RU2452498C2 - Method for preparing anti-tuberculosis drug - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing an anti-tuberculosis drug containing isoniaside and silver nanoparticles. The declared method consists in the fact that chitosan 1-5 wt %, isoniaside 6-10 wt % are successively dissolved in distilled water; the solution is heated up to 45-55°C with added stabiliser specified in polyethylene glycol or gelatin, in the amount of 5-40 wt %, and the ingredients are mixed to dissolve them completely. Then ammonium citrate in the amount of 1 g per 1 l of the solution is added in mixing; a silver anode is electrochemically dissolved for 10-30 min at silver nanoparticle yield in the water solution of stabilisers 2-15 mg per 1 l, and isoniaside if it has not been added previously.

EFFECT: invention provides a high-effective preparation for treating tuberculosis wherein the preparation ingredients show a synergetic effect and reduce resistance of tuberculosis mycobacteria to various antibiotics.

3 tbl, 1 dwg, 7 ex

Description

The invention relates to medicine, in particular to medicinal anti-TB drugs, and can be used to obtain drugs based on isoniazid for the treatment of drug-resistant forms of tuberculosis.

The main problems of treating tuberculosis include the limited arsenal of anti-TB drugs, the rapid development of resistant forms of mycobacterium tuberculosis (MBT) during treatment and, as a result, the increase in the number of patients infected with primary resistant MBT to the main anti-TB drugs. Therefore, there is a constant search for new anti-TB drugs, as well as ways to increase the effectiveness of known ones.

To eliminate drug resistance, anti-TB drugs are used in compositions with potentiating substances.

So, the drug RU 2003335 C1 is known (“A method for the treatment of destructive pulmonary tuberculosis with massive bacterial excretion”, YAKUT BRANCH OF SCIENTIFIC AND PRACTICAL ASSOCIATION “PHYSIOPULMONOLOGY”), which consists in the dissolution of anti-TB drugs in silver water. Silver water for this is obtained by electrolysis. Therefore, in the process of electrolysis metal ions pass into the water, which are extremely unstable in time, prone to oxidation and chemical interaction with the components of the anti-TB system.

CN 101225533 A describes a method for producing silver nanosized particles by electrochemical means with the addition of stabilizers, for example ammonium citrate. The method includes mixing silver nitrate with ammonium citrate, immersion in the specified mixture of two silver electrodes.

When using silver nitrate, part of the salt remains in the product - a solution of silver nanoparticles. Despite the stability of the nanosized particles of silver obtained in this way, the use of silver nitrate makes it impossible to use such particles in the composition of an anti-TB drug due to the toxicity of silver nitrate. In contrast to this method, our proposed method for the preparation of an anti-tuberculosis drug allows us not only to synthesize a colloidal solution of silver nanoparticles that does not contain extraneous impurities, but also to achieve its compatibility with isoniazid. In addition, the use of an anti-TB drug obtained by the claimed method allows to achieve the maximum therapeutic effect.

From WO2002085385 A2 it is known to add chitosan to antimicrobial nanocrystalline metal powders, in particular silver, which are used to treat tuberculosis. This family of patents discusses the inhalation use of antimicrobial nanocrystalline powders. In contrast to the said patent, the claimed method for preparing an anti-tuberculosis drug allows to obtain a product containing an aqueous solution of stabilized silver nanoparticles and isoniazid, which is acceptable for parenteral administration (injection). In addition, as already mentioned above, the claimed method allows to obtain a non-toxic drug containing no metal salts.

Closest to the claimed invention is the preparation RU 2008142461 A (Combined anti-tuberculosis drug, LLC NPO LIKOM), which consists in the combined use of isoniazid and silver nanoparticles. It should be noted that silver nanoparticles obtained from an aqueous solution by vacuum are used to prepare the anti-TB drug, which violates not only the shell formed from the stabilizer, but also the initial properties of silver nanoparticles.

The aim of the invention is the creation of a highly effective drug for the treatment of tuberculosis, containing, in addition to known chemotherapeutic agents, components that have a synergistic effect and reduce the resistance of tuberculosis mycobacteria to various antibiotics, as well as expanding its spectrum of action.

The goal is achieved as follows.

Organic stabilizers (5-40% wt.), Selected from gelatin, polyethylene glycols, and an inorganic stabilizer (monosubstituted ammonium citrate, 0.1% wt.), Chitosan (1-5% wt.) Are then dissolved in distilled water, then in the resulting the solution is an electrochemical dissolution of the silver anode. Isoniazid in an amount of 6-10% wt. is introduced either before electrolysis, or after it.

Obtaining an aqueous solution of silver nanoparticles by the electrochemical method in the presence of organic and inorganic stabilizers allows you to create a drug with high antimicrobial properties, which has a synergistic effect when combined with other drugs. In addition, silver nanoparticles have a developed mass transfer surface, which gives the maximum effect of suppressing microflora. The stabilizer shell prevents oxidation and chemical interaction with other components of the system, thereby increasing the useful life of the drug.

Introduction to the system of natural chitosan polysaccharide gives it unique properties. Chitosan is a derivative of chitin, easily soluble in water, has antimicrobial properties, significantly reduces the growth of bacteria, which allows to achieve the greatest therapeutic effect from the use of the claimed composition. In addition, it plays the role of a sorbent, which means it also performs a transport function, ensuring fast and accurate delivery of the drug to the target organ.

Example 1

In distilled water with stirring, dissolve isoniazid in an amount of 6% wt. and natural polysaccharide chitosan in an amount of 3% wt. Then, when heated to 45-55 ° C, polyethylene glycol with a molecular weight of 4000 in the amount of 10% is introduced and mixed until completely dissolved. Then it is cooled to a temperature of 20-25 ° C. Ammonium citrate is added to the resulting solution with stirring at the rate of 1.0 g per 1 liter of solution with stirring. Then, the electrode system is placed in the resulting medium, where the silver plate (GOST R ISO 9001-2001) serves as the anode, and the stainless steel plate (GOST 5582-95 grade 12X18H10T) serves as the anode. With stirring, stabilized direct current with a density of 10-20 A / sq is supplied to the electrodes. m and a voltage of 10-20 V. The electrochemical dissolution of silver is carried out for 10-30 minutes based on the yield of silver nanoparticles in an aqueous solution of stabilizers 2-15 mg per 1 liter of solution. The size of the resulting particles is 5-60 nm, which is confirmed by the analysis of variance obtained using the Nanotrac particle size analyzer (Fig. 1).

Example 2

A method of preparing an anti-tuberculosis drug according to claim 1, characterized in that isoniazid is taken in an amount of 8% by weight, chitosan in an amount of 5% by weight, and gelatin in an amount of 5% by weight is used as an organic stabilizer.

Example 3

A method of preparing an anti-tuberculosis drug according to claim 1, characterized in that chitosan is taken in an amount of 2% wt., And isoniazid in an amount of 10% wt. added at the end of the electrolysis process.

Example 4

A method of preparing an anti-tuberculosis drug according to claim 1, characterized in that chitosan is taken in an amount of 1% wt., Gelatin in an amount of 5% wt. Is used as an organic stabilizer, and isoniazid in an amount of 7% wt. added at the end of the electrolysis process.

Example 5

A method of preparing an anti-tuberculosis drug according to claim 1, characterized in that isoniazid is taken in an amount of 8% by weight, chitosan in an amount of 2% by weight, and polyethylene glycol with a molecular weight of 400 in an amount of 40% by weight is used as an organic stabilizer.

Example 6

A method of preparing an anti-tuberculosis drug according to claim 5, characterized in that isoniazid is taken in an amount of 6% by weight, chitosan in an amount of 1% by weight, and polyethylene glycol in an amount of 35% by weight.

Example 7. The prototype.

The study of the inhibition of the growth of drug-resistant MBT isolates was carried out by the method of absolute concentrations using a dense Levenshtein-Jensen nutrient medium. The material for the study was obtained in the laboratory of the State Health Institution of the regional tuberculosis hospital from 50 patients with various forms of pulmonary tuberculosis. All isolated MBT strains (740 pcs.) Had drug resistance to various anti-TB drugs. MBTO assessment of the results of the study was carried out by counting colonies in experimental samples (examples 1-4) in comparison with the control (example 5 prototype), where four degrees of inhibition of MBT growth were distinguished (complete, significant, moderate inhibition and lack of inhibition).

The research results are shown in table 1.

The data in table 1 confirm the increase in the positive effect of the use of the claimed composition in the treatment of drug-resistant forms of tuberculosis.

In addition to studying the overwhelming ability of the claimed drug, its antiviral properties were evaluated, in particular its effect on the course of HIV infection. In vitro studies were conducted on the basis of the Yaroslavl Regional Center for the Prevention and Control of AIDS, in which a blood test was taken from 8 registered patients. 6 of them had hepatitis C concomitant disease. Sample I was taken at the beginning of the study, samples II and III after 2 months and 6 months of using the claimed drug, respectively.

The research results are shown in tables 2 and 3.

According to Tables 2 and 3, after 2 months of research, a decrease in viral load and an increase in the number of CD-4 lymphocyte cells in the blood are observed.

It is important to note that the use of the inventive preparation did not have any side effects on the blood formula and biochemical parameters.

Table 1 The results of the test of the suppressive ability of anti-TB drugs on the growth of LU Office The degree of inhibition of the growth of LU strains of the Office Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Full% 82.5 73.8 77.5 81.4 79,4 77.1 37,4 Significant% fifteen 21,2 18.8 15.6 16.8 20,0 30,4 Moderate,% 2.5 3,7 3,7 2.7 3.3 2.9 9.7 Lack of. % 0 1.3 0 0.3 0.5 0 2.5 Total one hundred one hundred one hundred one hundred one hundred one hundred one hundred

table 2 Viral load before and after using the drug Patient No. Sample number Viral load example 1 example 2 example 3 example 4 example 5 example 6 example 7 one I 16754 16760 16757 16723 16754 16755 16750 II 15643 14233 15782 16007 15290 15673 16774 III 14712 12651 12227 13514 13741 14111 16723 2 I 5417 5403 5521 5629 5424 5479 5428 II 5128 5200 5478 5015 5138 5003 5410 III 4380 4456 4901 4781 4500 4289 5400 3 I 10982 10927 10956 10783 10789 10892 10990 II 9891 10113 9903 9567 9561 9800 10993 III 7562 8251 8560 8014 8001 8236 10981 four I 8814 8725 8898 8856 8821 8867 8913 II 8712 8400 8361 8224 8367 8009 8874 III 6065 5814 7511 6996 7006 7159 8870 5 I 50873 50096 51114 50527 50078 51001 50548 II 45620 42790 46573 43561 46081 47207 50500 III 32081 30098 34219 35800 34200 35692 50481 6 I 21863 21513 21744 21762 21901 21872 21007 II 18034 18001 19540 18992 18650 18900 20984 III 16590 15300 17016 16003 16520 17007 20900 7 I 30025 30112 30277 30671 31009 30810 30019 II 28549 29310 28780 29006 28379 27964 29921 III 25800 26755 25555 27953 26300 25019 29956 8 I 78923 78214 79900 78160 79042 78231 78615 II 73005 65060 72187 71923 73116 72000 78593 III 61290 58116 64253 67500 60067 63214 78550

Table 3 The number of CD-4 leukocytes before and after the use of the drug Patient No. Sample number The number of CD-4 cells example 1 example 2 example 3 example 4 example 5 example 6 example 7 one I 321 314 330 319 328 334 315 II 367 360 378 353 369 357 320 III 450 423 440 468 455 441 324 2 I 532 541 529 538 542 550 533 II 580 613 687 592 590 601 537 III 670 668 690 667 681 684 550 3 I 333 337 341 346 350 332 337 II 384 390 378 384 392 379 343 III 449 432 456 449 461 448 351 four I 478 463 444 435 459 442 430 II 521 512 498 470 507 515 447 III 584 567 558 555 572 564 479 5 I 200 205 207 221 237 216 214 II 267 254 267 281 265 254 226 III 312 353 300 378 362 320 250 6 I 316 323 317 311 320 327 315 II 347 378 389 376 383 367 327 III 479 456 428 420 431 448 349 7 I 298 301 274 293 311 281 285 II 349 370 338 328 338 348 299 III 401 427 390 411 420 420 307 8 I 187 190 188 191 205 183 201 II 253 215 227 249 254 240 215 III 302 284 300 305 308 300 229

Claims (1)

A method of preparing an anti-tuberculosis drug product, comprising mixing isoniazid and silver nanoparticles, characterized in that it additionally contains chitosan, silver nanoparticles are introduced in the form of their aqueous solution, and the preparation process consists in sequentially dissolving isoniazid in the amount of 6- 10 wt.% And chitosan in an amount of 1-5 wt.% Or chitosan in an amount of 1-5 wt.%, Heating to 45-55 ° C, adding stabilizers selected from polyethylene glycol and whether gelatin, in an amount of 5-40 wt.% and stirring until they are completely dissolved, adding ammonium citrate in an amount of 1 g per 1 liter of solution with stirring, electrochemically dissolving the silver anode for 10-30 minutes based on the yield of silver nanoparticles in water a solution of stabilizers 2-15 mg per 1 liter, the addition of isoniazid in an amount of 6-10% in the event that it was not previously added.

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