Assessment of 8-hydroxy-2-deoxyguanosine activity, gene expression and antioxidant enzyme activity on rainbow trout (Oncorhynchus mykiss) tissues exposed to biopesticide.

The goal of this study was to determinate toxicity mechanism of biopesticide with antioxidant enzymes parameters such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and malondialdehyde (MDA) levels, oxidative DNA damage (8-hydroxy-2-deoxyguanosine (8-OHdG)), transcriptional changes of heat shock protein 70 (HSP70), and cytochromes P4501A (CYP1A), sod, cat, and gpx in liver and gill tissues of Oncorhynchus mykiss. For this aim, plant-based (natural pesticides, azadirachtin (AZA)) and synthetic pesticides (deltamethrin (DLM)) were exposed on the fish at different concentrations (0.0005 and 0.00025ppm of DLM; 0.24 and 0.12ppm of AZA) for 21 days. According to the results of the study, the activity of SOD, CAT and GPx decreased, but malondialdehyde (MDA) level and activity of 8-OHdG increased in the gill and liver of rainbow trout (p<0.05). Additionally sod, cat and gpx were down regulated; HSP70 and CYP1A were up regulated for transcriptional observation. The downwards regulation of antioxidant (sod, cat and gpx) and the upregulation of HSP70 and CYP1A was obvious with doses of AZA or DLM (p<0.05). The findings of this study suggest that biopesticide can cause biochemical and physiological effects in the fish gill and liver by causing enzyme inhibition, an increase in 8-OHdG levels and changes in both transcriptional parameters (sod, cat, gpx, HSP70 and CYP1A). We found that excessive doses of plant-based pesticide are nearly as toxic as chemical ones for aquatic organisms. Moreover, 8-OHdG, HSP70 and CYP1A used as a biomarker to determinate toxicity mechanism of biopesticide in aquatic environment.

Functional characterization of 12 allelic variants of CYP2C8 by assessment of paclitaxel 6α-hydroxylation and amodiaquine N-deethylation.

Cytochrome P450 2C8 (CYP2C8) is one of the enzymes primarily responsible for the metabolism of many drugs, including paclitaxel and amodiaquine. CYP2C8 genetic variants contribute to interindividual variations in the therapeutic efficacy and toxicity of paclitaxel. Although it is difficult to investigate the enzymatic function of most CYP2C8 variants in vivo, this can be investigated in vitro using recombinant CYP2C8 protein variants. The present study used paclitaxel to evaluate 6α-hydroxylase activity and amodiaquine for the N-deethylase activity of wild-type and 11 CYP2C8 variants resulting in amino acid substitutions in vitro. The wild-type and variant CYP2C8 proteins were heterologously expressed in COS-7 cells. Paclitaxel 6α-hydroxylation and amodiaquine N-deethylation activities were determined by measuring the concentrations of 6α-hydroxypaclitaxel and N-desethylamodiaquine, respectively, and the kinetic parameters were calculated. Compared to the wild-type enzyme (CYP2C8.1), CYP2C8.11 and CYP2C8.14 showed little or no activity with either substrate. In addition, the intrinsic clearance values of CYP2C8.8 and CYP2C8.13 for paclitaxel were 68% and 67% that of CYP2C8.1, respectively. In contrast, the CLint values of CYP2C8.2 and CYP2C8.12 were 1.4 and 1.9 times higher than that of CYP2C8.1. These comprehensive findings could inform for further genotype-phenotype studies on interindividual differences in CYP2C8-mediated drug metabolism.

Pathway-dependent inhibition of paclitaxel hydroxylation by kinase inhibitors and assessment of drug-drug interaction potentials.

Paclitaxel is often used in combination with small molecule kinase inhibitors to enhance antitumor efficacy against various malignancies. Because paclitaxel is metabolized by CYP2C8 and CYP3A4, the possibility of drug-drug interactions mediated by enzyme inhibition may exist between the combining agents. In the present study, a total of 12 kinase inhibitors were evaluated for inhibitory potency in human liver microsomes by monitoring the formation of CYP2C8 and CYP3A4 metabolites simultaneously. For reversible inhibition, nilotinib was found to be the most potent inhibitor against both CYP2C8 and CYP3A4, and the inhibition potency could be explained by strong hydrogen binding based on molecular docking simulations and type II binding based on spectral analysis. Comparison of K(i) values revealed that the CYP2C8 pathway was more sensitive toward some kinase inhibitors (such as axitinib), while the CYP3A4 pathway was preferentially inhibited by others (such as bosutinib). Pathway-dependent inactivation (time-dependent inhibition) was also observed for a number of kinase inhibitors against CYP3A4 but not CYP2C8. Further studies showed that axitinib had a K(I) of 0.93 µM and k(inact) of 0.0137 min(-1), and the observed inactivation toward CYP3A4 was probably due to the formation of reactive intermediate(s). Using a static model, a reasonably accurate prediction of drug-drug interactions was achieved by incorporating parallel pathways and hepatic extraction ratio. The present results suggest that potent and pathway-dependent inhibition of CYP2C8 and/or CYP3A4 pathways by kinase inhibitors may alter the ratio of paclitaxel metabolites in vivo, and that such changes can be clinically relevant as differential metabolism has been linked to paclitaxel-induced neurotoxicity in cancer patients.

In vitro assessment of 36 CYP2C9 allelic isoforms found in the Chinese population on the metabolism of glimepiride.

Of the 57 reported CYP2C9 alleles, to date, 36 of them have been identified in the Chinese population. The aim of this study was to assess the catalytic characteristics of these allelic isoforms and their effects on the metabolism of glimepiride in vitro. Baculovirus-mediated expressing system was used to highly express wild-type and the 35 CYP2C9 allelic variants in insect cell microsomes. Then, the enzymatic characteristics of each variant were evaluated using glimepiride as the substrate. Reactions were performed at 37°C with the insect microsomes and 0.125-10 µM glimepiride for 40 min. After termination, the products were extracted and used for signal collection by LC-MS/MS. Of the 36 tested CYP2C9 allelic isoforms, only four variants (CYP2C9.40, CYP2C9.47, CYP2C9.51 and CYP2C9.54) exhibited similar relative clearance values to that of wild-type CYP2C9.1. In addition, one variant (CYP2C9.36) showed a higher intrinsic clearance value than the wild-type protein, while the remaining 30 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.1% to 87.2%) compared to CYP2C9.1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used antidiabetic drug, glimepiride. Our results indicate that most of the tested rare alleles significantly decrease the catalytic activity of CYP2C9 variants towards glimepiride hydroxylation in vitro.

Gene expression and growth as indicators of effects of the BP Deepwater Horizon oil spill on spotted seatrout (Cynoscion nebulosus).

The BP Deepwater Horizon oil spill has great potential to negatively affect estuarine fish populations. In order to assess possible impacts of this event, a series of sublethal lab experiments were performed, using the economically and ecologically important species spotted seatrout (Cynoscion nebulosus). Larval and juvenile spotted seatrout were exposed to sublethal concentrations of high energy water accommodated fraction (HEWAF), chemically enhanced water accommodated fraction (CEWAF), or dispersant alone in an acute exposure. Response to exposure was evaluated with quantative polymerase chain reaction (qPCR) to examine expression of cytochrome P-4501A (CYP1A). Growth of larvae and juveniles over the duration of the experiment was measured as an index of physiological response. Our data showed that the different life stages respond differently to crude and dispersed oil, with larval spotted seatrout affected most by CEWAF, while juvenile spotted seatrout were affected to a greater extent by HEWAF. In both cases, the treatment with the highest CYP1A levels resulted in the greatest reductions in growth.

Routine assessment of on-clopidogrel platelet reactivity and gene polymorphisms in predicting clinical outcome following drug-eluting stent implantation in patients with stable coronary artery disease.

OBJECTIVES: This study sought to assess the usefulness of clopidogrel-pathway genotyping and on-treatment platelet reactivity (OTR) testing in predicting major adverse cardiac events (MACE) in stable coronary artery disease (CAD) patients receiving drug-eluting stents (DES) under dual antiplatelet (clopidogrel plus aspirin) therapy. BACKGROUND: The role of pharmacogenetics and OTR in predicting MACE-death, myocardial infarction, or stent thrombosis-in stable CAD patients scheduled for DES implantation is still debated. METHODS: Patients with stable CAD treated by DES implantation (n = 1,432) were genotyped with a TaqMan OpenArray (Applied Biosystems, Carlsbad, California) and assessed for OTR with the VerifyNow P2Y12 test (Accumetrics Inc., San Diego, California). Genes tested were ABCB1, CYP1A2, CYP2B6*9, CYP2C8*3, CYP2C9*2, CYP2C19, CYP3A4, CYP3A5*3, P2RY12, and PON1CYP2C19. High OTR was defined as P2Y12 reaction units ≥230. The endpoint at 12-month follow-up was MACE occurring during antiplatelet therapy. RESULTS: All groups that were stratified for loss-of-function variants of the cytochrome P450 gene CYP2C19 had significant hazard ratios (HR) for MACE (genotypic HR: 1.41, 95% confidence interval [CI]: 1.06 to 1.89, p = 0.01; allelic HR: 1.56, 95% CI: 2.26 to 1.2, p = 0.01). Variants of other clopidogrel-pathway genes were not significantly associated with MACE. When OTR was assessed, clinical significance was found only in high-risk diabetic (HR: 2.11, 95% CI: 1.29 to 3.45, p < 0.001) and chronic kidney disease (HR: 2.03, 95% CI: 1.03 to 4.02, p = 0.04) patients. CONCLUSIONS: CYP2C19 metabolizer status is an independent predictor of MACE after DES implantation and can be used for prognostication in all stable CAD patients. High OTR, as assessed by the VerifyNow P2Y12 test, is an independent predictor of MACE only for high-risk subsets, that is, patients with diabetes or chronic kidney disease.

Assessment of effects of chronic hydrogen sulfide poisoning on cytochrome P450 isoforms activity of rats by cocktail approach.

Hydrogen sulfide (H2S) is one of the neurotoxic gases with suffocating and irritating. Its main target organs of toxic effects are the central nervous system and respiratory system. Cocktail method was used to evaluate the influence of chronic hydrogen sulfide poisoning on the activities of cytochrome P450 (CYP450) isoforms CYP1A2, CYP2C9, CYP2B6 and CYP2D6, which were reflected by the changes of pharmacokinetic parameters of 4 specific probe drugs phenacetin, tolbutamide bupropion and metroprolol, respectively. The experimental rats were randomly divided into two groups, control group and chronic hydrogen sulfide poisoning group. The chronic hydrogen sulfide poisoning group rats were inhaled 20 ppm for 1 h twice a day for 40 d. The mixture of 4 probes was given to rats through sublingual veins and the blood samples were obtained at a series of time-points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by liquid chromatography-mass spectrometry (LC-MS). In the experiment for chronic hydrogen sulfide poisoning and control group, there was a statistically significant difference in the area under the plasma concentration-time curve from zero to infinity (AUC(0-∞)), plasma clearance (CL) and maximum plasma concentration (C(max)) for phenacetin and bupropion, while there was no statistical pharmacokinetics difference for tolbutamide and metoprolol. Chronic hydrogen sulfide poisoning could induce the activity of CYP1A2 and CYP2B6 of rats.

Assessment of CYP2B6 activity in rats: a cocktail study with bupropion alone and in combined with tolbutamide.

A "cocktail"of numerous probe drugs to assess the metabolic activity of the corresponding cytochrome P450 enzymes requires that there is no problem of interaction among them. Some interactions among probe drugs can appear and may affect the rate of biotransformation of other ones. To develop a useful "cocktail" of probe drugs, our presented work was aimed on the influence of tolbutamide on cytochrome P450-mediated metabolism of bupropion. The biotransformation rates of bupropion administered either separately or in combined with tolbutamide were compared in this new study. The results revealed that tolbutamide had significantly decreased the rate of bupropion hydroxylation. Thus, due to shift in cytochrome P450 enzyme metabolic activity some extent differential results in the rate of P450-mediated metabolism can be observed when comparing assessment using combination of two model drugs with the common way (single marker use).

Genetic testing in patients with acute coronary syndrome undergoing percutaneous coronary intervention: a cost-effectiveness analysis.

BACKGROUND: The CYP2C19 genotype is a predictor of adverse cardiovascular events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) treated with clopidogrel. OBJECTIVES: We aimed to evaluate the cost-effectiveness of a CYP2C19*2 genotype-guided strategy of antiplatelet therapy in ACS patients undergoing PCI, compared with two 'no testing' strategies (empiric clopidogrel or prasugrel). METHODS: We developed a Markov model to compare three strategies. The model captured adverse cardiovascular events and antiplatelet-related complications. Costs were expressed in 2010 US dollars and estimated using diagnosis-related group codes and Medicare reimbursement rates. The net wholesale price for prasugrel was estimated as $5.45 per day. A generic estimate for clopidogrel of $1.00 per day was used and genetic testing was assumed to cost $500. RESULTS: Base case analyses demonstrated little difference between treatment strategies. The genetic testing-guided strategy yielded the most QALYs and was the least costly. Over 15 months, total costs were $18 lower with a gain of 0.004 QALY in the genotype-guided strategy compared with empiric clopidogrel, and $899 lower with a gain of 0.0005 QALY compared with empiric prasugrel. The strongest predictor of the preferred strategy was the relative risk of thrombotic events in carriers compared with wild-type individuals treated with clopidogrel. Above a 47% increased risk, a genotype-guided strategy was the dominant strategy. Above a clopidogrel cost of $3.96 per day, genetic testing was no longer dominant but remained cost-effective. CONCLUSIONS: Among ACS patients undergoing PCI, a genotype-guided strategy yields similar outcomes to empiric approaches to treatment, but is marginally less costly and more effective.

Pharmacokinetics and bioavailability comparison of generic and branded citalopram 20 mg tablets: an open-label, randomized-sequence, two-period crossover study in healthy Chinese CYP2C19 extensive metabolizers.

BACKGROUND: Citalopram is a selective serotonin reuptake inhibitor (SSRI) mainly prescribed to treat major depression. OBJECTIVE: The aim of this study was to compare the pharmacokinetic characteristics of a new and a branded citalopram 20 mg formulation to support the marketing authorization of the test formulation in China. METHODS: A single-dose, open-label, randomized-sequence, two-period crossover design was used in this study. Healthy Chinese male cytochrome P450 (CYP) 2C19 extensive metabolizers, aged 18-40 years, were eligible to participate. CYP2C19 poor metabolizers were excluded, based on genotyping of genomic DNA from blood samples. Twenty-four subjects were randomly assigned to receive the test formulation followed by the reference formulation, and then vice versa. A 2-week washout occurred between study periods. Blood samples were collected for up to 144 h post-dose. Quantification was carried out using a validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. Pharmacokinetic parameters were calculated and analysed statistically. The two formulations were considered pharmacokinetically equivalent if the 90 % confidence intervals (CIs) of the log-transformed ratios (test/reference) of the maximum plasma concentration (C(max)), area under the plasma concentration-time curve from time zero to the last measurable concentration (AUC(last)), and area under the plasma concentration-time curve from time zero to infinity (AUC(∞)) were within the predetermined acceptance range (70-143 % for C(max); 80-125 % for AUC(last) and AUC(∞)) according to China State Food and Drug Administration bioequivalence guidelines. Tolerability was monitored by clinical assessment, vital signs, laboratory analysis and interviews with participants about adverse events. RESULTS: A total of 24 participants, with a mean (SD) age of 26 (3) years (range 22-32 years), body weight of 65.2 (5.0) kg (range 53-73 kg), and height of 172.7 (4.9) cm (range 159-182 cm), were enrolled in this study. Both formulations showed similar pharmacokinetic profiles. Mean (SD) AUC(last), AUC(∞) and C(max) were 1436 (341) ng · h/mL, 1595 (381) ng · h/mL and 32.3 (5.9) ng/mL, respectively, for the test formulation, and 1444 (388) ng · h/mL, 1648 (504) ng · h/mL, 33.1 (7.4) ng/mL, respectively, for the reference formulation. Median (range) time to reach C(max) (t(max)) was 2 (1-12) hours (test) and 3 (1-6) hours (reference). The 90 % CIs of the treatment ratios for the ln-transformed values of C(max), AUC(last) and AUC(∞) were 92.5-103.6, 95.2-100.6 and 96.4-105.4, respectively. No significant difference was found between treatments with regard to pharmacokinetic parameters. Fifteen adverse events were reported during the study but none were considered serious. CONCLUSION: This single-dose study found that the test and reference citalopram 20 mg tablets met the regulatory criteria for assuming bioequivalence in the selected healthy Chinese male subjects. Both formulations were well tolerated.

Co-administration of vismodegib with rosiglitazone or combined oral contraceptive in patients with locally advanced or metastatic solid tumors: a pharmacokinetic assessment of drug-drug interaction potential.

PURPOSE: Vismodegib, a first-in-class oral hedgehog pathway inhibitor, is an effective treatment for advanced basal cell carcinoma. Based on in vitro data, a clinical drug-drug interaction (DDI) assessment of cytochrome P450 (CYP) 2C8 was necessary; vismodegib's teratogenic potential warranted a DDI study with oral contraceptives (OCs). METHODS: This single-arm, open-label study included two cohorts of patients with locally advanced or metastatic solid malignancies [Cohort 1: rosiglitazone 4 mg (selective CYP2C8 probe); Cohort 2: OC (norethindrone 1 mg/ethinyl estradiol 35 µg; CYP3A4 substrate)]. On Day 1, patients received rosiglitazone or OC. On Days 2-7, patients received vismodegib 150 mg/day. On Day 8, patients received vismodegib plus rosiglitazone or OC. The effect of vismodegib on rosiglitazone and OC pharmacokinetic parameters (primary objective) was evaluated through pharmacokinetic sampling over a 24-h period (Days 1 and 8). RESULTS: The mean ± SD vismodegib steady-state plasma concentration (Day 8, N = 51) was 20.6 ± 9.72 µM (range 7.93-62.4 µM). Rosiglitazone AUC(0-inf) and C(max) were similar with concomitant vismodegib [≤8% change in geometric mean ratios (GMRs); N = 24]. Concomitant vismodegib with OC did not affect ethinyl estradiol AUC(0-inf) and C(max) (≤5% change in GMRs; N = 27); norethindrone C(max) and AUC(0-inf) GMRs were higher (12 and 23%, respectively) with concomitant vismodegib. CONCLUSIONS: This DDI study in patients with cancer demonstrated that systemic exposure of rosiglitazone (a CYP2C8 substrate) or OC (ethinyl estradiol/norethindrone) is not altered with concomitant vismodegib. Overall, there appears to be a low potential for DDIs when vismodegib is co-administered with other medications.

A comprehensive assessment of repaglinide metabolic pathways: impact of choice of in vitro system and relative enzyme contribution to in vitro clearance.

Repaglinide is presently recommended by the U.S. Food and Drug Administration as a clinical CYP2C8 probe, yet current in vitro and clinical data are inconsistent concerning the role of this enzyme in repaglinide elimination. The aim of the current study was to perform a comprehensive investigation of repaglinide metabolic pathways and assess their contribution to the overall clearance. Formation of four repaglinide metabolites was characterized using in vitro systems with differential complexity. Full kinetic profiles for the formation of M1, M2, M4, and repaglinide glucuronide were obtained in pooled cryopreserved human hepatocytes, human liver microsomes, human S9 fractions, and recombinant cytochrome P450 enzymes. Distinct differences in clearance ratios were observed between CYP3A4 and CYP2C8 for M1 and M4 formation, resulting in a 60-fold M1/M4 ratio in recombinant (r) CYP3A4, in contrast to 0.05 in rCYP2C8. Unbound K(m) values were within 2-fold for each metabolite across all in vitro systems investigated. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system used. The repaglinide M4 metabolic pathway is proposed as a specific CYP2C8 probe for the assessment of drug-drug interactions.

Oral flurbiprofen metabolic ratio assessment using a single-point dried blood spot.

We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.

[Specific considerations on the prescription and therapeutic interchange of statins]. / Consideraciones específicas en la prescripción e intercambio terapéutico de estatinas.

OBJECTIVE: The pharmaceutical industry currently offers six different statins in Spain and there is one more soon to be available. Choosing the most appropriate drug and dose is determined by the therapeutic target (reduction in LDL-C levels). Statin doses that decrease LDL-C at the same percentage are considered equivalent. Evaluating the pharmacokinetic characteristics of each statin can be useful when setting selection criteria, helping to determine which statin may be more appropriate for a patient based on their individual characteristics and on the other co-administered drugs. METHODS: We reviewed the pharmacokinetics properties of each statin and its possible involvement in drug interactions. RESULTS: CYP3A4 was responsible for the metabolism of lovastatin, simvastatin and atorvastatin; fluvastatin depends on CYP2C9; P-glycoprotein is responsible for decreased atorvastatin, pravastatin, simvastatin and lovastatin concentrations. The OATPA1B1 transporter involved in all statins' access to the hepatocyte, except for fluvastatin, is essential for rosuvastatin and pravastatin. These circumstances cause those drugs inhibiting or inducing isoenzymes or transporters' activity not to have the same effect on the different statins. CONCLUSION: The pharmacokinetics is important when choosing the best statin and could be a limitation in the use of interchange therapeutic programmes when other drugs are present.

Gene, ethnic and gender influences predisposition of adverse drug reactions to artesunate among Malaysians.

CONTEXT: Artesunate (AS) and amodiaquine (AQ) are two prodrugs widely used as antimalarial agents and are metabolized by the CYP P450 2A6 (CYP 2A6) and CYP P450 2C8 (CYP 2C8) enzymes, respectively. OBJECTIVE: In this study, we aim to investigate the association of both genes on AS and AQ's tolerabilities in the hope of identifying a pharmacogenetic approach that could be useful in prediction and prevention of adverse drug reactions (ADRs) among Malaysian population. MATERIALS AND METHODS: In this randomized crossover study, loose and AS/AQ formulations were administered to normal healthy volunteers (n = 24) over two study phases. The drugs' tolerabilities (incidence of facial flushing, giddiness, headache, nausea, abdominal discomfort, progression of liver enzymes and neutrophil counts) were compared between the two treatment arms. Volunteers were also genotyped for the CYP2C8 and CYP2A6 variants. RESULTS: The frequency of the CYP2A6*1B, CYP2A6*4, CYP2A6*8 and CYP2A6*9 alleles were 54.2%, 16.7%, 4.2% and 10.4%, respectively. No mutations for CYP2C8 gene were, however, detected. Most (96%) of the subjects were of the Malay ethnicity. Subjects having the CYP2A6*1B variants responsible for ultra rapid metabolism of AS suffered a significantly higher incidence of ADRs. DISCUSSION: Our study is the first to report that CYP2A6 genotyping influences AS's ADR. Gender also plays a role where females reported more incidences of nausea (p < 0.05). CONCLUSION: It is concluded that genetic polymorphisms of CYP2A6 as well as gender influence the side effect profiles of subjects receiving AS among this Malaysian population.

Assessment of a novel ß2-adrenoceptor agonist, trantinterol, for interference with human liver cytochrome P450 enzymes activities.

The effect of a novel ß(2)-adrenoceptor agonist, trantinterol on the activities of cytochrome P450 (CYP450) was investigated with human liver microsomes and human cryohepatocytes in order to assess the potential for drug-drug interactions. The ability of trantinterol to inhibit CYP450 activities was evaluated in vitro in human liver microsomes. Trantinterol did not inhibit CYP2C19, CYP2D6, and CYP3A4/5 (IC(50)>100 µM). It acted as a weak inhibitor of CYP1A2 and CYP2C9 with IC(50) of 70.8 and 81.9 µM, respectively. No time-dependent inhibitions were observed in the present research. To evaluate CYP450 induction, human cryohepatocytes (n=3) were used and treated once daily for 3 days with trantinterol (0.01, 0.1, and 1 ng/ml), after which CYP450 activities were measured. At concentration of 0.01 ng/ml, which is close to the C(max) at maximal recommended doses (50 µg), trantinterol was about 8% as effective as omeprazole (CYP1A2 inducer) only with donor 2. At concentration of 1 ng/ml, trantinterol was about 3.6 ± 3.1% as effective as rifampin (CYP3A4/5 inducer). These in vitro results indicated that, at pharmacological relevant concentrations, trantinterol will not produce clinically significant CYP450 inhibition or induction.

Assessment of nonsteroidal anti-inflammatory drug-induced hepatotoxicity.

INTRODUCTION: Liver toxicity related to NSAIDs is of outstanding importance because of the wide use of these drugs. NSAIDs are responsible for roughly 10% of the total of cases of drug-induced hepatotoxicity. The assessment of NSAID-induced hepatotoxicity, presently based on clinical and analytical biomarkers, is critical for early diagnosis and immediate withdrawal of the causing drug. AREAS COVERED: The review presents an overview of current knowledge of the assessments of NSAID-induced hepatotoxicity with emphasis on the causative drugs, the NSAID-specific mechanisms involved, and a summary of genetic and non-genetic risk factors. Additionally, the authors discuss genetic factors which show NSAID-specific risk, namely CYP2C, UGT2B7, GSTM1 and GSTT1, as well as HLA alleles. The paper includes a list of the NSAID 'usual suspects' that cause hepatotoxicity based on the integrated information of drug-induced hepatotoxicity databases. EXPERT OPINION: The ultimate goal of this research is pre-prescription testing. Unfortunately, genetic testing, alone, is not sufficient to predict NSAID-induced hepatotoxicity. The development of genetic biomarkers capable of identifying at-risk individuals will not be complete until we develop the ability to fully characterize patients' phenomes and the phenome-genome interaction in patients with NSAID-induced hepatotoxicity. Additionally, a characterization of the metabolic profile of the causative drug in patients with NSAID-induced hepatotoxicity would add crucial information which is presently disregarded in most studies. The full development of robust biomarkers will require the combination of several disciplines including causal statistics, phenomics, genomics, transcriptomics and metabonomics.

In vitro-in vivo extrapolation of CYP2C8-catalyzed paclitaxel 6α-hydroxylation: effects of albumin on in vitro kinetic parameters and assessment of interindividual variability in predicted clearance.

OBJECTIVES: This study aimed to characterize the effects of bovine serum albumin (BSA) on the kinetics of CYP2C8-catalyzed paclitaxel 6α-hydroxylation in vitro; determine whether the addition of BSA to incubations improves the prediction of paclitaxel hepatic clearance via this pathway in vivo; and assess interindividual variability in predicted clearance. METHODS: The kinetics of paclitaxel 6α-hydroxlation by human liver microsomes (HLM) and recombinant CYP2C8 were characterized in incubations performed with and without BSA (2% w/v) supplementation, and the in vitro kinetic data were extrapolated to provide estimates of in vivo clearances. The Simcyp population-based ADME simulator was used to determine interindividual variability in the predicted clearances. RESULTS: Supplementation of incubations of HLM with BSA resulted in a 3.6-fold increase in the microsomal intrinsic clearance for paclitaxel 6α-hydroxylation, due mainly to a reduction in K(m) (7.08 ± 2.50 to 2.26 ± 0.39 µM), while addition of BSA to incubations of recombinant CYP2C8 resulted in an approximate doubling of intrinsic clearance. Mean values of predicted in vivo hepatic clearance were in good agreement with clinical data when in vitro data obtained in the presence of BSA were used for IV-IVE. Simcyp predicted 20- to 30-fold interindividual variability in in vivo paclitaxel hepatic clearance via the 6α-hydroxylation pathway. CONCLUSIONS: Human liver microsomal K(m) and intrinsic clearance values are over- and underpredicted, respectively, when incubations of the CYP2C8 substrate paclitaxel are performed without BSA supplementation. IV-IVE based on kinetic parameters generated in the presence of BSA improves the accuracy of predicted paclitaxel hepatic clearance.

Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities.

The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 µg/ml. The EC(50) value was calculated to be 72.0 µg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 µg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c(max) at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 µg/ml, which is close to c(max). The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 µg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 µg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 µg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 µg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K(i) values were determined. All K(i) values exceeded c(max) by at least a factor of 10-fold. According to FDA regulations 1>c(max)/K(i)>0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.