RU2635472C1 - Method for manufacture of microspheres containing medicinal substances as biologically active substance, enclosed in polymer matrix, using microspheres production by double emulsion method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: matrix is a copolymer of lactic and glycolic acid with a viscosity of 0.2-0.8 dl/g and a comonomer ratio of 47.0:53.0 to 85.0:15.0, respectively. The process for microspheres manufacture comprises the steps of obtaining of a water/oil type emulsion, then a double emulsion is obtained in the presence of an emulsifier as a sodium carboxymethyl cellulose emulsion stabiliser, mannitol, the resulting product in the form of a double water/oil/water emulsion is loaded into the third reactor containing water for injection, emulsifier as an emulsion stabiliser, and the microspheres are formed as particles, removing methylene chloride vapor solvent for a time in the range of 1 to 24 hours with a gradual increase temperature range up to 25-45°C and at constant stirrong to form a suspension of cured particle microspheres, coarse fraction with a particle size of more than 150 mcm and fine fraction with a particle size of less than 10 nm are removed by separation, fractionation, filtration, and the desired product with a particle size of 10 nm to 150 mcm is obtained.

EFFECT: this invention allows to obtain microencapsulated microspheres that are nontoxic, stable during storage, which provides prolonged action of active agents.

4 cl, 3ex, 1 tbl

Description

The invention relates to biotechnology and can be used to obtain microencapsulated dosage forms used to produce drugs. Specifically, the invention relates to a method for producing microspheres in the form of particles from a biodegradable copolymer of lactic and glycolic acid with a biologically active substance encapsulated in microspheres.

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

After oral administration, most protein and peptide drugs lose their active structures in the acidic environment of the stomach or undergo enzymatic decomposition. They are also absorbed through the mucous membrane of the stomach or intestines at very low levels. For these reasons, protein or peptide drugs are generally not administered orally, that is, they are usually administered by injection. Non-oral (parenteral) administration of protein or peptide drugs should be repeated, since most non-oral (parenteral) administered protein or peptide drugs exhibit short half-lives and low bioavailability in the body. In addition, in many cases, their administration can continue for a long period of time, for example, months. In order to avoid these problems, an active study of sustained release dosage formulations was carried out, which led to the use of biodegradable polymer carriers with protein or peptide drugs encapsulated in them, which can release protein or peptide drugs from them as the polymer carriers biodegrade [Heller, J . et al., Controlled release of water-soluble macromolecules from bioerodible hydrogels, Biomaterials, 4, 262-266, 1983; Langer R., New methods of drug delivery, Science, 249, 1527-1533, 1990; Langer R., Chem. Eng. Commun., 6, 1-48, 1980; Langer R.S. and Peppas N.A., Biomaterials, 2, 201-214, 1981; Heller, J., CRC Crit. Rev. Ther. Drug Carrier Syst, 1 (1), 39-90, 1984; Holland S.J. Tighe B.J. and Gould P. L., J. Controlled Release, 155-180, 1986]. Aliphatic polyesters, currently used as polymer carriers, in particular for protein or peptide drugs, have received FDA approval for their use, since their biological compatibility has been established. They are widely used as carriers for the delivery of medicinal substances or threads for suturing during operations.

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 drug substances 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.

However, dosage forms containing polyester microspheres with protein drugs encapsulated therein have the disadvantage of exhibiting an initial burst effect, an uncontrolled release rate over a period of time due to various factors, or an incomplete release of the encapsulated drug substance. For example, model protein drugs, such as bovine serum albumin, lysozyme, etc., are released in large quantities at the initial stage, but exhibit a final release of approximately 50% [Crotts G. and Park T. G., J. Control. Release, 44, 123-134, 1997; Leonard N.V., Michael L.H., Lee M.M.J. Pharm. Sci., 84, 707-712]. Regarding microspheres using aliphatic polyester carriers with recombinant human growth hormone encapsulated in them, they initially release the drug substance in an amount of 30-50%, however, 40 ~ 60% of the drug substance remains in the microspheres [Van C, et al., J. Control . Release, 32, 231-241, 1994; Kim N.K. and Park T.G., Biotechnol. Bioeng., 65, 659-667, 1999].

The initial burst of drug release is due to the fact that protein drug substances aggregated or adsorbed on the surfaces or voids of the microspheres are released through rapid diffusion at the initial stage. In the manufacturing process of the microspheres, proteinaceous drugs can denature at the interface between water and an organic solvent, and thus they can form irreversible aggregates, which lead to unstable release. In order to prevent the contact-induced denaturation of protein drugs, the use of surfactants (for example, nonionic surfactants such as Tween, Pluronic F68, Brij 35, etc.) and stabilizers (for example mannitol, gelatin, trehalose) is described in the preparation of microspheres , carboxymethyl cellulose, etc.) or an organic solvent that does not contain water [Gombotz WR, Healy M., Brown L, US patent No. 5019400].

One example of the successful use of deposited dosage forms is the use of drugs encapsulated in microspheres from a polylactide-polyglycolide (PL-PG) copolymer (Shtilman MI. Polymers in biologically active systems // Sorosovsky 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).

Specific examples of aliphatic polyesters include poly-L-lactic acid, polyglycolic acid, a copolymer of D-lactic acid and glycolic acid, a copolymer of L-lactic acid and glycolic acid, a copolymer of D, L-lactic acid and glycolic acid (hereinafter referred to as as "PLGA"), polycaprolactone, polyvalerolactone, polyhydroxybutyrate and polyhydroxyvalerate [Peppas, L. B., Int. J. Pharm., 116, 1-9, 1995]. With the development in recent years of high molecular weight peptides or proteins as new drugs, various attempts have been made to release them from polymer carriers in a controlled manner.

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 manufacturing 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 C.E. 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.

To increase the degree of encapsulation of hydrophilic molecules (active substance), 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 (XM Deng, XH Li, ML Yuan, CD Xiong 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.

In particular, from patent RU 2326655, C1, 06/20/2008, a method is known for encapsulating protein-containing substances in microspheres from a polylactide-polyglycolide copolymer by evaporation of a solvent, which includes 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 emulsion and hydrophilic phase consisting of an emulsion stabilizer and water, solvent evaporation, characterized in that as a stabilizer of the secondary emulsion This is used polyglukin. The known method contains the following sequence of operations:

(1) preparation of the primary (two-phase) emulsion: an aqueous solution of a protein-containing substance (bovine serum albumin) (hereinafter - BSA) is emulsified in an organic phase consisting of an organic solvent and a polymer dissolved in it;

(2) preparation of a secondary (three-phase) emulsion: the primary emulsion is emulsified in a 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) freeze drying the finished product.

The known method is intended mainly for encapsulating such a protein-containing substance as albumin. The optimal concentration of polyglucin in a dispersion medium, at which satisfactory microspheres are obtained, and at the same time, after freeze drying, it is possible to obtain a well-resuspended preparation with low microsphere aggregation, is only 40%. In this case, with a different amount of polyglucin in the dispersion medium, microspheres with a wide spread of the obtained microsphere diameters from 12.1 μm to 327.4 μm and being a toxic drug are obtained (see table 1 in RU patent No. 2226655), and the presence of aggregates in the suspension also increases.

The technical task of the claimed method for producing microspheres as a whole is:

- development of an affordable and easy for industrial implementation of an improved method for producing microencapsulated microspheres for use in the preparation of various pharmaceuticals used to produce various drugs, providing a more complete release of the encapsulated biologically active active substance (pharmaceutical substance, drug) in order to increase their arsenal funds;

- decrease in particle aggregation,

- ensuring high stability of the encapsulated agent, - reducing the toxicity of the encapsulated biologically active substance through the use of a biodegradable copolymer, which decomposes with the formation of non-toxic substances.

The stated technical problem and the technical result are achieved by a method of manufacturing microspheres containing biologically active active substances enclosed in a polymer matrix using microspheres using the double emulsion method, and including a peptide enclosed in a polymer matrix as an active active substance, representing a copolymer of lactic and glycolic acids with a viscosity of 0.2-0.8 dl / g and with a ratio of these comonomers, respectively, from 47 , 0: 53.0 to 85.0: 15.0, formed from a double emulsion of the type water / oil / water,

wherein the method includes the following stages:

a) first get the first simple emulsion of the type water / oil in the first reactor by mixing, homogenizing and emulsifying in the presence of an emulsifier emulsifier stabilizer solution of a copolymer of lactic and glycolic acids in methylene chloride in a ratio of from 1.0: 2.5 to 1.0: 5.0 with a solution of the peptide as a biologically active substance in water for injection or in an aqueous solution of acetic acid with a ratio of 1.0: 2.0 to 1.0: 8.0,

b) then get a double emulsion in the second reactor by mixing the first emulsion with a mixture containing water for injection, the emulsifier is an emulsion stabilizer sodium carboxymethyl cellulose, mannitol, carried out in the flow circulation mode, maintaining the temperature from -10 ° C to + 10 ° C and

c) further, the resulting product in the form of a double emulsion of the type water / oil / water is loaded into a third reactor containing water for injection and an emulsifier, as an emulsion stabilizer, a surfactant;

g) carry out the formation of microspheres with a particle size of 10 nm to 150 μm, removing pairs of methylene chloride solvent over a period of time from 1 hour to 24 hours with a gradual increase in temperature to 25-45 ° C and constant stirring to obtain a suspension of cured microspheres in particle form;

d) from the suspension, followed by separation of the microspheres into fractions, remove by separation, filtration a large fraction with a particle size of more than 150 microns and a small fraction with a particle size of less

10 nm and get the target product with a particle size of 10 nm to 150 microns.

In the claimed method of manufacturing microspheres, a copolymer of lactic and glycolic acids can be used, in particular with a viscosity of 0.3-0.5 dg / l and with a ratio of comonomers, respectively, from 47.0: 53.0 to 53.0: 47, 0.

In the inventive method, it is advisable to manufacture microspheres (microparticles), in particular with a particle size of 5-90 microns, removing by separation a large fraction with a particle size of more than 90 microns and a small fraction with a particle size of less than 5 microns.

According to the claimed method, it is desirable to carry out the step of producing a double emulsion in step (b), in particular in a flow circulation mode with a temperature of about 0 °.

Below is a general description of the process, as an example, illustrating the method, but not limiting it. The first container is loaded with 1.5 l of a solution of a copolymer of lactic and glycolic acids in dichloromethane (methylene chloride, methylene chloride, CH ₂ Cl ₂ ) (hereinafter - DCM) in a ratio of 1.0: 2.5 to 1.0: 5.0

In a second container, 0.1 l of a solution of a peptide, in particular, octreotide, is loaded as a biologically active active substance in water or in an aqueous solution of acetic acid (ratio from 1: 2 to 1: 8).

Solutions from the first and second containers are fed through sterilizing filters to the first reactor (Reactor No. 1). The total volume of the solution is 1.6 liters. The first reactor (reactor No. 1) is equipped with a stirrer, jacket using cooling / heating capabilities from minus 10 ° С to 150 ° С, with the possibility of connecting a SIP, CIP, breathing filter with a mesh size of not more than 0.22 μm, nitrogen, and equipped with a temperature sensor.

After loading the first reactor, the solution supply lines are closed and stirring is started for no more than 5 minutes, then the first flow homogenizer is turned on and the solution is mixed in the circulation mode in the presence of an emulsifier - emulsion stabilizer. The resulting product is an emulsion of type B / M (type water / oil) (primary emulsion).

Next, in the second reactor (reactor No. 2), water for injection (hereinafter - VDI) is loaded in an amount of about 30 l of VDI, about 430 g of an emulsifier — an emulsion stabilizer, for example sodium carboxymethyl cellulose, about 920 g of mannitol are added. Stirred for about 60 minutes, after stirring, the resulting solution was heated to a temperature of at least 121 ° C and kept to achieve sterilization of the product in the flow circulation mode, maintaining the temperature from -10 ° C to + 10 ° C, in particular about 0 ° C.

The second reactor (reactor No. 2) is equipped with a stirrer, jacket using cooling / heating capabilities from 0 ° С to 150 ° С, with the possibility of connecting SIP, CIP, a breathing filter with a mesh size of not more than 0.22 μm, nitrogen, and equipped with thermal sensor.

To the resulting solution in the second reactor (Reactor No. 2), the primary emulsion from the first reactor (Reactor No. 1) is added, they are mixed and transferred to a homogenization through a second flow homogenizer in the circulation mode, maintaining the product temperature from -10 ° С to + 10 ° FROM. The resulting product is a water / oil / water double emulsion (double emulsion).

About 200 l of VDI are loaded into the third reactor (Reactor No. 3), an emulsifier-stabilizer emulsion is added, for example, a surfactant (hereinafter referred to as surfactant), for example, polysorbate 80 is about 1% and sodium chloride with an approximate concentration of about 1 can be used, 5%. The solution is cooled to approximately 0 °.

The third reactor (Reactor No. 3) is equipped with a stirrer, a jacket, which provides the possibility of cooling / heating from 0 ° С to 150 ° С, with the possibility of connecting a SIP, CIP, breathing filter with a mesh size of not more than 0.22 μm, and connecting a nitrogen equipped with a temperature sensor .

To the resulting solution in the third reactor (Reactor No. 3), a double emulsion from the second reactor is added with constant stirring. Next, the process of forming microspheres is carried out for about 24 hours with a gradual increase in temperature to 24-25 ° C and constant stirring. During the formation of particles (microspheres), it is preferable to supply and extract sterile air or inert gas to more intensively remove DCM vapors. When removing DCM from the dispersed phase of the product, the particles (microspheres) gradually solidify. The finished product is a suspension in which the dispersed phase is the particles (microspheres) of the target product with a particle size of 10 nm to 150 μm.

Due to the fact that the obtained microspheres (microparticles) can have a fairly wide range of sizes, the process of isolating microspheres with the required (necessary) sizes is carried out. In this case, depending on the task, the first step may be the removal of a large fraction, for example, microspheres with a diameter of more than 150 microns, for example, more than 90 microns, using the filtration process, or the removal of a small fraction, for example, microspheres with a diameter of less than 10 nm, in particular, less than 5 microns using the filtering process and / or tangential filtering, or both of these processes in any sequence. It is possible to wash and / or concentrate the obtained product by filtration and / or tangential filtration.

The resulting suspension (as the dispersed phase, the target product is microspheres with a particle size of less than 150 microns, in particular 90 microns, and more than 10 nm, in particular more than 5 microns, sent to the filter No. 1. Characteristics of the filter No. 1 : equipped with a stirrer, equipped with a jacket that allows cooling / heating from 0 ° С to 150 ° С, has the ability to connect SIP, CIP, a breathing filter with a mesh size of not more than 0.22 μm, nitrogen, equipped with a temperature sensor, provides aseptic discharge of the product has a mesh size of not more than 5 microns, has the ability to carry out vacuum drying, pressure filtration, the ability to conduct cleaning and suspension washing.

The following is a breakdown of some of the abbreviations used and explanations:

The abbreviation SIP is “sterilization in place”, sterilization of an object and / or equipment in place. Sterilization is carried out through the connection to the object of a sterilizing agent. Sterilization can be carried out by any known and permitted method (gas sterilization, sterilization with hydrogen peroxide vapor, heat treatment). In our case, it is preferable to use "clean" steam.

The abbreviation CIP is “clean in place”, cleaning the facility and / or equipment on site. Cleaning can be carried out through the connection or supply to the object of a cleaning agent. It can be:

water of varying degrees of purity (purified water, water for injection, softened water);

water containing various detergents (for example, surfactants (hereinafter referred to as surfactants);

detergents, solutions of acids or salts.

As auxiliary substances for the manufacture of microspheres by the claimed method using the double emulsion method, various emulsion stabilizers — emulsifiers — are used. As emulsifying agents promoting the formation of a stable emulsion, for example, anionic surfactants (sodium surfactants) (sodium oleate, sodium stearate, sodium lauryl sulfate (sodium lauryl sulfate), etc.) are used; nonionic surfactants (polyoxyethylene sorbent fatty acid ester (polysorbate 80, Twen 80, Tween 80), polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, lecithin, gelatin. Such emulsifiers can be used either individually or in combination; a emulsifiers with a dispersing effect can also be used; thickeners (increase viscosity and control the release of biologically active substances).

As emulsifiers, as emulsion stabilizers, other cellulose derivatives are also used (hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose salts), various polyethylene glycols (PEG), for example, PEG 200, PEG 500, PEG 1000, PEG 6000, etc., buffering agents, isotonizing agents (sodium chloride, mannitol, sorbitol, glucose). All these emulsifiers ensure the stability of emulsions (its stability) and, in turn, make it possible to control the surface structure of microcapsules, which is a decisive factor in the rate of release of a biologically active active substance (from the matrix).

As a biologically active active substance in the implementation of the claimed method, peptides such as, for example, buserelin acetate, octreotide, leuprorelin, goserelin, triptorelin are used.

In this case, we used a biodegradable polymer of lactic and glycolic acids containing D, L-lactide and glycolide, in particular, in a ratio of 50:50. Use a copolymer with a ratio of comonomers in the General case from 47:53 to 85:15 D, L-lactide and glycolide, respectively. The viscosity of the polymer is from 0.2 to 0.8 dl / g, in particular, preferably from 0.3 to 0.5 dl / g.

The experimental data using the target product indicate:

a) about the delayed (prolonged) release in the body of active substances (in this case, various peptides, for example, octreotide, leuprorelin, goserelin, tryptorelin, buserelin acetate and others;

b) to reduce their degradation along the path to the biological target and a sufficiently high speed in relation to various microorganisms, infections, diseases of various types.

The microspheres obtained by the claimed method from a biodegradable copolymer of lactic and glycolic acids with a biologically active active substance encapsulated in microspheres (pharmaceutical substance, drug substance) provide a prolonged release of these substances. The resulting microspheres (in the form of nanoparticles) possess stability of the main indicators of the quality of the products during storage, preparations containing them (like themselves) are practically non-toxic, possess antimicrobial activity, expand the assortment of various pharmaceutical and medicinal products that provide for the controlled release of encapsulated biologically active substances.

Example 1

Immunochemical properties of the claimed microencapsulated drug

Laboratory animals (guinea pigs) were immunized with preparations obtained by the claimed method, prepared using various adjuvants. The preparation No. 1 was a peptide (octreotide) encapsulated in microspheres from PL-PG in a ratio of 50:50 (where PL-PG is a polylactide-polyglycalide copolymer) by evaporation of the solvent from the double emulsion using an aqueous solution of mannitol as a surfactant. Comparison preparations (Nos. 2, 3) contained identical amounts of the peptide in complex 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 of octreotide (peptide).

Three months after immunization in the blood serum of animals, the titer of antibodies to octreotide (peptide) was evaluated in RIGA with a erythrocyte antigen diagnostic laboratory preparation.

Table 1 presents the results of determining the immunochemical properties of the compared drugs.

The data in table 1 show that 3 months after immunization of animals, the highest titer of antibodies to the indicated peptide was detected in the blood serum of animals immunized with the drug octreotide, microencapsulated in PL-PG, or emulsified in PAF.

The geometric mean titer of antibodies to octride in animals immunized with octreotide adsorbed on HGA was approximately 4 times lower (the difference was significant for p = 0.95)

Example 2

Toxicity assessment of new drugs

Assessment of the toxicity of the resulting preparations (microspheres) specified in the invention (example 1) was carried out in accordance with the rules and methods of experiments in laboratory animals adopted in the Russian Federation.

Acute toxicity was determined in Balb-C mice and Wistar rats of both sexes. The study of the toxic effects of each dose was carried out on 10 animals (5 males and 5 females). To study acute toxicity, drugs were administered intramuscularly with a syringe, and orally through an atraumatic metal probe, gradually immersing it to the stomach. Large doses were achieved by repeated application of drugs with an interval of 30-60 minutes for 3-4 hours. We evaluated the effect of doses equivalent to effective single therapeutic doses of known medicinal substances (rifabutin, isoniazid, pyrazinamide, D-cycloserine, D-mannitol), as well as 5 and 10 times their superior.

It was found that for the obtained preparations (example 1) with octreotide for mice and rats with intramuscular injection is 2-3 g / kg, and with oral application - more than 12-15 g / kg. Innami words, new drugs are practically non-toxic. At autopsy of experimental and control animals, pathological changes in organs and tissues were not recorded.

Example 3

Evaluation of the antimicrobial activity of new drugs.

The sensitivity assessment of gram-positive, gram-negative and atypical bacteria in relation to new drugs (Example 1) was carried out in accordance with the recommendations of the National Committee for Clinical and Laboratory Standards of the USA (NCCLS) and the guidelines in force in the Russian Federation.

The minimum inhibitory concentrations of the preparations with respect to the test cultures were determined by the method of serial micro-dilutions in a liquid medium according to the recommendations of NCCLS. In addition, the growth rate of microorganisms in the presence of sub- and super-inhibiting concentrations of the studied drugs was evaluated by the dynamics of the optical density of bacterial suspensions. Dynamic measurement of optical density was carried out using a Bioscren multichannel spectrophotometer (Labsystems) at a wavelength of 610 nm with an interval of 20 min. Plates with bacterial suspensions were incubated at 37 ° C in a thermostatic module of the device. The initial concentration of microorganisms was 5 × 10 ⁴ CFU / ml. It was found that for the obtained preparations (example 1) with octreotide, high antimicrobial activity was recorded.

In connection with the foregoing, the claimed invention allows to obtain in an affordable manner highly effective drugs with antimicrobial action in the form of nanoparticles, which are proposed to be used to treat various diseases.

The conducted experimental data indicate:

a) about the delayed (prolonged) release in the body of active substances (in this case, various peptides, for example, octreotide, leuprorelin, goserelin, tryptorelin, buserelin acetate and others;

b) to reduce their degradation along the path to the biological target and a sufficiently high speed in relation to various microorganisms, infections, diseases of various types.

Thus, the microspheres obtained by the claimed method from a biodegradable copolymer of lactic and glycolic acids with a biologically active active substance encapsulated in microspheres (pharmaceutical substance, drug substance) provide a prolonged release of these substances. The resulting microspheres (in the form of nanoparticles) possess stability of the main indicators of the quality of the products during storage, preparations containing them (like themselves) are practically non-toxic, possess antimicrobial activity, expand the assortment of various pharmaceutical and medicinal products that provide for the controlled release of encapsulated biologically active substances.

Information sources

1. Patent RU 2326655, C1, 06/20/2008 (prototype).

Claims (10)

1. A method of manufacturing microspheres containing medicinal substances as a biologically active active substance, enclosed in a polymer matrix, using the preparation of microspheres by the double emulsion method, and including, as an active active substance, a peptide enclosed in a polymer matrix representing a milk copolymer and glycolic acids with a viscosity of 0.2-0.8 dl / g and with a ratio of these comonomers, respectively, from 47.0: 53.0 to 85.0: 15.0, formed from a double emulsion of the type water / oil / water, wherein the method includes the following stages: a) first get the first simple emulsion of the type water / oil in the first reactor by mixing, homogenizing, stabilizing and emulsifying in the presence of an emulsifier as a stabilizer of emulsions of a solution of a copolymer of lactic and glycolic acid in methylene chloride in a ratio of 1.0: 2.5 to 1 , 0: 5.0 with a solution of the peptide as a biologically active substance in water for injection or in an aqueous solution of acetic acid with a ratio of from 1.0: 2.0 to 1.0: 8.0, b) then get a double emulsion in the second reactor by mixing the first emulsion with a mixture containing water for injection, emulsifiers as emulsion stabilizers sodium carboxymethyl cellulose, mannitol, carried out in a flow circulation mode, maintaining the temperature from -10 ° С to + 10 ° С and c) then the resulting product in the form of a double emulsion of the type water / oil / water is loaded into a third reactor containing water for injection, an emulsifier as an emulsion stabilizer, and g) carry out the formation of microspheres with a particle size of 10 nm to 150 μm, removing pairs of methylene chloride solvent over a period of time from 1 hour to 24 hours with a gradual increase in temperature to 25-45 ° C and constant stirring to obtain a suspension of cured microspheres in particle form; d) from the suspension by subsequent separation of the microspheres into fractions, a coarse fraction with a particle size of more than 150 μm and less than 10 nm is removed by separation, filtration and the target product with a particle size of 10 nm to 150 μm is obtained. 2. A method of manufacturing microspheres according to claim 1, characterized in that they use a copolymer of lactic acid and glycolic acid with a viscosity of 0.3-0.5 dl / g and with a ratio of comonomers, respectively, from 47.0: 53.0 to 53 0: 47.0. 3. A method of manufacturing microspheres according to claim 1, characterized in that in step (e) a coarse fraction of more than 90 microns and a fine fraction with a particle size of less than 5 microns are removed by separation and the target product with a particle size of 5-90 microns is obtained. 4. A method of manufacturing microspheres according to claim 1, characterized in that in stage (b), the preparation of a double emulsion is carried out in a flow circulation mode with a temperature of about 0 ° C.