Drug Metabolism Letters - Volume 2, Issue 3, 2008

It is my great pleasure to introduce Drug Metabolism Letters (DML; www.bentham.org/dml) to the Readers as a unique journal in the field, which has gained recognition among its audience within a very brief span of time since its launch back in January 2007. The journal has already been accepted for coverage in Chemical Abstracts, EMBASE, EMBiology and Scopus! DML publishes short papers/letters on major advances in all areas of drug metabolism and disposition. Its publisher, Bentham Science, publishes more than 250 print and open access journals (www.bentham.org), (www.bentham.org/open) and books (www.bentham.org/ebooks). The journal board comprises nine Associate Editors, forty-five Editorial Board Members and forty-three Reviewers. Each Associate Editor of DML handles peer reviewing of papers received from his/her region on behalf of the Editor-in-Chief. The journal is also publishing one issue each year dedicated to submissions from the Editorial Board Members of DML. This is the first such issue and I congratulate to all the contributing Editorial Board Members for their positive response and hope they will continue to participate in each year's issue. I hope this will prove to be valuable for all readers by helping them to identify with some Editorial Board Members when submitting their work to DML. The contributions in this issue were subjected to the same peer review process as all manuscripts followed by desired revisions. I do hope the DML Reader will find all twelve papers included in this issue, to be quite interesting: Urs A. Boelsterli et al.. studied the bioactivation of leflunomide and the metabolic degradation to its major metabolite, A771726. Leflunomide was rapidly metabolized in human hepatocytes to A771726, but its toxicity was dependent on other, CYP-dependent intermediates. James F. Rusling and colleagues used rat liver microsomes attached to nanoparticles for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC50 values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were also estimated. Lee Jia et al. investigated the effects of CsA and ITZ on 1) intestinal permeability of amlodipine (a calcium channel blocker used as a cardiovascular agent) in isolated rat everted gut sac model, and 2) biliary excretion and pharmacokinetics of amlodipine in rats. Pretreatment of rats with ITZ increased plasma levels and biliary excretion of amlodipine in a dose-dependent manner. In contrast, pretreatment with CsA slightly decreased biliary excretion of amlodipine and made no changes in its plasma levels. Michael W. Sinz et al. demonstrated that the humanized SXR mouse can be used as a model to predict human CYP3A4 induction and the resulting pharmacokinetic changes of CYP3A4 substrates in humans. Jasminder Sahi et al. compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. They also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. Their data showed major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. David Rodrigues et al. worked on troglitazone (TGZ) induced hepatotoxicity, which has been linked to cytochrome P450 (CYP)- catalyzed reactive metabolite formation. They concluded that both CYP3A4/5 and CYP2C8 play a major role in the formation of TGZ adduct in HLM. However, the contribution of these CYPs varies depending on their relative expression and the concentration of TGZ.

We used immortalized human hepatocytes to study the bioactivation of leflunomide and the metabolic degradation to its major metabolic,A77 1726. Both leflunomide and A77 1726 caused a time-and concentration-dependent increase in LDH release. The cytotoxicity of leflunomide,but not that of A77 1726, was prevented by the pan -CYP inhibitor, 1-aminobenzotriazole,indicating that an oxidative metabolite(s) was responsible for the cell injury.LC/MS/MS analysis revealed that leflunomide was rapidly degraded in hepatocytes biphasically(t1/2a=1.5 h,t1/2b>24h),but much slower in cell-free medium(t1/2>24 h).In contrast,the generation of A77 1726 occured at a similar rate in cells and cell-free system s.In conclusion,leflunomide wsa rapidly metabolized in human hepatocytes to A77 1726, but its toxicity was dependent on other ,CYP-dependent intermedietly metabolized in human hepatocytes to A77 1726, but its toxicity was dependent on other ,CYP-dependent intermediidly metabolized in human hepatocytes to A77 1726, but its toxicity was dependent on other,CYP-dependent intermediidly metabolized in human hepatocytes to A77 1726,but its toxicity was dependent intermediates.

Rat liver microsomes attached to nanoparticles were used for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N -nitro so compounds.Using these biocolloids,turnover rates were measured within 2 min. Inhibitor IC 50 values for ketoconazole(KET) and 4-methylpyrazoie (4-MEP) were estimated.

Cyclosporin A (CsA) is a P-glycoprotein (P-gp) inhibitor clinically used as an immunosuppressant. Itraconazole (ITZ) functions as an inhibitor of both the P-gp and CYP3A and is used as afungistatic/fungicidal agent in human and veterinary medicine. The present studies were designed to investigate the effects of CsA and ITZ on 1 intestinal permeability of amlodipine (a calcium channel blocker used as a cardiovascular agent) in isolated rat everted gut sac modle, and 2) biliary excretion and pharmacokinetics of amlodipine in rats. The concentrations of amlodipine in biosamples were measured by the liquid chromatograph mass spectrometer (LC/MS). Both CsA ITZ significantly increased permeability of amlodipine in the ileum and jejunum of the rat everted gut sac modle,and ITZ showed more potent than CsA IN this modle. Pretreatment pf rats with ITZ increased plasma levels and biliary excretion of amlodipine and made no changes in its plasma levels. In conclusion, ITZ inreased in vitro permeability of amlodipine and bile in vivo. Whereas, CsA showed no significant efects on the levels of amlodipine in rat plasma and bile in vivo. Whereas, CsA showed no significant effects on the levels of amlodipine in rat plasma and bile probably due to the potent inhibition of ITZ against both CYP3A and P-gp.

The SXR humanized mouse modle was used to quantitatively assess an vivo induction response of the human PXR agoinst, rifampicin, Three days of rifampicin treatment increased RNA expression and microsomel enzyme activity of CYP3A, as wall as significantly reduced triazolam plasma exposure. These results indicate that the humanized SXR mouse can be used as a modle to prediict human CYP3A4 induction and the resulting pharma cokinetic changes of CYP3A4 substrates in humans.

Aldehyde oxidase (AO) is a cytosolic enzyme that contributes to the Phase I metabolism of xenobiotics in human and preclinical species. We compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. We also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. AO activity was measured using the formation of vanillic acid from vanillin. The rate of vanillic acid formation was 2 ± 0.2 nmol/min/mg in human liver cytosol and 0.79 ± 0.45 nmol/min/million cells in cryopreserved human hepatocytes. AO activity (Vmax,app) was highest in monkey and lowest in rat. Mouse liver cytosol had the lowest Km,app (1.44 ± 0.16 μM) and highest intrinsic clearance (8.97 ml/min/mg) and rat liver cytosol the highest Km,app (10.9 ± 1.2 μM) and lowest intrinsic clearance (0.47 ml/min/mg). There was a 4.25-fold difference in AO activity between the 5 human hepatocyte preparations. Drug interaction studies with the seven marketed drugs revealed marked species-specific inhibition. Our data indicates major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. Cryopreserved hepatocytes and cytosolic fractions from animals and humans provide qualitatively similar data within the species.

I do hope the DML Reader will find all twelve papers included in this issue, to be quite interesting: Urs A. Boelsterli et al.. studied the bioactivation of leflunomide and the metabolic degradation to its major metabolite, A771726. Leflunomide was rapidly metabolized in human hepatocytes to A771726, but its toxicity was dependent on other, CYP-dependent intermediates. James F. Rusling and colleagues used rat liver microsomes attached to nanoparticles for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC50 values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were also estimated. Lee Jia et al. investigated the effects of CsA and ITZ on 1) intestinal permeability of amlodipine (a calcium channel blocker used as a cardiovascular agent) in isolated rat everted gut sac model, and 2) biliary excretion and pharmacokinetics of amlodipine in rats. Pretreatment of rats with ITZ increased plasma levels and biliary excretion of amlodipine in a dose-dependent manner. In contrast, pretreatment with CsA slightly decreased biliary excretion of amlodipine and made no changes in its plasma levels. Michael W. Sinz et al. demonstrated that the humanized SXR mouse can be used as a model to predict human CYP3A4 induction and the resulting pharmacokinetic changes of CYP3A4 substrates in humans. Jasminder Sahi et al. compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. They also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. Their data showed major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. David Rodrigues et al. worked on troglitazone (TGZ) induced hepatotoxicity, which has been linked to cytochrome P450 (CYP)- catalyzed reactive metabolite formation. They concluded that both CYP3A4/5 and CYP2C8 play a major role in the formation of TGZ adduct in HLM. However, the contribution of these CYPs varies depending on their relative expression and the concentration of TGZ.

The influence of the enzyme source such as phospholipid and expression ratio of NADPHcytochrome P450 reductase on midazolam 1'-hydroxylation activity of cytochrome P450 3A4 was investigated in reconstituted systems and in membranes using bicistronic expressions. Preferable ratio of P450 reductase over human P450 3A4 would be a determinant factor for the drug metabolism studies.

The great emphasis on ethical and humane treatment of animals in biomedical research has culminated in the promulgation of the rule of 3Rs - Replacement, Reduction, and Refinement. We have proposed an addition to the 3Rs - a fourth R for Recycling the animal. In drug discovery single-dose pharmacokinetic studies in rats, each animal is generally used only once and then euthanized. A reduction in the number of rats used in this high-throughput era can be readily implemented by reusing animals, just as larger animals are reused in multiple pharmacokinetic studies, consequently reducing the overall number of animal lives sacrificed in research. We provide evidence here for the reproducibility of pharmacokinetic studies of tolbutamide and fluconazole, used as test compounds, in rats receiving once-weekly oral and intravenous doses for 4 weeks, proving that recycling rats for multiple single low-dose pharmacokinetic studies is a viable option.

We used protein-ligand docking and minimization to identify celecoxib as an allosteric modulator of SULT2A1-catalyzed estradiol sulfonation. Subsequent to celecoxib docking and complex minimization, conformational changes in SULT2A1 allowed estradiol docking to an alternative binding region with predicted preference for 17β-OH-E2 sulfonation over 3-OH-E2 sulfonation.

A fast and sensitive method was developed for the assessment of CYP induction in human hepatocytes cultured in 96-well plates. The effects on CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were examined by means of mass spectrometry and the well-known cocktail strategy. The performance of the method was tested by using prototypic inducers such as methylcolanthrene, phenobarbital and rifampicin. The method was shown to be robust and predictable for the in vitro induction potential studies of drugs.

UGT enzymes catalyze the formation of glucuronic acid conjugates. Specifically selected representative stable isotope (C13, N15) labeled peptide internal standards of each enzyme were employed to quantify UGTs 1A1 and 1A6 by LC-MS/MS using isotope dilution techniques. Inter-day variability (n=5) for human liver microsomes was ≤ 8.0 % for UGT1A1 and ≤ 19 % for UGT1A6. Comparison within a human liver microsomal library showed a strong correlation with Western blot for UGT1A1 concentrations (r=0.988). The data presented indicates that an accurate and reproducible method for UGT absolute quantification can be established using LC-MS/MS analysis of characteristic peptides within the protein.

The extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we have investigated the role of P-glycoprotein (P-gp) in the transport of tanshinone I (TSI), a major active constituent of S. miltiorrhiza. The TSI transport across Caco-2 monolayers was pH-, energy-, and temperature-dependent, but not sodium-dependent. TSI exhibited a polarized transport in Caco-2 monolayers which was attenuated by P-gp inhibitors. The permeability (Papp) values of TSI in the basolateral to apical direction were significantly higher in MDCK-II cells over-expressing MDR1, as compared to the wild-type control cells. Furthermore, TSI significantly inhibited the transport of digoxin in Caco-2 cells with an IC50 value of 0.53 ± 0.09 μM. TSI also moderately stimulated P-gp ATPase activity with Km and Vmax values of 31.70 ± 7.09 μM and 57.71 ± 5.26 nmol/min/mg protein, respectively. Our findings indicate that TSI is a substrate and inhibitor of Pgp, which has important clinical and toxicological implications.