JPH09151136A - Production of protein sustained release microsphere - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize a method for obtaining the subject microspheres having an excellent sustained release property by adding a specific protecting agent to the solution of a protein used as a physiologically active substance or an antigen used for vaccines, and further using a polymer capable of being orally administered or injected. SOLUTION: This method for producing the protein sustained release microspheres comprises preliminarily adding gelatin to a protein-containing solution, mixing and emulsifying the mixture with an organic solvent containing a biodegradable polymer, reemulsifying the emulsion in an aqueous phase containing a dispersant, and subsequently recovering the produced microspheres. The gelatin is desirably added to the protein solution in a concentration of >=4%. The addition of the gelatin to the preparation of the protein-containing microspheres in this method enables to encapsule the protein in the micropheres without denaturalizing or inactivating the protein with the organic solvent. When the microspheres are prepared at a freezing temperature, the protecting effect on the protein from the organic solvent is further improved. In addition to the above method, a method wherein the gelatin is added to the dispersant-containing aqueous phase (outer aqueous phase) at room temperature may be performed.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention comprises an orally or injectable biodegradable and biocompatible polymer or copolymer for sustained release of useful protein solutions such as antigens and bioactive substances used in vaccines. Microsphere (micr
ospheres) and the manufacturing method thereof.

[0002]

BACKGROUND OF THE INVENTION AND PRIOR ART Microspheres in the present invention mean hollow particles which can contain useful substances such as drug components in a polymer matrix.
As a typical example that is often used as a polymer that is a material for such microspheres, poly (lactide-
Glycolide) [hereinafter, PLGA] and the like are known. The polymer forming the microspheres is biodegradable, is already widely used as a surgical suture, and is nontoxic to the living body. Therefore, the microspheres formed from this polymer can be administered to animals or humans after encapsulating active ingredients such as proteins and suspending them in a suitable liquid.

By encapsulating useful proteins such as antigens or physiologically active substances used in vaccines in such microspheres, oral administration becomes possible, and improvement in safety and convenience of administration method can be expected. Furthermore, when microspheres encapsulating useful proteins such as antigens or physiologically active substances are orally administered to a living body or administered subcutaneously or intramuscularly by injection, useful proteins are gradually released from the microspheres. When the protein is an antigen, a preferable immune response is obtained by continuously stimulating immunocompetent cells [Eldridge et al., Mol. Immunol., 28, 287-294 (199).
1)]. The size of microspheres can be adjusted arbitrarily during the preparation process, but when administered orally, the size of Ig
It has been reported that G-class antibody response and local IgA-class antibody response can be selected [Eldridge et al.,
J. Controlled Release, 11, 205-214 (1990)]. on the other hand,
In the case of a physiologically active substance or the like, it is possible to reduce the number of administrations required to maintain its blood concentration. As described above, the microspheres using a biodegradable and biocompatible polymer as a matrix have been attracting attention as a new delivery system for useful proteins such as antigens or physiologically active substances used in vaccines.

[0004]

The methods for encapsulating useful proteins such as antigens or physiologically active substances used in vaccines in microspheres using biodegradable polymers are roughly classified into three types. These methods are coacervation or emulsion / phase separation methods, encapsulation methods by spray drying, solvent evaporation methods in organic or aqueous phases. However, in any of the preparation methods, it is essential to use an organic solvent such as methylene chloride or ethyl acetate in order to dissolve the polymer in the process of forming microspheres. As a result, there arises a problem that the useful protein to be encapsulated is denatured and insolubilized by the organic solvent, or the higher order structure of the protein is unnaturally changed to deactivate its antigenicity or physiological activity. Therefore, the application range of the microsphere as a drug delivery system has been significantly limited.

[0005]

Under the circumstances, as a result of intensive studies by the present inventors, a protein-containing solution and an organic solvent in which a biodegradable polymer is dissolved are mixed and emulsified, and then dispersed. Re-emulsified in an aqueous phase containing an agent, obtained by collecting the resulting microspheres, in the method for producing sustained-release microspheres in which the protein is encapsulated, by adding gelatin to the protein-containing solution, The inventors have found that it is possible to protect the protein to be encapsulated from denaturation by an organic solvent during microsphere formation, and have completed the present invention. That is, the present invention is obtained by a method of encapsulating a useful protein solution such as an antigen used in a vaccine or a physiologically active substance in a microsphere made of a biodegradable polymer without deactivating it with an organic solvent, and the method. The present invention provides a sustained-release protein microsphere.

[0006]

As a result of various studies on a protective agent for protecting a useful protein from denaturation of an organic solvent, it was found that gelatin has the most excellent protective effect. Gelatin is added to the protein solution before mixing the protein with the organic solvent in which the biodegradable polymer is dissolved. The concentration of gelatin added to the protein solution is preferably 4% or more.

When the microspheres are prepared under ice cooling, a more effective protein protection effect against organic solvents can be obtained. Further, at room temperature, gelatin may be contained in the aqueous phase (external aqueous phase) containing the dispersant.

The biodegradable polymer which is a material forming the microspheres used in the present invention is poly (lactide-
Glycolide), polylactide, polylactic acid, polyglycolic acid, poly (lactic acid-glycolic acid), poly (ethyl glycol-lactide), polylactic acid-polyethylene glycol copolymer, polycaprolactone, poly (glycolic acid-caprolactone), poly (Lactide-caprolactone), polyvalerolactone, polyhydroxybutyric acid, polyhydroxybutyrate, poly (hydroxybutyrate-
Hydroxyvalerate), polyisobutyl cyanoacrylate, polyalkyl cyanoacrylate, poly (ethylene oxide-butylene terephthalate), or
It is preferred to use enteric agents such as cellulose-acetate-terephthalate, or mixtures thereof.

The organic solvent used in the present invention is used for dissolving the above biodegradable polymer. Examples thereof include methylene chloride, chloroform, benzene, acetic acid ester and the like.

In the present invention, the protein encapsulated in the microsphere is a protein such as an antigen or a physiologically active substance used as a vaccine. According to the present invention, by adding gelatin during the preparation of protein-containing microspheres, the protein can be encapsulated in the microspheres without being denatured (deactivated) by the organic solvent.

The protein-containing microspheres obtained according to the present invention can gradually release proteins and can be used as a drug delivery system. Hereinafter, embodiments of the present invention will be described in detail with reference to examples.

[0012]

【Example】

<< Reference Example 1: HBc Antigen >> In order to examine the protein protective effect of the gelatin of the present invention, hepatitis B virus core antigen (HBc antigen) particles were selected as a model of the protein to be encapsulated. The HBc antigen is a simple protein composed of 183 amino acids, but a gene encoding this antigen was expressed in yeast to form viral particles (HBc particles) associated with 180 HBc antigens. Used [Mol. Imm
unol., 30, 221-231 (1993)]. This constituent protein, HB
If the c-antigen is denatured even a little, the stability of the HBc particles will drop rapidly and eventually the particles will disintegrate. Therefore, the structural change of the HBc antigen due to denaturation can be detected with high sensitivity in the form of particle-like decay.

<Reference Example 2: HBc antigen detection system by ELISA> As a detection system, an ELISA system using a mouse monoclonal antibody (Yc-3) was used. The mouse monoclonal antibody is the same as 80% of particulate HBc antigen.
It has a characteristic that it recognizes and binds to the three-dimensional structure near the position, but does not bind to that of the HBc antigen in the monomer state. Therefore, by examining the binding property of this monoclonal antibody to HBc particles by using an ELISA, the amount of HBc antigen in the unmodified particle state can be easily measured. First, 200-fold diluted anti-H in a 96-well microtest plate (Nunc MaxiSoap ^™ )
Bc rabbit antiserum (manufactured by Biomeda) was added at 50 μl / well and solidified by incubating at 4 ° C. overnight. Furthermore, 2% BSA (bovine serum albumin) solution 10
Masking was performed by adding 0 μl and incubating in the same manner. H was added to the antibody-immobilized plate thus prepared.
After adding Bc particles and incubating at 37 ° C. for 30 minutes,
It was washed 3 times with 1% Tween 20 / PBS. To this, 0.2 μg / m
l Yc-3 monoclonal antibody was added at 50 μl / well,
Incubated at 37 ° C for 30 minutes. After washing 3 times as above, 50 μl / well of peroxidase-labeled anti-mouse immunoglobulin antibody solution (Chemicon, diluted 5000 times) was added. After incubating at 37 ℃ for 30 minutes, 0.1% Tween20 / P
After washing 5 times with BS, TMBZ substrate solution was added, color was developed by a conventional method, and the absorbance was measured at a wavelength of 450 nm. Hereinafter, the effect of adding gelatin using HBc particles as a model will be described with reference to Examples.

Example 1 Particle Stability of HBc Particles H
The stability of HBc particles against pH, salt concentration, ultrasonic treatment, and freeze-thawing was examined in consideration of the encapsulation conditions of Bc particles in microspheres. As a result, in distilled water of pH6, 4 ℃, room temperature, 37 ℃
No decrease in HBc antigenicity was observed even after standing for 3 days, sonication for 60 seconds, which is usually used for preparation of microspheres, and repeated freeze-thaw three times. These results indicate that the HBc particles have physical stability to withstand encapsulation in microspheres.

Therefore, an attempt was made to encapsulate HBc particles in microspheres using HBc particles as a model antigen. 547μg / ml HBc particles 184μ
l was added to 10 ml of methylene chloride containing 500 mg of PLGA,
Ultrasound emulsified. 200 ml of this emulsion containing 0.5% polyvinyl alcohol (PVA) and 5% sodium chloride
Was dispersed in the outer aqueous phase of and was further stirred for 5 hours. The resulting microspheres were collected by centrifugation and freeze dried. HBc particles were re-extracted from this microsphere using methylene chloride, acetone, and acetonitrile, and the recovery rate was quantified using ELISA. However, HBc particles could not be recovered in the extract.

In order to pursue this cause, HBc particles and an organic solvent (methylene chloride) were mixed in equal amounts and sonicated for 1 minute, and then the aqueous phase was separated to quantify the amount of HBc antigen. . As a result, it was not possible to detect the antigenicity of HBc in the aqueous phase. ELIS for detection of HBc antigen
In A, a mouse monoclonal antibody (Yc-3) that specifically detects HBc antigen in a particle state was used. Therefore,
The inactivation of the antigenicity of the HBc particles is caused by the H that constitutes the HBc particles.
It is speculated that this is due to the result that the structure was changed by denaturation of the Bc protein, and thereby the HBc particles were disintegrated by the organic solvent (methylene chloride) used for the preparation before being encapsulated in the microspheres.

Example 2: Modification from organic solvent to HB
Selection of Additives that Protect c Particles >> Polyethylene glycol (PEG: # 2000 to # 6000) 2% and 20%, which are stabilizers for general antigen proteins, using HBc particles as a model protein
%, Carboxymethylcellulose 2%, cyclodextrin 2%, sugars and sugar alcohols (mannitol, lactose) 2%, amino acids (glycine, lysine) 2% and
Using 20% and 4% gelatin (manufactured by Nakarai Co., Ltd.), the effect of protecting HBc particles from denaturation by an organic solvent (methylene chloride) was examined. 0.1 ml of internal water phase containing HBc particles and protective agent
And 5.0 ml of methylene chloride were mixed, and this was sonicated for 1 minute to emulsify and shaken at room temperature or under ice cooling for 4 hours. Then, after adding 4.9 ml of phosphate buffer and stirring,
After centrifugation, the amount of HBc antigen in the supernatant was quantified by ELISA. As a result, only gelatin was able to inactivate the HBc antigen.
The protective effect was limited to about 20%, but none of the others showed a protective effect.

Therefore, the relationship between the concentration of gelatin added to the inner aqueous phase and its protective effect was examined in more detail. PH 7. containing 0-4% gelatin and 2.5 μg HBc particles at final concentration.
5 ml of methylene chloride was added to 0.1 ml of the phosphate buffer solution of 4,
It was sonicated for 1 minute to emulsify, and shaken at room temperature or under ice cooling for 2 hours or 4 hours. After shaking,
Add 4.9 ml of phosphate buffer, stir, and then add 5 at 3000 rpm.
Centrifugation was performed for minutes, and the amount of HBc antigen in the aqueous phase was quantified by ELISA (Table 1). The numerical values in the table represent the amount of HBc antigen remaining after emulsification and shaking of the inner aqueous phase and methylene chloride in percentage. As a result, a remarkable protective effect was observed with the addition of gelatin at a concentration of 4% or more, and the protective effect decreased as the concentration of gelatin decreased.

[0019]

[Table 1] ND means not done.

Example 3 Protective Effect of Gelatin against Various Organic Solvents 0.1 ml of an inner aqueous phase containing 8% gelatin and 25 μg / ml HBc particles was added to 5 ml of organic solvent, and ultrasonic waves were applied for 1 minute. After emulsifying, the mixture was shaken at room temperature for a specified time. To this, 4.9 ml of phosphate buffer was added, stirred well, then centrifuged at 3,000 rpm for 10 minutes, and the amount of HBc antigen in the aqueous phase was measured by ELISA (Table 2).

[0021]

[Table 2] +: Gelatin added, −: gelatin not added

When gelatin is not added, organic solvents (ethyl acetate, benzene, chloroform, methylene chloride)
Due to denaturation by HBc particles, the antigenicity of HBc particles is instantaneous (0 hours).
However, by adding gelatin 4
About 50% or more of the antigenicity was retained even after shaking for a while.
This result shows that by adding gelatin to the inner aqueous phase in advance, not only methylene chloride but also ethyl acetate, benzene,
It shows that denaturation of HBc particles by other organic solvents such as chloroform was prevented, and the antigenicity of HBc was protected.

Example 4 Confirmation of Encapsulation of HBc Particles in Microspheres 91 μl of an inner aqueous phase containing 25 μg of HBc particles and 4% gelatin at the final concentration was dissolved in 200 mg of PLGA.
After mixing with ml of methylene chloride solution, ultrasonic treatment was carried out for 1 minute to prepare an emulsion. 200 ml of this emulsion containing 5% polyvinyl alcohol (PVA) and sodium chloride
Was added to the outer aqueous phase of, and the mixture was stirred at 6,000 rpm for 1 minute and then slowly stirred for 5 to 6 hours. Collect the microspheres by centrifugation,
Microspheres encapsulating HBc particles were prepared by freeze-drying. In order to confirm that the HBc particles were taken up in the microspheres, methylene chloride, acetone and acetonitrile were used to try to extract the HBc particles encapsulated in the obtained microspheres. In order to avoid denaturation of the HBc particles during recovery, 4% gelatin was added during extraction (Table 3). The numerical values in the table represent the antigen amount of HBc recovered through the extraction from the encapsulation as a percentage.

[0024]

[Table 3] +: Gelatin added, −: gelatin not added

As a result, it was confirmed that the HBc antigen retaining the particulate property was encapsulated in the obtained microspheres. Considering the extraction efficiency of HBc particles in this example, it is estimated that the actual encapsulation rate of HBc particles is equal to or higher than the above recovery rate. Further, as a result of this Example, the higher the recovery rate of HBc antigen was that the addition of gelatin at the time of extraction showed that the gelatin has a protective effect not only on methylene chloride but also on denaturation of HBc particles by acetone and acetonitrile. It became clear to have.

Example 5 Examination of Various Conditions in Preparation of HBc Particle-Containing Microspheres 91 μl of an inner aqueous phase containing 0.5 mg of HBc particles and 4% or 8% of gelatin at the final concentration was 200 mg.
This was mixed with 5 ml of methylene chloride solution in which PLGA was dissolved, and then ultrasonicated for 1 minute to prepare an emulsion. 200m of this emulsion containing 5% PVA and 5% sodium chloride
l of the outer aqueous phase, and the mixture was stirred at 6000 rpm for 1 minute and then slowly stirred for 5 hours. The microspheres were collected by centrifugation at 8000 rpm for 10 minutes and lyophilized to prepare microspheres encapsulating HBc particles. At this time, the conditions were examined with and without the external water phase being ice-cooled and with or without the addition of 4% gelatin. Acetone was used and 4% gelatin was added to recover the HBc particles from the microspheres. The numerical values in the table represent the amount of HBc antigen recovered from the inclusion through extraction (Table 4).

[0027]

[Table 4]

From these results, in order to avoid the modification of HBc particles during the preparation of microspheres, the gelatin concentration in the inner aqueous phase was 4%.
8% is more preferable. In addition, cooling the outer aqueous phase with ice during stirring was effective for encapsulation while avoiding denaturation of the HBc particles. Further, in the preparation method of E, the recovery rate from encapsulation to extraction was about 60%, but considering that the extraction efficiency is not 100%, the actual encapsulation efficiency is expected to exceed 60%. It

Example 6: HB extracted from microspheres
Confirmation of particle property of c antigen >> E used in the process of leading the present invention
LISA is designed to detect only particulate HBc antigen. However, this reactivity is not always H
It is not a direct proof that the Bc antigen retains its particulate nature. Therefore, sucrose concentration gradient centrifugation was performed to prove that the HBc antigens extracted from the microspheres are particles.

Solution containing HBc antigen extracted with acetone from microspheres prepared according to condition C of Example 5
1.5 ml was layered on 11 ml of a 20% to 60% sucrose gradient solution and centrifuged at 30,000 rpm for 16 hours using an RPS40T rotor manufactured by Hitachi. After the centrifugation, 0.7 ml each was fractionated in order of increasing sucrose concentration, and the amount of HBc antigen in each fraction was plotted on a graph (Fig. 1). Using a yeast-expressing HBc particle preparation as a control, sucrose concentration gradient centrifugation was performed in the same manner (FIG. 2). As a result, the HBc antigen extracted from the microspheres formed a peak by ELISA at the position of almost the same sucrose concentration (about 43%) as that of the control HBc particles. Therefore, the HBc antigen extracted from the microspheres is
Have the same settling coefficient as the control HBc particles,
That is, it was confirmed that the particles exist in the state of particles. This result shows that the HBc antigen was encapsulated in the microspheres by the addition of gelatin without being denatured by the organic solvent.

Example 7 Sustained Release Study of HBc Antigen-Containing Microspheres In order to confirm that the HBc antigen encapsulated in the microspheres is released slowly, it was prepared under the condition E of Example 5. 3 mg of the microspheres were suspended in 5 ml of a phosphate buffer solution containing 0.02% Tween 80 and shaken at 37 ° C. After shaking, the supernatant was sampled over time, and HB released into the solution
The amount of c antigen was measured by ELISA (Fig. 3). as a result,
About 24% of the encapsulated HBc antigen was released into the phosphate buffer solution after 24 hours and reached a plateau after 50 hours.
The amount of HBc antigen released at that time was about 80%. From these results, it was confirmed that the microspheres of the present invention gradually release proteins. It was also found that gelatin does not adversely affect the sustained release of HBc antigen. The sustained release time of the protein can be easily adjusted by the preparation conditions of the microspheres and the like.

[0093]

As described above, according to the method for producing protein-containing microspheres of the present invention, by adding gelatin as a protective agent, the protein is not inactivated by the organic solvent used for preparing the microspheres. , It becomes possible to enclose in a microsphere. In the protein-containing microspheres obtained by the present invention, a preparation containing an antigen or a physiologically active substance used as a protein in a vaccine can be sustained-released in vivo after administration by oral administration or injection into the living body. It is extremely useful as a drug product.

[Brief description of the drawings]

FIG. 1 shows the results of sucrose gradient centrifugation of HBc antigen extracted from PLGA microspheres.

FIG. 2 shows the results of sucrose gradient centrifugation of HBc particle preparations.

FIG. 3 shows the results of investigation of sustained release of PLGA microspheres containing HBc antigen.

Front page continuation (72) Inventor Katsuhiko Fukada 2000-38 Toyooka, Koshimachi, Kumamoto Prefecture Kumamoto Prefecture No. 203 Suzuka Heights No. 203 (72) Inventor Yukio Tokichi 3-14-19 Wakaba, Kumamoto City, Kumamoto Prefecture

Claims (9)

[Claims] 1. A method of mixing a protein-containing solution and an organic solvent in which a biodegradable polymer is dissolved to emulsify, re-emulsifying this in an aqueous phase containing a dispersant, and recovering the formed microspheres. A method for producing the protein sustained-release microspheres obtained by the method, wherein gelatin is contained in the protein-containing solution. 2. The production method according to claim 1, wherein the microspheres are prepared under ice temperature. 3. The method according to claim 1, wherein gelatin is contained in the aqueous phase containing the dispersant. 4. The biodegradable polymer is poly (lactide-glycolide), polylactide, polylactic acid, polyglycolic acid, poly (lactic acid-glycolic acid), poly (ethyl glycol-lactide), polylactic acid-polyethylene. Glycol copolymer, polycaprolactone, poly (glycolic acid-caprolactone), poly (lactide-caprolactone), polyvalerolactone, polyhydroxybutyric acid,
Polyhydroxybutyrate, poly (hydroxybutyrate-hydroxyvalerate), polyisobutylcyanoacrylate, polyalkylcyanoacrylate, poly (ethylene oxide-butylene terephthalate), or an enteric agent such as cellulose-acetate-terephthalate, or these The method according to any one of claims 1 to 3, which is selected from the mixture of 5. The production method according to claim 1, wherein the organic solvent is selected from methylene chloride, chloroform, benzene, and acetic acid ester. 6. The method according to any one of claims 1 to 5, wherein the protein is an antigen used as a vaccine. 7. The production method according to claim 1, wherein the protein is a physiologically active substance. 8. A sustained-release microsphere containing a gelatin-added protein solution obtained by the production method according to claim 1. 9. A preparation comprising the sustained-release microsphere according to claim 8.