RU2418585C1 - Rifampicin-based medication with prolonged action for treatment of resistent forms of tuberculosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of pharmacology and medicine and represents rifampicin-based medication of prolonged action for treatment of resistant tuberculosis forms, which is different because it represents stable nanoparticles and contains rifampicic, biodegradable polymer of lactic acid or copolymer of lactic and glycolic acid, as well as surface active substance, cryoprotector, components in medication being in specified ratio in wt %.

EFFECT: invention ensures reduction of risk of toxic effects, prolonged action and reduction of intake factor in treatment.

6 cl, 3 ex, 1 tbl, 1 dwg

Description

The invention relates to the field of pharmacology and medicine, specifically to a new generation of antimicrobial and anti-TB drugs of controlled action based on 3 - [[(4-methyl-1-piperazinyl) imino] methyl] rifamycin or 5,6,9,17,19, 21-hexahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8- [N- (4-methyl-1-piperazinyl) formimidoyl] -2,7-epoxypentadec [1, 11.13] trienimino) naphtho- [2,1-b] furan-1,11 (2H) -dione-21-acetate (rifampicin).

According to the WHO, in 2006 there were 9.2 million new cases of tuberculosis (139 cases per 100,000 people). This is 100 thousand more than in 2005. In 2006, 1.7 million people died from tuberculosis. In Russia in 2007, the number of multidrug-resistant tuberculosis patients among the newly diagnosed was 4149 people (9.8% of the total number of newly diagnosed). In total, it is registered at the end of 2007 - 24,445 people for every 100 thousand people.

A similar picture is observed with the incidence of the gastrointestinal tract, upper and lower respiratory tract, etc., caused by various microorganisms that are resistant to most modern antibiotics.

One of the modern antibiotics with a wide antibacterial and bactericidal spectrum of action is a semi-synthetic drug - rifampicin. Rifampicin is also active against Mycobacterium tuberculosis and therefore is widely used to treat tuberculosis in combination with other drugs. The antibiotic is also used for infections of the skin, soft tissues, treatment of pneumonia, lung abscesses, cholecystitis, etc.

The resistance of mycobacteria to rifampicin develops rapidly, which is a big drawback of antibiotics of this series.

Another negative property of rifampicin is its poor absorption when taken orally, as a result of which it has to be used parenterally.

Rifampicin, like other antibacterial and bactericidal drugs, has a low selectivity of action and when an antibiotic is administered to patients, only a small dose of it gets into the target cell. Most of it undergoes biotransformation and is excreted from the body without having a therapeutic effect. In this regard, in the treatment of rifampicin, especially its prolonged use, numerous undesirable effects are recorded: allergic reactions (skin rashes, eosinophilia), nausea, vomiting, increased liver transaminases in the blood, headache, arthralgia, tubular necrosis, visual impairment, development is possible thrombophlebitis and others. Rifampicin is contraindicated in patients with impaired liver and kidney function, pregnant women and with lactation [1].

To solve the problems associated with the toxicity of drugs, the presence of contraindications, resistance, etc., it is necessary to solve the problem of increasing the effectiveness of drugs and, therefore, reducing the therapeutic dose and toxic effects. The most significant of the possible ways to achieve this goal is the development of drugs or dosage forms of directed action, ensuring delivery and localization of the drug in target cells in doses effective for therapy. At the same time, it is desirable that this property be combined with a prolonged gradual release of the substance (prolonged action), which allows creating more comfortable conditions for patients who are forced to regularly and continuously use drugs.

A new technical result is achieved by the totality of all existing signs of the created drug based on rifampicin, which allows to achieve a positive pharmacodynamic effect of the drug at lower doses and thereby reduce the risk and degree of occurrence of toxic effects, while achieving the effect of prolonging the action, reducing the frequency of administration during treatment.

The technical result of the invention is achieved by the inclusion in a new drug based on rifampicin, commercially available biodegradable polymers that are approved for medical use, to obtain nanoparticles. As such, they are used: a polymer of lactic acid (PLA), a copolymer of glycolic and lactic acids (PLGA) and a copolymer of glycolic and lactic acids with a free terminal carboxyl group (PLGA-COOH).

To obtain nanoparticles, polymers were used with a molecular weight of 10 to 300 kDa and a molar ratio of lactic and glycolic acid residues from 25: 75% to 50: 50%. Surfactants: polysorbate 80, polyvinyl alcohol (PVA), poloxamer 188, serum albumin and cryoprotectants: mannitol, lactose, glucose and others are also used to obtain a stable dosage form representing 200-500 nm nanoparticles and having a prolonged release of the antibiotic. Sahara. The proposed tool is obtained by the known method of simple (single) emulsification (water / oil) [2]. Sorption of rifampicin inside the polymer nanoparticles occurs when the organic solvent is removed from the resulting emulsion and during the formation of the suspension.

It should be noted that previously similar studies were carried out by Indian, Japanese and Iranian researchers [3-7]. The work of Indian scientists [3, 4] describes the preparation of nanopreparations based on PGA with rifampicin, isoniazid, protionamide and ethambutol incorporated into the polymer matrix by ultrasonic emulsification. The drugs showed high anti-tuberculosis activity in vivo for 10 days, while in our case, high activity continued even 21 days after the start of the experiment. In [3, 4], there are no data on the toxic properties of drugs and therefore it is difficult to judge the prospects of their further use.

In an article by Iranian authors [5], data are presented on the preparation of nanosomal preparations (209–260 nm) based on PGA with rifampicin and a study of their physicochemical properties. Brief data on the in vitro study of antibacterial activity in cell cultures are also provided. In vivo experiments are not shown.

In [6], experiments were described on the production of microparticles (less than 3 μm) based on PGA and anti-TB drugs (isoniazid, rifampicin and pyrazinamide) by the double emulsification method. The distribution of drugs in various organs of experimental animals after the administration of these drugs over time was studied. It was shown that the concentration of rifampicin and its activity decreased 9 days after administration. Information on the more distant effects of drugs is not given. Similar information is provided by Barrow E.L.W. et al. [7], which describes a method for producing microspheres (7.5–15 μm) from polymers of PLGA of various compositions with sorbed rifampicin. Data on the presence of activity on Mycobacterium tuberculosis within 6 days are given [7].

Thus, according to the totality of all existing signs of the created drug: the presence of a prolonging effect, the presence of high specific in vivo activity in experiments on animals infected with resistant forms of tuberculosis, even after 3 weeks, reduced toxicity, a new technical result is achieved.

The invention is illustrated by the following examples.

Example 1. Obtaining polymer nanoparticles with rifampicin incorporated in them by the method of simple emulsions

A mixture consisting of a solution of 5 ÷ 60% polymer (PLA, PLGA or PLGA-COOH) and 1 ÷ 10% rifampicin in an organic solvent (chloroform, methylene chloride), as well as a 0.5 ÷ 2% aqueous solution of the polymer emulsion stabilizer in bulk the ratio to the organic solvent 5: 1 is homogenized at 24 thousand rpm on an Ultra-Turrax T-25 homogenizer (IKA ^® , Germany) 3 times for 1 minute with breaks of 1 minute. The emulsion is stirred for 1 hour at 40 ° C (if chloroform was used) or 1.5 hours at room temperature (using methylene chloride) until the organic solvent is completely removed. This operation must be carried out with exhaust ventilation operating. The resulting suspension is filtered through a coarse glass filter (por. 40 ÷ 110 μm), 1 ÷ 3% (vol.) Cryoprotectant (D-mannitol) is added to the filtrate, frozen and lyophilized. The average particle size, determined by autocorrelation spectroscopy on a Coulter N4MD submicron laser spectrometer (USA), ranges from 250 to 500 nm, which contributes to efficient absorption in the gastrointestinal tract [8]. The size of the resulting particles depends on the type of polymer, the stabilizer of the emulsion, their concentration, as well as on the conditions of homogenization. The degree of inclusion of rifampicin in polymer particles is from 15 to 75%.

Composition 1 (as in example 1) wt.% PLA 59.2 Rifampicin 8.5 Polyvinyl alcohol 15,2 D-mannitol 17.1 Particle size 378 ± 84 nm Composition 2 (as in example 1) wt.% PLGA 50: 50 58.3 Rifampicin 8.3 Polyvinyl alcohol 16.6 Lactose 16.8 Particle size 281 ± 73 nm Composition 3 (as in example 1) wt.% PLGA 50:50 58.5 Rifampicin 8.4 Albumen 15.8 D-mannitol 17.3 Particle size 324 ± 57 nm

The optimal dilution of the product in water corresponds to a ratio of 1:20 to 1:10, which can be used to adjust the dosage of the drug depending on the individual characteristics of the patient.

The drug obtained according to claim 1 can be made in the form of a tablet, capsule, granule, powder or other oral form based on known technologies [9, 10].

Example 2. Comparative pharmacological efficacy of the original drug prolonged action based on rifampicin

The effect of the developed drug based on rifampicin on the survival and plating of M. tuberculosis in the lungs was studied on the model of acute tuberculosis by introducing a lethal dose (M. tuberculosis strain H37Rv at a dose of 5 · 10 ⁶ / mouse). The introduction of a drug of prolonged action based on rifampicin (PH) obtained in example 1, began one day after the reproduction of tuberculosis. The drug was administered for 3 days. As a comparison, experiments were carried out under similar conditions, where instead of a new rifampicin preparation, either physiological saline (K) or the substance of rifampicin (P) was used. After the experiment was completed, the animals were euthanized with hexenal, the lungs were removed and homogenized, and culture was performed to determine the number of viable M. Tuberculosis. The data obtained are presented in table 1 and in the drawing.

Table 1 Comparative pharmacological efficacy of a prolonged-release drug based on rifampicin A drug Identification Dose Inoculation of M. tuberculosis in the lungs, the number of mycobacteria, CFU * / mouse 7th day 14th day 21st day Control (saline solution) TO 1.0 ml (3.7 ± 1.22) × 10 ⁵ (1.6 ± 0.06) × 10 ⁷ (2.6 ± 0.68) × 10 ⁹ Rifampicin substance R 20 mg / mouse (7,3 ± 0,06) × ¹⁰ March (1.7 ± 0.17) × 10 ⁵ (4.6 ± 0.56) × 10 ⁷ twenty Rifampicin Based Medication PH 20 mg / mouse (1.2 ± 0.2) × 10 ³ (7.9 ± 0.9) × 10 ³ (2.03 ± 0.08) × 10 ⁵ twenty Rifampicin substance R 10 mg / mouse (2.3 ± 0.08) × 10 ⁴ (1.3 ± 0.16) × 10 ⁶ (1.8 ± 0.65) × 10 ⁹ 10 Rifampicin Based Medication PH 10 mg / mouse (6,3 ± 0,07) × ¹⁰ March (5.3 ± 0.16) × 10 ⁴ (1.06 ± 0.06) × 10 ⁶ 10 Note. In the control, physiological saline was administered in a volume equal to the volume of the administered drug; * CFU - colony forming unit.

From the data shown in table 1 and in the drawing, it can be seen that the rifampicin-based preparation obtained in Example 1, having a prolonging effect, also significantly exceeds the initial substance rifampicin in activity. This is especially evident after the 14th and 21st day after the start of the experiment, as well as at a concentration of 10 mg / mouse, when rifampicin (substance) practically ceases to act on the 21st day. This can reduce the therapeutic dose of rifampicin and thereby reduce the level of toxic side effects. The treatment of acute experimental tuberculosis in mice with this drug according to the developed scheme allows to reduce the content of mycobacteria in lung tissue by more than 100 times, compared with free rifampicin, to reduce the frequency of administration to 3 injections due to the prolonged action of the developed drug.

Example 3. Toxicological characteristics of the original drug prolonged action based on rifampicin

A comparative study of the acute toxic effects of rifampicin and the developed polymer preparation based on it (composition according to example 1) was carried out on male Balb mice weighing 19 ÷ 21 g at the time of testing. The mice were kept in standard cages No. 4, in conditions of unlimited access to water and food, with a natural change in illumination. To test the toxic effects of drugs started after a two-week quarantine of animals. The preparations were administered orally into the stomach in saline for warm-blooded animals using an atraumatic probe. Observations of the effect of the drugs were made within 14 days from the time of administration. As a control, the corresponding mass of the polymer composition obtained without the active substance was introduced in an equivalent volume of physiological saline.

The determination of the average lethal dose of LD ₅₀ (at n = 7 for each of the tested doses, the calculations were carried out according to the method of Litchfield and Wilcoxon [11]) showed that the LD ₅₀ value for the developed drug based on rifampicin prolonged action in example 1 was 700 mg / kg, and for the substance rifampicin 500 mg / kg. Consequently, the mean toxic dose for the new dosage form of rifampicin was significantly higher (by 40%), and the toxicity was correspondingly lower than that of the initial rifampicin.

Thus, a nanoparticle-derived rifampicin-based agent with a prolonged action exhibits a high specific activity against Mycobacterium tuberculosis with reduced toxicity compared to the parent drug substance. This result allows us to conclude that the increase in therapeutic breadth and increase in the therapeutic index while reducing the frequency of administration of the drug due to the prolonged action.

The developed drug is offered for release in the form of enteric-soluble tablets or in capsules.

Information sources

1. Radar - Encyclopedia of drugs / Ch. ed. Yu.F. Krylov, 8th ed. M .: "RLS-2001", 2000. S.766-767.

2. Fang D., Singh M., O'Heygan D., Hora M. Compositions of microparticles and methods for their preparation // RF Patent No. 2257198 C2. - Publ. 07/27/2004

3 Pandey R., Khuller G.K. Oral nanoparticle-based antituberculosis drug delivery to the brain in an experimental model // Journal of Antimicrobial Chemotherapy 2006, V. 57 (6). P.1146-1152.

4. Pandey R., Sharma A., Zahoor A. et al. Poly (DL-lactide-co-glycolide) nanoparticle-based inhalable sustained drug delivery system for experimental tuberculosis // Journal of Antimicrobial Chemotherapy 2003. V.52 (6). P.981-986.

5. Esmarili F., Hosseini-Nasr M., Rad-Malekshahi M. et al. Preparation and antibacterial activity evaluation of rifampicin-loaded poly lactide-co-glycolide nanoparticles // Nanomedicine: Nanotechnology, Biology and Medicine 2007, V.3 (2). P. 161-167.

6. Ul-Ain Q., Sharma S., Khuller G.K. Chemotherapeutic Potential of Orally Administered Poly (Lactide-Co-Glycolide) Microparticles Containing Isoniazid, Rifampin and Pyrazinamide against Experimental Tuberculosis // Antimicrobial Agents and Chemotherapy 2003. V.47 (9). P.3005-3007.

7. Barrow E.L.W., Winchester G.A., Staas J.K. et al. Use of Microsphere Technology for Targeted Delivery of Rifampin to Mycobacterium tuberculosis-Infected Macrophages // Antimicrobial Agents and Chemotherapy 1998. V.42 (10). P.2682-2689.

8. Wranks A., Demoutier M., Deleer M. Pharmaceutical composition containing nanocapsules, and a method for its production // RF Patent No. 2145498 C1. - Publ. 02/20/2000

9. Pharmaceutical technology: Technology of dosage forms: Textbook for universities / Ed. I.I.Krasnyuk and G.V. Mikhaylova. Ed. 3rd reslave. and add. M .: Publishing house "Academy", 592 S.

10. Microencapsulation: Methods and Industrial Applications / Ed. S. Benita. 2nd ed. Taylor & Francis Group. 2006.747 p.

11. Belenky M.L. Elements of a quantitative assessment of the pharmacological effect / 2nd ed. reslave. and add. L .: Medgiz, 1963. P.81-106.

Claims (6)

1. Long-acting drug for the treatment of resistant forms of rifampicin-based tuberculosis, characterized in that it is stable nanoparticles and contains rifampicin, a biodegradable lactic acid polymer or a copolymer of lactic and glycolic acids, as well as a surfactant, cryoprotectant in the following ratio of components in wt.%:
rifampicin 1-10 biodegradable lactic acid polymer or a copolymer of lactic and glycolic acids 20-60 surfactant and cryoprotectant rest 2. The drug according to claim 1, characterized in that as a biodegradable polymer contains a polymer of lactic acid or a copolymer of lactic and glycolic acids or a copolymer of lactic and glycolic acids with a free carboxyl group. 3. The drug according to claim 1, characterized in that as a cryoprotectant contains D-mannitol or lactose, or glucose. 4. The drug according to claim 1, characterized in that as a surfactant contains polyvinyl alcohol or polysorbate 80, or poloxamer 188, or serum albumin. 5. The drug according to claim 1, characterized in that it is a nanoparticle in size from 250 to 500 nm. 6. The drug according to claim 1, characterized in that it is made in the form of a tablet or capsule, or granule, or powder, or other oral form.

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