Evaluation Of The Antibacterial Activity Of OBP-4 Against Clostridioides Difficile And Its Pharmacokinetics Study In Rats

OBP-4 is a new oxazolidinone-fluoroquinolone hybrid with excellent antibacterial activities and good safety.The present study evaluated the in vitro and in vivo activities of OBP-4 against Clostridioides difficile(C.difficile),and the absorption,distribution,metabolism and excretion(ADME)of OBP-4 and the potential effect of OBP-4 on CYP450 enzymes in rats.The main results are as follows:1.The in vitro and in vivo activities of OBP-4 against C.difficile were evaluated by the agar dilution method and C.difficile infection(CDI)mouse model,respectively.The results showed that OBP-4 had good antibacterial activities against C.difficile(MICs,0.25-1 mg/L),and completely inhibited the spore formation of C.difficile(ATCC BAA-1803)at the concentration of 0.5×MIC.In the mouse CDI model,5-day oral administration of 10 mg/kg OBP-4 provided a complete protection for the infected mice with 100%survival.Furthermore,the mice treated with OBP-4 have no CDI recurrence after the stoppage of treatment,while the CDI relapse was observed in the mice treated with vancomycin and cadazolid.This suggested the efficacy of OBP-4 was superior to the comparators in the mouse CDI model.2.A liquid chromatography tandem mass spectrometry(LC-MS/MS)analytical method for the determination of OBP-4 in rat biological samples was established and confirmed.The results showed that this method has good specificity and high sensitivity,and the linear concentation range was 1/2-1000 ng/mL(R~2>0.99).The extraction recovery of OBP-4 in all biological samples was greater than85.2%,and the matrix effect was not obvious.The intra-day and iner-day precisions were less than9.7%,and intra-day and iner-day accuracies were between 9.5%and 9.0%.Moreover,OBP-4 showed good stability under different treatment conditions.3.The absorption,tissue distribution and excretion of OBP-4 in rats were studied with the established LC-MS/MS method.The results showed that OBP-4 presented a relative rapid absorption and elimination,and an extremely low bioavailability after oral administrations of 5 mg/kg,10 mg/kg and 20 mg/kg in rats.Moreover,OBP-4 possessed a nonlinear pharmacokinetic characteristic within the dose range of 5-20 mg/kg.After a single oral administration of OBP-4 at 10 mg/kg in rats,the highest concentration level of OBP-4 was observed in small intestine,followed by stomach,while the heart,liver,lung and kidney have a extremely low OBP-4 exposure.In addition,OBP-4 was not detected in spleen and brain.Following a single oral administration of OBP-4,OBP-4 was mainly excreted from feces in the unchanged form with a recovery of 92.8%,and the recovery of OBP-4 in urine accounted for only 0.015%.4.The in vitro and in vivo metabolic stability of OBP-4 was investigated by a LC-MS/MS method.The results showed that OBP-4 was almost non-metabolized when incubated with rat liver microsome(RLM)in vitro for 90 min.Following a single intravenous administration of OBP-4,only two?phase metabolites M1 and M2 were detected in rat plasma,urine and feces samples.M2 can only be detected in rat feces,while M1 can be found in rat plasma,urine and feces.Furtherore,the relative proportion of M1 in plasma and urine was significantly higher than that of OBP-4.However,OBP-4 was predominant in feces,and the relative proportion of M1 and M2 was very low.5.A rapid and reliable LC-MS/MS analytical method was established for the simultaneous determination of phenacetin,omeprazole,testosterone,tolbutamide and chlorzoxazone in rat plasma,and applied to investigate the effect of OBP-4 on rat cytochrome P450(CYP450)enzymes.The results showed that the low dose of OBP-4 induced CYP1A2 activity,while the high dose of OBP-4 inhibited CYP1A2 activity,but none were statistically significant.Meanwhile,the low and high doses of OBP-4showed slight induction effect on CYP2D1,and a slight inhibitory effect on CYP2C11 and CYP2E1 in a dose-dependent manner.In conclusion,OBP-4 exhibited excellent in vitro and in vivo activities against C.difficile,and a favorable pharmacokinetic feature,which are conducive to the further development of OBP-4 as a promising candidate against CDI.