Antibacterial Activity Of Pleuromutilin Derivative NPDM Against MRSA And Pharmacokinetics In Mice

Methicillin Resistant Staphylococcus aureus（MRSA）infection can directly lead to a variety of inflammations,such as meningitis,endocarditis,pneumonia,etc.,and even lead to systemic infections such as septicemia and sepsis.It is extremely harmful to livestock and humans.In recent years,the detection rate of MRSA in various regions has increased significantly.The resistance mechanism of MRSA is complex.It is resistant to cephalosporin antibiotics such as methicillin.Vancomycin is currently the drug for the treatment of MRSA,and researchers are constantly developing new drugs to cope with this resistant strain.The pleuromutilin has an unique antibacterial mechanism and has little cross-resistance with commonly used antibacterial drugs,and has received extensive attention.A series of pleuromutilin derivatives with novel chemical structures were synthesized in our laboratory.In this study,the in vitro antibacterial activity of 13 compounds was determined and it was found that NPDM has excellent antibacterial activity in vitro.In order to evaluate the safety and efficacy of NPDM,this study explored the in vitro antibacterial activity,toxicity,in vivo efficacy and pharmacokinetics of NPDM.Firstly,the MIC/MBC,in vitro time-killing curve and post-antibacterial effect（PAE）of NPDM were studied.The results showed that NPDM had superior in vitro antibacterial activity against Staphylococcus aureus（S.aureus）,for the MIC and MBC of MRSA were both 0.125μg/m L.The time-killing curve indicated that NPDM was a fungicide against MRSA,and the antibacterial effect was time-dependent.The PAE of MRSA exposed to 2×MIC NPDM for 1 h and 2 h were 2.58 h and 3.62 h respectively,and the PAE were 3.11 h and 4.03 h respectively when MRSA exposed to 4×MIC NPDM for 1 h and 2 h.Then the cytotoxicity and oral acute toxicity of NPDM was evaluated.The cytotoxicity of NPDM to BRL-3A cells was determined by MTT assay,and the half-inhibitory concentration(IC₅₀)of the cells was 9.64μg/m L.Five different doses of NPDM were used to conduct acute oral toxicity tests in mice.It was found that mice in the5000 mg/kg dose group died within one day and had severe tumbling behavior during their lifetime.Some dead mice have swelling in the abdomen and back,and strong wheezing can be heard during life.According to the modified Karber’s method,the LD₅₀of NPDM was3764.49 mg/kg,and the 95%confidence interval was 3006.67～4713.31 mg/kg.NPDM is a low toxicity compound.In vivo antibacterial efficacy of single-dose NPDM on MRSA was studied in the neutrophil-reducing mice thigh infection model and the mice peritonitis model.The ED₅₀of the compound was determined by Galleria mellonella（G.Mellonella）infection model.The results showed that the 20 mg/kg dose of NPDM significantly reduced the number of MRSA in the thigh muscle of mice infected with bacteria（P<0.001）.At a dose of 30 mg/kg,NPDM achieved 70%protection against immunosuppressive mice with intraperitoneal infection of MRSA.The survival curves of G.Mellonella suffered MRSA were obtained by the G.Mellonella infection model,and the ED₅₀of NPDM against MRSA in G.Mellonella was 50.53 mg/kg according to the modified Karber’s method.This study established a method for the determination of the concentration of NPDM in mice plasma by LC-MS/MS.Healthy female mice plasma samples were collected for a certain period of time after intravenous injection at a single dose（10 mg/kg body weight）of NPDM to determine the drug concentration in plasma,and the pharmacokinetic parameters were analyzed using a non-compartmental model of Win Nonlin 5.2 software.The main pharmacokinetic parameters were T_1/2:0.22±0.04 h,AUC:3.66±0.37 h?μg/m L,MRT:0.31±0.05 h,CL:2.76±0.31 L/h?kg.