Study On Antibacterial Activity In Vivo, Toxicity And Pharmacokinetics Of ATTM

14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin(ATTM) is a new pleuromutilin derivative firstly synthesized in our lab with excellent antibacterial activity and may be served as a possible lead compound for the development of antibacterial drug. This report describes a subsequent pharmacological investigation of ATTM, containing antibacterial activity in vivo in mice, acute toxicity, subchronic toxicity and pharmacokinetic studies.1.The synthesis of ATTMThe 22-hydroxyl of pleuromutilin was activated by p-toluenesulfonyl chloride, followed by reaction with KI under the condition of alkali. Then ATTM was obtained in good yield by the nucleophilic attack of 2-amino-5-mercapto-1, 3, 4,-thiadiazole on intermediate 14-O-(iodine acetyl) mutilin under alkaline conditions. 2.Antibacterial activity of ATTM in vivoA staphylococcal systemic infection model was used to evaluate ATTM efficacy by measurement the survival of mice after a lethal challenge of methicillin-resistant Staphylococcus aureus(MRSA). Tiamulin fumarate was chosen as reference drug. The results show that the 50% effective dose(ED50) of ATTM and tiamulin fumarate are 5.74 and 5.95 mg/kg body weight(the confidence level of 95% was 3.75 to 8.78 mg/kg and 3.59 to 9.87 mg/kg), respectively. Thus, ATTM was more active against MRSA in the mice systemic model and showed slight higher activity as compared to tiamulin fumarate. 3.Acute oral mice toxicity of ATTMAccording to the modified Kaber method, thirty male and thirty female SPF Qunming mice were stratified by weight and randomly assigned to six groups: five treatment groups and one vehicle control group, 5 male and 5 female mices for each group. Mices were observed twice daily for symptoms and mortality for14-days. The vehicle control group was recorded at the same time. The 50% lethal dose(LD50) was calculated by Bliss method on 15 th day. All the animals were euthanized and their vital organs were individually observed for overt pathology by necropsy. The results of the oral single-dose toxicity study show an approximate LD50 in mice was 2304.4 mg/kg by Bliss method, and the confidence level of 95% was 1861.4 mg/kg to 2870.5 mg/kg. This shows that ATTM has a relatively low toxicity profile and has a high security as a new drug. 4.Subchronic oral toxicity of ATTMA total of 100 healthy male and female SD rats(140~160 g) were selected and randomly divided into five groups after one week acclimatization. One group was assigned to positive group and received DMSO and another group was assigned to negative group and received physiological saline. The rest of three groups were assigned to ATTM test groups and received via oral gavage at doses of 5, 25 and 125 mg/kg b. w. per day(the same time each day ± 1 h), respectively. No toxicologically significant effect was observed on body weight, food consumption and hematological examination. The high-dose groups are associated with additivity for a number effects: increasing in liver organs weights and increasing of ALP, CR and GLU in biochemical analysis. Histopathology examination of liver, spleen and kidney in high-dose groups showed some toxicological effect. We deduced that the target organs are liver, kidney and spleen. 5 Pharmacokinetic studies of ATTMA sensitive, specific, accurate, and reproducible reversed phase high performance liquid chromatography(HPLC) method for the determination of 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl]mutilin(ATTM) in broiler chickens plasma was developed. The intra- and inter-day precisions were in the range of 92~99 %. The average recoveries of ATTM were 92.70, 98.27, and 98.76 % for ATTM of 0.122, 1.22 and 12.2 μg/mL. The analytical method was also successfully applied to the pharmacokinetic study of ATTM in broiler chickens for the first time. ATTM was conducted in healthy broiler chickens after intravenous(i.v.), intramuscular(i.m.) and oral administrations at the dosage of 30 mg/kg body weight. Pharmacokinetic parameters of ATTM were analyzed by non-compartmental analysis using the PK solver program. The plasma profile of ATTM exhibited favorable pharmacokinetic characteristics in broiler chickens, such as wide distribution, and rapid absorption and elimination.