The Investigation Of The Liver Injury Associated With Pyrazinamide And The Effects Of Intestinal Flora On The Metabolism Of Pyrazinamide

OBJECTIVES:1.Investigation of the liver injury associated with pyrazinamide（PZA）and the effects of the amidase inhibitor bis（p-nitrophenyl）phosphate（BNPP）on the liver injury associated with PZA.2.Development and validation of liquid chromatography-tandem mass spectrometry（LC-MS/MS）method for simultaneous determination of pyrazinamide（PZA）and its metabolite pyrazinoic acid（POA）in culture medium to investigate the effects of rat intestinal flora on the in vitro metabolism of PZA.3.Development and validation of LC-MS/MS method for simultaneous determination of PZA and POA in rat plasma to investigate the effects of rat intestinal flora on the pharmacokinetic parameters of PZA and POA.METHODS:1.30 rats were randomly divided into the control group and 4 test groups（A1,A2,B1 and B2）,with 6 rats in each group,half male and half female.Among them,groups A1 and A2 were used for the investigation of the liver injury associated with PZA,and groups B1 and B2 for the investigation of the effects of amidase inhibitor on the liver injury associated with PZA.The control group was given with normal saline;Group A1 with isoniazid（INH）,ethambutol（EMB）,rifampicin（RIF）and PZA;Group A2 with INH,EMB and RIF;Group B1 with the large dose of PZA;Group B2 with BNPP and the large dose of PZA,by intragastric administration once a day for 8 weeks.Then,the liver index,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and alkaline phosphatase（ALP）levels in serum of the rats were examined.2.The LC-MS/MS method for the simultaneous determination of PZA and POA in the culture medium was developed using phenacetin as internal standard（IS）.The separation was performed on Agilent ZORBAX SB-Aq column by gradient elution with 0.2%formic acid（containing 6 mmol/L ammonium acetate）-methanol at the flow rate of1.2 ml/min.The column temperature was 40℃.And the injection volume was 10μl.The determination was performed with an electrospray ionization（ESI）source in positive mode（500℃）and a multiple reaction monitoring（MRM）.PZA was co-cultured with intestinal flora incubation solution of normal rats,inactivated intestinal flora incubation solution of rats and intestinal flora incubation solution of pseudo germ-free rats at37℃in an anaerobic environment in vitro.Concentrations of PZA and POA in the culture medium were determined by the validated LC-MS/MS method.3.The LC-MS/MS method for the simultaneous determination of PZA and POA in rat plasma was developed using phenacetin as IS.The separation was performed on Agilent ZORBAX SB-Aq column by gradient elution with 0.2%formic acid（containing 8 mmol/L ammonium acetate）-methanol（gradient elution）at the flow rate of 1 ml/min.The column temperature was 30℃,and the injection volume was 10μl.The determination was performed with an ESI source in the positive mode（500℃）and an MRM.16 rats were randomly divided into two groups,with 8 rats in each group,half male and half female.While one group was given mixed antibiotics（streptomycin sulfate+neomycin sulfate）intragastrically to construct pseudo germ-free rat model,the remaining one was given with normal saline.After modeling,both groups were given pyrazinamide intragastrically（150 mg/kg）.Blood samples were collected at different collection time points.Concentrations of PZA and POA in the rat plasma were determined by the validated LC-MS/MS method.The pharmacokinetic parameters were calculated and compared by using DAS 2.1.1 software.RESULTS:1.Compared with the control group,both ALT and AST levels in test group A1,A2,B1 and B2 increased.Both ALT and AST levels in group A1 were significantly higher than those in group A2（P<0.05）.The liver injury in group B2 was significantly improved compared with that in group B1（P<0.05）.2.The linearity ranges of PZA and POA were 50–5000 ng/ml（r=0.9997）and 200–12500 ng/ml（r=0.9961）.The lower limits of quantitation（LLOQ）were 50 and 200 ng/ml,respectively.Intra-batch and inter-batch accuracy were 93.07%–103.70%,and relative standard deviations（RSDs）of intra-batch and inter-batch precision and matrix effect tests were all lower than or equal to 7.15%（n=6 or n=3）.The concentration of PZA decreased significantly in the intestinal flora incubation solution of normal rats,while the concentration of its metabolite POA increased significantly.However,there was no significant change in the concentration of PZA either in inactivated rat intestinal flora incubation solution or pseudo germ-free rat intestinal flora incubation solution,and only a small amount of POA was determined in the system.3.The linear ranges of PZA and POA were 25–5000 ng/ml（r=0.9976）and 100–12500 ng/ml（r=0.9990）.The LLOQ were 25 and 100ng/ml,respectively.Intra-batch and inter-batch accuracy were 92.93%–100.50%,and RSDs of intra-batch and inter-batch precision and matrix effect tests were all lower than or equal to 8.42%（n=6 or n=3）.Compared with the normal rats,time to peak concentration(t_max)of PZA in the pseudo germ-free rats was prolonged significantly（P<0.01）,area under the curve（AUC）and half-life(t_1/2)of PZA was increased,and the AUC of POA was decreased,but the difference was not significant（P>0.05）.CONCLUSIONS:1.PZA can induce strong liver injury in rats,amidase inhibitors can decrease liver injury associated with PZA.2.PZA can be converted to POA by rat intestinal flora in vitro.3.The change of intestinal flora in rats delays the absorption of PZA and may affect the in vivo exposure of PZA and POA.