CN1660412A - A kind of preparation method of interferon polylactic acid-glycolic acid copolymer PLGA microsphere - Google Patents

Abstract

A microspherical interferon polylactate-glycollate copolymer (PLGA) is prepared from the polylactate-glycollate copolymer (PLGA) as microspherical carrier and the recombinant human interferon-alpha as the coated object by remulsifying-solvent evaporating method.

Description

A kind of preparation method of interferon polylactic acid-glycolic acid copolymer PLGA microsphere

technical field

The invention relates to a method for preparing biodegradable controlled-release microspheres of bioactive drugs, and belongs to the field of cross research between biomedical polymer materials and controlled-release preparations of bioactive drugs.

Background technique

In recent years, with the rapid development of biotechnology, more and more peptide and protein drugs have been developed, and have shown many unique curative effects in clinical applications, such as insulin, hepatitis B vaccine, interferon, etc. However, due to the short biological half-life and poor stability of peptide and protein drugs, they are easily metabolized by enzymes in the body and difficult to absorb in the gastrointestinal tract, so long-term and frequent injections are required, which hinders their convenient, extensive and effective clinical application. use.

Recombinant human interferon-α is a product prepared by biotechnology. It is a milky white powder made of highly purified and freeze-dried engineering bacteria with human interferon gene. It has strong immune regulation function. It can inhibit tumor proliferation, anti-virus, and enhance the immune regulation function of the body. It is mainly used in the treatment of rheumatoid arthritis, tumors and viral diseases. Due to oral inactivation of interferon, injection is required, and the half-life in the blood is short, poor stability, easy to be metabolized by enzymes in the body, and will be cleared or degraded soon after injection. In order to achieve curative effect, frequent, large doses and long-term Time administration, the usual usage is daily intramuscular injection of 500,000-2,000,000 IU, 3 months as a course of treatment.

The use of biodegradable polymer microparticle systems to transport protein drugs is a major method of current research at home and abroad. It has been reported that the human growth hormone microsphere preparation with polylactic acid-glycolic acid copolymer as the carrier has a drug effect that is higher than that injected once a day. The effect of continuous injection for 28 days is better. Making interferon into polylactic acid microspheres can achieve the purposes of improving drug stability, prolonging action time, and being convenient to use. Abroad promptly had the report of interferon microsphere preparation in the nineties of 20th century, and its carrier material has albumin, gelatin and polylactic acid-glycolic acid copolymer (PLGA) etc., and PLGA microsphere is relatively long because of drug release time. ideal.

At present, when scientists at home and abroad are developing sustained-release microsphere preparations, the most widely used biodegradable polymer material is polylactic acid-glycolic acid copolymer, because of its good biodegradability, compatibility and absorbability, it has been accepted FDA approved as a pharmaceutical excipient. Moreover, polylactic acid sustained-release microsphere products such as luteinizing hormone-releasing hormone and leuprolide have been officially launched abroad. Since polylactic acid-glycolic acid copolymer has not been produced in China as a medicinal material, and genetically recombinant human interferon-α is produced by many manufacturers in my country, and the price is reasonable, if interferon-α is made into polylactic acid-glycolic acid The copolymer sustained-release microsphere preparation will greatly increase its added value, which can not only promote the development of biotechnology drugs, but also promote the industrialization process of pharmaceutical excipients.

Contents of the invention

The object of the present invention is to adopt polylactic acid-glycolic acid copolymer (PLGA) as carrier, make interferon-alpha into microsphere preparation, reach and increase drug stability, prolong drug action time, reduce injection frequency and consumption, improve drug curative effect The purpose of the economic benefit is provided, and a preparation method of the interferon microsphere is provided.

The preparation method of the interferon polylactic acid-glycolic acid copolymer (PLGA) microspheres of the present invention adopts a double emulsion-solvent evaporation method. Dissolve polylactic-co-glycolic acid (PLGA) in an organic solvent, dissolve lyophilized recombinant human interferon for injection in a PBS solution containing a certain amount of stabilizer to form an internal water phase, and then inject the internal water phase into the organic solvent. phase, stir and emulsify into colostrum (W/O). Then the colostrum is added into the PVA aqueous solution (outer water phase) under the condition of stirring, and emulsified into double emulsion (W/O/W). Then stir in the sodium chloride solution at a low speed for a certain period of time to completely evaporate the organic solvent. The microspheres are centrifuged, washed, collected, and freeze-dried to obtain the interferon polylactic-co-glycolic acid (PLGA) microsphere powder.

Interferon polylactic acid-glycolic acid copolymer (PLGA) microsphere preparation method is characterized in that preparation method comprises the following steps:

(1) Preparation of polylactic acid-glycolic acid copolymer (PLGA) solution

Dissolving polylactic acid-glycolic acid copolymer (PLGA) in dichloromethane to form an organic phase, the concentration of PLGA is 1% to 60% (g/ml);

(2) Preparation of interferon PBS solution

Dissolve lyophilized recombinant human interferon α for injection in PBS (pH7.4 phosphate buffer) solution containing a certain amount of stabilizer to form an internal water phase, the concentration of interferon is 0.1% to 10% (g/ml) , the concentration of stabilizer is 1%～60% (g/ml);

(3) Preparation of colostrum

Inject the inner water phase into the organic phase to form colostrum (W/O), the emulsification speed is 1000-10000rpm, the emulsification time is 1-10 minutes, and the volume ratio of the organic phase to the inner water phase is 2:1 to 50:1 ;

(4) Preparation of double emulsion

Add colostrum to a certain concentration of PVA (polyvinyl alcohol) and NaCl aqueous solution (external water phase) while stirring, emulsify into double emulsion (W/O/W), the volume ratio of colostrum to external water phase It is 1: 5 to 1: 600; the stirring speed of double emulsion formation is 1000～2900rpm, the concentration of PVA solution is 0.2～10% (g/ml), the concentration of NaCl solution is 0.2～10% (g/ml), The emulsification time is 5-20 minutes.

(5) Formation of microspheres

Transfer the double emulsion solution to NaCl aqueous solution, stir at a low speed until the organic solvent evaporates completely, collect the microspheres after centrifugation and washing. The NaCl concentration is 0.2-10% (g/ml), the volume is 2-6 times that of the double emulsion, the stirring speed is 200-2000rpm, the centrifugal speed is 500-8000rpm, the centrifugal time is 10-30 minutes, and the washing times are 2-2000rpm. 6 times.

(6) Freeze drying

The positive effect of the present invention is: the interferon polylactic acid-glycolic acid copolymer (PLGA) microsphere that the present invention adopts double emulsion-solvent volatilization method to prepare, microsphere preparation process is stable, feasible, and the form of microsphere is round, and the surface Smooth, good fluidity, uniform particle size distribution, average particle size of 30 μm, drug loading of more than 8%, encapsulation rate of about 40%, and its in vitro drug release performance conforms to the characteristics of long-acting preparations. The interferon-α is made into a slow-release microsphere preparation, which can prolong the action time of the drug and improve the curative effect and economic benefit of the drug.

Drawings and Description

Fig. 1 adopts the optical microscope and the scanning electron microscope photo of the interferon PLGA microsphere prepared by the present invention, observes the surface morphology.

Figure 2 is the DSC curves of PLGA blank microspheres (1) and interferon PLGA drug-loaded microspheres (2).

Detailed ways

The following examples further describe the present invention so that those skilled in the art can further understand the present invention, but do not limit the present invention in any form.

Example 1 Preparation of interferon poly(lactic-co-glycolic acid) (PLGA) microspheres

(1) Preparation of polylactic acid-glycolic acid copolymer (PLGA) solution

400 mg of poly(lactic-co-glycolic acid) (PLGA) was dissolved in 2 ml of dichloromethane to form an organic phase (20% g/ml).

(2) Preparation of interferon PBS solution

Dissolve 40 mg of lyophilized recombinant human interferon-α for injection in 300 μl of 1% (g/ml) gelatin solution to form an inner aqueous phase;

(3) Preparation of colostrum

Inject the inner aqueous phase into the organic phase, stir and emulsify at 9000rpm for 1 minute to form colostrum (W/O);

(4) Preparation of double emulsion

Add colostrum to 300ml containing 1% (g/ml) PVA and 1% (g/ml) NaCl solution (outer water phase) under the situation of stirring at 2000rpm, emulsify for 10 minutes, emulsify into double emulsion (W/O /W);

(5) Formation of microspheres

Transfer the double emulsion solution to 3 times the amount of 1% (g/ml) NaCl aqueous solution, stir at a low speed of 1000rpm until the organic solvent is completely volatilized, centrifuge at 1500rpm for 15 minutes, wash with distilled water 3 times, and collect the microspheres after washing;

(6) Freeze-drying conditions: -40°C, 4h; -25°C, 10h; -10°C, 20h; -4°C, 48h.

The prepared microspheres are freeze-dried, and the white loose powder interferon polylactic acid-glycolic acid copolymer microspheres are obtained under the above-mentioned freeze-drying conditions.

The shape of the obtained interferon microspheres is round, the surface is smooth, the fluidity is good, the particle size distribution is uniform, the average particle size is 10.71 μ m, the drug loading is 6.96%, and the encapsulation rate is 32.90%. feature.

Example 2 The preparation of interferon poly(lactic-co-glycolic acid) (PLGA) microspheres

(1) Preparation of polylactic acid-glycolic acid copolymer (PLGA) solution

600 mg of poly(lactic-co-glycolic acid) (PLGA) was dissolved in 2 ml of dichloromethane to form an organic phase (30% g/ml).

(2) Preparation of interferon PBS solution

Dissolve 60 mg of lyophilized recombinant human interferon-α for injection in 300 μl of 30% PEG400 solution to form an inner aqueous phase;

(3) Preparation of colostrum

Inject the inner aqueous phase into the organic phase, stir and emulsify for 3 minutes at 1000rpm to form colostrum (W/O);

(4) Preparation of double emulsion

Add colostrum to 200ml containing 3% (g/ml) PVA and 0.5% (g/ml) NaCl solution (outer water phase) under the situation of stirring at 1600rpm, emulsify for 20 minutes, emulsify into double emulsion (W/O /W);

(5) Formation of microspheres

Transfer the double emulsion solution to 2 times the amount of 1% (g/ml) NaCl aqueous solution, stir at a low speed of 1000 rpm until the organic solvent is completely volatilized, centrifuge at 1200 rpm for 20 minutes, wash with distilled water 3 times, and collect the microspheres after washing;

(6) Freeze-drying conditions: -40°C, 4h; -25°C, 10h; -10°C, 20h; -4°C, 48h.

The prepared microspheres are freeze-dried, and the white loose powder interferon polylactic acid-glycolic acid copolymer microspheres are obtained under the above-mentioned freeze-drying conditions.

The shape of the obtained interferon microspheres is round, the surface is smooth, the fluidity is good, the particle size distribution is uniform, the average particle size is 12.36 μ m, the drug loading is 6.06%, and the encapsulation rate is 36.80%. feature.

Claims (5)

1, a kind of preparation method of interferon polylactic acid-glycolic acid copolymer microsphere, carry out according to the following steps: Dissolve polylactic acid-glycolic acid copolymer in organic solvent; dissolve lyophilized recombinant human interferon for injection in PBS solution containing stabilizer to form internal water phase; then inject internal water phase into organic phase, stir and emulsify into colostrum (W/O); then add colostrum to PVA aqueous solution (outer water phase) under stirring conditions, emulsify into double emulsion (W/O/W); then stir at low speed in the solution containing electrolyte , to completely evaporate the organic solvent; centrifuge, wash, collect the microspheres, and freeze-dry to obtain the interferon polylactic acid-glycolic acid copolymer PLGA microsphere powder. 2. The method for preparing interferon polylactic acid-glycolic acid copolymer microspheres as claimed in claim 1, characterized in that: the preparation method comprises the following steps: (1) Preparation of polylactic acid-glycolic acid copolymer PLGA solution Dissolving polylactic acid-glycolic acid copolymer PLGA in an organic solvent to form an organic phase, the concentration of PLGA is 1% to 60% (g/ml); (2) Preparation of interferon PBS solution Dissolve lyophilized recombinant human interferon α for injection in PBS solution containing a certain amount of stabilizer to form an internal water phase, the concentration of interferon is 3% to 90% (g/ml), and the concentration of stabilizer is 1% to 60% (g/ml); (3) Preparation of colostrum Inject the inner water phase into the organic phase to form colostrum (W/O), the emulsification speed is 1000-10000rpm, the emulsification time is 1-10 minutes, and the volume ratio of the organic phase to the inner water phase is 2:1 to 50:1 ; (4) Preparation of double emulsion Add the colostrum into a certain concentration of PVA and NaCl aqueous solution (outer water phase) while stirring, and emulsify into double emulsion (W/O/W); the stirring speed for forming double emulsion is 1000-2900rpm, The concentration is 0.2-10% (g/ml), the concentration of NaCl solution is 0.2-10% (g/ml), the emulsification time is 5-20 minutes, and the volume ratio of colostrum and external water phase is 1:5-1 :600; (5) Formation of microspheres Transfer the double emulsion solution to the NaCl aqueous solution, stir at a low speed until the organic solvent is completely evaporated, collect the microspheres after centrifugation and washing; the concentration of the NaCl solution is 0.2-10% (g/ml), and the volume is 2-6 times that of the double emulsion , the stirring speed is 200-2000rpm, the centrifugal speed is 500-8000rpm, the centrifugal time is 10-30 minutes, and the washing times are 2-6 times; (6) Freeze drying 3. the method for preparing interferon polylactic acid-glycolic acid copolymer microspheres as claimed in claim 1 or 2, is characterized in that: described organic solvent is methylene chloride. 4. The method for preparing interferon polylactic acid-glycolic acid copolymer microspheres as claimed in claim 1 or 2, characterized in that: the stabilizer for interferon is gelatin or PEG400. 5. The preparation method of interferon polylactic acid-glycolic acid copolymer microspheres as claimed in claim 1 or 2, characterized in that: the conditions for freeze-drying the prepared microspheres are: -40°C, 4h; -25°C , 10h; -10°C, 20h; -4°C, 48h.