Cytochrome P450 and the immune response to prolonged administration of isoniazid, nevirapine and paracetamol in a rat model

[摘要] English: Drug-induced liver injury is a major adverse drug reaction that presents duringisoniazid (INH) and nevirapine (NVP) treatment, and after paracetamol (PAR)overdose. It was postulated that after these drugs are metabolised, reactivemetabolites are formed which attack cellular proteins and result in the formation ofantigenic metabolite-protein adducts. Subsequently, the immune system starts toeliminate hepatocytes expressing these adducts, and this leads to the developmentof overt drug-induced liver injury. As such, the effect of prolonged administration ofINH, NVP and PAR on the cytochrome P450 (CYP450) and immune response wasinvestigated here.A high performance liquid chromatography (HPLC) method for the simultaneousdetermination of INH, NVP and PAR in plasma was developed. Sample preparationinvolved protein precipitation with zinc sulphate and methanol, followed by solidphase extraction. The mobile phase was 0.06% trifluoroacetic acid (A) andacetonitrile (B) and run by a gradient programmer over a C18 (4.60 x 250 mm) 5 μanalytical column at 1 ml/min, while the eluent was detected by UV at 260 nm. INH,PAR, internal standard and NVP eluted at retention times of 3.1, 9.9, 10.6 and 11.6minutes, respectively. The average 5 day calibration curves of INH, NVP and PARwere linear with regression equations and correlation coefficients of y = 0.029x +0.025 (r2 = 0.9954), y = 0.043x + 0.127 (r2 = 0.9968) and y = 0.097x + 0.070 (r2 =0.9997), respectively. The method was used successfully to monitor INH, NVP andPAR in rat plasma.The CYP450 and immune response to prolonged administration of INH, NVP andPAR were investigated using an SPD rat model. For each drug, the animalexperiment was divided into three phases. In phase I, rats were orally administeredsaline solution (S), INH (20 mg/kg), NVP (200 mg/kg) or PAR (500 mg/kg), while forphase II, rats received S, INH, NVP or PAR in combination with an immunestimulant, levamisole (LMS; 2.5 mg/kg), and lastly, during phase III, rats received S,INH, NVP or PAR along with a CYP450 inducer, carbamazepine (CBZ; 60 mg/kg). Ineach phase, five rats per group were sacrificed after 2, 7, 14, 28 and 42 days. Blood was analysed for full blood count, CD4 and CD8 counts, liver function, renal function,IL-2, IL-10, IgG, IgM and drug concentrations. A piece of liver was sent forhistopathology testing, and the activity of rat CYP1A2, CYP2E1 and CYP3A2 wereanalysed.During administration of the test drugs alone, both INH and NVP triggered an earlyTh1 immune response that was associated with liver injury, and counteracted by alater Th2 immune response which was associated with healing. Overall, the liverinjury correlated with low concentrations of NVP, but high INH concentrations. Thatsaid, INH increased CYP2E1 activity, while NVP increased that of CYP3A2.When LMS (immune stimulant) was co-administered, INH liver injury wasexacerbated, while for NVP it was the same. Again, INH and NVP provoked a Th1response (injury) that was counteracted by a Th2 response (healing). Here, the liverinjury was also associated with low NVP concentrations, and high INHconcentrations.During co-administration of CBZ (CYP450 inducer), INH and NVP caused the sameimmune response, and resulted in improvement of the liver injury. Again, the liverinjury was associated with low NVP concentrations, and high INH concentrations.Also, INH increased CYP2E1 activity and NVP increased CYP3A2 activity, but not tothe same extent as the test drugs alone.PAR did not exhibit a distinct pattern of immune response by which to associate itwith the liver injury, most probably because the concentrations were too low forgeneration of toxic metabolites.In conclusion, the pattern of immune response to prolonged administration of INHand NVP shows that the immune system is involved in the drug-induced liver injury,probably as a protective buffer to prevent further drug toxicity.

[发布机构] University of the Free State