RU2318513C1 - D-cycloserine-base drug, nanoparticles-containing preparation of prolonged effect, method for its preparing - Google Patents

Abstract

FIELD: medicine, pharmacology, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a new generation of antibacterial preparations based on D-cycloserine that comprises nanoparticles and possessing the regulated effect. Method for preparing this preparation involves heating an agent containing a mixture consisting of D-cycloserine, PLGA 50/50, D-mannitol, polysorbate-80 and dimethylsulfoxide taken in the definite ratio at temperature 50-60^oC to obtain uniform clear liquid followed by its cooling to room temperature, dilution with water, and formed suspension containing nanoparticles of size 3.0 ± 0.5 nM (~20% of the total amount of particles) and ~200 nM (~80% of the total amount of particles) are used by designation. Invention provides preparing the preparation possessing high antibacterial activity, prolonged effect, relative harmless and low toxicity.

EFFECT: improved preparing method, improved and valuable medicinal properties of agent.

3 cl, 4 tbl, 5 ex

Description

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

Cycloserine - D-4-amino-3-isoxazolidinone - has a wide spectrum of antibacterial activity and is prescribed for patients with chronic forms of tuberculosis, in which the previously used main drugs ceased to have an effect [1]. In this regard, cycloserine is used both for the treatment of active pulmonary tuberculosis and extrapulmonary tuberculosis (including kidney tuberculosis), and for the treatment of acute urinary tract infections caused by sensitive strains of gram-positive and gram-negative bacteria, in particular Klebsiella / Enterobacter and Escherichia coli.

However, in the treatment of cycloserine, there are side effects associated with the toxic effect of the drug on the central nervous system. So, when taking cycloserine more than 500 mg per day in patients, convulsions, drowsiness, headache, tremor, dizziness, memory loss, psychosis were recorded, possibly with suicide attempts, character changes and increased irritability.

The reduction of the toxic effect of cycloserine is achieved by the appointment of either glutamic acid (0.5 g 3-4 times a day), or by intramuscular injection of the sodium salt of adenosine triphosphoric acid (1 ml of a 1% solution daily), or by intramuscular injection of pyridoxine (1- 2 ml of 5% solution per day). An original method has been patented for overcoming the toxicity of cycloserine by its use in gelatin capsules containing 2-5 wt.% Glutamic acid [2]. This is the closest analogue, which does not make it possible to obtain a tool in the form of nanoparticles, which give a prolonged effect.

Cycloserine is rapidly absorbed from the gastrointestinal tract after ingestion, creating detectable plasma levels within one hour. It is freely distributed in body fluids and tissues. About 66% of the dose is found unchanged in the urine within 24 hours, 10% is excreted within the next 48 hours. About 35% is metabolized, the half-life of cycloserine ranges from 8-12 hours.

In its molecular structure, cycloserine resembles D-alanine and is a potent inhibitor of alanine racemase. It also inhibits ATP-dependent D-alanyl-D-alanine synthetase, which is necessary for the biosynthesis of peptidoglucan of bacterial cell walls, in particular Micobacterium tuberculosis [3-4].

It should be noted that in solutions, cycloserine exists in neutral and dipolar forms, has amphoteric properties, pKa 4.4 and 7.3 [5], and is unstable in neutral and acidic aqueous media [6].

From the analysis of the above information materials, the purpose of the present invention is formulated naturally, namely: the creation of a medicinal product and a preparation based on cycloserine that reduces the toxicity of an active substance with a prolonged action while maintaining antimicrobial activity stable during storage and simplifying the preparation.

The goal is achieved by using cycloserine in a preparation containing nanoparticles, and is based on modern advances in pharmacology and medicine. So, after the penetration of M. tuberculosis into the human body, phagocytosis develops as the first protective reaction. The process of interaction of pulmonary macrophages with these bacteria is very complicated [7]. At the same time, the bio-distribution of an antimicrobial drug can be optimized only by reducing its accumulation in the liver. A decrease in the effectiveness of phagocytosis in the liver is achieved through the use of nanoparticles of a drug with nonionic surface-active substances (surfactants), for example, polysorbate 80, which allows to reduce the accumulation of particles in the liver and increase the time of their circulation in the body ("stealth effect") [ 8]. In this regard, significant results were achieved in the development of the nanosomal dosage form of rifampicin on polybutyl cyanoacrylate [9]. There are other data on the high effectiveness of nanosomal drugs in the treatment of, in particular tuberculosis [10, 11]. Abroad, anti-oncological preparations based on nanoparticles of lactic and glycolic acid copolymers (PLGA) have been developed [12], which under the names decapeptyl, zoladex, sandostatin and somatulin are approved for use in medical practice of the Russian Federation. The PLGA copolymer [50/50 Poly (DL-lactide-co-glycolide) (nominal)] is non-toxic and undergoes biodegradation in the human body with the formation of lactic and glycolic acids, the catabolism of which ends with the formation of carbon dioxide and water [13].

Another aspect of this problem is associated with contraindications for the use of cycloserine in patients with severe renal failure (creatinine clearance less than 250 ml / min) [1], which is overcome with the help of osmotic diuretics, for example, D-mannitol [14].

Thus, from the above data it follows that the new drug and the drug based on cycloserine should potentially contain a biodegradable polymer (PLGA), surfactant (Polysorbate 80) and D-mannitol. The patent literature describes many methods for producing nanoparticles containing drugs or diagnostics on biodegradable polymers. To date, these methods are summarized in two fundamental patents: emulsification followed by freeze drying [15] and mechanical crushing in the presence of a grinding medium [16]. In this case, special attention is drawn to the fact that in order to achieve effective absorption of drugs in the gastrointestinal tract, it is necessary that the particle diameter be less than 500 nm [17].

Testing the known methods of nanotechnology in relation to cycloserine has not led to success due to its instability in neutral and acidic aqueous media [6]. In this regard, we have developed a new method for producing a controlled-action drug based on cycloserine containing nanoparticles. The proposed method is based on the ordered structure of dimethyl sulfoxide (DMSO), which is destroyed in the temperature range 40-60 ° C and resumed at 20-25 ° C [18]. Moreover, DMSO is capable of coordination solvation with a number of organic and inorganic compounds, stabilizing labile substances [19]. This fully applies to D-cycloserine, which exists in a dipolar form [5], and to PLGA, and to D-mannitol. The deficit of electron density on the carbon atom and the nucleophilicity of the oxygen atom of the carbonyl groups of PLGA contribute to the formation of chain structures in liquid DMSO. Obtaining an ordered matrix becomes a determining event with the integrated use of DMSO, cycloserine, PLGA and D-mannitol due to the occurrence of hydrogen bonds due to the last ingredient. The system is stable and can be used for its intended purpose. The need for the use of nonionic surfactants (Polysorbate 80) was previously considered. Upon dilution of the obtained product with water, a stable, uplifting suspension is formed containing nanoparticles of sizes 3.0 ± 0.5 nm (~ 20% of the total number of particles) and ~ 200 nm (~ 80% of the total number of particles), which can be effectively absorbed in the gastrointestinal tract according to the above data regarding particle sizes [17].

It should be emphasized that such ratios of cycloserine, PLGA, D-mannitol, polysorbate 80 and DMSO were found experimentally that produce a liquid that is uniform, uniform, transparent and stable for over a year under room conditions. In assessing the stability of the quality of the samples of the preparation obtained then, classical methods of visual control and refractometric analysis were used. Refractometry is an integral method for assessing the authenticity and purity of substances, allows you to determine the concentration of substances in solutions and has been used in the Pharmacopoeia for more than a decade [20].

When discussing the composition of the agent and drug under consideration, one cannot ignore the question of the prospects for the use of DMSO in medicine. So, intramuscular administration of disulfiram to alcohol-dependent patients with DMSO leads to a long-term sensitizing effect [21]; for external use, DMSO is used in the complex treatment of rheumatoid arthritis, ankylosing spondylitis, discoid lupus erythematosus, thrombophlebitis, furunculosis and eczema [22]; for the treatment of amyloidosis, DMSO is administered orally to 100-200 ml of a 3-5% solution in water per day [23].

In general, summarizing the foregoing, for the first time a drug of controlled action based on cycloserine containing nanoparticles has been proposed. First, a solution of cycloserine, PLGA, D-mannitol and polysorbate 80 in DMSO is obtained. Then an aliquot of this mixture is diluted with water and get a stable colloidal system, consisting of nanoparticles, and ready for use.

The optimal composition of the new tool is as follows, wt.%:

D-cycloserine 0.75-2.50 PLGA 50/50 1.25-7.50 D-mannitol 1.25-7.50 Polysorbate 80 0.5-1.0 DMSO rest

With other ratios of ingredients, it was experimentally established that there is either a loss of antimicrobial activity, or an increase in toxicity, or undesirable changes in the size of polymer particles. 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 invention is illustrated by the following examples.

Example 1. A method of obtaining funds based on D-cycloserine

In a three-necked flask equipped with a stirrer, reflux condenser and thermometer, at a temperature of 20-25 ° C, D-cycloserine, PLGA 50/50, D-mannitol, Polysorbate 80 and DMSO are successively charged. The mixture is stirred and heated to 50-60 ° C for 15-20 minutes until the solid phase is completely dissolved, and then cooled to room temperature in 20-30 minutes.

The composition of the boundary preparations is presented in tables 1 and 2.

Table 1 The composition of drugs No. 1 No pp Component Name wt.% one. D-Cycloserine 0.75 ± 0.05 2. PLGA 50/50 7.50 ± 0.10 3. D-mannitol 7.50 ± 0.05 four. Polysorbate 80 0.75 ± 0.05 5. Dimethyl sulfoxide 83.50 ± 0.20

table 2 The composition of drugs No. 2 No pp Component Name wt.% one. D-Cycloserine 2.50 ± 0.10 2. PLGA 50/50 1.25 ± 0.10 3. D-mannitol 1.25 ± 0.10 four. Polysorbate 80 0.80 ± 0.05 5. Dimethyl sulfoxide 94.20 ± 0.20

The above examples of the product are transparent homogeneous liquids of light yellow color, stable when stored for more than a year. When water is added to them in a ratio of 1: 20-1: 10, stable opalescent suspensions are formed, the determination of particle sizes of which are given in Example 2.

Example 2. Analysis of particle sizes of drugs.

Particle size and particle size distribution were determined by autocorrelation spectroscopy on a Coulter N4MD submicron laser spectrometer from Coulter Electronics (France-USA). The measurement algorithm is based on the CONTIN program.

Samples of the preparations, when diluted with water No. 1 and No. 2 cycloserine in amounts of 3.5 mg, were added to a cuvette with distilled water (3 ml) and subjected to ultrasonic treatment for 1 min using an ultrasonic bath (Westwood Ultrasonics Ltd, Model 90S). Then made measurements. The results are presented in table 3.

Table 3 The results of the analysis of particle sizes of samples of drugs No. 1 and No. 2 No. of composition Unimodal Analysis Particle distribution Particle Size Distribution Nm size value Nm standard deviation The average value of nm Nm standard deviation The coefficient of variation % Nm size Nm standard deviation Quantity% one 145 47 126 81 64 3.0 0.5 21 158 33 79 2 148 31 133 94 71 3.0 0.5 23 172 95 77

Example 3. Elimination of ingredients from drugs.

A comparative study of the elimination of ingredients from preparations was carried out under interchangeable dialysis in phosphate-buffered saline (pH 7.5) at room temperature.

PBS Tablets phosphate buffered saline (AMRESCO) tablets were used to prepare the phosphate buffered saline (PBS) (pH 7.5). One tablet per 100 ml of distilled water.

Dialysis tubing, high retention tape was used as dialysis bags. Size: 23 mm × 15 mm (Sigma-Aldrich). The exclusion limit is 12 kDa. Preliminarily, sections of the dialysis tape (10 cm) were boiled in a ~ 0.001 M solution of ethylenediaminetetraacetic acid for 1 h.

Dialysis vessels were 250 ml measuring cylinders with caps. Mixing of the dialysis fluid in laminar mode was carried out using magnetic stirrers in a Teflon shell and an RH-KT / S magnetic stirrer (IKA ^® , Germany).

The optical densities of the solutions were measured on a Hitachi 557 spectrophotometer (Japan).

For weighing, a VIBRA analytical balance (max / d 220 / 0.0001 g) (Japan) was used.

A pipette (1-5 ml) and plastic tips (BIOHIT) (Finland) were used for sampling.

The following drugs were dialyzed:

(1) DMSO (1.33 g).

(2) A solution of cycloserine in water (2.5 mg cycloserine +0.34 ml of water).

(3) Composition No. 1 (0.3361 g).

The prepared dialysis bags (capacity of about 5-6 ml) were pipetted into the liquid samples listed. The bags sealed with clamps were placed in dialysis vessels with 250 ml of buffer solution, fixed with a thread, closed with a lid and stirring was turned on.

Dialysis samples (3 ml each) were periodically taken into 10 ml glass tubes. The samples were measured optical density at λ 207 and 226 nm against a pure solution of PBS. Then the samples were returned back to the dialysis vessels. 2 dialysate shifts were performed. The duration of the experiments was 1-2 days.

Installed:

1. The elimination of DMSO from the drug (1) was completed after 3 hours, and the dialysis of cycloserine from the drug (2) stopped after 2 hours.

2. Elimination of cycloserine and DMSO from drug No. 1 did not end even after a day.

Thus, it is legitimate to believe that the prolongation of the action of cycloserine will increase up to 10 times in the case of the use of drug No. 1 in comparison with individual cycloserine.

The same data were obtained for the preparation No. 2.

Example 4. Evaluation of the antimicrobial activity of drugs

Determination of the antimicrobial activity of drug samples in relation to test cultures of gram-positive, gram-negative and atypical bacteria was carried out by the method of serial micro-dilutions in a liquid medium in accordance with the recommendations of NCCLS / 24 / for visual registration of visible growth. 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. The results obtained are summarized in table 4.

Table 4 Antibacterial activity of drug samples A drug The concentration of the drug,% Zones of growth inhibition of microorganisms, mm Staphylococus aureus ATCC 906 Escherichia coli ATCC 25022 Salmonella spp. (atypical form) DMSO one hundred 6.5 8 8 10 - 7 7 5 - 7 - Cycloserine 2.5% solution in DMSO one hundred 19 twenty twenty 10 - 7 eleven 5 - 7 - Drug No. 1 one hundred - - - 10 - - - 5 - - - Drug No. 2 one hundred 24 eighteen 19 10 - eleven eleven 5 - 10 7

Example 5. Assessment of toxicity of drugs.

Determination of the toxicity of drug samples was carried out according to generally accepted methods [25].

In studies on 2 species of animals of both sexes - Balb-C mice and Wistar rats, it was found that a ready-to-use preparation (10% solution of means No. 1 and No. 2) with a single oral administration at a dose equivalent to an effective single therapeutic dose of cycloserine for human (250 mg), as well as in doses of 5 and 10 times its superior, did not produce toxic effects.

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Claims (3)

1. The drug has an antimicrobial effect, characterized in that it contains D-cycloserine, a copolymer of lactic and glycolic acids (PLGA 50/50), D-mannitol, polysorbate 80, dimethyl sulfoxide (DMSO) in the following ratio, wt.% : D-Cycloserine  0.75-2.50 PLGA 50/50  1.25-7.50 D-mannitol  1.25-7.50 Polysorbate 80  0.5-1.0 Dimethyl sulfoxide  Rest 2. A drug of prolonged action, characterized in that it contains the tool according to claim 1, which when diluted with water in a ratio of 1:20 to 1:10 is a suspension of nanoparticles with a particle size of from 3 to 200 nm, in an effective dose. 3. The method for producing the preparation according to claim 2, characterized in that D-cycloserine, PLGA 50/50, D-mannitol, polysorbate 80 and DMSO are sequentially loaded into the container, the mixture is stirred and heated to 50-60 ° C for 15- 20 minutes until the solid phase is completely dissolved, then cooled to room temperature in 20-30 minutes, when water is added to the mixture in a ratio of 1:20 to 1:10, a suspension of nanoparticles with a particle size of from 3 to 200 nm is obtained.