Сельскохозяйственная биология, 2012, № 2, с. 96-101.

doi: 10.15389/agrobiology.2012.2.96eng

УДК 636.32/.38:619:615.284

INVESTIGATION OF THE GEMACS DRUG EFFICIENCY UNDER THE GASTROINTESTINAL STRONGYLATOSIS IN SHEEP

M.Kh. Dzhafarov, I.V. Zavarsin, M.N. Mirzaev, Z.A. Devrishova, Yu.A. Yusupov

On sheep during their keeping in the pasturable conditions the authors investigated the anti-helmint efficiency of Gemax, the new drug on the base of avermectin hemisuccinate as substance, first synthesized by the authors by means of acylation of avermectin B_1а. The data indicate high efficiency of Gemax as anti-helmint drug under the gastrointestinal strongylatosis of sheep.

Keywords: the Gemacs agent, avermectin B_1а hemisuccinate, anti-helmint efficiency, strongylatosis, substance, synthesis of а new drug substance.

Parasites of plants, animals and humans pose a serious threat and contribute to significant economic losses (human disablement, reduce in productivity of livestock and fur-bearing animals, low yields of agricultural crops, worsened quality of crop production and wood, etc.).
Chemical insecticides still remain the most effective and cheapest method of pest control even despite the current advance in decoding genomes of many species (including pathogens and parasites) and attempts to develop a vaccine. Along with it, treatment of many parasitoses (tropical malaria, Chagas disease, filariasis, echinococcosis, cysticercosis, etc.) needs improved drugs (1, 2).
Another global problem is a development of drug resistance to biocides (antibiotics, anthelmintics, etc.) previously highly effective against agents of infectious diseases, which necessitates finding new broad-spectrum biocidal medications (3, 4).
In earlier studies, the authors have performed a screening of semisynthetic derivatives of 16-membered macrocyclic lactones (avermectins) along with N- and S-containing steroid compounds, which has resulted in selecting several promising biocides for developing a new antiparasitic drug (5-8).

Avermectins were discovered in 1976 in the U.S. by the research group of Merck & Co. Inc. in the culture of Streptomyces avermitilis MA-4,680 (NRRL8165) isolated by researches of Kitasato Institute (Tokyo) from soil samples obtained in the vicinity of Kawana (Ito City, Shizuoka Prefecture of Japan). Their results of in vivo tests of the derived samples in mice infected with nematodes Nematospiroides dubius have set a start point for a new class of anthelmintic drugs. Later it was established a wide spectrum biocidal action of avermectins on nematodes, insects and mites. Since the most pronounced antiparasitic activity is caused by B_1a component, there were synthesized a group of B₁-based avermectins included in commercial agricultural antiparasitic drugs: abamectin (a mixture of B_1a and B_1B avermectins, respectively 80 and 20%) and ivermectin - a dehydrated derivative of avermectin B₁ (9).  There was also observed an antitumor activity of avermectins (8, 12).
The purpose of this work was testing anthelmintic action of a new developed drug Gemax under the gastro-intestinal strongylatosis of sheep farmed at a pasture grazing.
Technique. The active substance of Gemax was produced using the method proposed by the authors (13). Elemental analyzes were performed on Perkin Elmer 2400, Series II, CHNS/O analyzer (“Perkin Elmer”, USA), IR spectra were recorded on the spectrometer UR-20 (Germany). Mass spectra were recorded on MicrOTOF II (“Bruker Daltonics GmbH”, Germany) high resolution mass-spectrometer with electrospray ionization method  (ESI), scanning range - from m/z 50 to m/z 3000, ion polarity - positive (capillary voltage 4500 V) or negative (capillary voltage 3200 V), syringe injection of a substance; solvents - acetonitrile, methanol, flow rate of the solution - 3 ml/min, interface temperature 180 °C, spraying gas - nitrogen (4/0 l / min). ¹H-NMR spectra were recorded on devices Bruker WM- 300 (300 MHz) and Bruker  DRX-500 (500 MHz) (“Bruker Daltonics GmbH”, Germany) at 303 °K. The melting point of avermectin hemisuccinate (AI of Gemax) was determined using a polarizing optical microscope NAGEMA-K8 equipped with a heating table Boetius (“NAGEMA”, Germany), heating rate 4 °C/min (not adjusted).
Antiparasitic effects of Gemax was assessed during the applied science research in sheep farms of Babayurtovsky district of the Republic of Dagestan (LLC “Kolos” and the owner-operated farm “Shaula”), as well as in SUE "Leonovsky" of K.I. Srkyabin Moscow State Academy for Veterinary Medicine and Biotechnology (Moscow province, Konobeevo settlement) on pasture-grazed sheep aged 1,5-2,0 years. Among the animals spontaneously invaded with gastro-intestinal strongylatosis there were selected a group of individuals (50 animals, I experiment) upon the results of laboratory faecal tests based on Fulleborn’s indicator of invasion intensity. These animals were divided into two groups of 25 individuals; sheep of groups I and II were injected subcutaneously with Gemax at doses of, respectively, 1,0 and 1,5 ml/50 kg body weight. A control group of 6 animals was not administered any drug. In II experiment, three groups (9 sheep in each one) were the subject in comparative tests of known commercial de-worming drugs - Niatsid-plus (“Agrovet”, “Ekobiovet”, Russia) and Ivermek (“Nitafarm”, Russia) (injected according to the attached manufacturer’s instructions) and Gemax for treating intestinal strongylatosis. All the test animals were kept under identical conditions and constant surveillance.
Deworming effect was assessed upon the results of faecal tests performed in 28 days after the use of anthelmintic drugs. Extensiveness of invasion (EI - total proportion of infected animals, %) and intensity of invasion (II - the number of eggs of strongilyate nematodes in 1 g feces determined as described by Fulleborn before and after the treatment of sheep) were assessed. Therapeutic effect was evaluated by extensive efficacy (EE - the proportion of recovered sheep, %).
General condition of animals’ health was monitored using the data of biochemical and morphological blood tests performed on the analyzers Olympus AU400 (Germany) and PCE-90vet (USA).
The obtained data were statistically treated in the program Statistica.
Results. The active ingredient of Gemax is hemisuccinate of avermectin B1a synthesized through an acilation of a natural avermectin B₁ (B_1acontent not less than 98 %) with succinic anhydride in organic solvents against the presence of amine type catalysts as proposed in the authors’ method (13). This way of synthesis differs from other known similar methods (14) in its simplicity and cheapness. The yield of the desired product amounts to 96 %. The identity of avermectin hemisuccinate structure was confirmed by results of ¹H-NMR spectroscopy (additional signal detected at 2,75 ppm region, which corresponds to the signals of -CH₂-groups of succinyl residue), IR spectroscopy (the presence in the spectrum of 3440 cm^-1 signal related to acidic -OH groups) as well as mass spectrometry (the mass spectrum showed a peak of the toned molecular ion 973,5169; calculated value for the gross formula C52N76O17 – 973,5155) and elemental analysis (obtained values: C – 64,28, H – 7,76 and O – 27,87 %); melting temperature of avermectin  hemisuccinate 158-161 °C.
Comparative tests of the abovementioned anthelmintics Gemax, Ivermec and Niatsid-plus has shown (Table 1) their deworming effect in, respectively, 8; 9 and 9 of 9 treated animals – so, extensive efficacy (EE) of these drugs amounted to 89,9; 100 and 100 %. Infectiousness of the control animals did not change significantly during the experiment.
These results indicate high antiparasitic efficacy of the compared drugs: all experimental animals became almost free from parasites in 28 days after the treatment (EE varied within 84,0-100 %).

This is a noteworthy fact that Gemax didn’t cause any negative effects on the organism of experimental animals, as was evidenced by major morphological and biochemical blood parameters in sheep on the 28th day of the experiment (Table 2). For example, blood levels of hemoglobin, erythrocytes and leukocytes significantly varied in control and experimental sheep – respectively, from 77,52±5,84 to 89,60 ± 6,20 g/l, from 8,19±0,57Ѕ10¹²/l to 9,14±0,78Ѕ10¹²/l and from 6,89±0,70Ѕ10⁹/l to 7,49±0,53 Ѕ10⁹/l, which fluctuations though were within the physiological norm.
There was also observed an absence of negative impact on liver function from Gemax: aminotransferase activity (AST and ALT) in control and experimental sheep were similar. It is known that metabolism in the liver is clearly reflected by such important characteristic as the ratio of transferase activity (AST / ALT), or de Rytis ratio. Normally, it equals 1,33. In control animals, this value was 1,43, while in the experimental groups it varied in the range 1,34-1,49 (Table 2).
The analysis of the presented data in terms of modern ideas about the adaptive development of new systems under continually changing environmental conditions allows assuming the adaptive nature of the abovementioned changes in transamination enzyme activity in experimental animals treated with the drugs, because it provides an appropriate metabolic status and levels adverse effects of anthelmintic drugs.
Previously, M.Kh. Dzhafarov et al. (6) have established LD₅₀ of avermectin B1 hemisuccinate in white mice equal to 35,96 mg/kg and LD₁₀₀ – 69,44 mg/kg (“hemisuccinate avermectin” in this work and “adermectin” in the cited article are names-synonyms) and these values correspond to similar parameters of the original avermectin (6).
Thus, the new developed anthelmintic drug Gemax synthesized by means of the original authors’ method of chemical modification of avermectin B_1a, has relatively low toxicity and provides high antiparasitic effect under the gastro-intestinal strongylatosis of sheep.
 

REFERENCES

1. Klinicheskaya farmakologiya po Gudmanu i Gilmanu (Goodman & Gilman’s Clinical Pharmacology), Gilman A.G., Ed, Moscow, 2006.
2. Mirzaev M.N., Voronin E.S., Devrishov D.A. and Savchenkov S.N., Preparat dlya bor’by s vrednymi nasekomymi i gel’mintami (Agent against Harmful Insects and Helminthes), Patent RF № 02210362 (A61K31/00, A01N25/00 or RU 2210362 C2),  Published 20.08.2003. http://ntpo.com/patents_medicine/medicine_6/medicine_1337.shtm.
3. Sangster N.C., Anthelmintic Resistance: Past, Present and Future, Int. J. Parasitol., 1999, vol. 29, no. 1, pp. 115-124.
4. European Strategic Action Plan on Antibiotic Resistance. EUR/RC61/14,World Health Organization Regional Committee for Europe, Sixty-First Session, Baku, Azerbaijan, 12-15 September 2011. + EUR/RC61/Conf.Doc./7. http://www.euro.who.int/_data/assets/pdf_file/0008/147734/
wd14E_AntibioticResistance_111380.pdf. 5. Dzhafarov M.Kh., Mirzaev M.N., Zavarzin I.V., Mel’nitskaya T.I., Zaitsev S.Yu., Maksimov V.I., Kamernitskii A.V., Chertkova V.V., Chernoburova E.I., Krayushkin M.M. and Yarovenko V.N., Synthesis and Biological Activity of Steroid Dihydropyrazoles, Rossiiskii immunologicheskii zhurnal, 2008, vol. 2, no. 11/2-3, p. 192.
6. Dzhafarov M.Kh., Mirzaev M.N., Urazaev D.N., Zaitsev S.Yu. and Maksimov V.I., Antiparasitic Activity of Adermectin and Compounds of a Steroid Nature, Dokl. RASKhN, 2010, vol. 2, pp. 45-46.
7. Dzhafarov M.Kh, Mirzaev M.N. and Zavarzin I.V., Antiparasitic Activity of Famectin and Some Compounds of Different Chemical Nature, S.-kh. biol., 2011, no. 2, pp.  108-112.
8. Dzhafarov M.Kh., Zaitsev S.Yu. and Maksimov V.I., Steroidy. Stroenie, poluchenie, svoistva i biologicheskoe znachenie. Primenenie v meditsine i veterinarii (Steroids. Structure, Synthesis, Properties and Biological Role. Application in Medicine and Veterinary), St.Petersburg, 2010, pp. 200-215.
9. Macrolide Antibiotics. Chemistry, Biology and Practice. 2nd Edition, Omura S., Ed., New York: Elsevier Science, 2002.
10. Burg R.W., Miller B.M., Baker E.E. et al., Avermectins, a New Family of Potent Anthelmintic Agents: Producing Organism and Fermentation, Antimicrob. Agents Chemother., 1971, vol. 5, no. 9, pp. 361-367.
11. Hafner E.W., Holdom C.S., Li Sh.-J.E., Sposob polucheniya avermektina i shammy Streptomyces avermitilis – produtsenty avermektina (Process for Production of Avermectin and Cultures of Streptomyces avermitilis — Producents of Avermectin), Patent RF 1 2096462, Published 20.11.1997,
Bull. № 1.
12. Mosin V.A., Drinyaev V.A., Kokoz Yu.M. et al., Sredstvo, usilivayuschee protivoopukholevuyu aktivnost’ khimiopreparatov, i sposob lecheniya onkologicheskikh zabolevanii (Medication Enhancing Antitumor Activity of Chemotherapy and Method for Treatment of Oncological Diseases), RF Patent 1 2250775, Published 27.04.2005 (A61K35/70, A61P35/00 or RU 2250775 C2).
13. Zavarzin I.V., Dzhafarov M.Kh., Mirzaev M.N., Kolobov A.V., Chernoburova E.I. and Bobova T.A., 5-O-Succinilavermektin B_1a, sposob ego polucheniya i antiparazitarnoe sredstvo na ego osnove (5-О-Succinylavermectin В_1a, Method for Its Production and Antiparasitic Drug Based on It), Patent Application 1  20011118586, Applied 11.05.2011.
14. Mrozik H., Eskola Ph., Fisher M.H. et al., Avermectin Acyl Derivatives with Anthelmintic Activity, J. Med. Chem., 1982, vol. 25, pp. 658-663.