Synthesis and antituberculosis activity of N’-(2-(5-((theophylline-7’-yl) methyl)-4-R-4Н-1,2,4-triazole-3-ylthio)acetyl)isonicotinohydrazides

The paper shows the results of clinical, pathological and histological studies of tuberculosis inflammation and non-specific changes in guinea pigs organs in the experimental model of tuberculosis during the comparative isoniazid and GKP-305 (N’-(2(5-((theophylline-7’-yl)methyl)-4-ethyl-4Н-1,2,4-triazole-3-ylthio)acetyl)-isonicotinohydrazide) treatment. The optimum location for the GKP-305 injection is found.

Primary resistance appears when a person gets infected with a drug-resistant strain of tuberculosis. The man who has no drug resistance during the treatment may develop secondary (acquired) resistance. It may occur because of improper treatment or failure to maintain prescribed regime accurately or taking substandard medicine. Resistant tuberculosis is a serious public health problem in many developing countries [1,2]. The treatment of the tuberculosis takes longer and requires more expensive drugs. Multidrug resistant tuberculosis (MDR-TB) is TB, which does not affected by the two most effective drugs: rifampicin and isoniazid.
The problem of side effects of xenobiotics and countering its toxic manifestation is extremely important. The problem of treatment of tuberculosis patients has significant scientific and social importance in the global epidemic of this disease in the world, including Ukraine [4,7,8]. Antibacterial drugs, paints, acids and other chemicals are factors of Mycobacterium tuberculosis variability, inducing pigment in cultures of microorganisms, the reduction of sticks with the bacterial wall defect (L-shaped), the accelerated formation of granular forms, acid resistance loss [5,6].
The work presents the urgent issues of comparative effectiveness of isoniazid and GKP-305 treatment of TB patients in a laboratory model (guinea pigs infected with pathogenic strains of Mycobacterium tuberculosis).

Purpose of the work
To study the modelling process and the features of tuberculosis in guinea pigs for the use of its results in further medical experiments and research practice.
Research is performed in the laboratory of Histology, Immunocytochemistry and Pathomorphology of Scientific Research Center of Biosafety and Environmental Resources Control in agro-industrial complex of Dnipro State Agrarian and Economic University (DSAEU), in educational and scientific laboratory of epizootology and infection process research of tuberculosis and mycobacterioses on DSAEU animals. For the experiment, 18 guinea pigs with an average weight of 250 g were taken to form six groups of three animals each.
According to the guidelines for the diagnosis of tuberculosis of animals and poultry two guinea pigs were used for bioprocessing.
Duration of treatment was 90 days. The control group: guinea pigs without treatment (survival test) and clinically healthy animals. The procedure of infecting animals was carried out with subcutaneous injection of M. bovis passage 100, wet weight 0,01 mg, in 0,5 cm 3 volume of saline sodium chloride.
During the dissection of animals, TB macroscopic lesions in USD for each individual pig were estimated. Regional infected lymph nodes, pieces of spleen, liver, lung, and kidney were placed in 10 % formalin solution for histological examination of each pig. The autopsy was performed by total evisceration method initiated by G. Shore. The material was taken immediately after examination for histopathological research carried out by hematoxylin and eosin coloration. Obtained histoagent was studied using Leica DM 1000 microscope. The histoagent photofixation was processed with digital camera Leica DFC 295.
In carrying out researches we used cryogenic epizootic strain M. bovis 100 passage, isolated from responding to PPD-tuberculin for mammal's cow. For infecting animals we used suspension of mycobacteria 8-10 mg bacterial mass, which was removed a spatula from the surface of a dense nutrient medium and transferred to a sterile penicillin bottle with rubber stopper, which was previously weighed. Then the flacon was weight again on an analytical scale and the number of selected cultures of mycobacteria determined the difference in weight. In flack with 1 cm 3 of bacterial mass an equal amount of isotonic solution was added.
Each animal was inject with a suspension of 1cm 3 -1 million international units.
Experimental data was processed by the software package for statistical analysis of Excel 2003 (Microsoft corp.) with integrated data analysis software add-in AtteStat. Data with continuous distribution represented as average and error average, and discretely distributed data-in the form of median and interquartile scale. The reliability of the differences between the experimental groups was assessed by the Student's t-test (for continuously distributed data and data with a normal distribution) and Wilcoxon signedrank test (for discretely distributed data), considering the differences reliable at P < 0.05.
In the 1 H NMR spectra of compounds 4-6, the signaling of methyl groups appears in the form of singlets at 2.88-3.15 and at 3.65-3.85 ppm, in the form of a triplet at 1.65 ppm, signals of methylene groups -in the form of singlet at 3.77-3.85 and at 5.00-5.75 ppm, protons of the pyridine ring in the form of two doublets at 7.50-9.15 ppm. The NH signal is within the range of 8.32-9.65 ppm in the form of a singlet ( Table 1).
As a result of infecting laboratory animals with Mycobacterium pathogenic strains guinea pigs of a control group visually demonstrated ulcer at the site of M. bovis culture injection.
Lungs, liver, kidneys and spleen were marked with significant specific inflammatory process and the evolving of Besnier-Boeck-Schaumann syndrome with granulomas in polynuclear cells. Caseous-necrotic and degenerative changes were observed.
There were no pathological changes in clinically healthy animals.
The specific inflammation centers consist mostly of epithelioid and lymphoid cells, including single giant polynuclear Langhans-Pirogov cells. In addition to this, there can be seen histiocytes and plasma cells with eccentrically placed nuclei, single mononuclear macrophages.
In liver specimen there were degenerative changes of hepatocytes, specific inflammation centers with caseous necrosis. Lymphoid and epithelioid infiltrates and giant multinuclear macrophages were found at the periphery of them. Severe degenerative changes of epithelial direct tubules. A significant TB progression in kidneys is indicated by giant multinuclear Langhans-Pirogov cells. Spleen tissue contains numerous inflammation lesions in the form of caseous necrosis.
At the periphery of lesions there are large multinuclear macrophages in Langhans-Pirogov cells, indicating severe specific inflammation. Pathological changes in animal organs infected with 100 passage M. bovis are put in the Table 2.
The data shows that animal organisms infected with M. bovis passage 100 (control group) underwent featured pathological changes -lungs demonstrated primary symptoms of pneumonia with granuloma necrosis in the center, perifocal inflammation and tuburcles. Liver

Basic research
modification revealed fatty degeneration, diffuse and nodule histolymphocytic infiltrates, unspecific vasculitides. Spleen tissue contained numerous foci of tuberculosis inflammation and caseous necrosis. At the periphery of lesions large multimacrophage Pirogov-Langhans cells are found, indicating specific inflammation. Spleen was featured with tuburcle (miliary tuberculosis) large foci changes, splenomegaly tuberculosis, amyloidosis, lesions in lymph nodes and kidneys. Thus, the lymph nodes are rich with the giant elongated cells of Pirogov-Langhans type and epithelial cells, typical for infectious granulomas. Kidneys showed globocellular cell infiltration, connective tissue growth zone capsules, vascular sclerosis, hyalinosis, granular and fatty degeneration tubules. Changes are characterized with nodular hystolymphocyte infiltrates, glomerular infiltration and capillar epithelial necrosis.
We conducted a comparative analysis of the impact of a 1 % solution of isoniazid and GKP-305 on the body of guinea pigs infected with M. bovis passage 100 by different methods of administration (subcutaneous and intra). As a result of observation for 90 days we found that for the internal use of isoniazid in lung cells of primary pneumonia -in the granulomas of necrosis phenomena, and around there perifokalne inflammation and tubercles; liver fatty degeneration of hepatocytes; small cells in the spleen caseous necrosis, splenomegaly. In the lymph nodes showed the presence of inflammatory foci of giant cells Pirogov-Langhans; kidney fatty convoluted tubules.

Discussion
In our opinion, this method results in the use of isoniazid intoxication sick animals, although discovered tuberculostatic effect in relation to the control group (infected animals). So isoniazid treatment significantly reduced the intensity of tuberculous lesions, but not completely eliminated, which was confirmed by the presence of small foci of tuberculous lesions in the lungs, lymph nodes and spleen. The use of isoniazid subcutaneously on animals infected M. bovis 100 passage led to permanent tuberculostatic exposure: lung, spleen, lymph nodes were found as pathologic changes characteristic to tuberculosis lesions, although found in liver fatty degeneration of hepatocytes and kidney dystrophy protein winding tubules. Positive results were obtained using the agent GKP-305 as only 1 % solution used internally affects tuberculostatically, except for liver and kidneys with imperceptible fatty hepatocyte and convoluted tubules degeneration.
When GKP-305 agent was used subcutaneously, it showed greater tuberculostatic effect compared with isoniazid, featured with the absence of pathological changes in lungs, liver, spleen, lymph nodes and kidneys (Fig. 2).

Conclusion
The examples illustrate the results of a comparative analysis of isoniazid and GKP-305 treatment based on clinical, pathological and histological studies of tuberculosis inflammations symptoms and nonspecific changes in the organs of guinea pigs with the usage of experimental model of tuberculosis. It is found that subcutaneous injection of GKP-305 at a dose of 10 mg/kg of animal mass causes the absence of specific and non-specific symptoms of inflammation in lungs, liver, kidneys and spleen.
Conflicts of Interest: authors have no conflict of interest to declare. Конфлікт інтересів: відсутній.