Primary Preclinical Pharmacology Study Of ZTW-41,A Novel Anti-MRSA Compound

BackgroundMethylillin-resistant Staphylococcus aureus(MRSA)is one of the most common drug-resistant bacteria in the clinic.It has strong toxicity,wide spectrum of drug resistance and fast transmission speed,which bring severe serious challenges to clinical treatment.Previous studies have shown that the 9-bromo substituted indolizinoquinoline-5,12-dione derivative-ZTW-41 has strong anti-MRSA activity,which was superior to vancomyc:in.However,there are still few pharmacodynamic studies on ZTW-41.The pharmacokinetics and safety evaluation of ZTW-41 have not been reported yet.In view of its good pharmacodynamic activity,further pharmacological studies of ZTW-41 is necessary.This article intends to study the preclinical pharmacology of ZTW-41,including pharmacodynamics,pharmacokinetics and preliminary safety evaluation,and thus lay the foundation for the development of a new generation of anti-MRSA drug.MethodsThe minimum inhibitory concentration(MIC)of ZTW-41 was determined by micro-broth dilution method.The minimum bactericidal concentration C(MBC)and the time-kill curves were determined by colony counting method.Also,the MIC values in different culture conditions were determined.The in vivo antibacterial activity of ZTW-41 was evaluated using a mouse sepsis systemic infection model.The concentration of ZTW-41 in rat plasma was determined by established LC-MS/MS analysis method,and the pharmacokinetic parameters were calculated by WinNonlin software.The protein binding rate of ZTW-41 was eveluated by rapid equilibrium.The toxic effect of ZTW-41 on HepG2 cells was determined by CCK-8 method.The acute toxicity in mice was observed after intraperitoneal injection.The effect of ZTW-41 on the nervous system in mouse and the respiratory and cardiovascular system.in rats were also determined.ResultsPharmacodynamics:ZTW-41 was found to be highly active against Gram-positive pathogens,however,Gram-negative bacteria were not susceptible to ZTW-41.The MIC50 value of ZTW-41 against methicillin-resistant Staphylococcus aureus(MRSA,n=200),methicillin sensitive Staphylococcus aureus(MSSA,n=100),Enterococcus faecalis(E.faecalis,n=32)and Enterococcus faecium(E.faecium n=32)were 0.125、0.063、0.063 and 0.016μg/mL,respectively.The MBC50 values for ZTW-41 to MRSA,MSSA,E.faecalis and E.faecium were 0.5、0.25、0.25 and 4 μg/mL,respectively.Overall,ZTW-41 was the most potent agent against 364 clinical Gram-positive bacteria tested compared to vancomycin,oxacillin,ciprofloxacin,levofloxacin,moxifloxacin,linezolid and ampicillin.The bactericidal activity of ZTW-41 was stronger than vancomycin,oxacillin and ampicillin.The in vitro time-kill experiments suggested that ZTW-41 had bactericidal activity against MRSA,MSSA strains and had bacteriostatic activity agninst E.faecalis strains.Different medium pH,bacterial inoculum and serum protein concentration could affect the antibacterial activity of ZTW-41.The ED50 of ZTW-41 was 6.59 mg/kg.Our results indicated that the efficiency was comparable to vancomycin.Pharmacokinetics:A simple and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was developed and validated for quantitation of ZTW-41 in rat plasma.The validated method has been successfully applied to a pharmacokinetic study of ZTW-41 in rats following intravenous administration at the dose of 20、40 and 80 mg·kg-1.The reslts showed that the half-life of ZTW-41 were 15.92±7.76、15.17±3.67and 6.40±2.34 h,respectively.The protein binding rate of ZTW-41 in rat and human plasma was 97.87%and 97.80%,respectively at 2000 ng/mL,which indicated ZTW-41was a high protein binding rate drug.Preliminary safety assessment:The IC50 values of ZTW-41 for HepG2 cells was 8.97 μg/mL,ZTW-41 showed good selectivity indices(SI)ranging from 1.12-71.76 against clinical isolates,demonstrating excellent therapeutic selectivity in MRSA,MSSA and E.faecalis strains.ZTW-41 did not show significant weight loss,death or signs of toxicity in the dose range of 50 mg/kg after administering intraperitoneally in Kunming mice during 14-days experient.ZTW-41 had no significant effect on the spontaneous activity of mice at a single dose of 6,20and 60 mg/kg,and had no significant effect on the coordination ability of mice.Except for the high dose of 60 mg/kg and the sub-hypnotic dose of pentobarbital sodium,there was obvious synergistic hypnosis effect.The other dose groups had no obvious synergistic hypnotic effect with the sub-hypnotic dose of pentobarbital sodium.Significant changes in systolic blood pressure,diastolic blood pressure,mean arterial pressure,and heart rate were observed in a single intraperitoneal injection of 30 mg/kg ZTW-41when there were no significant changes observed in 3 and 10 mg/kg group.3,10 and 30 mg/kg intraperitoneal injection of ZTW-41 had no significant effect on respiratory rate in mice.Conclusion1.Pharmacodynamic studies showed that ZTW-41 had good antibacterial activity against Gram-positive bacteria in vitro and in vivo.2.A rapid,sensitive,accurate and reliable LC-MS/MS method was established to determine the concentration of ZTW-41 in rat plasma.The validated method has been successfully applied to a pharmacokinetic study of ZTW-41 in rats.3.In vitro experiments showed that ZTW-41 was relatively safe.In vivo experiments suggested that ZTW-41 had certain toxic effect on the central nervous system and cardiovascular system.