RU2326655C1 - Method of incapsulation of protein bearing materials in microspheres from copolymer polylactide-polyglycolide - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: microspheres represent a polylactide-polyglycolide copolymer - polylactic and polyglicolic acids. It breaks up in an organism to natural metabolites - milk and glycolic acids, is nontoxic, does not invoke inflammatory responses in an injection place, is stable at storage. The incapsulation method of protein bearing materials in microspheres from a polylactide-polyglycolide copolymer includes: preparation of primary pap by an emulsification of an aqueous solution of reacting material in an organic phase; reception of secondary pap by means of bond of primary pap and the hydrophylic phase consisting of an aqueous solution of the pap stabiliser; evaporation of an organic dissolvent. As the stabiliser of secondary pap Polyglucinum resolved to application as a part of injected medical products was used.

EFFECT: can be used for reception of microencapsulated forms of medicinal preparations.

2 tbl, 3 ex

Description

The invention relates to biotechnology and can be used to obtain microencapsulated forms of drugs.

The treatment and prevention of endocrine, oncological, immune, infectious, hereditary and a number of other diseases often require long-term maintenance of the necessary concentration of appropriate drugs in the patient's body, which necessitates the development of dosage forms that provide a prolonged effect of the active principle of the drug.

The advantages of the deposited dosage forms over the non-deposited ones are the continuity of the therapeutic or prophylactic effects, a stable predictable concentration of drugs in the blood plasma, the possibility of using lower therapeutic doses, reducing the risk of side effects, and the absence of the need for repeated administration. As part of many developed depot preparations, the active substance is in soluble polymer microcapsules. The duration of action of such drugs depends, inter alia, on the size and material of the microcapsules used.

One example of the successful use of deposited dosage forms is the use of preparations encapsulated in microspheres from a polylactide-polyglycolide (PL-PG) copolymer (Shtilman M.I. Polymers in biologically active systems // Sorosovskiy Obrazovatelny. Zhurn. - 1998. - No. 5. - S. 48-53). PL-PG - polyester of polylactic and polyglycolic acids. It is destroyed in the body to natural metabolites - lactic and glycolic acids, non-toxic, does not cause inflammatory reactions at the injection site, is stable during storage, technologically advanced in manufacture, and is characterized by good reproducibility of the quality of the final product. The ratio of PL and PG in the composition of the copolymer determines the rate of its biodegradation in vivo. Absorbable bone implants and surgical sutures made from this material have a long history of safe use in medicine (V.V. Mikhailov, R. A. Khamitov, B. V. Kravtsov. Immobilized preparations in virology and vaccinology // Journal. Vopr. Virology. - 1995. - No. 5. - S.194-197).

There are various methods for microencapsulation of the active substance using PL-PG, however, one of the most common of them is the method of evaporation of the solvent.

A known method of preparing microspheres by evaporation of a solvent from a simple (two-phase) emulsion, which is a special case of the phase reversal method (Beck LR, Pope VZ, Flowers CE et al. Poly (DL-lactide-co-glycolide) / Norethisterone Microcapsules: An injectable Biodegradable Contraceptive // Biology of reproduction. - 1983. - No. 28. - P.186-195). The method is based on the use of two phases: a hydrophobic organic phase, consisting of an organic solvent, a polymer and the active substance dissolved in it, and a hydrophilic dispersion medium, consisting of water and an emulsion stabilizer (surfactant), which is usually used as polyvinyl alcohol (PVA). The organic phase is added to the dispersion medium and subjected to emulsification, after which the organic solvent is evaporated from the resulting emulsion with continuous stirring. In the process of evaporation of the solvent, the droplets of the organic phase solidify, the emulsion turns into a suspension. The resulting suspension of microspheres is precipitated by centrifugation. In order to wash the obtained microspheres from the surfactant, the centrifugation operation, followed by resuspension in distilled water, is repeated three times. After washing, the microsphere preparation is subjected to freeze drying. Thus obtained microspheres are characterized by a spherical shape, relative homogeneity and small size (about 10 microns). The disadvantage of the above method is that for its use it is necessary that the encapsulated preparation is, at least partially, soluble in an organic solvent. This is the main limitation of the use of this method for encapsulating hydrophilic compounds, such as peptides, proteins or nucleic acids.

Common essential features of the described method with the claimed method are:

1. The inclusion of the active substance in the microspheres from a biodegradable carrier;

2. Evaporation of the organic solvent from the organic phase, as a result of which the emulsion turns into a suspension.

To increase the degree of encapsulation of hydrophilic molecules, alternative methods have been developed, the most common of which is the method of preparing microspheres by evaporation of an organic solvent from a double (three-phase) emulsion (X.M.Deng, XHLi, MLYuan, CDXiong et al. Optimization of preparative conditiones for poly-DL-lactide-polyethylene glycol microspheres with entrapped Vibrio cholera anti-genes // Jomal of Controlled Release. - 1999. - N58. - P.123-131). In this case, the aqueous solution of the hydrophilic compound is primarily emulsified in an organic polymer solution. Then the primary emulsion is poured into a large volume of water containing surfactant. An organic solvent is evaporated from the resulting three-phase emulsion with continuous stirring. Thus obtained microspheres are washed from the surfactant and freeze-dried. When using the double emulsion technique, as a rule, good encapsulation of hydrophilic compounds is achieved, however, the particle size is usually larger than when using the method based on a simple emulsion.

The reasons that impede the achievement of the technical result indicated below include the fact that when using the method of preparing microspheres by evaporation of an organic solvent from a double (three-phase) emulsion adopted as a prototype, the active substance is incompletely loaded into microspheres as a result of its partial transition to a dispersion medium in the process hardening microspheres. The loss can reach 80% or more of the active ingredient used for loading. In addition, when using this method, there remains a need to wash the finished microspheres from the dispersion medium, which makes the technology of preparing microspheres difficult, cumbersome and expensive.

Common essential features of this method with the claimed method are:

1. The inclusion of the active substance in the microspheres from a biodegradable carrier;

2. The use of an emulsion consisting of three phases: aqueous, organic, aqueous;

3. Evaporation of the organic solvent from the organic phase, as a result of which the emulsion turns into a suspension.

The objective of the invention is to improve the method of microencapsulation of protein-containing substances.

The technical result of the invention is an increase in the amount of active substance in the composition of the final preparation and simplification of the procedure for preparing a liquid semi-finished vaccine preparation for the freeze-drying stage.

The specified technical result in the implementation of the invention is achieved by the fact that in the proposed method as a stabilizer of the emulsion instead of PVA it is proposed to use polyglucin. Polyglukin is a dextran drug, a 6% aqueous solution of which is used in medical practice as a plasma substitute for improving the rheological properties of blood. It is a partially hydrolyzed glucose polymer with a molecular weight of about 60,000 D. Since polyglucin is a drug officially approved for parenteral use, there is no need to exclude it from the composition of the dosage form, as is the case with PVA. In addition, polyglucin, as is known, has been successfully used as a cryoprotectant for freeze drying of drugs.

Thus, the claimed method is the following sequence of manipulations:

1. Preparation of the primary (two-phase) emulsion: an aqueous solution of a protein-containing substance is emulsified in an organic phase consisting of an organic solvent and a polymer dissolved in it.

2. Preparation of the secondary (three-phase) emulsion: the primary emulsion is emulsified in the hydrophilic phase, consisting of water and polyglucin dissolved in it.

3. Evaporation of the organic solvent from the organic phase, as a result of which the emulsion turns into a suspension.

4. Lyophilic drying of the finished product.

Between the set of essential features of the proposed method and the technical result achieved, there is a causal relationship, namely: the use of polyglucin as an emulsion stabilizer made it possible to optimize the microencapsulation of protein-containing substances by eliminating the stage of washing the microspheres and simplifying the stage of preparation for freeze drying. In this case, the active substance released into the dispersion medium from the microspheres during the evaporation of the solvent remains in the final preparation in its free form and is not lost, as is the case in the prototype method.

The possibility of carrying out the claimed invention is shown by the following examples. All of the studies below were performed using bovine serum albumin (BSA) as a model of encapsulated active ingredient.

Example 1. The scheme for the preparation of microspheres by evaporation of the solvent from the double emulsion using polyglucin as stabilizer

To prepare the preparation, 0.2 ml of a 1% aqueous solution of BSA was added to the organic phase, consisting of 0.4 g of PL-PG dissolved in 1.8 ml of dichloromethane, and vigorously emulsified on a magnetic stirrer for 1 minute. After that, the resulting primary emulsion was added to 12 ml of an aqueous solution of polyglucin. The secondary (three-phase) emulsion was stirred on a magnetic stirrer for 1 minute at maximum speed, after which the mixing speed was reduced to a minimum (~ 100 rpm) and such conditions were maintained for about 2 hours until dichloromethane was completely evaporated. The resulting suspension of microspheres was placed in penicillin vials in a volume of 2.5 ml and subjected to freeze drying.

Example 2. Physico-chemical properties of the microencapsulated preparation BSA

To assess the effect of the concentration of polyglucin in the dispersion medium on the size of the microspheres, on the degree of their loading and on the characteristics of the freeze-dried preparation by the proposed method (see example 1), a number of microsphere preparations loaded with BSA were prepared. In this case, an aqueous solution containing a different amount of polyglucin was used as a dispersion medium. In lyophilized preparations, weight loss upon drying was evaluated. Upon rehydration of the dried preparations by visual method, the rate of resuspension and the presence of aggregates were evaluated. In rehydrated preparations, the average diameter of the microspheres was determined by the microscopic method by measuring 200 microspheres. The degree of loading of the microspheres was determined spectrophotometrically at a wavelength of 280 nm, the optical density of the extract from the microspheres was compared with the optical density of a reference preparation containing the same amount of BSA that was used to prepare the prototype. The research results are presented in table 1.

As can be seen from the data presented in table 1, with an increase in the concentration of polyglucin in the dispersion medium, the average size of the microspheres decreases, while the degree of their BSA loading decreases. The optimal concentration of polyglucin in a dispersion medium, which provides satisfactory characteristics of the microspheres, and at the same time, after freeze drying, it is possible to obtain a well-resuspended preparation with low microsphere aggregation, is 40%.

Example 3. Immunochemical properties of microencapsulated BSA

Laboratory animals (guinea pigs) were immunized with BSA preparations prepared using various adjuvants. The preparation No. 1 was a BSA encapsulated in microspheres from PL-PG (50:50) by evaporation of the solvent from the double emulsion using a 40% aqueous solution of polyglucin as a surfactant. Comparison preparations (Nos. 2, 3) contained identical amounts of BSA in combination with respectively oil (Freund's complete adjuvant, PAF) and mineral (aluminum hydroxide gel, HGA) adjuvants.

Immunization of guinea pigs (20 animals per group) was carried out subcutaneously in the inner thigh in a volume of 0.2 ml at a dose of 20 μg BSA.

3 months after immunization in the blood serum of animals, the titer of antibodies to BSA in RNGA was evaluated with an erythrocyte antigen diagnostic laboratory preparation.

Table 2 presents the results of determining the immunochemical properties of the compared drugs.

The data in table 2 show that 3 months after immunization, the highest titer of antibodies to BSA was detected in the blood serum of animals immunized with BSA, microencapsulated in PL-PG, or emulsified in PAF. The geometric mean titer of antibodies to BSA in animals immunized with BSA adsorbed on GTA was approximately 4 times lower (the difference was significant for p = 0.95).

Table 1 The effect of the concentration of polyglucin in a dispersion medium on the characteristics of microspheres The concentration of polyglucin in a dispersion medium,%

, мкмDiameter of microspheres,

μm The degree of loading of the microspheres,% Loss in mass upon drying of the drug after lyophilic dehydration,% Dry suspension resuspension rate,

min The presence of aggregates in suspension 10 327.4 63.5 0.1 four + fifteen 280.5 50.3 0.1 7 + twenty 120.5 41.8 0.2 13 + 25 45.7 28.3 0.5 17 ± thirty 14.3 24.3 0.9 23 - 40 12.1 25,4 1.4 26 -

table 2 Immunochemical activity of microencapsulated BSA The composition of the drug The number of animals with seroconversion, percentage

BSA antibody titer in animal blood,

BSA + PL-PG 95

BSA + PAF (control 1) 90

BSA + GGA (control 2) 70

Notes: 1. BSA dose - 20 mcg per animal; 2. Drugs were administered subcutaneously in a volume of 0.2 ml.

Claims (1)

A method of encapsulating protein-containing substances in microspheres from a polylactide-polyglycolide copolymer by evaporation of a solvent, comprising preparing a primary emulsion by emulsifying an aqueous solution of the active substance in the organic phase, obtaining a secondary emulsion by combining the primary emulsion and the hydrophilic phase, consisting of an emulsion stabilizer and water, evaporating the solvent characterized in that polyglucin is used as a stabilizer of the secondary emulsion.