Evaluation of acute toxicity of pidotimod synthesized in vietnam – Quach Thi Ha Van

Journal of military pharmaco-medicine n 0 9-2018 91 EVALUATION OF ACUTE TOXICITY OF PIDOTIMOD SYNTHESIZED IN VIETNAM Quach Thi Ha Van1; Nguyen Hong Hai1; Nguyen Thi Thu Hang2 Nguyen Hai Nam2; Tran Viet Hung3 SUMMARY Objectives: In this study, the acute toxicity of pidotimod synthesized in Vietnam have been evaluated. Materials are synthetic pidotimod offered by Hanoi University of Pharmacy. Equipments and chemicals used for the study was calibrated according to ISO/IEC 17025. Method: Acute toxicity was evaluated according to the acute toxicity determination method (Medical Publishing House 1996) and the OECD guidelines (OECD guidelines for testing of chemicals. Acute oral toxicity - fixed dose procedure OECD 420, 2001). Results: Acute toxicity of pidotimod by oral administration in mice, LD50 was 22.041 ± 0.649 g/kg. Conclusion: The synthesized pidotimod sample (P120513) belongs to the class of non-toxic substances. * Keywords: Pidotimod; Acute toxicity. INTRODUCTION Pidotimod is an immunostimulant that has been synthesized since the late 1980s. Pidotimod increases 100% of T lymphocytes and 133% of NK cell activity in immune deficiency individuals. On some volunteers, pidotimod also increases the number of white blood cells and macrophages [5]. In addition to effects on the immune system, studies have shown that pidotimod has no effect on other organ systems in laboratory animals, safe for infants even after long periods of treatment [5]. Pidotimod has been used extensively in foreign countries, but in Vietnam market, axil (400 mg/7 mL) is the only circulation of pidotimod, but is imported at high prices. Step by step self-sufficiency of pharmaceutical raw materials for domestic drug production is one of the key tasks of the pharmaceutical industry in Vietnam. Hanoi University of Pharmacy has built the process of synthesizing and purifying. Pidotimod reached purity of 98.0%. In order to make the appropriate dosage forms, therapeutic and safety results, we have studied acute toxicity of pidotimod. MATERIALS AND METHOD 1. Materials. Pidotimod synthesis at Department of Pharmaceutical Chemistry, Hanoi University of Pharmacy. Experimental animals: Swiss black mice administered by National Institute of Hygiene and Epidemiology. Weight: 18 - 20 g, number: 80, raised in cool air conditioners, hygiene, diet according to the needs of the mouse. 1. Vietnam Military Medical University 2. Hanoi University of Pharmacy 3. National Institute of Drug Quality Control Corresponding author: Nguyen Hong Hai (honghaik85@gmail.com) Date received: 31/07/2018 Date accepted: 14/11/2018 Journal of military pharmaco-medicine n 0 9-2018 92 2. Method. Acute toxicity was evaluated according to the acute toxicity determination method (Medical Publishing House 1996) and the OECD guidelines (OECD guidelines for testing of chemicals. Acute oral toxicity - fixed dose procedure OECD 420, 2001). - Indicators: + Safe dose. + Maximum oral dose. + The dose causing visible toxicity. + The lowest dose that can kill the venom (if any); + LD50 dose (if detectable) (calculated according to Behrens formula); + Typical symptoms of poisoning can be observed in animals and their resilience (if any). * Preliminary test: - Preparation of the test sample: Use a suspension contain 0.5 g/mL in starch 3%. (suspension A). - Probe dose at not killing mice: To carry out on 4 groups of mice. For each group of 5 mice administered 0.4; 0.5; 0.6; and 0.7 mL of suspension A equaled to 10.0; 12.5; 15.0 and 17.5 g/kg. - Probe dose at 100% killing mice: To carry out on 4 groups of mice: each group of 5 mice with 1.0; 1.1; 1.2 and 1.3 mL of suspension A corresponding to the dose of 25.0; 27.5; 30.0 and 32.5 g/kg. * Official test: - Mice were fasted for 15 hours before the test, drinking water on demand. Check their weight before the test. - Administration: Take the test sample as an oral suspension. Take the sample volume directly into the stomach. - Based on the results of the preliminary test, 60 mice were officially tested, divided into six groups according to the administrated dose. The test groups were given a test suspension at the dose and dose levels shown in table 1. Table 1: Volume and dosage of test sample used in mice. Dose level Dosage (mL sample/20 g mouse) Dosage (g sample/kg mouse) Number of mice ML 1 0.6 mL 15.0 g/kg 10 ML 2 0.7 mL 17.5 g/kg 10 ML 3 0.8 mL 20.0 g/kg 10 ML 4 0.9 mL 22.5 g/kg 10 ML 5 1.0 mL 25.0 g/kg 10 ML 6 1.1 mL 27.5 g/kg 10 - Tracking calendar: Track expression of mouse activity after drinking, 24 hours and 7 days. Journal of military pharmaco-medicine n 0 9-2018 93 RESULTS 1. The results of the probe not killing mice. After 24 hours, the mice which received a test suspension of 10.0, 12.5 and 15.0 g/kg were not killed, while at the dose of 17.5 g/kg one mouse was killed. So no lethal dose should not exceed 15.0 g/kg. 2. Results of probe dose at 100% killing mice study. After a 24-hour follow-up, the mice at the dose of 25.0 g/kg killed four fifth of the group mice, while at 27.5, 30.0 and 32.5 g/kg doses, 100% of the mice killed. So the lethal dose of 100% of the mice should therefore be no less than 27.5 g/kg of sample mice. Thus, the LD50 was determined in the range of 15.0 to 27.5 g/kg. 3. Results of monitoring feed and oral consumption of mice. After oral ingestion at ML1 dose level, mice showed normal activity. At the ML2, ML3, ML4, ML5 and ML6 doses, after administration of test, mice exhibited decreased physical activity. Mice that did not die at the above dose levels reduced feed intake during the follow-up period. 4. Results of observation signs of poisoning. At the ML2, ML3, ML4, ML5 and ML6 doses, after administration of test suspension, mice demonstrated exhibited decreased activity and fatigue. Mice died for a period of 2 to 24 hours. Mice that did not die at the above dose levels showed decreased activity, fatigue during follow-up. Mice at ML1 dose did not find evidence of toxicity. Monitoring the mortality/alive mice ratio when given a test at the dose levels was shown in table 2 and figure 1. Table 2: Results of monitoring dead and alive mice. Dosage level Dose (g sample/kg mouse) Number of dead/alive mice Dead/alive mice % death ML 1 15.0 0/10 0/33 0 ML 2 17.5 1/9 1/23 4.17 ML 3 20.0 3/7 4/14 22.22 ML 4 22.5 5/5 9/7 56.25 ML 5 25.0 8/2 17/2 89.5 ML 6 27.5 10/0 27/0 100 Journal of military pharmaco-medicine n 0 9-2018 94 Figure 1: Chart of correlation of dose and proportion dead mouse. Calculating the LD50 by Behrens formula lead to the value of 22.041 ± 0.649 g/kg. After administration of the drug, mice exhibited the following symptoms: At low doses (15.0 g/kg of mice), there was no evidence of toxicity; at doses from 17.5 to 27.5 g/kg, mice exhibited fatigue, decreased activity. Mice died in the 2 to 24 hours and the death rate was dependent on the oral dose. In this trial, the non-killing dose found was 15.0 g/kg samples mice. The killing dose of 100% of the test mice was 27.5 g/kg samples mice. Based on actual data, we found the dose of LD50 = 22.041 ± 0.649 g/kg. The substances whose LD50-grade toxicity are greater than 5,000 mg/kg mice administered orally are considered to be non-toxic. CONCLUSION The acute toxicity level of the synthesized pidotimod sample was studied. The results showed that the LD50 value was 22.041 ± 0.649 g/kg. Thus, based on the results of this test, it can be concluded that pidotimod sample (P120513) belongs to the class of non-toxic substances. REFERENCES 1. Do Trung Dam. Methods of toxicity determination of drugs. Medical Publishing House. 2014, pp.101-112. 2. Vietnam Ministry of Health. Circular 03/2012/TT-BYT Guidelines for Clinical Trials on Drugs. 2012. 3. K. Adams et al. Genotoxicity testing of pidotimod in vitro and in vivo. Arzneimittel- Forschung. 1994, 44 (12A), pp.1454-1459. 4. Globally Harmonized System of Classification and Labelling of Chemicals. 2017. 5. Magni A., Signorelli G., Bocchiola G. Arzeim-Forsch/Drug Res. Synthesis and preliminary pharmacological evalution of pidotimod, its enantiomers, diastereomers and carboxamido derivatives. 1994, 44 (II), 12A, pp.1402-1404.