or physical impact force is applied and the solution does not come into contact with the organic solvent.

That is, the present invention is characterized in that a mixed aqueous solution of a polypeptide and polyethylene glycol is freeze-dried, and an organic solvent which does not dissolve the polypeptide but dissolves the polyethylene glycol is added to the obtained solid. To a method for micronizing a polypeptide.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, first, a mixed aqueous solution containing a polypeptide and polyethylene glycol (hereinafter, simply referred to as a mixed aqueous solution) is prepared.

Examples of the polypeptide include various peptides or proteins which have useful physiological activities for mammals and can be used clinically. The polypeptide has a molecular weight of, for example, 1000 as a monomer.
~ 200,000, particularly preferably 50
Those having a size of 00 to 70000 are widely used. These polypeptides also include macromolecules classified as proteins that are said to have higher-order structures in the field of biochemistry. The type of the polypeptide used in the present invention is not particularly limited as long as the object of the present invention is achieved, but it is preferable that the polypeptide has a certain water solubility. 01 mg / ml or more, preferably about 1 mg / ml
That is all.

Specific examples of polypeptides include, for example, hormones, enzymes, cytokines and growth factors. More specifically, for example, the following polypeptides can be mentioned.

Examples of hormones include growth hormone (GH), growth hormone releasing factor (GRF), insulin, glucagon, gastrin, prolactin, adrenocorticotropic hormone (ACTH), and thyroid stimulating hormone (TS).
H), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), calcitonin and the like.

Examples of enzymes include tissue plasminogen activator (tPA), urokinase (U
K), streptokinase, protein C, metalloproteases, superoxide dismutase (SO
D), Factors VIII and IX, peroxidase and the like.

Examples of cytokines include interferons (IFN-α, -β, -γ, etc.), interleukins (IL-1 to IL-11, etc.), cachectin, oncostatin, colony stimulating factors (G- , M
−, GM-CSF, etc.), thrombopoietin (TP
O), erythropoietin (EPO) and the like.

Examples of growth factors include nerve growth factors (NGF-1, NGF-2, etc.) and neurotrophic factors (NT
F), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1,
IGF-2, IGF-3, etc.), fibroblast growth factor (aFGF, bFGF), osteogenic growth factor (BMP-
1, BMP-2, BMP-3, BMP-4, etc.), atrial natriuretic factor (ANP), cartilage inducing factor and the like.

Of these, particularly preferred polypeptides include, for example, growth hormone (eg, 22 kilodalton human growth hormone, 20 kilodalton human growth hormone, etc.), interferons, erythropoietin and the like.

These polypeptides may have a sugar chain, and their sugar chain structures may be different. In addition, they also include muteins, derivatives, analogs and active fragments.

The polypeptide used in the present invention may be either naturally occurring or obtained by genetic recombination techniques.

The polypeptide used in the present invention may be a metal salt. Examples of the metal in such a metal salt include alkaline earth metals (for example, calcium and magnesium) and zinc (II). , Iron (II
(Valent, III-valent), copper (II-valent), tin (II-valent, IV-valent), aluminum (II-valent, III-valent) and the like. , Polypeptide.

The polyethylene glycol used in the present invention is not particularly limited as long as it is commonly used in the field of pharmaceutical technology. Such polyethylene glycol has an average molecular weight of 400 to 500,000, preferably 2000 to 100,000. And particularly preferably 6000 to 70,000.

The aqueous mixed solution may be prepared by dissolving both the polypeptide and polyethylene glycol in water at one time, or dissolving one of them and dissolving the other. A glycol aqueous solution may be mixed. In short, it is only necessary that the polypeptide and polyethylene glycol are finally dissolved in water, and the preparation method is not particularly limited.

The polypeptide concentration in the mixed aqueous solution is
There is no particular limitation as long as it is within the range in which the polypeptide dissolves.
ml, preferably 1 to 100 mg / ml.

The concentration of polyethylene glycol in the mixed aqueous solution is not particularly limited as long as it is within the range in which the polyethylene glycol is dissolved. However, if it is too high, the peptide may precipitate in the aqueous solution, which is not preferable. A preferred concentration of polyethylene glycol is, for example, 0.025 to 200 mg / ml, preferably 0.25 to 100 mg / ml.

Next, the mixed aqueous solution is freeze-dried, and to the obtained solid, an organic solvent which does not dissolve the polypeptide but dissolves the polyethylene glycol is added, and the polyethylene glycol is dissolved by adding an organic solvent. Peptide microparticles are obtained as an organic solvent suspension.

In freeze-drying the mixed aqueous solution,
First, the mixed aqueous solution is frozen, and the freezing is preferably performed under such conditions that the temperature of the mixed aqueous solution gradually decreases rather than rapidly. Such conditions can be easily achieved by freezing using a commonly used freezer or freeze dryer having a set temperature of −20 to −60 ° C. If these conditions are specified more specifically, The initial cooling rate (the temperature of the mixed aqueous solution per minute after the start of cooling) is about 2 to 40 ° C./min, preferably about 10 to 30 ° C./min.
Minutes.

The frozen mixed aqueous solution may be subsequently decompressed and sublimated in a freeze dryer in accordance with a conventional method.

In the present invention, in the freezing process of the freeze-drying step, coagulation is first started from water in the mixed aqueous solution, so that the concentration of the polypeptide and polyethylene glycol in the non-ice crystal portion occurs, and as a result, Phase separation occurs between the polypeptide-rich phase and the polyethylene glycol-rich phase.

This phase separation phenomenon occurs when the polypeptide-rich phase forms microdroplets in the polyethyleneglycol-rich phase, and the polyethyleneglycol phase becomes microdroplets in the polypeptide-rich phase. May be formed, and which of the phase separation forms is determined by the concentration ratio of polyethylene glycol / polypeptide in the mixed aqueous solution. If this concentration ratio is equal to or greater than a certain value (referred to as phase inversion ratio) determined by the type of polypeptide and the molecular weight of polyethylene glycol, the phase rich in polypeptide is very small in the phase rich in polyethylene glycol. A droplet will be formed. In general, when the concentration ratio is equal to or higher than the phase inversion ratio, spherical fine particles having a smaller particle diameter are obtained, and when the concentration ratio is equal to or less than the phase inversion ratio, particles having a slightly larger particle diameter and irregularly shaped particles are obtained. .
Therefore, in order to obtain fine particles having a smaller particle diameter, for example, fine particles having an average particle diameter of 10 μm or less, it is preferable that the concentration ratio is equal to or higher than the phase inversion ratio.

The phase inversion ratio varies depending on the type of polypeptide used and the molecular weight of polyethylene glycol. By appropriately preparing fine particles using mixed aqueous solutions having various concentration ratios, and examining the particle diameter and shape thereof, , Can be easily determined. However, when polyethylene glycol having a molecular weight of 6000 or more is used, the phase inversion ratio has almost no effect on the molecular weight, and therefore, the phase inversion ratio is determined only by the type of polypeptide used. Variation of the phase inversion ratio depending on the type of polypeptide is considered to be largely due to the molecular weight of the polypeptide. Therefore, the phase inversion ratio when using polyethylene glycol having a molecular weight of 6000 or more can be estimated to some extent by the molecular weight of the polypeptide used. . That is, as polyethylene glycol, a molecular weight of 600
In the case where 0 or more is used, the following relational expression T = 10 ^2.7 / M ^0.68 holds approximately between the molecular weight (M) of the polypeptide and the phase inversion ratio (T). At
By substituting the molecular weight of the polypeptide to be used, the phase inversion ratio can be determined.

As described above, the phase inversion ratio varies depending on the type of the polypeptide to be used and the like. However, if the concentration ratio is set so as to obtain fine particles having an average particle size of 10 μm or less, it is preferably 0.25 or more, Is 0.5 or more, more preferably 1 or more.

The organic solvent used in the present invention is such that the polypeptide cannot be dissolved but the polyethylene glycol can be dissolved, for example, the polypeptide has a solubility of 1 mg / ml or less at 20 ° C. There is no particular limitation as long as the solubility of the glycol is 1 mg / ml or more, and it may be appropriately selected according to the type of polypeptide used. To exemplify specific examples of such an organic solvent, for example, methylene chloride, chloroform, halogenated hydrocarbons such as carbon tetrachloride, methyl acetate, ethyl acetate, acetates such as butyl acetate, methanol, ethanol Such as lower alcohols, aromatic hydrocarbons such as benzene, toluene, xylene and cumene; ethers such as ethyl ether and tetrahydrofuran; ketones such as acetone; and pentane, acetonitrile and pyridine. Among them, methylene chloride, chloroform, acetone, acetonitrile, ethyl acetate, toluene, methanol and ethanol are particularly preferred.
You may mix and use more than one kind.

Further, among the above-mentioned organic solvents, those which are volatile and have low solubility in water, such as methylene chloride, chloroform, toluene, and ethyl acetate, and which can dissolve the biodegradable polymer can be obtained by the present method. When the obtained polypeptide microparticles are used in a microsphere preparation, the microparticle preparation step and the microsphere preparation step can be performed continuously.

In the present invention, since the polypeptide is already dispersed as fine particles in polyethylene glycol at the stage of the solid obtained by freeze-drying, it is only necessary to dissolve polyethylene glycol using an organic solvent. Polypeptide microparticles are obtained, and no means for miniaturization such as grinding or ultrasonic irradiation is required.

The obtained polypeptide fine particle suspension is, if necessary, dissolved in the fine particle suspension by solvent exchange using known means such as dialysis, centrifugation / washing, filtration / washing, and the like. Removal of the polyethylene glycol can also be performed. It is also possible to remove fine particles as powder by removing the solvent using a means such as filtration or centrifugation and drying.

The obtained polypeptide microparticles can be formed into a spray preparation, a microsphere preparation and the like by known means.

The spray preparation is prepared by using the polypeptide fine particles taken out as a powder from the suspension alone or by adding a surfactant to improve the dispersibility of the fine particles. Examples of the surfactant include sorbitan trioleate, soy lecithin, oleyl alcohol, hydrogenated castor oil derivative, and the like, and the compounding amount is in the range of 0.001 to 5% (w / w) based on the weight of the polypeptide. And preferably 0.05 to 2% (w / w).

If necessary, stabilizers and excipients may be added. Examples of the stabilizer include human serum albumin, gelatin and the like, and the compounding amount thereof is preferably 0.01 to 200% (w / w) based on the weight of the polypeptide. Examples of the excipient include sugars or sugar alcohols such as lactose, maltose, sorbitol, trehalose, xylose, mannitol, sorbitol, and xylitol, and the amount thereof is 0.01 to 200% (w) based on the weight of the polypeptide. / W), preferably 1 to 50% (w / w).

Further, mineral ions which are originally required for maintaining the osmotic pressure existing in a living body, such as calcium ions, magnesium ions, sodium ions, potassium ions,
Chloride ions and the like may be appropriately compounded,
In order to suppress irritation to the living body, a surfactant derived from the alveoli may be added as necessary.

The polypeptide composition for a spray formulation prepared as described above may be used in the oral cavity or in the nasal cavity as it is or by dispersing compressed air, compressed carbon dioxide gas or the like as a spray gas or in a suitable propellant. Inhaled into the pharynx or lungs via Usable propellants include chlorofluorocarbons (CFC 11, 1
2, 114), chlorofluorocarbons containing hydrogen (such as Freon 123, 124, 141b), and fluorocarbons containing no chlorine (such as Freon 125, 134a).

In the case of preparing a microsphere preparation, an “oil phase” in which polypeptide fine particles are dispersed in a volatile organic solvent in which a biodegradable polymer is dissolved and which has low solubility in water is prepared. It may be prepared by solidifying the biodegradable polymer in the oil phase. When two or more biodegradable polymers are present in the oil phase, they may be phase-separated from each other to form an O / O emulsion.

In the oil phase, a volatile organic solvent having low solubility in water and a biodegradable polymer, which cannot dissolve the polypeptide but dissolve the biodegradable polymer, are added to the polypeptide fine particle powder. Alternatively, in a solid obtained by freeze-drying a mixed aqueous solution of a polypeptide and polyethylene glycol, the polypeptide cannot be dissolved, but polyethylene glycol and the biodegradable polymer can be dissolved in water due to its volatile nature. It can be prepared by adding an organic solvent having low reactivity. When the oil phase is prepared by the former method, a high content of polypeptide microspheres is obtained because polyethylene glycol is not mixed in the microspheres,
When the oil phase is prepared by the latter method, there is no need for an operation of extracting the polypeptide fine particles as a powder, which is advantageous in production.

Specific examples of volatile organic solvents having low solubility in water that can dissolve the biodegradable polymer without dissolving the polypeptide include methylene chloride, chloroform, ethyl acetate, and toluene. Of these, methylene chloride and chloroform are particularly preferred, and these organic solvents may be used in combination of two or more.

Specific examples of the volatile organic solvent having low solubility in water which can dissolve the polypeptide but not polyethylene glycol and the biodegradable polymer include methylene chloride, chloroform and ethyl acetate. ,
Among them, methylene chloride and chloroform are particularly preferred.
Mixtures of more than one species may be used.

Any biodegradable polymer may be used as long as it has no biological activity and has the property of decomposing and disappearing in vivo. For example, homopolymers such as lactic acid, glycolic acid, malic acid and hydroxybutyric acid, and copolymers thereof. In particular, polylactic acid having an average molecular weight of 1,000 to 500,000 and lactic acid glycolic acid copolymer are preferred.

The content of the polypeptide relative to the biodegradable polymer can be arbitrarily selected and varies depending on the type of the polypeptide, the desired pharmacological effect and the release time, but is about 0.01 to about 40% (w / W), especially 0.01
~ 20% is preferred.

The concentration of the biodegradable polymer in the oil phase is:
Depending on the molecular weight of the biodegradable polymer and the type of organic solvent, it is usually about 0.01% (w / w) to about 8%.
0% (w / w), preferably about 0.5% (w / w).
It is from 1% (w / w) to about 70% (w / w), particularly preferably from about 1% (w / w) to about 60% (w / w).

Means for solidifying the biodegradable polymer in the oil phase include O / W type, O / O / W type emulsions in liquid, phase separation, and spray drying. Hereinafter, these methods will be described.

(A) O / W type emulsion drying method in liquid:
In order to prepare microspheres by this method, an O / W emulsion is prepared by adding the oil phase prepared by the above method to an aqueous phase, emulsifying the emulsion, and drying the obtained emulsion in a liquid. .

The volume of the aqueous phase may be, for example, about 1 to about 10,000 times the volume of the oil phase, and preferably about 2 to about 5 times.
000 times, particularly preferably about 5 times to about 2000 times.

An aggregating agent can be added to the aqueous phase to prevent coalescence of the oil phase and aggregation of the formed microspheres. Any coagulation inhibitor may be used as long as it is generally used. Examples thereof include polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, gum arabic, chitosan, gelatin, serum albumin, and a surfactant. The concentration of the anticoagulant in the aqueous phase is 0.01 to 10% (w / v), particularly 0.1 to 2% (w / v).
/ V) is preferred. By changing the type and concentration of the emulsion stabilizer, the particle size and the particle size distribution of the microsphere can be controlled.

Further, in the aqueous phase, a pH adjuster (for example,
Acetic acid, hydrochloric acid, sodium hydroxide, etc., a preservative (eg, paraoxybenzoic acid, etc.), and an osmotic pressure adjusting agent (eg, salts such as sodium chloride, saccharides such as mannitol, etc.) may be added.

The emulsification operation can be easily carried out by a propeller type stirrer, a turbine type emulsifier, an ultrasonic disperser, a high-pressure emulsifier or the like.

Drying in liquid can be carried out by a conventional method such as a heating method or a reduced pressure method. For example, in the heating method, the temperature is raised while stirring the emulsion with a propeller type or turbine type stirrer, and the solvent is distilled off. . The stirring speed slightly varies depending on the apparatus and the charged amount, but is about 10 to about 25,000 rpm
m, particularly preferably 50 to 10,000 rpm.
The temperature may be raised over a period of about 0.5 to about 4 hours. The initial temperature is 0 to 25 ° C, and the maximum temperature after rising is 25 to 50
C is preferred. In the decompression method, the solvent is distilled off by gradually reducing the pressure of the emulsion with a suitable decompression device such as a rotary evaporator to about 0.1 to 50 mm / Hg.

The microspheres obtained have an average particle diameter of from 0.01 μm to 500 μm, and are generally obtained by increasing the ratio of the amount of organic solvent to the amount of polymer in the oil phase. Has a fine particle size.

The microspheres thus obtained are separated by centrifugation or filtration, and the emulsion stabilizer and the like adhering to the surface of the microspheres are repeatedly washed and removed several times with distilled water. And lyophilized by adding additives such as mannitol, if necessary. Thereafter, if necessary, the water and the organic solvent in the microspheres may be removed by heating under reduced pressure. As the heating conditions, for example, the microspheres were heated at a heating rate of 10 to 20 ° C. per minute by about 5 ° C from the midpoint glass transition temperature of the biocompatible polymer determined by a differential scanning calorimeter.
After heating the microspheres to a predetermined temperature, the temperature may be maintained within one week, preferably within a few days, more preferably about one to 24 hours. .

(B) O / O / W type emulsion drying method: To prepare microspheres by this method,
An oil phase is prepared using at least one kind of biodegradable polymer. (Here, the two kinds of polymers are dissolved in an organic solvent, but the phases are not compatible with each other and are separated. This is emulsified to form an O / O emulsion, and the O / O emulsion is added to an aqueous phase as in (a) and emulsified to prepare an O / O / W emulsion, What is necessary is just to dry the obtained emulsion in liquid.

The combination of the two polymers used here is such that when the respective organic solutions are mixed, the respective phases are not compatible, and one polymer (first polymer) is alloyed in the other polymer (second polymer). Any combination can be used as long as the combination forms polylactic acid and lactic acid-glycolic acid copolymer. These first and second polymers can be used in the form of a mixture of two or more polymers.

In general, the first polymer and the second polymer are formed by forming a continuous layer in the O / O type emulsion by using a polymer having a large weight to be used as the second polymer, and by using a polymer having a small weight in the O / O type emulsion. Since the fine particles are dispersed in the continuous layer of the emulsion to form the first polymer and become the first polymer, the first polymer can be selected as an index. The weight ratio of the first polymer and the second polymer to be used may be determined based on the above-mentioned index, and there is no particular limitation. For example, the first polymer is 1 and the second polymer is 1 to 10. Can be Among them, a preferable weight ratio is one in which the first polymer is 1 and the second polymer is 2 to 4. For example, in the case of a combination of a polylactic acid and a lactic acid / glycolic acid copolymer, when the molecular weights are the same and the weight ratio is 2 for the polylactic acid and 1 for the lactic acid / glycolic acid copolymer, the lactic acid / glycolic acid copolymer forms fine droplets. And becomes the first polymer. In the case of the above combination, when the weight ratio is opposite, polylactic acid forms microdroplets and becomes the first polymer. Further, the polypeptide fine particles are unevenly distributed in one of the polymer solutions due to a difference in affinity for the first or second polymer.
Therefore, in the present method, by utilizing the above relationship, it is possible to obtain a microsphere having an excellent sustained release property by including a polypeptide in a polymer forming a microdroplet.
For example, when the polypeptide fine particles are unevenly distributed in the continuous phase (that is, the second polymer) in the O / O emulsion, the polypeptide fine particles can be contained in the fine droplets by changing the weight ratio of the polymer. This makes it possible to easily select a combination of a polypeptide and a polymer. In general, when the viscosity increases, coalescence between the particles is suppressed, so that the O / O emulsion is stabilized, and microspheres having a small particle diameter in the inner minute region can be manufactured, and two types of microspheres can be produced. By adopting a polymer having a high molecular weight as one of the biodegradable polymers, microspheres having a small particle diameter in the internal microregion may be manufactured in the same manner as described above, and the inside of the microsphere may be produced. Can control the particle size of the fine region.

Therefore, the microsphere preparation obtained by the present method comprises two or more biodegradable polymers, and one of the biodegradable polymers (first polymer)
Is a polynuclear microsphere preparation having an internal structure dispersed in a region composed of the other biodegradable polymer (second polymer) and containing a polypeptide in the microregion.

The effects of the first polymer and the second polymer in the present method are as follows. The first polymer has a higher affinity for the polypeptide than the second polymer, and thus can selectively retain the polypeptide. The second polymer has two major effects. First, when the O / O emulsion prepared by the present method is further emulsified in an aqueous phase to form an O / O / W emulsion, the effect of not releasing the polypeptide held by the first polymer into the aqueous phase. Having. This results in a high uptake rate.
Second, when the microspheres come into contact with body fluids, the internal phase alloy composed of the first polymer holding the polypeptide prevents direct contact with water, thereby controlling the release control such as the suppression of initial burst-like elution. Has an effect.

The obtained microspheres may be washed and lyophilized in the same manner as in (a).

(C) Phase separation method (coacervation method): When microcapsules are produced by this method, a microsphere is precipitated and solidified by gradually adding a coacervation agent to the oil phase while stirring. Good.

The coacervation agent is added in an amount of about 0.01 times to about 2,000 times the volume of the oil phase, preferably about 0.05 times to about 500 times, and particularly preferably about 0.05 times to about 500 times. .1 times to about 200 times the volume. The coacervation agent is not particularly limited as long as it is a compound which dissolves a biodegradable polymer, such as a polymer, a mineral oil or a vegetable oil miscible with the organic solvent used in the oil phase. Specifically, for example, silicon oil, sesame oil, soybean oil, corn oil,
Cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane, n-heptane and the like are used. These may be used as a mixture of two or more.

The microspheres obtained are collected by centrifugation or filtration, washed repeatedly with heptane or the like to remove the coacervation agent, further washed in the same manner as in (a), and then frozen. It may be dried.

(D) Spray drying method: In the case of producing microspheres by this method, the oil phase is sprayed into a drying chamber of a spray drier (spray drier) using a nozzle.
The organic solvent in the spray droplets is volatilized in a very short time.

As the nozzle, for example, a two-fluid nozzle type,
There are a pressure nozzle type and a rotating disk type.

If necessary, the obtained microspheres may be heated under reduced pressure in the same manner as in (a) to remove water and organic solvents in the microspheres.

Among the methods (a) to (d), from the viewpoints of the incorporation rate of the polypeptide, the efficiency of suppressing the initial burst phenomenon, the recovery rate, etc., it is prepared by the in-liquid drying method from the O / O / W emulsion. Is most preferred.

The obtained microspheres can be directly administered to a living body as an implant. It can also be used as a raw material for producing various preparations.
Such preparations include, for example, injections, oral administration,
Transdermal preparations, suppositories, transnasal preparations, buccal preparations, intraocular preparations, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0077]

EXAMPLES Example 1 Bovine Serum Albumin (Molecular Weight 67000, Sigma) 5
% (W / v) aqueous solution, polyethylene glycol 600
0 (manufactured by Wako Pure Chemical Industries), 5% (w / v) aqueous solution and purified water are appropriately mixed, and a bovine serum albumin concentration of 10 mg / ml,
1 ml of an aqueous solution having a polyethylene glycol concentration of 0 to 30 mg / ml (concentration ratio of 0 to 3) was prepared.
After freezing these aqueous solutions at -45 ° C, they are freeze-dried using a freeze dryer (KYOWA VAC RLE-52ES: manufactured by Kyowa Vacuum Technology) (vacuum degree: about 0.02 torr,-
(20 ° C. for 3 hours, 20 ° C. for about 12 hours), and 2 ml of methylene chloride was added to the obtained solid to obtain a bovine serum albumin fine particle suspension.

Laser diffraction particle size distribution analyzer (SAL
D-1100: manufactured by Shimadzu Corporation), and the average particle diameter of the obtained fine particles was measured. The result is shown in FIG.

When the concentration ratio was 0.25 or more, fine particles having an average particle diameter of 10 μm or less were obtained.

When the fine particles obtained at the concentration ratios of 0.5 and 0.1 were observed with a microscope, as shown in FIG. 2, when the concentration ratio was 0.5, the fine particles were spherical. As shown in FIG. 3, when the concentration ratio is 0.1,
The particles were irregularly shaped particles with circular holes. The circular holes observed in the particles obtained at a concentration ratio of 0.1 seem to be traces of the polyethylene glycol phase which became the dispersed phase during phase separation.

In FIG. 1, the concentration ratio is 0.2 to 0.25.
When the concentration ratio is equal to or more than the value of the inflection point, fine particles having an average particle diameter of 10 μm or less are obtained, whereas when the concentration ratio is equal to or less than the value of the inflection point, the obtained particles are obtained. Is significantly larger, it is estimated that the phase inversion ratio is this inflection point, that is, 0.2 to 0.25.

Example 2 In Example 1, polyethylene glycol 2000 and 400 were used in place of polyethylene glycol 6000.
0, 20000 (all manufactured by Katayama Chemical) and 70
000 (manufactured by Wako Pure Chemical Industries, Ltd.), a similar experiment was performed, and the phase inversion ratio when polyethylene glycol of each molecular weight was used was determined in the same manner as in Example 1, and the relationship between the polyethylene glycol molecular weight and the phase inversion ratio was plotted. The results are shown in FIG.

Polyethylene glycol 20000, 70
The phase inversion ratio when using 000 is almost the same as the phase inversion ratio when using polyethylene glycol 6000,
When polyethylene glycol having a molecular weight of 6000 or more is used, the change in the phase inversion ratio due to the difference in the molecular weight seems to be small.

In any case where polyethylene glycol of any molecular weight was used, fine particles of about 10 μm or less were obtained by setting the concentration ratio to the phase inversion ratio or more.

Example 3 In Example 1, gelatin D (molecular weight: 3000, manufactured by Nippi), gelatin A (molecular weight: 7000, manufactured by Nippi), Bowman-Birk inhibitor (molecular weight: 8000, manufactured by Sigma), soybean were used instead of bovine serum albumin. Trypsin inhibitor (molecular weight 20100, manufactured by Sigma), superoxide dismutase (molecular weight 32000, manufactured by Sigma), horseradish peroxidase (molecular weight 4000)
0, manufactured by Wako Pure Chemical Industries, Ltd.), and the phase inversion ratio when each polypeptide was used was determined in the same manner as in Example 1.
FIG. 5 shows the relationship between the polypeptide molecular weight and the phase inversion ratio.

As is clear from FIG. 5, the logarithm of the polypeptide molecular weight and the logarithm of the phase inversion ratio have a linear relationship.
Therefore, between the molecular weight (M) of the polypeptide and the phase inversion ratio (T), a relationship approximately represented by the expression LogT = −0.68 LogM + 2.7 is established, and therefore, the phase inversion ratio (T) is expressed by the polypeptide. It was found that T = 10 ^2.7 / M ^0.68 was represented by the molecular weight (M).

Further, when any of the polypeptides is used, the concentration ratio is set to be equal to or higher than the phase inversion ratio.
Fine particles of about 10 μm or less were obtained.

Example 4 250 mg of bovine serum albumin (manufactured by Sigma) and 250 mg of polyethylene glycol 6000 (manufactured by Wako Pure Chemical Industries) were dissolved in 10 ml of purified water to prepare a mixed aqueous solution. After freezing this aqueous solution at -45 ° C, a freeze dryer (KYOW)
AVAC RLE-52ES: manufactured by Kyowa Vacuum Engineering Co., Ltd. was freeze-dried (vacuum degree: about 0.02 torr, -20 ° C for 3 hours, 20 ° C for about 12 hours), and 10 ml of methylene chloride was added to the obtained solid substance. A suspension of bovine serum albumin microparticles was obtained. From this suspension, bovine serum albumin microparticles were collected by filtration using a membrane filter (manufactured by Millipore) having a pore size of 0.22 μm.
Washed twice with each l. The obtained fine particles were dried overnight under reduced pressure to obtain bovine serum albumin fine particles as a powder.

A laser diffraction type particle size distribution analyzer (SAL
D-1100: manufactured by Shimadzu Corporation), and the average particle diameter of the bovine serum albumin fine particles was measured to be 4.77 μm.
Met.

The amount of protein in the obtained microparticles was measured, and the content of bovine serum albumin in the microparticles was determined to be 99.2%, which was extremely high.

Example 5 Horseradish peroxidase (manufactured by Wako Pure Chemical) 5 mg, polyethylene glycol 6000 (manufactured by Wako Pure Chemical) 5% (w
/ V) 0.1 ml of an aqueous solution and 0.9 ml of purified water are mixed,
A mixed aqueous solution was prepared. This aqueous solution was frozen at -45 ° C, and then freeze-dried (KYOWA VAC RLE-5).
2ES: manufactured by Kyowa Vacuum Technology) and freeze-dried (vacuum degree: about 0.02 torr, -20 ° C for 3 hours, 20 ° C for about 12 hours). 2 ml of methylene chloride was added to the obtained solid to obtain a suspension of horseradish peroxidase fine particles.

Laser diffraction particle size distribution analyzer (SAL
D-1100: manufactured by Shimadzu Corporation), and the average particle diameter was 4.08 μm.

Further, the solvent (methylene chloride) was distilled off from the obtained fine particle suspension, and the residue was dissolved in purified water, and its peroxidase activity was measured. Was not found.

Example 6 Bovine serum albumin (manufactured by Sigma) (25 mg), polyethylene glycol 6000 (Wako Pure Chemical Industries) (15 mg) and purified water (1 ml) were mixed to prepare a mixed aqueous solution. After freezing this aqueous solution at -45 ° C, a freeze dryer (KYOWA V)
Freeze drying using AC RLE-52ES (manufactured by Kyowa Vacuum Technology) (vacuum degree: about 0.02 torr, -20 ° C for 3 hours, 2 hours)
0 ° C. for about 12 hours).
20 (lactic acid glycolic acid copolymer, molecular weight 2000
0, the molar ratio of lactic acid and glycolic acid is 50:50, 184 mg of PLA0020 (polylactic acid, molecular weight 20,000, manufactured by Wako Pure Chemical Industries) 248.4 mg, and 1.85 g of methylene chloride are added. Of polyethylene glycol, PLGA5020 and PLA0020,
After dispersing the polypeptide microparticles, PLA00110
(L-form polylactic acid, molecular weight 110000, manufactured by Boehringer Ingelheim) 27.6 mg was added and dissolved to obtain O /
An O-type emulsion was prepared. This O / O emulsion was added to 4 ml of a 0.25% (w / w) aqueous solution of methylcellulose at 15 ° C., and emulsified using a Polytron homogenizer (8000 rpm, 5 minutes, manufactured by Kinematica) to obtain O / O / W. A type emulsion was prepared, and
At 400 ° C and 400 ml of purified water, and 400 rpm using a four-wing paddle (Three One Motor, manufactured by Shinto Kagaku)
While stirring at, the temperature was raised to 15 to 30 ° C. over 3 hours to perform drying in the liquid to form microspheres. After the obtained microsphere dispersion liquid was sieved with a 150 μm mesh, the microspheres were collected by filtration with a 20 μm mesh and freeze-dried.

[0095] 50 mg of the obtained microspheres are placed in a test tube with a stopper, 10 ml of eluate (PBS containing 0.02% (w / v) sodium azide) is added, and the mixture is placed in a rotary incubator (25 rpm, 37 ° C). Incubate. The test tube is removed from the incubator over time and centrifuged (2000
(rpm, 5 minutes) to remove 9 ml of the supernatant, and then added a new eluate to continue the incubation. The amount of protein in the collected supernatant was measured, and the amount of bovine serum albumin released from the microspheres after a certain period of time was determined. FIG. 6 shows the results.

As apparent from FIG. 6, the obtained microspheres released bovine serum albumin at a constant rate over about 21 days, and the initial burst rate was very low.

Example 7 500 mg of bovine serum albumin (manufactured by Sigma) and 500 mg of polyethylene glycol 6000 (manufactured by Wako Pure Chemical Industries) were dissolved in 20 ml of purified water to prepare a mixed aqueous solution. After freezing this aqueous solution at -45 ° C, a freeze dryer (KYOW)
A VAC RLE-52ES: manufactured by Kyowa Vacuum Engineering Co., Ltd. was freeze-dried (vacuum degree: about 0.02 torr, -20 ° C. for 3 hours, 20 ° C. for about 12 hours), and 20 ml of methylene chloride was added to the obtained solid. A suspension of bovine serum albumin microparticles was obtained. From this suspension, bovine serum albumin microparticles were collected by filtration using a membrane filter (manufactured by Millipore) having a pore size of 0.22 μm.
Washed twice with each l. The obtained fine particles were dried overnight under reduced pressure to obtain bovine serum albumin fine particles as a powder.

190 mg of PLGA5020 (lactic acid / glycolic acid copolymer, molecular weight: 20,000, molar ratio of lactic acid to glycolic acid: 50:50, manufactured by Wako Pure Chemical Industries, Ltd.)
A0020 (polylactic acid, molecular weight: 20,000, manufactured by Wako Pure Chemical Industries) 256.5 mg was dissolved in 1.85 g of methylene chloride, and the bovine serum albumin fine powder 2 was added thereto.
After dispersing 5 mg, 28.5 mg of PLA00110 (1-polylactic acid, molecular weight 110000, manufactured by Boehringer Ingelheim) was added, and the mixture was dissolved to prepare O / O.
A mold emulsion was prepared. This O / O emulsion was added to 4 ml of a 0.25% (w / w) methylcellulose aqueous solution at 15 ° C., and the mixture was mixed with a polytron homogenizer (8).
000 rpm, 5 minutes) to prepare an O / O / W emulsion, which was transferred into 400 ml of purified water at 15 ° C., and stirred for 3 hours at 400 rpm with a four-bladed paddle. The temperature was raised to 15 to 30 ° C. to carry out drying in the liquid to form microspheres. After the obtained microsphere dispersion liquid was sieved with a 150 μm mesh, the microspheres were collected by filtration with a 20 μm mesh and freeze-dried.

50 mg of the obtained microspheres were placed in a test tube with a stopper, 10 ml of an eluate (PBS containing 0.02% (w / v) sodium azide) was added, and the mixture was placed in a rotary incubator (25 rpm, 37 ° C.). Incubate. The test tube is removed from the incubator over time and centrifuged (2000
(rpm, 5 minutes) to remove 9 ml of the supernatant, and then added a new eluate to continue the incubation. The amount of protein in the collected supernatant was measured, and the amount of bovine serum albumin released from the microspheres after a certain period of time was determined. FIG. 7 shows the results.

As is clear from FIG. 7, the obtained microspheres released bovine serum albumin at a constant rate over about 21 days, and the initial burst rate was very low.

[0101]

According to the present invention, polypeptides can be micronized without denaturation because they do not involve heat or physical collision and do not come into contact with an organic solvent in an aqueous solution. The obtained polypeptide fine particles have very high purity and good redispersibility.

Further, according to the present invention, almost no loss occurs during the collection of fine particles, so that the collection rate is almost 10%.
0%.

Further, by using the polypeptide microparticles obtained by the present invention, a polypeptide can be released at a constant rate over a long period of time, and a high-content microsphere preparation having an extremely low initial burst rate can be easily obtained.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the concentration ratio of a mixed aqueous solution of polyethylene glycol 6000 and bovine serum albumin and the particle size of the obtained bovine serum albumin fine particles.

FIG. 2: Polyethylene glycol 6 with a concentration ratio of 0.5
000, a micrograph of bovine serum albumin microparticles obtained using a mixed aqueous solution of bovine serum albumin.

FIG. 3 Polyethylene glycol 6 at a concentration ratio of 0.1
000, a micrograph of bovine serum albumin particles obtained using a mixed aqueous solution of bovine serum albumin.

FIG. 4 is a graph showing the relationship between the molecular weight of polyethylene glycol and the phase inversion ratio.

FIG. 5 is a graph showing the relationship between the molecular weight of a polypeptide and the phase inversion ratio.

FIG. 6 is a graph showing the results of an in vitro elution test of the microspheres containing bovine serum albumin obtained in Example 6.

FIG. 7 is a graph showing the results of an in vitro elution test of the microspheres containing bovine serum albumin obtained in Example 7.

Claims (16)

[Claims] 1. A mixed aqueous solution of a polypeptide and polyethylene glycol is freeze-dried, and an organic solvent which does not dissolve the polypeptide but dissolves the polyethylene glycol is added to the obtained solid. Peptide micronization method. 2. A mixed aqueous solution of polyethylene glycol /
2. The method according to claim 1, wherein the concentration ratio of the polypeptide is not less than the phase inversion ratio.
A method for micronizing the polypeptide according to the above. 3. A mixed aqueous solution of polyethylene glycol /
2. The polypeptide according to claim 1, wherein the concentration ratio of the polypeptide is 0.25 or more.
A method for micronizing the polypeptide according to the above. 4. A polypeptide having a molecular weight of 5,000 to 70.
4. The method for micronizing a polypeptide according to claim 1, 2, or 3. 5. Polyethylene glycol having a molecular weight of 40
5. The method for micronizing a polypeptide according to claim 1, wherein the number is from 0 to 500,000. 6. The polyethylene glycol having a molecular weight of 20.
The method for micronizing a polypeptide according to claim 1, 2, 3, or 4, wherein the number is from 100 to 100,000. 7. The polyethylene glycol having a molecular weight of 60
The method for micronizing a polypeptide according to claim 1, 2, 3, or 4, wherein the number is from 00 to 70000. 8. The polyethylene glycol having a molecular weight of 60
And the concentration ratio of polyethylene glycol / polypeptide in the mixed aqueous solution is 10 ^2.7 / M.
^{2. The method according to} claim 1, wherein the molecular weight is ^0.68 or more (M: molecular weight of the polypeptide).
Or a method for microparticulating the polypeptide according to 4. 9. An organic solvent in which a polypeptide cannot be dissolved but polyethylene glycol can be dissolved, methylene chloride,
9. One or more selected from the group consisting of chloroform, acetone, acetonitrile, ethyl acetate, toluene, methanol and ethanol.
A method for micronizing a polypeptide according to any one of the above. 10. A polypeptide microparticle suspension obtained by the method according to claim 1. 11. The polypeptide fine particles having an average particle size of 1
The polypeptide microparticle suspension according to claim 10, which has a diameter of 0 µm or less. 12. A polypeptide fine particle powder obtained by removing polyethylene glycol and an organic solvent from a polypeptide fine particle suspension obtained by the method according to any one of claims 1 to 9. 13. A biodegradable polypeptide microparticle powder obtained by removing polyethylene glycol and an organic solvent from a polypeptide microparticle suspension obtained by the method according to any one of claims 1 to 9. An oil phase is prepared by adding a volatile organic solvent having low solubility in water capable of dissolving the polymer and a biodegradable polymer, and the biodegradable polymer in the oil phase is solidified. Of microsphere preparation. 14. A solid obtained by freeze-drying a mixed aqueous solution of a polypeptide and polyethylene glycol,
The polypeptide is insoluble, but polyethylene glycol and the biodegradable polymer are soluble.A volatile organic solvent having low solubility in water and a biodegradable polymer are added to prepare an oil phase, and the oil phase is prepared. A method for producing a microsphere preparation, comprising solidifying a biodegradable polymer in a phase. 15. The biodegradable polymer is at least two kinds, the oil phase is an O / O type emulsion, and the biodegradable polymer is solidified by dispersing the O / O type emulsion in water. The method for producing a microsphere preparation according to claim 13 or 14, which is a method for drying an O / O / W emulsion in a liquid, comprising preparing an / O / W emulsion and then drying the emulsion in a liquid. 16. A microsphere preparation obtained by the method according to claim 13, 14, or 15.