Sanja Dragovic

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

Drug‐induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF‐α). HepG2 cells were treated with diclofenac followed by TNF‐α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF‐α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF‐α‐mediated enhancement involved activation of caspase‐3 through a caspase‐8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c‐Jun N‐terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF‐α‐induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF‐α‐mediated nuclear factor kappaB (NF‐κB) translocation oscillation in association with reduced NF‐κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF‐α‐induced phosphorylation of the inhibitor of NF‐κB alpha (IκBα). Finally, inhibition of IκB kinase β (IKKβ) with BMS‐345541 as well as stable lentiviral short hairpin RNA (shRNA)‐based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF‐α‐induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac‐mediated stress signaling suppresses TNF‐α‐induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;)

AMAP, the alleged non-toxic isomer of acetaminophen, is toxic in rat and human liver.

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.

Human Precision-Cut Liver Slices as an ex Vivo Model to Study Idiosyncratic Drug-Induced Liver Injury

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Because of the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict IDILI in humans. According to the inflammatory stress hypothesis, the effects of inflammation interact with the effects of a drug or its reactive metabolite, precipitating toxic reactions in the liver. As a follow-up to our recently published mouse precision-cut liver slices model, an ex vivo model involving human precision-cut liver slices (hPCLS), co-incubated for 24 h with IDILI-related drugs and lipopolysaccharide (LPS), was developed to study IDILI mechanisms related to inflammatory stress in humans and to detect potential biomarkers. LPS exacerbated the effects of ketoconazole and clozapine toxicity but not those of their non-IDILI-related comparators, voriconazole and olanzapine. However, the IDILI-related drugs diclofenac, carbamazepine, and troglitazone did not show synergistic toxicity with LPS after incubation for 24 h. Co-incubation of ketoconazole and clozapine with LPS decreased the levels of glutathione in hPCLS, but this was not seen for the other drugs. All drugs affected LPS-induced cytokine release, but interestingly, only ketoconazole and clozapine increased the level of LPS-induced TNF release. Decreased levels of glutathione and cysteine conjugates of clozapine were detected in IDILI-responding livers following cotreatment with LPS. In conclusion, we identified ketoconazole and clozapine as drugs that exhibited synergistic toxicity with LPS, while glutathione and TNF were found to be potential biomarkers for IDILI-inducing drugs mediated by inflammatory stress. hPCLS appear to be suitable for further unraveling the mechanisms of inflammatory stress-associated IDILI.

Role of Human Glutathione S-Transferases in the Inactivation of Reactive Metabolites of Clozapine

The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.

Characterisation of human cytochrome P450s involved in the bioactivation of clozapine

Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized. The results of the present study show that 7 of 14 recombinant human P450s were able to bioactivate clozapine to a glutathione-reactive nitrenium ion. CYP3A4 and CYP2D6 showed the highest specific activity. Enzyme kinetical characterization of these P450s showed comparable intrinsic clearance of bioactivation, implicating that CYP3A4 would be more important because of its higher hepatic expression, compared with CYP2D6. Inhibition experiments using pooled human liver microsomes confirmed the major role of CYP3A4 in hepatic bioactivation of clozapine. By studying bioactivation of clozapine in human liver microsomes from 100 different individuals, an 8-fold variability in bioactivation activity was observed. In two individuals bioactivation activity exceeded N-demethylation and N-oxidation activity. Quinidine did not show significant inhibition of bioactivation in any of these liver fractions, suggesting that CYP2D6 polymorphism is not an important factor in determining susceptibility to hepatotoxicity of clozapine. Therefore, interindividual differences and drug-drug interactions at the level of CYP3A4 might be factors determining exposure of hepatic tissue to reactive clozapine metabolites.

Role of residue 87 in the activity and regioselectivity of clozapine metabolism by drug-metabolizing CYP102A1 M11H: application for structural characterization of clozapine GSH conjugates.

In the present study, a site-saturation mutagenesis library of drug-metabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by 1H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.

Evidence-based selection of training compounds for use in the mechanism-based integrated prediction of drug-induced liver injury in man

The current test systems employed by pharmaceutical industry are poorly predictive for drug-induced liver injury (DILI). The ‘MIP-DILI’ project addresses this situation by the development of innovative preclinical test systems which are both mechanism-based and of physiological, pharmacological and pathological relevance to DILI in humans. An iterative, tiered approach with respect to test compounds, test systems, bioanalysis and systems analysis is adopted to evaluate existing models and develop new models that can provide validated test systems with respect to the prediction of specific forms of DILI and further elucidation of mechanisms. An essential component of this effort is the choice of compound training set that will be used to inform refinement and/or development of new model systems that allow prediction based on knowledge of mechanisms, in a tiered fashion. In this review, we focus on the selection of MIP-DILI training compounds for mechanism-based evaluation of non-clinical prediction of DILI. The selected compounds address both hepatocellular and cholestatic DILI patterns in man, covering a broad range of pharmacologies and chemistries, and taking into account available data on potential DILI mechanisms (e.g. mitochondrial injury, reactive metabolites, biliary transport inhibition, and immune responses). Known mechanisms by which these compounds are believed to cause liver injury have been described, where many if not all drugs in this review appear to exhibit multiple toxicological mechanisms. Thus, the training compounds selection offered a valuable tool to profile DILI mechanisms and to interrogate existing and novel in vitro systems for the prediction of human DILI.

From Face-to-Face training to blended learning in the postgraduate program SafeSciMET : a case study

&NA; This commentary concerns a postgraduate education and training initiative within the pharmaceutical sciences with the purpose to inspire future course organizers. It reports on the experiences obtained from preparing and evaluating two blended learning courses organized under the European Modular Education and Training Programme SafeSciMET. The blended courses included an eLearning part, followed by a face‐to‐face part of 2.5 days involving interactive case studies, and an examination. The purpose was to reduce the time the students spent away from work and home by 2 days. The feedback from the students and teachers is presented.