EA012121B1 - Pharmaceutical composition for the treatment of tuberculosis and diseases mediated by helicobacter pylori based on polymer nanoparticles, method for preparing thereof and methods of treatment - Google Patents

Abstract

The invention relates to medicine and pharmacy, in particular, to a pharmalogical composition for the treatment of tuberculosis and diseases mediated by Helicobacter pilory based on polymer nanoparticles comprising rifabutin in therapeutically effective amount and auxiliaries applicable for intravenous injection to a patient in need thereof, to a method for preparing thereof, to a method for the treatment of tuberculosis and diseases mediated by Helicobacter pylori comprising intravenous administration of the pharmalogical composition to a patient in need thereof.

Description

The present invention relates to the field of medicine and pharmacy, in particular, to a pharmaceutical composition of rifabutin based on nanoparticles containing a polymer / polymers of lactic acid and / or a copolymer / copolymers of lactic and glycolic acids, and suitable for intravenous administration to a patient in need of a therapeutically effective quantity, to the method for its preparation and to the method for the treatment of tuberculosis and diseases mediated by Neusobac! er ρνίοπ. comprising intravenously administering a pharmaceutical composition in accordance with the present invention in a therapeutically effective amount to a patient in need thereof.

Rifabutin (1 ', 4-didehydro-1-deoxy-1,4-dihydro-5' - (2-methylpropyl) -1-oxorifamycin XIV) is a semi-synthetic broad-spectrum antibiotic. Effective against intracellular and extracellular located microorganisms. Selectively inhibits DNA-dependent RNA polymerase of bacteria. It has a bactericidal effect. Highly active against Musoabas! (Muso-bac! Exsti 1 and bacillus arteriosus, Muso-bac! Exstus auscultus, including those located intracellularly) and other atypical mycobacteria. Between 1/3 and 1/2 of the Mycobacterium rhampocin resistant strains of rifampicin are susceptible to rifabutin, indicating incomplete cross-resistance between these antibiotics. It is also active against many gram-positive microorganisms. With monotherapy, resistance develops rapidly.

It is indicated for chronic multiresistant pulmonary tuberculosis caused by rifampicin-resistant strains of Muscular bacillus tuberculosis and other diseases. Neysobac! Er ru1op (especially strains resistant to clarithromycin).

Rifabutin is rapidly and completely absorbed in the gastrointestinal tract, the time to reach the maximum concentration of the substance in the blood plasma (T _max ) is 2-4 hours, the time to reach the 50% concentration of the substance in the blood plasma (T _1/2 ) is 35-40 hours; it penetrates well into organs and tissues (the highest concentration is created in the lungs); subjected to biotransformation in the liver, excreted in urine and bile.

In connection with the growing incidence of tuberculosis in the world caused by the emergence of drug-resistant strains and the spread of diseases of the immune system, including AIDS, it is necessary to create new anti-tuberculosis drugs. According to the World Health Organization, by 2020, the number of newly infected with tuberculosis will reach 1 billion, 200 million people will become ill and 35 million will die from tuberculosis if new, more effective treatments are not found.

Unfortunately, the high gastrointestinal toxicity of rifabutin has been repeatedly shown in the literature, and therefore its oral administration is risky. In addition, tuberculosis is most widespread in the Russian Federation and the CIS countries, in particular in prisons, where prisoners often avoid taking medications administered orally. In this regard, the problem arises of creating a dosage form of rifabutin different from solid oral dosage forms.

The prior art discloses Eurasian patent No. 9878, which discloses a composition of biodegradable microparticles for inhalation, which is applicable for specific delivery of drugs to targets for the treatment of pulmonary tuberculosis, and this composition includes two anti-TB drugs selected from the group consisting of rifabutin, rifapentin, isoniazid, pyrazinamide and ethambutol, and a biodegradable polymer for drug delivery, the ratio of drugs: polymer is from 1: 2 to 2: 1, a method for its preparation and method are also described. treatment of tuberculosis, including inhalational administration of the composition to a patient in need thereof a pharmaceutically effective amount. However, the use of the inhalation route of administration of rifabutin is not always convenient, and therefore there is a need to create dosage forms of rifabutin based on such colloidal delivery systems (micro- and nanoparticles) that could be administered to the patient intravenously.

US Pat. No. 6,264,991 is known from the prior art, which discloses a method for treating and / or preventing intracellular infections (including tuberculosis), comprising intravenously administering in a therapeutically effective amount of microspheres with a diameter of 1-5 or 5-10 microns made on the basis of a biocompatible polymer (preferably based on lactic acid polymers or copolymers of lactic and glycolic acid) containing a therapeutically effective amount of an anti-TB drug (including rifabutin). At the same time, formulations suitable for parenteral use with rifabutin, characterized, as indicated above, by almost complete insolubility in aqueous media, are not given, and therefore the task associated with the development of such formulations is solved in the present invention.

The present invention thus relates to a pharmaceutical composition for the treatment of tuberculosis and Neusobac! E p1op mediated diseases based on polymer nanoparticles containing rifabutin in a therapeutically effective amount and excipients suitable for intravenous administration to a patient in need thereof, characterized in that as

- 1 012121 auxiliary substances contains a polymer / polymers of lactic acid and / or a copolymer / copolymers of lactic and glycolic acid with a glycolic acid content in copolymers of up to 50 mol% with or without an additional carboxyl group at the end of the molecule or copolymers of lactic acid polymers or copolymers of lactic or glycolic acid with polyethylene glycol; the molecular weight (MM) of the polymers and copolymers is from 2 to 200 kDa, and the proposed pharmaceutical composition is a lyophilisate with a particle size of 0.1-0.8 μm, and when water or physiological solution is added, a stable suspension with particle size 0.1-0.8 microns; the pharmaceutical composition further comprises a water-soluble natural or synthetic polymer stabilizer with M.M. not more than 70 kDa and may contain a lipid plasticizer and fillers in the following ratio of components, wt.%:

Polymer / polymers of lactic acid and / or copolymer / copolymers

lactic and glycolic acids 10-50 Rifabutin 3-40 Polymer stabilizer 10-30 Plasticizer 0-10 Fillers rest.

The invention also relates to a method for producing a pharmaceutical composition and to methods for treating tuberculosis and diseases mediated by Hepatobacter pylori, including intravenously administering the composition in a therapeutically effective amount.

As a plasticizer, the pharmaceutical composition contains lecithin, lipoic acid or α-tocopheryl succinate, and as a water-soluble natural or synthetic polymer stabilizer emulsion with M.M. not more than 70 kDa contains monoglyceride esters, serum albumin.

As fillers in the proposed tool can be used sugars with cryoprotective properties (for example, glucose, lactose, mannitol, trehalose), and salts (for example, sodium chloride, sodium citrate).

The rifabutin pharmaceutical composition is prepared as follows: by the known method of manufacturing a simple water / oil emulsion by single or multiple homogenization of the organic and aqueous phases containing the components of the claimed agent, followed by evaporation of the organic solvent, a suspension of lactic acid polymers or lactic and glycolic acid copolymers with a separate size is obtained particles 0.1-0.8 microns. Rifabutin is sorbed inside the polymer particles during the formation of a suspension of nanoparticles by removing the organic solvent from the emulsion.

A system consisting of a 2-20 wt.% Solution of a polymer / polymers of lactic acid (PBA) and / or a copolymer / copolymers of lactic and glycolic acids (PBA), 0.5-5 wt.% A solution of rifabutin and 0.005-1 wt.% plasticizer (for example, lecithin, lipoic acid, α-tocopheryl succinate) in an organic solvent (usually in methylene chloride or chloroform) and 0.5-5 wt.% aqueous solution of the polymer emulsion stabilizer (volume ratio of organic to aqueous phases 1: 5), homogenize before receiving the emulsion. The organic solvent from the resulting emulsion is removed by evaporation under reduced pressure using a rotary evaporator. Then the resulting suspension is filtered, add 0-10 wt.% Filler - cryoprotectant (for example, glucose, lactose, mannitol, trehalose) and lyophilized. The degree of inclusion of rifabutin is 70-98%. The average particle size is 100-800 nm (depending on the conditions of homogenization and the emulsion stabilizer used).

The following are examples (compositions 1-10) obtained in accordance with the above method and are used solely to illustrate the claimed invention, and not to limit the scope of claims.

1. Composition 1 (particle size 207 nm):

RACA 50: 50 * M.M. 2300 Yes

Rifabutin

CSA ** 68kDa

38.5 wt.%

3.9 wt.%

19.1 wt.%

Mannitol

38.5 wt.%

- 2 012121

2. Composition 2 (particle size 516 nm):

RIOA 50:50 M.M. 2300 Yes

37.2 wt.%

Rifabutin

Soya lecithin

3.7 wt.%

1.9 wt.%

CSA 68kDa

Mannitol

3. Composition 3 (particle size 191 nm):

RIOA 50:50 M.M. 2300 Yes

20.0 wt.%

37.2 wt.%

37.2 wt.%

Rifabutin

Lipoic acid

3.7 wt.%

1.9 wt.%

CSA 68kDa

20.0 wt.%

Mannitol

4. Composition 4 (particle size 259 nm):

RLOA-COOH 50:50 M.M. 20 kDa

37.2 wt.%

38.5 wt.%

Rifabutin CSA 68kDa

3.9 wt.%

19.1 wt.%

Mannitol

5. Composition 5 (particle size 176 nm):

RSA 50:50 M.M. 20 kDa

38.5 wt.%

38.5 wt.%

Rifabutin

3.9 wt.%

Soya lecithin

1.9 wt.%

CSA 68kDa

19.1 wt.%

Mannitol

6. Composition 6 (particle size 198 nm):

RBCA 50:50 M.M. 20 kDa

38.5 wt.%

36.4 wt.%

Rifabutin

9.0 wt.%

CSA 68kDa

18.2 wt.%

Mannitol

7. Composition 7 (particle size 152 nm):

RIOA 50:50 MM 20 kDa

36.4 wt.%

34.5 wt.%

Rifabutin

13.8 wt.%

CSA 68kDa

17.2 wt.%

Mannitol

8. Composition 8 (particle size 407 nm):

RIOA-PEG *** 5000 70: 300 M.M. 100 kDa

34.5 wt.%

36.4 wt.%

Rifabutin

9.0 wt.%

CSA 68kDa

18.2 wt.%

Mannitol

9. Composition 9 (particle size 0.35 μm):

RLOA 50:50, M.M. 2.3 kDa

36.4 wt.%

25.6 wt.%

Rifabutin

CSA 68kDa

25.6

12.8 wt.% Wt.%

A-tocopheryl succinate

25.6 wt.%

Glucose

10.4 wt.%

- 3 012121

10. Composition 10 (particle size 0.6 μm):

RBSA 50:50, M.M. 2.3 kDa 40.0 wt.% Rifabutin 4.0 wt.% Lecithin 4.0 wt.% CSA 68kDa 20.0 wt.% Mannitol 32.0 wt.%

Conventions, abbreviations * РЬСЛ 50:50 - a copolymer of lactic and glycolic acids with a glycolic acid content in copolymers of up to 50 mol.% With an additional carboxyl group or without an additional carboxyl group at the end of the molecule;

** CSA - human serum albumin;

*** PBA-PEG is a copolymer of lactic and glycolic acids with polyethylene glycol.

The following are the results of comparative studies of the anti-tuberculosis activity of the rifabutin composition for intravenous administration (in accordance with the present invention) and the reference preparation of rifabutin for enteral administration according to the method for determining the bacteriostatic active concentration (LH) (1), as well as the anti-tuberculosis activity of rifabutin in comparison with the reference the drug ίη νίΐτο (item 2).

Materials and research methods.

The study material was blood samples of rats after administration of the studied drugs: rifabutin for intravenous administration (Κν), reference rifabutin (E) and soluble rifabutin when administered orally (Co).

The whole blood of animals was used, which was taken from the hyoid vein 30 minutes, 1, 2, 3, 6, and 24 hours after drug administration. The drugs were administered once at a dose of 2.5 mg / kg of animal weight. Preparations for oral administration were administered intragastrically using a special probe. Rifabutin for intravenous administration was injected into the tail vein.

LHC was determined in accordance with the guidelines "Modification of the determination of bacteriostatic activity of blood of patients with tuberculosis in a solid nutrient medium by serial dilution method", 1983

Serial dilutions were prepared from blood samples and plated on Levenshtein-Jensen nutrient medium together with tuberculosis mycobacteria. The test strain used a laboratory strain of MusoBas (stst (biccioconi Η37Κν (MBT), sensitive to all anti-tuberculosis drugs. The growth inhibition of MBT is recognized as the complete absence of colonies or the presence of single (up to 20 small colonies) in a dense nutrient medium.

The results of the study.

Accounting for the growth of culture of mycobacteria was carried out 3 weeks after seeding. The results of determining the LHC are presented in table. one.

Table 1 LHC values depending on the drug and time of blood sampling

Sampling time blood after drug administration Lack of MVT growth in blood dilutions Κν To E 0.5 hour 1:16 1: 4 1: 4 1 hour 1: 4 1: 4 1: 4 2 hours 1: 4 1: 8 1: 8 3 hour 1: 4 1: 4 1: 4 6 hour 1: 2 1: 2 1: 2 24 hour MW growth * MW growth * MW growth *

* The growth of colonies of MBT as in control, i.e. without adding drugs.

As follows from the table, with intravenous administration of rifabutin, LHC was different from that with enteral administration of drugs. The maximum value of blood dilution (1:16) was observed with blood sampling after 0.5 h with the intravenous method of administration of rifabutin. Then the LHC value fell sharply and remained at this level (1: 4) up to 3 hours. When blood was taken after 6 hours, the absence of MVT growth was determined with a 1: 2 blood dilution; by 24 h, the increase in MVT was in all blood dilutions, as in the control. At

- 4 012121 intragastric administration of rifabutin, both a reference preparation and a new form of rifabutin, LHC values increased gradually from 1: 4 after 0.5 h after administration to 1: 8 to 2 h. After 3 h, the LHC level decreased to 1: 4; after 6 hours - up to 1: 2. After 24 hours, the LHC was not determined. There were no differences in the dynamics of changes in LHC when using rifabutin reference (E) and soluble rifabutin.

Conclusion

With the intravenous administration of a new dosage form of rifabutin, higher concentrations of the drug are created, which provide significant bacteriostatic blood activity (LHC) against MVT. A further sharp decrease in LHC values is due to the distribution of the drug in the body tissues, which is especially important for the treatment of tuberculosis infection. There were no differences in the dynamics of changes in the LHC between the reference drug and the new form of rifabutin during their intragastric administration.

Materials and research methods.

To determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of rifabutin for intravenous administration compared with the reference drug rifabutin for enteral administration, the standard method recommended for preclinical studies ((Guide to the experimental (preclinical) study of new pharmacological substances) , M., 2000, S. 287-292).

Comparison of the anti-tuberculosis activity of the studied drugs was performed on several strains of mycobacteria:

1) the standard laboratory strain Musobacillus 1bbci1o515 Η37Ρν ATCC 25618 (catalog number of the international collection Atepsai Ture Cuigué Soyesjoi - VeShekba, Mb.), Sensitive to anti-tuberculosis drugs;

2) strain No. 1274, isolated from a patient with tuberculosis, sensitive to anti-TB drugs;

3) strain No. 33 isolated from a patient with tuberculosis, with multiple drug resistance, with resistance to 40 μg / ml rifampicin;

4) strain No. 239 isolated from a patient with tuberculosis, with multiple drug resistance, with resistance to 80 μg / ml rifampicin.

All stages of the ίη νίίτο experiment were conducted under strict sterility conditions.

1. Preparation of a standard bacterial suspension.

Mycobacterium tuberculosis cultures grown on solid nutrient medium for 3-4 weeks were removed with a platinum spatula, ground in a porcelain mortar and suspended in a 0.9% sodium chloride solution. The resulting suspension was transferred to a test tube and kept at room temperature for 30 minutes to spontaneously precipitate the largest conglomerates of mycobacteria.

Then the supernatant was transferred to another tube, an isotonic sodium chloride solution was added, comparing with the optical turbidity standard No. 5. The bacterial suspension thus prepared corresponds to a content of 5x10 ⁸ CFU in 1 ml of liquid. The inoculum dose of the bacterial suspension was 0.2 ml, which corresponds to 10 ⁸ CFU.

2. Determination of ίη νίίτο minimum bacteriostatic concentration.

The bacteriostatic concentration of the studied drugs was determined by the method of serial dilutions. Stock solutions of preparations were prepared as follows: rifabutin for intravenous administration (0.9 mg) was dissolved in 1.1 ml of sterile distilled water; a sample of rifabutin for enteral administration was first dissolved in ethyl alcohol (mother liquor), and then the initial concentration was prepared using Shkolnikova’s medium. Then they were introduced in a volume of 2 ml into the experimental row of tubes, making a series of concentrations from 8 to 0.03 μg / ml. In each test series, 2 control tubes with 2 ml of culture medium containing no preparation were provided.

0.2 ml of bacterial suspension containing 5x10 ⁸ CFU / ml was added to all tubes. The tubes were closed with silicone stoppers and incubated at 37 ° C for 14-20 days until the visible deep growth of mycobacteria was obtained in the form of a flocculent or granular precipitate.

The results were evaluated by microscopic examination of smears obtained from the pellet from each tube and stained with Ziehl-Nielsenen. The value of the inhibitory concentration of the test drug was determined by the number of acid-resistant mycobacteria and the intensity of the formation of microcolonies according to the following scheme:

- 5 012121

The number of fields of view The number of mycobacteria Growth rate one hundred - 0 one hundred 1-9 + one hundred 10-100 one 1-9 -n- one 10 and more +++

or the presence of microcolonies

Based on the results obtained, the minimum bacteriostatic concentration of the drug was determined, leading to partial (±) IPC ₅₀ (IPC, inhibiting the growth of 50% of the microorganisms of the studied population) or full (0) IPC ₉₀ (IPC, inhibiting the growth of 90% of the microorganisms of the studied population), inhibiting the growth of mycobacteria .

3. Determination of the bactericidal activity of the drug ίη νίίτο.

To determine the bactericidal activity of the preparations, the cultivation of mycobacteria and the preparation of various concentrations of the preparations were carried out according to the experimental scheme described above. To determine the viability of the studied test cultures, they were exposed to the preparations for 0 days, after which the MBT sediments washed twice from the preparations by centrifugation in a sterile isotonic sodium chloride solution were resuspended in 1 ml of a 0.9 wt.% Solution No. 1C1. seeded in 2 test tubes with egg medium Levenshtein-Jensen and incubated for 2.5 months. at 37 ° C.

The growth rate of MVT was evaluated according to the following scheme:

- lack of growth;

+ - 1-20 colonies of MBT in 2 tubes;

++ - 20-100 colonies of MBT in 2 test tubes;

+++ - more than 100 colonies of MBT in 2 test tubes.

The absence of growth of 99% of the microbial population (0) was regarded as the presence of bactericidal activity in the studied drugs. The minimum bactericidal concentration (MBC) of the drug was determined according to the total data of cultural and microscopic studies.

The results of the study.

The results of the determination of MPC and MBK of rifabutin for intravenous administration and a reference preparation for enteral administration are presented in table. 2 and 3.

table 2

MIC and MBC (in μg / ml) of rifabutin for intravenous administration in relation to various strains of MBT

MVT strains IPC _m * IPC, and ** MBK Η37Κν 0,03 0.06 0.125 No. 1274 0,03 0.06 0.125 Number 33 0.125 0.25 0.25 No. 239 1,0 2.0 8.0

Table 3

MIC and MBC (in μg / ml) of reference rifabutin for enteral administration in relation to different strains of MBT

MVT strains MPKm * IPC, about ** MBK Η37Κν 0,03 0.06 0.125 No. 1274 0,03 0.06 0.125 Number 33 0.125 0.25 0.25 No. 239 1,0 2.0 8.0

* IPC, inhibiting the growth of 50% of the microorganisms of the studied population. ** IPC, inhibiting the growth of 90% of the microorganisms of the studied population.

Conclusion

Thus, the antituberculous activity of the rifabutin composition for intravenous administration according to the level of BMD and MBC determined in the ίη νίίτο experiments does not differ from that of the reference rifabutin for enteral administration. The value of BMD and MBC for both drugs depended on the sensitivity of mycobacterium tuberculosis. The least sensitive to the studied drugs was strain No. 239, multiresistant, with a resistance of 80 μg / ml rifampicin.

The proposed pharmaceutical composition of rifabutin (in the form of a dosage form for intravenous administration) can be successfully used to treat tuberculosis in cases where the use of an oral form of the drug is impossible or difficult.

Claims (8)

CLAIM 1. A pharmaceutical composition for the treatment of tuberculosis and diseases mediated by Neuenobacter pylori. based on polymer particles of 100-800 nm in size containing rifabutin in a therapeutically effective amount and excipients suitable for intravenous administration to a patient in need thereof, characterized in that it contains polymer / polymers of lactic acid and / or copolymer / copolymers lactic and glycolic acids with a glycolic acid content in copolymers of up to 50 mol% with or without an additional carboxyl group at the end of the molecule or copolymers of lactic acid mers or copolymers of lactic or glycolic acid with polyethylene glycol; the molecular weight (MM) of the polymers and copolymers is from 2 to 200 kDa, the composition being a lyophilisate with a particle size of 0.1-0.8 microns, and when water or physiological solution is added, a stable suspension with a particle size of 0.1-0.8 microns; the composition additionally contains a water-soluble natural or synthetic polymer stabilizer with M. M. no more than 70 kDa, if necessary, a lipid plasticizer and fillers in the following ratio of components, wt.%: Lactic acid polymer / polymers and / or lactic and glycolic acid copolymer / copolymers or lactic and glycolic acid copolymer / copolymers with polyethylene glycol 10-50 weight. % Rifabutin 3-40 weight. % Polymer stabilizer 10-30 weight. % Plasticizer 0-10 weight. % Fillers rest 2. The pharmaceutical composition according to claim 2, characterized in that the polymer stabilizer is selected from esters of monoglycerides or human serum albumin. 3. The pharmaceutical composition according to claim 1, characterized in that the plasticizer is selected from lecithin, lipoic acid and α-tocopheryl succinate. 4. The pharmaceutical composition according to claim 1, characterized in that the excipients are selected from the group comprising glucose, lactose, mannitol, trehalose, sodium chloride and sodium citrate. 5. A method of obtaining a pharmaceutical composition according to any one of claims 1 to 4, characterized in that: A) homogenize to form an emulsion 2-20 wt.% Solution of the polymer / polymers of lactic acid and / or copolymer / copolymers of lactic and glycolic acids, 0.5-5 wt.% Solution of rifabutin and 0.005-1 wt.% Solution of plasticizer in organic solvent; B) remove the organic solvent from the resulting emulsion by evaporation under reduced pressure using a rotary evaporator; C) the resulting suspension is filtered; D) add to the filtered suspension 0-10 wt.% Filler; D) lyophilized. 6. The method according to claim 5, characterized in that the organic solvent is selected from methylene chloride or chloroform. 7. A method of treating tuberculosis, comprising intravenously administering a pharmaceutical composition according to any one of claims 1 to 4 in a therapeutically effective amount to a patient in need thereof. 8. A method for the treatment of diseases mediated by Neurolabe ruby, comprising the intravenous administration of a pharmaceutical composition according to any one of claims 1 to 4 in a therapeutically effective amount to a patient in need thereof.