Pharmacodynamic And Pharmacokinetic Studies Of DPTM

14-O-[?4,6-Diaminopyrimidine-2-yl?thioacetyl]mutilin?DPTM?,a new pleuromutilin derivative,firstly synthesized in our lab.Previous studies have shown that DPTM showed excellent antibacterial activity in vitro.However,its clinical application potential is still unknown.Therefore,the preclinical pharmacodynamics and pharmacokinetics of this compound were further studied.The main research contents and results are showed as follows:1.Effect of DPTM on a mice systemic infection model with Pasteurella multocida?P.multocida?Minimum inhibitory concentration?MIC?value and bactericidal curve of DPTM against P.multocida were firstly tested in vitro.The mice were randomly divided into 5 groups?n=15?.Mice in each group were anesthetized by intraperitoneal injection,and then intranasally injected with 60?L of 4.0×10⁹CFU/mL bacterial suspension to establish mice P.multocida pneumonia infection model.Then,the P.multocida mice infection model was used to assess the therapeutic effects of DPTM on P.multocida by testing the survival rate and lung wet-dry weight?W/D?ratio,analyzing the lung tissue pathological changes,and detecting the inflammatory cytokines in the lungs and the growth of P.multocida in the lungs and blood.These results showed that DPTM displayed a good inhibitory effect on P.multocida in vitro with MIC value 0.781?g/mL,significantly reduced the mortality of mice,alleviated the pathological damage of the lungs,decreased the secretion of TNF-?,IL-6,IL-1?and MCP-1inflammatory cytokines in the lungs,and reduced the load of bacteria in the lungs and blood.2.Effect of DPTM on skin infection model of methicillin-resistant Staphylococcus aureus?MRSA?miceThe mice were randomly divided into 6 groups?n=15?.Mice in each group were cut a vertical incision at a depth of about 1.5 cm from the median spine of the back to the sarcolemma using a sterile scissors.MRSA43300(0.1 mL,10¹² CFU/mL)in the logarithmic growth phase was evenly smeared to the entire wound to establish a deep skin sensation model in mice.Then the topical skin infection model with MRSA43300 was used to evaluate DPTM efficacy by measurement the changes of white blood cells and bacterial counts,the levels of IL-6,TNF-?,IL-1?and VEGF in the plasma and the pathological changes using HE and the Masson trichrome staining.The results showed that DPTM could promote the healing of MRSA43300 skin infection,reduce the number of infected skin MRSA43300 and decrease the secretion of IL-6,TNF-?,IL-1?inflammatory cytokines in plasma.3.Inhibitory effect of sub-inhibitory concentration of DPTM on MRSA?-hemolysinThe sub-inhibitory concentration of DPTM were co-cultured with MRSA43300 to evaluate the inhibitory effect of DPTM on MRSA43300?-hemolysin by detecting the hemolysis capacity of the bacterial supernatant after DPTM treatment,the secretion level of?-hemolysin and the HIA and agrA mRNA of the bacterial cells.The results showed that the sub-inhibitory concentration of DPTM could inhibit the secretion of?-hemolysin of MRSA43300.4.Protective effect of sub-inhibitory concentration of DPTM on RAW264.7 macrophage injury induced by MRSAThe sub-inhibitory concentrations of DPTM,MRSA43300 and RAW264.7 macrophages were co-cultured to study the protective effect of DPTM on MRSA43300-induced cell injury by measurement the release of LDH and NO,the secretion of IL-6,IL-1?and TNF-?,the protein expression Phospho-p65,iNOS,Cox-2 and the NF-?B nuclear transport.The results showed that sub-inhibitory concentration of DPTM could protect RAW264.7 macrophage from MRSA43300-induced injury.5.Pharmacokinetics,tissue distribution,urine and fecal excretion of DPTM in ratsDPTM?10?25 and 75 mg/kg?was administered intravenously,intramuscularly and orally to study the pharmacokinetics,tissue distribution and excretion characteristics in rats.The results showed that DPTM could rapid absorb and eliminate,then widely distributed in the various tissues examined,and mainly excreted with feces.6.Effect of DPTM on CYP450 enzyme and gene expression in liver microsomes and HepG2 cellsCocktail probe drug method was used to evaluate the inhibitory effect of DPTM on rat liver microsomes CYP450 enzyme.DPTM was co-cultured with HepG2 cells for 2,4 and 12 h to study the regulation of CYP450s mRNA in HepG2 cells by Real-time PCR method.The results showed that DPTM showed inhibitory effect on CYP3A4 and CYP2E1 enzyme activity but no obvious effect on CYP2D6,CYP1A2and CYP2C enzyme activity.Furthermore,DPTM could significantly induce CYP3A4,CYP2C9 and CYP1A2 mRNA expression,however,the expression of CYP2E1 and CYP2D6 mRNA were inhibited with the prolongation of incubation time.In conclusion,DPTM showed a protective effect on P.multocida lung-infected mice and could significantly alleviated lung pathological damage.DPTM ointment promoted the healing of MRSA43300 skin infection,significantly inhibited the secretion of MRSA43300?-hemolysin and protected RAW264.7 macrophage from injury caused by MRSA43300.Furthermore,the drug showed good pharmacokinetic parameters and extensive tissue distribution,and selective inhibited or induced CYP450 enzyme.Therefore,DPTM has great value in the clinical development and application.