RU2327459C1 - Antimicrobial medicinal agent, method of production of directional medicinal agent containig nanoparticles - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: to produce composition of common agent, substance or combination of substances, copolymer of lactic and glycolic acid, polysorbate 80 and dimethyl sulfoxide taken in specified ratio are heated at 50÷60°C and mixed to produce homogeneous transparent liquid, which is cooled to room temperature and watered. Produced suspension containing nanoparticles sized 200-400 nm is used according to its intended purpose.

EFFECT: provided technologically available production method of medicinal agents of new generation based on common high-performance and almost non-toxic substances.

4 cl, 4 ex, 5 tbl, 1 dwg

Description

The invention relates to the field of pharmacology and medicine, specifically to a method for producing a new generation of directed drugs based on substances permitted for use in medical practice of the Russian Federation (substances with pharmacological activity and intended for the manufacture and manufacture of drugs [1]).

The aim of the present invention is to develop a technologically affordable method for producing new generation drugs based on known substances, highly effective, practically non-toxic drugs containing nanoparticles.

This goal was achieved using modern advances in pharmacology and medicine, taking into account the dominance in the nanoworld of the phenomena of self-ordering and self-organization, reflecting the manifestation of the effects of matrix copying and synergistic processes [2].

Among the medicinal substances used are water-soluble and water-insoluble substances that have simple and chemically complex molecular structures. To illustrate the invention in theoretical and experimental terms, the most socially significant pharmaceutical group of drugs for the treatment of infectious diseases was selected.

It is known that along with viruses, the causative agents of infections are microorganisms that were discovered in 1676 by Anthony van Levenguk, and only two centuries after the work of Robert Koch on the study of anthrax, their determining role in the occurrence of epidemics became clear. Success in treating infectious diseases has been achieved through the use of antibiotics (non-protein substances of originally natural origin), sulfamides, diamino-pyrimidines, quinolones and other groups of antibacterial chemotherapy drugs. Unfortunately, an epidemiological shift has now taken place, when many infectious diseases are returning again in new quality, as a result of resistance acquired by microorganisms to known drugs [3].

Today, tuberculosis has become one of the most common infectious diseases. According to the World Health Organization (WHO), approximately 3.5 million people fall ill with tuberculosis each year, and by 2010 more than 70 million people on the planet will die from this disease if new, more effective treatments are not found [4]. However, over the past 10 years, the arsenal of antimicrobials for the treatment of tuberculosis has not changed. Isoniazid, rifampicin, ethambutol and a number of other known substances or their compositions are still used. As a rule, treatment of patients with tuberculosis is not brought to a complete recovery, which causes the return of the disease, the emergence of secondary resistance to drugs. At the same time, the frequency of administration of drugs during the day affects the effectiveness of treatment. Data has been published that with 3-fold administration of drugs, only 37.7% of patients adhere to the dosing regimen, with 68% of patients with 2-fold administration, and 79.6% with a single administration [5]. Obviously, the best results can be achieved when taking drugs once a day. To avoid violations of the dosage regimen and the duration of the selection of effective drugs, WHO recommends abandoning mono- and combination therapy and indicates the need to create new combined anti-TB drugs containing a specially selected set of drug substances with fixed doses in one tablet (capsule, ampoule or vial), which provide optimal therapeutic effect when taking the drug once a day. Particular attention is paid to ensuring adequate bioavailability of substances and, in particular, rifampicin [6].

In this regard, the corresponding combined antimicrobial preparations containing a mixture of rifampicin, ethambutol and isoniazid as an active principle have been developed [7]; rifampicin, ethambutol, isoniazid, pyrazinamide and pyridoxine [8]; isoniazid and pyrazinamide or ethambutol hydrochloride [9]; isoniazid, rifampicin, pyrazinamide, ethambutol hydrochloride and pyridoxine hydrochloride [10]; lomefloxacin, protionamide, pyrazinamide, ethambutol hydrochloride and pyridoxine hydrochloride [11]; lomefloxacin, isoniazid, pyrazinamide, ethambutol hydrochloride and pyridoxine hydrochloride [12]; pyrazinamide, kanamycin and (or) ethambutol, maxavin and protionamide [13]; rifampicin, ethambutol and cycloserine [14]; isoniazid and rifampicin [15], isoniazid, rifampicin, pyrazinamide and ethambutol in Mayrin-P preparation [16]; cycloserine, rifabutin, and cycloserine, protionamide in combination with glutoxim [17]. Apparently, the search for new combinations continues on a larger scale.

At the same time, even when using the above-mentioned modern combined antimicrobial agents, only a small fraction of the drugs enter the target organ / cell, and most of them undergo biotransformation without any antibacterial effect. In this regard, serious side effects occur in the form of impaired hematopoiesis, liver and kidney function. One of the ways to overcome these negative consequences is the development of targeted drugs based on immobilized polymers. For example, antifungal agents are known for rifampicin containing cellulose as a polymer base [18], for isoniazid starch [19] and dextran [20, 21], for ampicillin, gentamicin, rifampicin and their mixtures polyalkyl cyanoacrylates [22]. Polymer-containing systems provide intracellular localization of drugs in alveolar macrophages in amounts effective for therapy and have a prolonged effect by adjusting the rate of release of the substance from the polymer matrix. There is a decrease in toxic effects. Moreover, delayed-release dosage forms of active substances allow a patient to take a single daily dose of the drug. Data were obtained on the high clinical efficacy and safety of fluoroquinolones in dosage forms with sustained (prolonged) release of ciprofloxacin and ofloxacin [23], which are two-layer tablets coated with a polymer coating.

There are various ready-made dosage forms of prolonged antibacterial drugs with the gradual release of the active ingredient, namely: in the form of tablets [24, 25], in the form of gels [26, 27], in the form of solutions for inhalation [28] and in the form of solutions for injection [ 29]. Liposome forms of drugs are used [30], the disadvantage of which is low stability due to the easy oxidation of the phospholipids that make up the liposomes.

By analyzing the above data, the desire to create new drugs suitable for single use and providing a delayed release of antibacterial drugs becomes justified and justified. At the same time, an increase in the effectiveness of treatment can be achieved using biodegradable polymers and a combination of active ingredients. Since the main object of the therapeutic effect is cells, and often macromolecules (receptors and enzymes), the particle size of the new generation of drugs must be of the same order as the size of the biological targets, that is, in the nanometer range. Nanopreparations entering the body should act only on the target, not linger and not degrade along the way. It was found that for effective absorption of drugs in the gastrointestinal tract (GIT), it is necessary that the particle diameter be less than 500 nm [31]. The nanoobject must pass through the pores of the capillaries and have dimensions less than 700 nm [32].

There is no doubt that in order to achieve significant results when creating new-generation drugs, an adequate choice of biodegradable polymers is important. In medicine, a significant number of polymer materials are used [33]. So, as a surgical adhesive, polyalkyl cyanoacrylates are used; polycaprolactones are used for suture material; for coating tablets - polymethacrylates (Eudragits); as food additives - chitosans (aminoglucans or polysaccharides), nutrients - solid lipids; based on polyvinylpyrrolidones produce plasma-exchange and detoxification solutions; homopolymers of lactic acid (PLA) and glycolic acid (PGA), as well as copolymers of lactic acid and glycolic acid (PLGA) are medical plastics. Recent nanomedical studies are mainly based on biodegradable polyalkyl cyanoacrylates, PLA, PGA homopolymers and PLGA type copolymers.

As the closest analogue of the claimed invention according to claim 1, the composition described in [22] is proposed. It should be noted that the composition described in this patent contains polyalkyl cyanoacrylates, which are characterized by very high decomposition rates in a living organism, which significantly reduces the duration of the release of drugs from the polymer matrix. Moreover, in the Russian Federation there are no examples of permits for their use as components of medicines for internal use. At the same time, preparations of decapeptil, zolatex, sandostatin and somatulin, which contain, in addition to cytostatics, nanoparticles of lactic and glycolic acid copolymers, are approved for use in Russian medical practice [34]. The PLGA copolymer [50/50 Poly (DL-lactide-co-glycolide)] 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 [33].

To optimize the biodistribution of the antimicrobial drug, as well as the substances of other pharmaceutical groups, is possible only by reducing its accumulation in the liver. A decrease in the activity 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") [ 35]. Degradation of a substance on its way to a biological target is sharply reduced.

Among the water-soluble antimicrobials of simple structural formulas, cycloserine, isoniazid and pyrazinamide are the most well-known, and rifabutin is a water-insoluble substance of complex molecular structure. When treating with the use of these substances, there are impaired liver and kidney function, allergic reactions and dyspeptic phenomena, and when taking cycloserine more than 500 mg per day, convulsions, psychoses, increased irritability and other disorders of the central nervous system are additionally observed [36, 34 - P.766 ].

Above were considered options for overcoming the toxicity of drug substances. The occurrence of renal failure can additionally be overcome by incorporating osmotic diuretics, in particular, D-mannitol, into the drugs being created [37]. However, in all cases, to avoid side effects, new-generation drugs must have the geometric parameters of the nanopreparation.

The patent literature describes many methods for producing nanoparticles containing drug substances, biodegradable polymers and surfactants. To date, they are summarized in two fundamental patents - these are various emulsification options followed by freeze drying [38] and mechanical crushing in the presence of a grinding medium [39], which is proposed as a prototype according to claim 3. Despite the obvious successes in the field of nanomedicine, the introduction of the achievements of nanotechnology in the industry is associated with significant financial costs for ordering new generation apparatus and equipment. The technical result of the claimed invention is the development of an affordable and easy for industrial implementation of a method for producing medicines based on known substances, highly effective preparations 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 resumes at 20 ÷ 25 ° C [40]. Moreover, DMSO stabilizes unstable (labile) substances due to its ability to coordinate solvation with organic and inorganic compounds [41]. Moreover, in medical practice, DMSO has found application in the complex treatment of ankylosing spondylitis, rheumatoid arthritis, discoid lupus erythematosus, thrombophlebitis, eczema, furunculosis, amyloidosis, etc. [42, 43]. It is advisable to note that in the treatment of amyloidosis, 100–200 ml of a 3–5% aqueous DMSO solution per day is taken orally for many months. Together with the drug disulfiram, DMSO leads to a long-term sensitizing effect in patients with alcohol dependence [44]. The radioprotective and antitumor activity of polyene antibiotics in combination with DMSO increases many times, contributing to prolongation of life [45].

The DMSO molecule is highly polar. According to X-ray spectral studies and quantum-chemical calculations, the positive charge on the sulfur atom is in the range from +0.5 to +0.7, and the negative pole of the dipole is localized on the oxygen atom [40]. Various methods show that in liquid DMSO there are aggregates of a chain structure due to oxygen bonds. Associations of native DMSO are easily destroyed by the addition of substances that are proton donors, with the formation of structures including hydrogen and oxygen bonds. Medicinal substances containing hydroxy and amine groups are capable of such transformations. The deficit of electron density on the carbon atom and the nucleophilicity of the oxygen atom of the carbonyl groups PLGA (or PLA, or PGA) promotes the formation of complex structures in liquid DMSO. The preparation of a self-organized and self-ordered matrix, characteristic of the nanoworld [2], becomes a decisive event with the combined use of DMSO, PLGA, surfactant, D-mannitol (osmotic diuretic) and other drug substances that are proton donors. Such systems are stable during storage and can be used for their intended purpose.

Based on changes in the ordering of the structure of DMSO in the temperature range from 20 to 60 ° C and its high activity in coordination solvation, experimentally we found such ratios of the known drug substance (or combination of substances), PLGA (50/50), polysorbate 80 and DMSO, in which liquid, homogeneous, transparent and stable under room conditions products are formed (example 1). These concentrates with a volume of up to 5 ml contain a therapeutic dose (or doses) of the substance (or combination of substances) necessary for taking once a day [51]. When diluting the obtained products with water in ratios from 1:20 to 1:10 (or 5 ml of a concentrate of 50 ÷ 100 ml of water), stable opalescent suspensions are formed containing nanoparticles above the effective sizes indicated above [31, 32] (Example 2). The ability to dilute funds with water can be used to adjust the dosage of drugs depending on the individual characteristics of the patient.

In experiments on laboratory animals (mice and rats of both sexes, oral and intramuscular administration), it was shown that the preparations obtained are practically non-toxic (example 3). If the survival of animals according to the prototype [22] was evaluated within 7 days, then in this case - 14 days and even at doses 5 and 10 times higher than the therapeutic values of side effects or deaths were not recorded. Studies of drugs for antimicrobial activity against gram-positive, gram-negative and atypical bacteria (Example 4) confirm the fact that the superiority of new nanopreparations is significant in comparison with the prototype [22] and with the control, which is a standard (usual) form of a known substance (D-cycloserine). In this case, the standard dosage form of the antibiotic at a concentration of 5% affects only one of the seven test cultures of microorganisms, and the prototype drug [22] is introduced into the bacterial environment three times to obtain an antimicrobial effect. New nanopreparations affect all used test cultures with a single introduction into the biological medium.

The experimental data of these two examples convincingly indicate a slow release of active substances in the body, a decrease in their degradation along the path to the biological target, and a sufficiently high speed with respect to microorganisms of various types.

The following examples illustrate various aspects of the invention. However, these examples in no way limit the invention or enumeration of specific medicinal substances, dosage values, test procedures or other parameters that should be used solely for carrying out the invention.

Example 1

A method of obtaining new funds based on known substances

In a three-necked glass flask equipped with a stirrer, driven by an electric motor, a thermometer and an air (return) refrigerator, at 20 ÷ 25 ° С the ingredients shown in Tables 1, 2 and 3 are successively loaded in the indicated percentages. The mixture is stirred and heated on a mantle at 50 ÷ 60 ° C until the solid phase is completely dissolved, and then cooled to room temperature for 20-30 minutes. Finished products are stored in a sealed container of orange glass.

Table 1
The composition of the funds №1 No. p / p Component Name wt.% one Rifabutin 2.95 ÷ 3.05 2 PLGA 50/50 2.95 ÷ 3.05 3 D-mannitol 2.95 ÷ 3.05 four Polysorbate 80 0.95 ÷ 1.00 5 Dimethyl sulfoxide rest table 2
The composition of the funds №2 No. p / p Component Name wt.% one D-Cycloserine 4.95 ÷ 5.05 2 PLGA 50/50 2.95 ÷ 3.05 3 D-mannitol 2.95 ÷ 3.05 four Polysorbate 80 0.95 ÷ 1.00 5 Dimethyl sulfoxide rest Table 3
The composition of the funds №3 No. p / p Component Name wt.% one Rifabutin 2.63 ÷ 2.70 2 D-Cycloserine 4.40 ÷ 4.50 3 Isoniazid 2.63 ÷ 2.70 four Pyrazinamide 4.40 ÷ 4.50 5 PLGA 50/50 2.63 ÷ 2.70 6 D-mannitol 2.63 ÷ 2.70 7 Polysorbate 80 0.95 ÷ 1.00 8 Dimethyl sulfoxide rest

Note. The ratio of components in funds No. 1, No. 2, No. 3 was selected taking into account

- therapeutic doses of drugs;

- homogeneity of funds in the temperature range of 20 ÷ 25 ° C;

- stability of the main indicators of the quality of funds during storage.

The above funds No. 1, No. 2, No. 3 are transparent homogeneous liquids that are stable when stored for more than a year. When water is added to them in a ratio of 1:20 to 1:10, stable opalescent suspensions are formed (preparations No. 1, No. 2, No. 3), the determination of particle sizes of which is given in Example 2.

Thus, the claimed method of obtaining the drug in comparison with the prototype is simpler by reducing the number of stages and the absence of a stage of mechanical crushing.

Example 2

Determination of particle sizes of new drugs in the aquatic environment

Particle sizes and particle size distribution by fractions 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.

Pre-mixed with water means No. 1, No. 2 and No. 3 in a ratio of 1:20 were introduced into a cell with distilled water (3 ml) in an amount of 10 ÷ 20 μl and subjected to ultrasonic treatment for 1 min using an ultrasonic bath (Weswood Ultrasonics Ltd , Model 90S). Then made measurements. The results are presented in table 4.

Table 4
The results of the analysis of the sizes of polymer-containing particles of drugs No. 1, No. 2, No. 3 A DRUG Unimodal Analysis Particle size distribution Particle Size Distribution Size value, nm Standard deviation, nm The average value, nm Standard deviation, nm The coefficient of variation, % Size, nm Standard deviation, nm Amount, % No. 1 348 nar 288 73 25 288 73 one hundred Number 2 238 broad 137 170 130 3.0 0.5 52 281 73 48 Number 3 191 broad 149 170 120 3.9 one 47 275 80 53 Notes, broad - in a wide range; nar - within narrow limits.

From table 4 it is seen that the particles of the preparations have a base size of 200-400 nm (for drug No. 1 - 100%), while fractions with a particle size of 2.5-5 nm are fixed in preparations 2 and 3.

Example 3

Toxicity assessment of new drugs

Assessment of the toxicity of the obtained preparations indicated in Example 2 was carried out in accordance with the rules and methods of experiments on laboratory animals adopted in the Russian Federation [46, 47].

Acute toxicity was determined in Balb-C mice and Wistar rats of both sexes. Studies of the toxic effects of each dose were performed on 10 animals (5 males and 5 females). To study acute toxicity, the 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. The effect of doses equivalent to effective single therapeutic doses of known drug substances (rifabutin, isoniazid, pyrazinamide, D-cycloserine, D-mannitol was evaluated [34, 36, 37, 51]), as well as 5 and 10 times their superior.

It was found that for the preparations obtained (example 2), the LD ₅₀ for mice and rats with intramuscular administration is 2–3 g / kg, and with oral application more than 12–15 g / kg. In other words, the new drugs are practically non-toxic. At autopsy of experimental and control animals, pathological changes in organs and tissues were not recorded.

Example 4

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 2) was carried out in accordance with the recommendations of the National Committee for Clinical and Laboratory Standards of the USA (NCCLS) [48] and guidelines in force in the Russian Federation [49].

The minimum inhibitory concentrations of the preparations with respect to the test cultures were determined by serial microdilution in a liquid medium according to the recommendations of NCCLS [50]. 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. The results are summarized in table 5 and in the drawing.

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

Table 5
Antimicrobial activity of drugs No. 1, 2 and 3 A DRUG The concentration of funds,% Zones of growth inhibition of microorganisms, mm Escherichia coli ATCC 1257 Staphylococcus aureus 906 (LVA + hemolytic) Staphylococcus spp. (hemolytic) Enterococcus faecalis ATCC 29212 Pseudomonas aeruginosa ATCC 10145 Salmonella spp. (apathetic form) Shigella sonnei, spp. No. 1 10 21 b > 35 b > 35 b 16 b 18 b 14 b 15 b 5 15 b > 35 b > 35 b 15 b 15 b 12 b 12 b Number 2 10 8 bps 13 bps 11 b 16 b 12 b 8 bps 7 bps 5 7 bps 12 bps 10 bps 15 b 10 bps 7 bps 7 bps Number 3 10 9 bps 26 b 28 b 20 bps 9 bps 9 bps 9 bps 5 8 bps 24 b 24 b 18 bps 8 bps 8 bps 8 bps THE CONTROL 10 7 bps no no no no 11 bps no 5 7 bps no no no no no no Notes. CONTROL - (cycloserine + DMSO); ATCC - American type culture collection; spp. - "wild" strain (clinical isolate); b / s - bacteriostatic effect; b - bactericidal effect.

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Claims (4)

1. An antimicrobial drug, characterized in that it contains an active substance or a combination of active substances, a copolymer of lactic and glycolic acids (PLGA 50/50), D-mannitol, polysorbate 80 and dimethyl sulfoxide (DMSO) in the following ratio, wt. %: Active substance (or a combination of active substances) 2.95 ÷ 14.40 PLGA 50/50 2.95 ÷ 3.05 D-mannitol 2.95 ÷ 3.05 Polysorbate 80 0.95 ÷ 1.00 DMSO rest 2. The antimicrobial drug, characterized in that it contains the tool according to claim 1, which when diluted with water in a ratio of from 1:20 to 1:10 is a suspension of nanoparticles with a size of 200-400 nm, containing the active substance in an effective dose. 3. The method of obtaining the drug according to claim 2, characterized in that the known substance or combination of substances, PLGA 50/50, D-mannitol, polysorbate 80 and DMSO is successively loaded into the container, the mixture is heated at 50 ÷ 60 ° C and stirring until complete dissolution of the solid phase, after which it is cooled to room temperature, when water is added to the resulting mixture in a ratio of 1:20 to 1:10, a suspension of nanoparticles with a size of 200-400 nm is obtained. 4. The drug and preparation according to claim 1 or 2 of antimicrobial action, characterized in that they contain either rifabutin or D-cycloserine, or a combination of rifabutin, D-cycloserine, pyrazinamide and isoniazid as a substance.