RU2403041C2 - D-cycloserin-based medication of plolonged action with dosed release in target organs for treatment of resistant forms of tuberculosis - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of pharmacology and medicine, namely to new generation of antimicrobial D-cycloserin-based medications of regulated action. Described is medication of anti-microbial action with prolonged release into target organs, characterised in that it represents stable nanoparticles and includes D-cycloserin, PLGA 50/50, D-mannit and polyvinyl alcohol, with the following component ratio, wt %: D-cycloserin from 1 to 15; PLGA 50/50 from 20 to 40; D-mannit from 5 to 40; polyvinyl alcohol from 5 to 15. Described medication has considerably lower neurotoxicity and is able to produce bactericidal effect with respect to Mycobacterium tuberculosis, without producing toxic effect and possessing high therapeutic efficiency.

EFFECT: application of elaborated medication allows to achieve highly selective dosed release of active substance into target organs-cells (macrophages).

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Description

The invention relates to the field of pharmacology and medicine, specifically to a new generation of controlled antimicrobial agents based on D-cycloserine.

D-Cycloserine is an analogue and a competitive antagonist of β-D-alanine and, by its chemical structure, is D-4-amino-3-isoxazolidinone, has a wide spectrum of antibacterial activity, and is used to treat chronic forms of tuberculosis [1], including pulmonary tuberculosis ( active form), extrapulmonary tuberculosis (including kidney disease) as part of combination therapy, acute urinary tract infections. D-Cycloserine inhibits the activity of two enzymes involved in the synthesis of the bacterial cell wall in the early stages: Z-alanine racemase (converts Z-alanyl to -D-alanine) and D-alanyl-D-alanine synthetase (ensures the inclusion of D-alanine in the pentapeptide necessary for the formation of peptidoglycans). Effective against gram-positive, gram-negative bacteria, Mycobacterium tuberculosis, etc. It exhibits a bacteriostatic or bactericidal effect depending on the concentration in the focus of infection and the sensitivity of microorganisms. The resistance of M. tuberculosis to cycloserine develops slowly and rarely; after 6 months of therapy, up to 20-30% of resistant strains are released. Cross-resistance with other anti-TB drugs has not been identified. Effectiveness is shown in chronic forms of tuberculosis caused by mycobacteria resistant to other anti-tuberculosis drugs and atypical mycobacterioses caused by the complex M.avium-intracellulare, M. xenopi, etc.

The main difficulty in using existing dosage forms of cycloserine is the presence of a pronounced negative central effect on the state of the central nervous system (CNS). By the mechanism of action, cycloserine is classified as an agonist of glutamate NMDA receptors; in chemical structure, it resembles the psychoactive substance of amanita - muscimol [2]. High doses of cycloserine cause hallucinatory psychosis with disorientation, euphoria or aggressiveness, nightmares, memory impairment. In the treatment with cycloserine, especially its prolonged use, such undesirable effects from the nervous system and sensory organs as headache, tremor, dysarthria, dizziness, convulsions, drowsiness, semi-unconscious state, confusion of thoughts, disorientation, accompanied by memory loss, psychosis are recorded suicidal attempts, character change, increased irritability, aggressiveness, paresis, hyperreflexia, paresthesia, bouts of clinical seizures, coma. In order to reduce complications from the central nervous system, an original drug was created in gelatin capsules, containing simultaneously with cycloserine 2-5 wt.% Glutamic acid [3].

The essence of the present invention is to create a drug based on D-cycloserine, which allows to achieve a positive pharmacodynamic effect of cycloserine at lower doses, lower blood concentration and increased concentration of cycloserine directly in the target organ cells of the pathological effect (in the case of tuberculosis - macrophages), and thereby reducing the risk of toxic effects while simultaneously achieving the effect of prolonging the action, reducing the frequency of administration during treatment and maintaining stability during storage.

This essence of the invention is achieved by the inclusion in a new drug based on D-cycloserine, which has a metered and sustained release, biodegradable polymers approved for medical use {(lactic acid polymer (PLA), glycolic and lactic acid copolymer (PLGA) and glycolic and free terminal carboxylic acid lactic acid (PLGA-COOH)}, surfactants {polysorbate 80, polyvinyl alcohol (PVA), poloxamer 188} and cryoprotectant (D-mannitol) to obtain . Nia active nanoparticles for preparation of nanoparticles using polymers having a molecular weight of 10 to 300 kDa and a molar ratio of residues of lactic and glycolic acid of 25: 75 to 50%: 50%.

The invention is illustrated by the following examples.

Example 1. Obtaining polymer nanoparticles with D-cycloserine incorporated in them by double emulsification based on PLGA

A mixture consisting of a 5-20% polymer solution in an organic solvent (chloroform) and a 1-10% aqueous solution of D-cycloserine (volume ratio of organic to aqueous phases from 3: 1 to 3: 2) is homogenized at 24000 rpm on an Ultra-Turrax T-25 homogenizer 3 times for 1 min with 1 min intervals to obtain a primary emulsion. The primary emulsion is mixed with a 0.5-2% aqueous solution of a polymer emulsion stabilizer (PVA) in a volume ratio of 1: 5 and homogenized repeatedly under the same conditions. The double emulsion is stirred for 1 hour at 40 ° C. until the organic solvent is completely removed. The resulting suspension is filtered, 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 is from 250 to 500 nm, which contributes to efficient absorption in the gastrointestinal tract [4]. 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 incorporation of β-D-cycloserine into polymer particles is from 10 to 35%.

Example 2. Obtaining polymer nanoparticles with D-cycloserine incorporated in them by a double emulsification method based on PLGA-COOH

A mixture consisting of a 5-20% solution of PLGA-COOH in an organic solvent (chloroform) and a 1-10% aqueous solution of D-cycloserine (volume ratio of organic to aqueous phases from 3: 1 to 3: 2) are homogenized at 24,000 vol. / min on an Ultra-Turrax T-25 homogenizer 3 times for 1 minute with 1 minute intervals to obtain a primary emulsion. The primary emulsion is mixed with a 0.5-2% aqueous solution of a polymer emulsion stabilizer (PVA) in a volume ratio of 1: 5 and homogenized repeatedly under the same conditions. The double emulsion is stirred for 1 hour at 40 ° C. until the organic solvent is completely removed. The resulting suspension is filtered, 1-3% (vol.) Cryoprotectant (D-mannitol) is added to the filtrate, frozen and lyophilized. The average particle size is from 200 to 350 nm. The degree of incorporation of β-D-cycloserine into polymer particles is from 10 to 20%.

Example 3. Obtaining polymer nanoparticles with included D-cycloserine by the method of double emulsification based on PLA

A mixture consisting of a 5-20% solution of PLA in an organic solvent (chloroform) and a 1-10% aqueous solution of D-cycloserine (volume ratio of organic to aqueous phases from 3: 1 to 3: 2) is homogenized at 24000 rpm on an Ultra-Turrax T-25 homogenizer 3 times for 1 min with 1 min intervals to obtain a primary emulsion. The primary emulsion is mixed with a 0.5-2% aqueous solution of a polymer emulsion stabilizer (PVA) in a volume ratio of 1: 5 and homogenized repeatedly under the same conditions. The double emulsion is stirred for 1 hour at 40 ° C. until the organic solvent is completely removed. The resulting suspension is filtered, 1-3% (vol.) Cryoprotectant (D-mannitol) is added to the filtrate, frozen and lyophilized. The average particle size is from 350 to 600 nm. The degree of inclusion of D-cycloserine in polymer particles is from 5 to 15%.

The following are the optimal compositions of the proposed funds obtained according to example 1 in wt.%:

D-Cycloserine from 1 to 15% PLGA 50/50 from 20 to 40% D-mannitol from 5 to 40% PVA from 5 to 15%

With other compositions and ratios of ingredients, it was experimentally established that undesirable changes in the size of polymer particles occur, a decrease in the degree of incorporation of β-D-cycloserine, and a decrease in antimicrobial activity and an increase in toxicity associated with them. 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.

Example 4. Comparative pharmacological efficacy of an original sustained release drug with a metered release into target organs based on D-cycloserine having significantly lower neurotoxicity

The influence of the developed drug based on D-cycloserine on the survival and seeding 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 based on sustained release D-cycloserine with a metered release into the target cell organs (table 1) began one day after tuberculosis was reproduced simultaneously with the administration of a pure substance and at a dose almost 10 times lower than the dose of the administered pure substance - D-cycloserine. The drug was administered for 3 days.

Table 1 Comparative pharmacological efficacy of a sustained release drug with dosed release of D-cycloserine A drug Dose Average life in relation to control,% Inoculation of M. tuberculosis in the lungs,% (relative to the content in the control group) 7th day 14th day 21st day The control 1.0 ml one hundred one hundred one hundred one hundred D-Cycloserine substance 100 mg / kg 101.5 78 85 82 D-cycloserine substance 250 mg / kg one hundred 63 60 70 D-Cycloserine substance 25 mg / kg 99.9 99 101 95 D-cycloserine-based drug 5 mg / kg 107 60 55 49 -D-cycloserine-based drug 12.5 mg / kg 120 fifteen 17.5 21 D-cycloserine-based drug 25 mg / kg 115 fourteen 17.0 19 Note: in the control, physiological saline was administered in a volume equal to the volume of the administered drug.

Example 5. Pharmacokinetics of an original sustained release drug with a metered release into target organs based on D-cycloserine having significantly lower neurotoxicity.

It was shown that after oral administration of immobilized D-cycloserine in a dose of 250 mg, it is rapidly and almost completely (70-90%) absorbed from the gastrointestinal tract. The maximum concentration (C _max ) of cycloserine in the blood is reached after 3-8 hours. After taking a dose of 250 mg every 12 hours, C _max is 25-30 μg / ml. D-Cycloserine does not bind to blood proteins, is well distributed over tissues and body fluids, including cerebrospinal fluid, lymphoid tissue, lungs, pleural and ascitic fluid, sputum, bile. D-Cycloserine passes the BBB, the placental barrier, penetrates into breast milk (concentration in the cerebrospinal, pleural fluid, fetal blood and breast milk approaches the plasma level). -D-Cycloserine partially (35%) is biotransformed in the liver to unidentified metabolites. The half-life is 8-12 hours.

The use of the developed prolonged dosage form of D-cycloserine in target organs, in this case macrophages, with a content of D-cycloserine of 125 mg in one capsule (vial) allows to achieve a high concentration of D-cycloserine (capture of -D-cycloserine occurs mainly by macrophages) macrophages, while in the blood the concentration does not exceed 10 μg / ml. The maximum concentration in the blood of 10 μg / ml is achieved on the 7-8th day after taking the prolonged form and is maintained for 6 days. This allows you to significantly reduce the frequency of administration, the prolonged form of D-cycloserine with dosage release should be prescribed 1 tablet once every seven days, while the antituberculous activity of the drug is completely preserved.

It was shown that the developed form of D-cycloserine in the proposed therapeutic dose of 12.5 mg / kg of the animal’s mass does not cause the development of orientation disorders and sensory-motor reactions, which were manifested when the substance D-cycloserine was administered in the same dose of 12.5 mg / kg of the mass and became more pronounced with increasing doses to 25 and up to 100 mg / kg of animal weight.

A decrease in the concentration of D-cycloserine in the blood is the basis of a sharp decrease in the development of the neurotoxic effect of the drug.

Thus, it was possible to obtain the effect of directed dosed bactericidal action and prolonged maintenance of the level of anti-TB drug in macrophages with resistant forms of tuberculosis. In medical practice, such a drug has not been previously used for this purpose.

The technical result is achieved solely by the ability of nanocarriers based on biodegradable polymers in combination with plasticizers and cryoprotectants to carry out a highly selective and metered release of the active substance into target cell organs (macrophages) and then completely removed from the body.

The technical result is also achieved due to the fact that the developed tool has high pharmacological activity and low neurotoxicity.

Using the invention will allow to obtain the following technical result:

- increase the effectiveness of treatment of resistant forms of tuberculosis;

- develop optimal measures to prevent the development of resistant forms of tuberculosis;

- achieve rapid positive dynamics in the treatment of resistant forms of tuberculosis;

- develop a rational management strategy for patients with resistant forms of tuberculosis, which will significantly increase the effectiveness of treatment and the patient's life expectancy;

- reduce dosage, increase effectiveness and reduce the time and development of undesirable effects when using chemotherapy for tuberculosis.

The obtained therapeutic effect is persistent, since it is achieved by stable long-term maintenance of the therapeutic dose of the drug in the macrophages of the patient.

The developed product is offered for release in glass or plastic bottles or ampoules containing a clear liquid or white lyophilized powder, or in the form of enteric-soluble white tablets or in capsules.

A single dose of the drug contains the active principle of the anti-tuberculosis drug D-cycloserine, when released into the blood, the maintenance dose does not exceed 25 mg per day.

LIST OF USED SOURCES

1. Mashkovsky M.D. Medicines In two parts. Part 2/12th ed., Rev. and add. M .: Medicine, 1993. - S. 381-382.

2. Bespalov A.Yu., Zvartau E.E. Neuropsychopharmacology of NMDA receptor antagonists. St. Petersburg: Nevsky Dialect, 2000 .-- 297 p.

3. Gaie E.F., Cundliffe E., Reynolds P.E. et al. The Molecular Basis of Antibiotic Action. / Wiley. - New York. - 1972. - P.61-71.

4. Wranks A., Demustier M., Deleer M. Pharmaceutical composition containing nanocapsules, and a method for its preparation. / RF patent No. 2145498 C1. - 1994

Claims (1)

Sustained-release antimicrobial drug in the target organs, characterized in that it is stable nanoparticles and includes D-cycloserine, PLGA 50/50, D-Mannitol and polyvinyl alcohol, in the following ratio, wt.%:
D-cycloserine 1 ÷ 15 PLGA 50/50 20 ÷ 40 D-mannitol 5 ÷ 40 Polyvinyl alcohol 5 ÷ 15