Drug Metabolism Letters - Volume 4, Issue 1, 2010

A concrete study is still fragmentary to correlate the effect of Nandrolone decanoate, a anabolic steroid, on the cholanthrene induced Glutathione-s-transferases (GST) activities in liver tissues. Administration of Nandrolone and 3- Methylcholanthrene in alone and combination is found to increase the activity of hepatic GST activity significantly (p< 0.01) suggesting Nandrolone as a potent inducer of GST activity.

A 47-year-old woman ingested an overdose of 8000 mg quetiapine. The treatment had been initiated 3 weeks before. The current medications were lamuvidine, ritonavir, atazanavir and tenofovir for an HIV infection. The patient presented a deep coma and sustained hypotension as main complications. The toxicokinetic data revealed a markedly prolonged elimination half-life for quetiapine (62.4 hours) and the relationship with antiretroviral therapy is discussed.

Desethylamiodarone was reported to inactivate human CYP1A1. To assess this, two protocols were implemented employing dilution and non-dilution of the preincubation mixture. Inactivation studies performed with diluted preincubation mixtures showed no inactivation of CYP1A1 by desethylamiodarone. However there was evidence for a mixed competitive inhibition (competitive and the uncompetitive inhibition constants of 2.1 μM and 9.6 μM, respectively) of CYP1A1 by desethylamiodarone. NADPH addition and/or replenishment were found to be important factors in determining the control activity in inactivation studies.

The aim of this study was to assess the impact of a P-glycoprotein and CYP3A inhibitor, verapamil on the pharmacokinetics of two methylxanthines, pentoxifylline and lisofylline in male CD-1 mice. To differentiate the effects of verapamil, both methylxanthines were also given to male CF-1 mdr1a (-/-) and mdr1a (+/+) mice. CD-1 mice received a single dose (50 mg/kg) of pentoxifylline or lisofylline intravenously, whereas mutant animals were given the same dose of both compounds intravenously and orally. Blood and tissue samples were collected at different time points following drug administration and concentrations of pentoxifylline and lisofylline were measured by a chiral HPLC method. Verapamil significantly increased concentrations of both methylxanthines in murine serum and tissues. In contrast to lisofylline, pentoxifylline concentrations were also significantly higher in mutant mice 30 min following intravenous administration. Due to the fact that pentoxifylline is not a good P-glycoprotein substrate, a possible mechanism of this interaction might be that in the presence of verapamil, pentoxifylline elimination is inhibited by its metabolites that are normally eliminated through P-glycoprotein-mediated transport. This hypothesis was supported by the outcomes of pharmacokinetic analysis. In conclusion, the interaction between verapamil and pentoxifylline is, at least partially, P-glycoprotein-mediated, whereas alterations in lisofylline pharmacokinetics are caused by inhibition of drug metabolising enzymes.

The results of an evaluation study of ondansetron binding to human cytochromes P450 CYP3A4 and CYP2D6 is reported. The methodology includes NMR spectroscopic measurements of substrate to heme iron distances together with molecular modelling of the enzyme-substrate interactions. It is shown that there is a generally good agreement between the experimental and calculated binding affinities for ondansetron towards CYP2D6 and CYP3A4 enzymes, based on interactive docking studies. Moreover, the modelled binding orientations for ondansetron in CYP2D6 and CYP3A4 are largely consistent with the NMR data and with the known routes for P450-mediated metabolism of this compound.

The present manuscript describes the development of a cell-based reporter transcriptional activation assay for evaluating induction of UGT1A1. A reporter construct (pGL-UGT1A1-Luc) encompassing the proximal promoter (nucleotide -254 to +38) and distal enhancer (-3483 to -3194) regions of the human UGT1A1 gene was generated by PCR cloning, and co-transfected with a previously generated PXR construct (pSG5-PXR) into HepG2 cells. The system was then validated using known ligands of PXR, rifampicin (RIF), clotrimazole (CLOT) sulfinpyrazone (SPZ) and phenobarbital (PB), which produced dose dependent induction of UGT1A1 luciferase activity by 4.4, 5.3, 4.7 and 3.7 fold, respectively, relative to the vehicle control, 0.1 % dimethylsulfoxide (DMSO). Aryl hydrocarbon receptor (AhR) ligands α- naphthoflavone (α-Naph), β-naphthoflavone (β-Naph) and 3-methylchloranthene (3-MC) increased UGT1A1 luciferase activity in a concentration dependent manner resulting in 17.2, 11.3 and 6.1 fold, respectively, at their highest concentrations, suggesting that endogenous AhR is also involved in the regulation of the UGT1A1 reporter construct in HepG2 cells. For comparison with transcriptional regulation of endogenous UGT1A1, 10 μM RIF, 50 μM SPZ, 10 μM CLOT, 4 μM 3-MC, 10 μM β-Naph and 25 μM α-Naph also induced UGT1A1 mRNA in human primary hepatocytes by 2.5, 2.8, 3.2, 3.7, 3.9 and 4.3 fold, respectively. In summary, by co-transfecting the UGT1A1 reporter and PXR constructs into HepG2 cells, we have developed a cellular model for evaluating induction of UGT1A1. Data from the reporter gene assay correlated with that generated in human primary hepatocytes. Based on these data, we suggest that this reporter gene assay can be used as a screening tool in the early stages of drug discovery, to evaluate potential induction of UGT1A1 by new chemical entities and to aid in lead selection and optimization.

Chemosensitization of HepG2 cells to doxorubicin by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol neither impinged on downregulation of P-glycoprotein expression nor on severe impairment of its activity. Moreover, differently from verapamil, a potent P-glycoprotein inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitized HepG2 cells in a fashion that was insensitive to the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone. At concentrations exceeding the one employed for chemosensitization, 1-phenyl-2-decanoylamino-3- morpholino-1-propanol was by itself strongly toxic to HepG2 cells, and also this effect was insensitive to the pancaspase inhibitor. These results suggest that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, at subtoxic concentrations, might synergize with scarcely toxic doxorubicin doses to propagate a caspase-independent cytotoxic response, such that P-glycoprotein-dependent drug resistance is circumvented.

Currently used combinations of anti-HIV drugs, known as Highly Active Antiretroviral Therapy (HAART), have considerably reduced the mortality in patients with AIDS. However, HAART medications such as efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) often cause adverse drug-drug interactions (DDIs) that result from changes in the expression and activity of drug metabolizing enzymes. Since EFV is most commonly used with ATV and RTV, the known CYP inhibitors, we evaluated the effects of combinations of these agents on the CYP3A4 induction by EFV. We determined the induction of CYP3A4 by EFV, RTV, ATV, EFV+RTV, EFV+ATV, EFV+RTV+ATV and rifampicin (RIF) employing primary human hepatocytes from three donors. Also, concentration dependent activation of human Pregnane X Receptor (hPXR) which is a key transcriptional regulator of CYP3A4 by EFV, RIF and RTV was estimated in transiently transfected LS180 cells. CYP3A4 activity (testosterone-6β-hydroxylation) was induced by EFV (3 fold) and RIF (4 fold), but was significantly suppressed in the presence of RTV and ATV. All treatments significantly induced the CYP3A4 transcripts (3-25 fold) as quantitated by RT-PCR. hPXR activation data in LS180 cells were consistent with the induction of transcripts and the estimated EC50 values were 0.87 μM, 0.44 μM and 3.7 μM for RIF, RTV and EFV, respectively. However, in primary hepatocytes the net effect was suppression of EFV mediated CYP3A4 induction by RTV and ATV. This observation corresponds to the clinical observations of attenuated CYP3A4 induction by EFV in the presence of RTV and other protease inhibitors (PIs). Our results underscore the limitation of transcriptional activation assays in predicting the net outcome for compounds that exhibit complex interactions resulting from induction and inhibition of CYP enzymes.

Objective: To investigate the effects of black seed on the metabolic activities of CYP3A4 and CYP2D6 in human liver microsomes and in human subjects using dextromethorphan as a probe drug. Methods: CYP2D6-mediated Odemethylation and CYP3A4-mediated N-demethylation of dextromethorphan (DEX) to dextrorphan (DOR) and 3- methoxymorphinan (3-MM), respectively, were utilized to assess the metabolic activities of the two enzymatic pathways. In the in vitro experiments, DEX was incubated with microsomes and NADPH in absence or presence of black seed extract (10-100 μg/ml) and the formation of the metabolites were measured by HPLC. In the clinical study, four healthy volunteers received a single oral dose of DEX 30 mg alone in phase I, and along with last dose of black seed (2.5 g twice daily for seven days) in phase II. Activities of the two enzymes were evaluated based on the urinary metabolic ratios (MRs), which were calculated from eight-hour urine collections. DEX and its metabolites were assayed in urine samples by HPLC following a liquid-liquid extraction. Results: Black seed extracts significantly inhibited the formation of both metabolites in microsomes. The maximum inhibition was observed at the highest extract concentration (i.e., 100 μg/ml), which was about 80% and 60% for DOR and 3-MM, respectively. In the clinical study, the urinary MRs of DEX/DOR and DEX/3-MM increased by factors of 127 and 1.6-fold, respectively, after consumption of black seed. Conclusion: Black seed significantly inhibited CYP2D6 and CYP3A4 mediated metabolism of DEX in human liver microsomes and healthy human volunteers indicating that it has the potential to interact with CYP2D6 and CYP3A4 substrates.