Protective Effect Of Bicyclol On Drug - Induced Liver Injury And Its Mechanism

I Protective effect of bicyclol on drug induced liver injury and related mechanismsDrug induced liver injury (DILI), which is also called drug induced liver disease, is cuased by drugs or/and their metabolites in the process of drug therapy. The incidence of DILI is increasing with the application of new drugs as well as the drug combination in clinic. To date, approximately 40% of hepatitis and 25% of acute hepatic failure were induced by DILI, which represents 10%-15% of drugs’ adverse reaction.The initiation and development of DILI is a multifactoral process, including various genetic and drug factors, and its pathogenesis is closely related to oxidative stress, cytokine release, mitochondrial dysfunction and apoptosis. Withdrawal of drugs and administration of hepatoprotective agents are effective for the treatment of DILI.Bicyclol is a novel anti-hepatitis drug for the treatment of chronic hepatitis (CH). It has been shown to improve the clinical symptoms and damaged liver function in CH patients, with low rebounding rate and less adverse effects. Previous pharmacological studies indicated that bicyclol had hepatoprotective action against acute and chronic experimental liver injury caused by certain chemical toxins and alcohol. The mechanisms were related to the clearance of reactive oxygen species, improvement of mitochondrial dysfunction, inhibition of immflamatory cytokines and apoptosis etc.Recent clinical data revealed that bicyclol had been used in the treatment of DILI induced by anti-psychotic, anti-tumor and anti-tuberculous drugs. The damaged liver function in patients with DILI was improved by bicyclol administration. However, the mechanisms of the protective effect of bicyclol against DILI are not clear at present. Therefore, liver injury models induced by anti-TB drugss (rifampicin, isoniazid and pyrazinamide) and atorvastatin were established to study the protective effect of bicyclol and related mechanisms, so as to provide experimental evidences for its clinical application1. Protective effect of bicyclol on anti-TB drugs induced liver injury in ratsOral administration of anti-TB drugss (INH,100 mg/kg; RIF,250 mg/kg; PZA, 50mg/kg) for 30 days caused liver injury as evidenced by the elevation of serum ALT, AST, AKP and TBIL levels, and liver pathological changes (increased cellular size, inflammatory cell infiltration and degeneration in hepatocytes), which reflected alterations of DILI in rats. Bicyclol (50,100,200 mg/kg) treatment significantly protected against anti-TB drugs induced liver injury by reducing the elevated serum ALT, AST, AKP and TBIL levels in a dose dependent manner. Moreover, bicyclol markedly improve the liver pathological changes in certain extents.Oxidative stress is one of the major pathogenetic factor in anti-TB drugs induced liver injury. The increasing of liver MDA formation (2.28 fold of control) and decreasing of hepatic GSH content (32.42% of control) were found in rats after administration of anti-TB drugs for 30 days. Treatment with bicyclol (50,100,200 mg/kg) significantly inhibited the elevation of MDA content and of depletion of liver GSH. In addition, a notable change of liver SOD, CAT, and GSH-px was also observed as indicated by the decreasing of enzyme activity (51.92%,53.46%, and 28.89%). Bicyclol (200 mg/kg) significantly inhibited the decrease of SOD, CAT and GSH-px activity.TNF-α and IL-1β are two key inflammatory cytokines in liver injury. Hepatic TNF-a and IL-1β levels were significantly increased (1.72 and 1.99 fold of control) in anti-TB drugs intoxicated rats. Bicyclol (100,200 mg/kg) treatment remarkably inhibited the overexpression of TNF-α and IL-1β indicated by the decreasing of serum and hepatic TNF-α and IL-1β levels.Mitochondrial dysfunction, particularly mitochondrial respiratory chain (MRC) deficiency, plays an importment role in the physiopathology of liver injury. Hepatic MRC I and IV were significantly inhibited by 38.6% and 63.4% in rats after treatment with anti-TB drugs for 30 days, respectively. Treatment of bicyclol (200 mg/kg) can reverse the decreasing of hepatic MRC complex Ⅰ&Ⅳ activity to normal levels. Besides, the isolated mitochondria had a rapid onset of membrane permeability transition (MPT) indicating by the decreasing of the uptake of cationic dye Rodamine 123 and the lowering sensitivity to Ca2+ after anti-TB drugs treatment. Bicyclol (200 mg/kg) remarkably improved anti-TB drugs induced mitochondrial dysfunction mentioned above.Hepatocyte growth factor (HGF) represents the first line of defense against hepatotoxicity. It was found that the hepatic HGF protein expression was increased 1.4-fold in anti-TB drugs intoxicated rats and was further up-regulated by the treatment of bicyclol (1.89 fold of control).CYP2E1, an isoform of CYP450, is known to be involved in the metabolism of isoniazid, and the oxygen species (ROS) formed during the metabolic process has been confirmed to be highly correlated with isoniazid induced hepatotoxicity in both rats and human. In addition, CYP2E1 can be induced by alcohol and several drugs such as rifampicin. Both in vitro and in vivo studies indicated that hepatic CYP2E1 was significantly induced by anti-TB drugs at both activity and protein levels. Co-administration with bicyclol (200 mg/kg) resulted in 30.83% decrease of CYP2E1 activity and prevented the up-regulation of protein expression in certain extent.Pharmacokinetic study in vivo indicated that the AUC0-t and Cmax of isoniazid were slightly decreased in anti-TB drugs treated rats compared with control group, and the reductions were significantly prevented by co-administration of bicyclol (200 mg/kg). Moreover, treatment of bicyclol did not affect the plasma pharmacokinetics of rifampicin, pyrazinamide and pyrazinoic acid in rats.In addition, the influence of bicyclol on the in vitro metabolism of anti-TB drugs was studied and results revealed that:1) Bicyclol (25 μM) did not inhibit the metabolism of rifampicin, isoniazid and pyrazinamide in rat liver microsomes. Furthermore, bicyclol at higher concentrations (50, 100 μM) was found to have inhibitory effects on the metabolism of rifampicin and isoniazid in vitro, however, the concentrations were much higher than the Cmax of efficacy dose.2) Bicyclol (25,50,100μM) has no inhibitory effect on the metabolism of pyrazinamide in rat liver cytoplasm in vitro.3) Bicyclol (25,50,100μM) showed no inhibitory effect on the metabolism of rifampicin and pyrazinamide in human liver microsomes in vitro.The results above suggested that bicyclol did not exhibit significant influence on the in vitro metabolism of rifampicin, isoniazid and pyrazinamide.In conclusion, bicyclol showed significant protective effect on anti-TB drugs induced liver injury indicating by the biochemical makers and pothalogical examinations. The mechanism of hepatoprotection was found to be partly due to the attenuation of oxidative stress, suppression of cytokine overexpression, induction of HGF, inhibition of mitochondrial dysfunction, and modulation of CYP2E1 at both activity and protein levels. No significant influence of bicyclol was found on the in vitro metabolism and in vivo pharmacokinetics of anti-TB drugs.2. Protective effect of bicyclol on atorvastatin induced liver injury in hamsters fed with high fat dietOral administration of atorvastatin (12 mg/kg) to hamsters fed with high fat diet for 9 days caused significant liver injury as evidenced by the elevation of serum ALT, AST and TBIL levels, and liver pathological changes (disordered hepatic lobule structure, liver beam malalignment, narrow liver sinusoid, inflammatory cell infiltration and degeneration in hepatocytes), which reflected alterations of DILI in hamsters. Pretreatment and treatment of bicyclol (50,100,200 mg/kg) significantly protected against atorvastatin induced liver injury by reducing the elevation of serum ALT, AST and TBIL levels in a dose dependent manner. Moreover, bicyclol markedly improve the liver pathological changes in certain extents.The increasing of liver MDA formation (1.47 fold of high fat diet control) and decreasing of hepatic GSH content (44.03% of high fat diet control) were found in hamsters after administration of atorvastatin for 9 days. Pretreatment and treatment with bicyclol (50,100,200 mg/kg) significantly inhibited the elevation of MDA and of depletion of liver GSH contents. In addition, a notable change of liver SOD, CAT, and GSH-px was also observed as indicated by the decreasing of enzyme activity (42.53%, 39.03%, and 35.04% of high fat diet control). Pretreatment and treatment of bicyclol (200 mg/kg) significantly inhibited the decrease of SOD, CAT and GSH-px activitives.The levels of hepatic TNF-α and IL-1β were significantly elevated (4.04 and 8.64 fold of high fat diet control) in atorvastatin intoxicated hamsters fed with high fat diet. Pretreatment and treatment of bicyclol (50,100,200 mg/kg) remarkably inhibited the overexpression of TNF-α and IL-1β indicated by the decreasing of both serum and hepatic TNF-α and IL-1βlevels.Hepatic MRC I and IV were significantly inhibited by 41.61% and 36.06% in rats after treatment of atorvastatin, respectively. Pretreatment of bicyclol (200 mg/kg) can reverse the decreasing of hepatic MRC complex I&IV to normal levels. In addition, the rapid onset of MPT with decreased uptake of cationic dye Rodamine 123 and lower sensitivity to Ca2+ were also found in isolated mitochondria after atorvastatin treatment. Pretreatment and treatment of bicyclol (200 mg/kg) remarkably improved atorvastatin induced mitochondrial dysfunction as mentioned above.An atorvastatin induced cell injury model was established for investigating the effect of atorvastatin on cell apoptosis in the present study using HepG2 cells. The results were summarized as follows:1) Addition of atorvastatin (100μM) induced the cell injury as evidenced by the decrease of cell viability (48.7% of control), and cellular morphology changes (cell shrinkage and nucleus swelling). Treatment of bicyclol (1,50,100 μM) significantly inhibited the decreasing of cell viability in a dose dependent manner. Moreover, bicyclol markedly improve the cellular morphology changes in certain extents.2) The activity of Caspase-3/7, which is a terminal protein-cutting enzyme involved in the apoptosis of cell, was significantly elevated (1.51 fold of control) in HepG2 cells treated with atorvastatin (100 μM). Bicyclol (100 μM) significantly inhibited the increase of Caspase-3/7 activity.3) The results from the examinations of cell mitochondrial membrane potentials assessed by JC-1 and AO/EB double fluorescence staining revealed early stage of apoptosis of intoxicated HepG2 cells. Bicyclol at 100 μM can significantly reversed the above markers, repectively.4) The protein expression of cytoplasm RhoA was remarkably increased (1.86 fold of control) in HepG2 cells after treatment of atorvastatin (100 pM), and the activity of cellular RhoA was decreased (48.46% of control) correspondingly. Treatment with bicyclol (100 μM) significantly inhibited the elevation of RhoA protein expression in cytoplasm and decreasing of cellular RhoA activity.The results above indicated that bicyclol had significant protective effect on atorvastatin induced liver injury in hamsters fed with high fat diet and HepG2 cell injury. The mechanism of hepatoprotection was found to be partly due to the attenuation of oxidative/nitrative stress, suppression of cytokine overexpression, inhibition of mitochondrial dysfunction and apoptosis, and modulation of RhoA at both activity and protein levels.In conclusion, bicyclol had a notable protective effect on DILI caused by anti-TB drugs and atorvastatin, indicating by the significant inhibition on the elevation of serum transaminase, AKP and TBIL, and histopathological changes. In addition, bicyclol markedly inhibited the HepG2 cell apoptosis induced by atorvastatin. The possible mechanisms included:1. Regulation on metabolizing enzyme:inhibition of CYP 2E1 activity and protein expression.2. Inhibition of oxidative stress:attenuation on the lipid peroxidation and depletion of GSH content, and improvement of the activity of antioxidant enzymes including SOD, GSH-px, and CAT.3. Inhibition of nitrosative stress:reducing the elevation of NO content, and inhibiting the activity of iNOS and TNOS.4. Modulation on inflammatory cytokines:down-regulation of overexpression of TNF-α and IL-1β.5. Attenuation of mitochondrial injury:Protection on the mitochondrial membrane integrity and normal membrane potential, as well as MRCⅠ and MRC Ⅳ activity.6. Regulation on expression of apoptosis-related and hepatic protective proteins: increasing the activity of cellular RhoA, and up-regulate hepatic HGF.With the results mentioned above, the present study will provide valuable experimental evidences for the further investigation on the hepatoprotective effect of bicyclol and the possibility of clinical application in the treatment of DILI.II Interaction of the metabolite of F18 (M3), a anti HIV compound and UGTs in vitroF18, a synthesized natural product by chemical modification and structural transformation, is a novel anti-HIV compound. The anti-HIV activity and therapeutic index of F18 are more than 10 times higher than that of original natural product and its activity is comparable with that of marked drug-Nevirapine. It was notable that F18 showed inhibitory effect on Y181C (not sensitive to Nevirapine), which is the major clinical resistant strain, with ECso less than 1nM. The toxicity of F18 was relatively lower based on the cute toxicological study. F18 could be potentially used for the patients afflicted with HIV in clinical trial.In our previous study, four phase Ⅰ metabolites of F18 were detected in both human liver microsomes (HLMs) and rat liver microsomes (RLMs) in vitro. M3, the dechlorination and hydroxylation product of F18, was the most abundant primary metabolite in both RLMs and HLMs. It was found to subsequently undergo conjugation through UGTs pathway to produce M3-0-glucuronide (M3-G) after oral administration of F18. In this study, we aimed to identify the UGT isozymes involved in the metabolism M3 and to investigate the inhibitory effects of M3 on the activities of human UGTs using HLMs and recombinant human UGTs. The interactions of M3 with UGTs substrates (MPA and AZT) that would be frequently used in combination with F18 were also evaluated in vitro.The results were summarized as follows:1) UGT1A1 was the major isoform catalyzing the glucuronidation of M3, while UGT1A4, UGT1A9, and UGT2B7 provided less contribution in vitro.2) M3 significantly inhibited the glucuronidation of propofol and AZT (P<0.01) in HLMs at both 10 and 100 μM, which reflected its inhibitory effect on UGT1A9 and UGT2B7. Otherwise, less inhibition of M3 (100 μM) on UGTIA1 (23.9%) and UGT1A4 (17.5%) was observed in HLMs. In addition, the inhibition of M3 on recombinant human UGT1A9 and UGT2B7 was much more remarkable than that in HLMs. M3 has no significant inhibitory effect on recombinant human UGT1A3 and UGT1A6.3) M3 showed potent inhibition on MPA glucuronidation with IC50 value of 0.39± 0.06 μM or 0.58 ±0.14 μM in the presence or absence of 2% BSA, respectively. Further inhibition kinetic study revealed a mixed inhibition of M3 on MPA glucuronidation with Ki value of 16.60 ±2.31 μM without BSA. Addition of BSA (2%) to the incubations resulted in 92.83% reduction of Ki value. Nevertheless, MPA had no effect on M3 glucuronidation in HLMs.4) Both AZT and M3 showed inhibition on the glucuronidation each other by a "mixed-type" mechanism with Ki of 902.0 ±21.1 μM for AZT on M3 glucuronidation and 16.8±1.2 μM for M3 on AZT glucuronidation, respectively.In summary, our results demonstrated that glucuronidation of M3 was primarily mediated by UGT1A1. M3 exhibited potent inhibitory effects on UGT1A9 and 2B7 at activitive levels in vitro. Furthermore, M3 could potentially interfere with the hepatic glucuronidation of MPA and AZT via the inhibition of UGT1A9 and 2B7, which provides the basis for further clinical studies on DDI potential of M3 in vivo.