The role of the immune system in nevirapine induced hepatotoxicity in a rat model

English: Nevirapine is associated with hypersensitivity reactions such as skin rash and hepatotoxicity, hampering its use for HIV prophylaxis. The mechanism of nevirapine induced hepatotoxicity remains unknown and was postulated to be immune mediated. As recently reported, several drugs have shown to induce toxicity by immune activation. Therefore, the aim of this study was to determine the role of the immune system in nevirapine induced hepatotoxicity. A high performance liquid chromatography method for the determination of nevirapine in rat plasma was developed. It involved protein precipitation with perchloric acid, followed by solid phase extraction on C18 cartridges. The mobile phase was tetraethylammoniumphosphate (TEAP) buffer and acetonitrile (60:40, v/v) and was run over a Luna C18 (4.60x150 mm) 5μ analytical column at 1 ml/min. The eluent was detected by UV at 210 nm. Nevirapine and chlorzoxazone eluted at 2.6 and 5.2 minutes, respectively. The average 5 day calibration curve (0 – 10 μg/ml) was linear with a regression equation of y=0.012x+0.051, and the correlation coefficient (r2) was 0.9985. The method was used to measure nevirapine concentrations in rat plasma. The animal experiment consisted of an acute and chronic phase. During the acute phase male Sprague-Dawley rats were divided into 3 groups of 10 rats each. Groups were dosed as follows: S+NVP, LPS+S and LPS+NVP. From each group, 5 rats were sacrificed at 6 and 24 hours after dosing. During the chronic phase, animals were divided into 2 groups of 15 rats each, to which nevirapine was administered daily. From each group, 5 rats were administered with LPS or saline, just before the daily nevirapine dose on days 7, 14 and 21. The animals were sacrificed 24 hours later. Blood was tested for ALT, full blood count, IL-2, IFN-γ, TNF-α and nevirapine concentrations. Liver sections were sent for histopathology testing. In the acute phase, the S+NVP group exhibited increased ALT at 6 and 24 hours. Liver injury was characterised by mild cloudy swelling (6 hours), and hepatocellular vacuolar degeneration (24 hours). Although LPS+S led to increased ALT at 6 hours, which normalised by 24 hours, abnormal liver histology was observed on both occasions as swollen cytoplasm and narrow sinusoids. The LPS+NVP group showed increased ALT at 6 hours, which returned to normal by 24 hours. Interestingly, liver histology was normal on both occasions, indicating that LPS+NVP prevented hepatotoxic effects of either drug. Whereas all three groups caused increased IL-2, it was higher in the LPS+NVP group, implying that LPS was responsible for the increase during the acute phase. Nevirapine concentrations at 6 hours were higher in the S+NVP group, but by 24 hours they were higher the LPS+NVP group. During the chronic phase, S+NVP caused liver injury on days 7 and 14 as demonstrated by increased ALT with centrilobular hepatocellular degeneration on day 7. However, by day 14 and 21 the ALT had normalised and liver histology was unaffected. Whereas the LPS+NVP group exhibited increased ALT on days 7 and 14, the corresponding liver histology was normal on all three occasions, illustrating further that LPS attenuates nevirapine induced hepatotoxicity. By day 21, S+NVP caused profound lymphocytosis, while LPS+NVP caused neutrophilia. S+NVP and LPS+NVP exhibited increased IL-2 and IFN-γ, but both cytokines were higher in the S+NVP group. TNF-α was elevated in both groups on day 7, and thereafter fell, remaining higher in the LPS+NVP group. Nevirapine concentrations were higher in the LPS+NVP group than in the S+NVP group. In conclusion, it was demonstrated that nevirapine is a slow onset immune stimulant, which caused nevirapine induced hepatotoxicity, and LPS prevented the hepatotoxicity. These observations suggest that immune manipulation may help to prevent nevirapine induced hepatotoxicity.