Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between approaches. Drug Metab

This paper is available online at http://www.dmd.org ABSTRACT: Relative activity factors (RAFs) and immunoquantified levels of cytochrome P450 (CYP) isoforms both have been proposed as scaling factors for the prediction of hepatic drug metabolism from studies using cDNA-expressed CYPs. However, a systematic comparison of the two approaches, including possible mechanisms underlying differences, is not available. In this study, RAFs determined for CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 in 12 human livers using lymphoblast-expressed enzymes were compared to immunoquantified protein levels. 2C19, 2D6, and 3A4 RAFs were similar to immunoquantified enzyme levels. In contrast, 1A2 RAFs were 5-to 20-fold higher than CYP1A2 content, and the RAF:content ratio was positively correlated with the molar ratio of NADPH:CYP oxidoreductase (OR) to CYP1A2. The OR:CYP1A2 ratio in lymphoblast microsomes was 92-fold lower than in human liver microsomes. Reconstitution experiments demonstrated a 10-to 20-fold lower activity at OR:CYP1A2 ratios similar to those in lymphoblasts, compared with those in human livers. CYP2B6-containing lymphoblast microsomes had 29-and 13-fold lower OR:CYP and cytochrome b 5 :CYP ratios, respectively, than did liver microsomes and yielded RAFs that were 6-fold higher than CYP2B6 content. Use of metabolic rates from cDNA-expressed CYPs containing nonphysiologic concentrations of electron-transfer proteins (relative to human liver microsomes) in conjunction with hepatic CYP contents may lead to incorrect predictions of liver microsomal rates and relative contributions of individual isoforms. Scaling factors used in bridging the gap between expression systems and liver microsomes should not only incorporate relative hepatic abundance of individual CYPs but also account for differences in activity per unit enzyme in the two systems. The cloning and heterologous expression of the drug-metabolizing human CYPs 1 has resulted in the commercial availability of cDNAexpressed CYPs for use as reagents for in vitro quantitative phenotyping, and bridging the gap between cDNA-expressed cytochromes and human liver microsomes has been the subject of several recent reviews The turnover number of CYPs in human liver microsomes is affected by several factors that are biochemically distinct from the CYP enzyme itself. Examples include the accessory electron-transfer proteins NADPH cytochrome P450-oxidoreductase (OR) and cytochrome b 5 , membrane lipid composition, and ionic strength of the in vitro incubation matrix The relative contribution of each isoform can then be determined as follows: Enzyme kinetic studies using cDNA-expressed human CYPs are being increasingly used to define the functions v i (s) for whatever individual CYPs biotransform a drug. Such analyses indicate the relative affinities and capacities of the CYPs contributing to a biotransformation. However, the relative contribution of each enzyme cannot be estimated unless the results incorporate the scaling factors (A i ). Although immunoquantified levels of CYP isoforms in human liver microsomes RAFs are determined for specific CYPs by comparing the rate of an isoform-specific index reaction at saturating substrate concentrations in human liver microsomes (V max for liver microsomes) to the rate of the same reaction catalyzed by the specific cDNA-expressed CYP under identical conditions (V max for cDNA-expressed enzyme): Mean V max for isoform-specific reaction in human liver microsomes V max for isoform-specific reaction by cDNA expressed isoform When the numerator is expressed in picomoles metabolite per milligram of protein per minute and the denominator as picomoles of metabolite per picomole of CYP per minute, the units of RAF are picomoles of CYP per milligram of protein, which can be compared with the immunoquantified CYP contents. RAFs have been used as estimates of A i in reaction phenotyping Materials and Methods Human Liver Microsomes and Heterologously Expressed CYP Isoforms. Liver samples, obtained from the International Institute for the Advancement of Medicine (Exton, PA) or the Liver Tissue Procurement and Distribution Service (University of Minnesota), were from 12 different transplant donors (L1-L12) with no known liver disease. The donor population (median age 27 years, range 3-50 years; 5 females and 7 males) was balanced with respect to age, sex, race, smoking habits, and alcohol consumption. The tissue was partitioned and kept at Ϫ80°C until the time of microsome preparation as described previously Microsomes from cDNA-transfected human lymphoblastoid cells expressing CYP 1A2, 2B6, 2C19, 2D6, or 3A4 (Crespi, 1995) were purchased from Gentest Corporation (Woburn, MA), aliquoted and stored at Ϫ80°C, and thawed on ice before use. Lymphoblast-expressed CYPs 3A4 and 2D6 used in the study were coexpressed with OR, whereas the activities of lymphoblast- expressed CYPs 1A2, 2B6, and 2C19 were supported by endogenous levels of reductase native to the host cell line. Microsomal protein concentrations and CYP content were provided by the manufacturer. Antibodies and Quantitative Western Blotting. Concentrations of CYP1A2, 2B6, 2C19, 2D6, and 3A4/5 in human liver microsomal preparations from livers L1 to L12 were determined by quantitative Western blotting In Vitro Metabolic Incubations and Metabolite Analysis. Incubations of index substrates with human liver microsomes and lymphoblas

Zolpidem and Gender: Are Women Really At Risk?

BACKGROUND: In 2013 the Food and Drug Administration (FDA) claimed the existence of new data showing women to be at risk for excessive daytime sedation and impaired driving proficiency following bedtime doses of zolpidem. The putative explanation was the reduced metabolic clearance of zolpidem and higher morning blood concentrations in women compared to men. The FDA acted to reduce the recommended dosage for women down to 50% of the dose for men. No other regulatory agency worldwide has taken similar action. METHODS: Gender effects on zolpidem pharmacokinetics, pharmacodynamics, adverse effects, clinical efficacy, and driving performance were evaluated through a further analysis of data from a previous study, together with a literature review. RESULTS: Women had on average 35% lower apparent clearance of zolpidem than men (236 vs 364 mL/min, P \u3c 0.001). This difference was not explained by body weight. In some laboratory studies, women had greater functional impairment than men taking the same dose, but in all studies active drug was not distinguishable from placebo at 8 hours after oral dosage. On-the-road driving studies likewise showed no evidence of driving impairment in men or women at 8 hours after 10 mg of oral immediate-release zolpidem. No clinical trial demonstrated a gender-related difference in clinical efficacy or adverse reactions, and there was no evidence of a particular risk to women. CONCLUSIONS: Dosage reduction in women is not supported by available scientific evidence, and may in fact lead to underdosing and the consequent hazard of inadequately treated insomnia

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1To determine kinetic parameters (Vmax, Km) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model ( F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes

Inhibition of cytochrome P450 by nefazodone in vitro: studies of dextromethorphan O- and N-demethylation

Nefazodone (NEF), a 5-HT2A/2C antagonist antidepressant, is extensively metabolized in the human body to hydroxy NEF (OH-NEF), p-hydroxy NEF (pOH-NEF), a dione metabolite, and via cleavage of the molecule to m-chlorophenyl-piperazine (mCPP) and BMY-33604. The latter is further metabolized to BMS-183695-01 (BMSa) and BMS-183562-01 (BMSb). To investigate the potential of NEF and its metabolites to interfere with the metabolism of other drugs, we tested these compounds for their ability to alter dextromethorphan (DMO) O-demethylation to dextrorphan (DOP; an index reaction for CYP2D6) and N-demethylation to 3-methoxy morphinan (MEM, a recently proposed index reaction of CYP3A3/4). The assay was performed in an in vitro system with human liver microsomes from three different donors. NEF, OH-NEF, pOH-NEF, mCPP and BMSb were weak inhibitors of DMO O and N-demethylation, with average Ki values ranging from 18 to 50 μm for DOP formation, and from 21 to >200 μm for MEM formation. The dione metabolite and BMSa did not produce detectable inhibition of either pathway. The findings for DMO O-demethylation, well-established as a CYP2D6-mediated reaction, indicate that NEF and metabolites are weak inhibitors of this reaction, with Ki values at least 100 times higher than fluoxetine (Ki=0.1 μm±0.09). The implications of results on DMO N-demethylation are not clear. In vivo data, as well as in vitro data based on ‘pure’ CYP3A3/4 substrates, provide evidence for clinically relevant CYP3A3/4 inhibition by NEF, OH-NEF, and pOH-NEF. Thus, formation of MEM by N-demethylation of DMO may not constitute a suitable index reaction to probe CYP3A3/4 activity

Population Pharmacokinetics of Methylphenidate in Children with Attention Deficit Hyperactivity Disorder

Sources of individual variation in plasma methylphenidate (MP) concentrations during usual clinical use are not established. This was evaluated in a series of patients receiving clinical treatment with MP. A single plasma MP concentration was determined in each of 273 children and adolescents ages 5 to 18 years (mean: 11.1 years) who were clinically good responders to MP for the treatment of attention-deficit hyperactivity disorder. MP was given on a twice-daily schedule (mean dose: 25 mg/day) in 40% of patients and three times daily (mean dose: 39.3 mg/day) in 60%. A nonlinear regression model was applied to estimate overall population values of MP clearance and elimination half-life (t1/2), assuming a one-component model with first-order absorption and elimination, and further assuming that clearance is linearly related to body weight. The model incorporated each patient\u27s dosage size and schedule, body weight, and time of the plasma sample. Iterated solutions of best fit were: t1/2, 4.5 hours (95% confidence interval [CI]: 3.1–8.1 hours), and apparent clearance, 90.7 ml/min/kg (95% CI: 74.6–106.7 ml/min/kg). The model explained 43% of the overall variance in MP concentrations (r2 = 0.43, p \u3c .001). In a small subsample (N = 16), a second plasma sample was drawn at the same time of day and at the same dose; the correlation between the two concentration values was 0.83. The relatively noninvasive approach used in this study allows the assessment of pharmacokinetic properties of medications under conditions of appropriate clinical use in special populations such as children, adolescents, and the elderly

Population Pharmacokinetics of Methylphenidate in Children with Attention Deficit Hyperactivity Disorder

Sources of individual variation in plasma methylphenidate (MP) concentrations during usual clinical use are not established. This was evaluated in a series of patients receiving clinical treatment with MP. A single plasma MP concentration was determined in each of 273 children and adolescents ages 5 to 18 years (mean: 11.1 years) who were clinically good responders to MP for the treatment of attention-deficit hyperactivity disorder. MP was given on a twice-daily schedule (mean dose: 25 mg/day) in 40% of patients and three times daily (mean dose: 39.3 mg/day) in 60%. A nonlinear regression model was applied to estimate overall population values of MP clearance and elimination half-life (t1/2), assuming a one-component model with first-order absorption and elimination, and further assuming that clearance is linearly related to body weight. The model incorporated each patient\u27s dosage size and schedule, body weight, and time of the plasma sample. Iterated solutions of best fit were: t1/2, 4.5 hours (95% confidence interval [CI]: 3.1–8.1 hours), and apparent clearance, 90.7 ml/min/kg (95% CI: 74.6–106.7 ml/min/kg). The model explained 43% of the overall variance in MP concentrations (r2 = 0.43, p \u3c .001). In a small subsample (N = 16), a second plasma sample was drawn at the same time of day and at the same dose; the correlation between the two concentration values was 0.83. The relatively noninvasive approach used in this study allows the assessment of pharmacokinetic properties of medications under conditions of appropriate clinical use in special populations such as children, adolescents, and the elderly

Role of NADPH-Cytochrome P450 Reductase and Cytochrome-b5/NADH-b5 Reductase in Variability of CYP3A Activity in Human Liver Microsomes

NADPH-cytochrome P450 reductase (CPR) and cytochrome- b 5 ( b 5 ) together with NADH- b 5 reductase ( b 5 R) play important roles in cytochrome P450 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contributes to the known interindividual variability in CYP3A activity. CPR and b 5 were measured in human liver microsomes (HLMs) by spectrophotometry and immunoblotting. HLMs from elderly (≥46 years) male donors ( n = 11) averaged 27% ( P = 0.034) and 41% ( P = 0.011) lower CPR levels than young (≤45 years) male donors ( n = 21) for spectrophotometric and immunoblot values, respectively. Similarly, HLMs from elderly male donors averaged 43% ( P = 0.034) and 47% ( P = 0.011) lower b 5 levels than young male donors for spectrophotometric and immunoblot values, respectively. α-Lipoic acid and 6-propyl-2-thiouracil were evaluated for selectivity of inhibition of CPR and b 5 R activities, respectively, using recombinant enzymes and HLMs, as well as for effects on CYP3A-mediated triazolam hydroxylation in HLMs with either NADH or β-NADPH. The results indicate that both compounds are relatively nonselective inhibitors of CPR and b 5 R activities. Finally, we used multivariate regression analysis and showed that variability in CPR or b 5 expression between HLMs does not contribute significantly to variability in CYP3A-mediated midazolam hydroxylation. Consequently, while aging is associated with decreased CPR and b 5 expression in human livers, this effect does not contribute to CYP3A variability

Escitalopram (Scitalopram) and its metabolites in vitro: cytochromes mediating biotransformation, inhibitory effects, and comparison to R-citalopram,” Drug Metabolism and Disposition

This paper is available online at http://dmd.aspetjournals.org ABSTRACT: Transformation of escitalopram (S-CT), the pharmacologically active S-enantiometer of citalopram, to S-desmethyl-CT (S-DCT), and of S-DCT to S-didesmethyl-CT (S-DDCT), was studied in human liver microsomes and in expressed cytochromes (CYPs). Biotransformation of the R-enantiomer (R-CT) was studied in parallel. S-CT was transformed to S-DCT by CYP2C19 (K m ‫؍‬ 69 M), CYP2D6 (K m ‫؍‬ 29 M), and CYP3A4 (K m ‫؍‬ 588 M). After normalization for hepatic abundance, relative contributions to net intrinsic clearance were 37% for CYP2C19, 28% for CYP2D6, and 35% for CYP3A4. At 10 M S-CT in liver microsomes, S-DCT formation was reduced to 60% of control by 1 M ketoconazole, and to 80 to 85% of control by 5 M quinidine or 25 M omeprazole. S-DDCT was formed from S-DCT only by CYP2D6; incomplete inhibition by quinidine in liver microsomes indicated participation of a non-CYP pathway. Based on established index reactions, S-CT and S-DCT were negligible inhibitors (IC 50 > 100 M) of CYP1A2, -2C9, -2C19, -2E1, and -3A, and weakly inhibited CYP2D6 (IC 50 ‫؍‬ 70-80 M). R-CT and its metabolites, studied using the same procedures, had properties very similar to those of the corresponding S-enantiomers. Thus S-CT, biotransformed by three CYP isoforms in parallel, is unlikely to be affected by drug interactions or genetic polymorphisms. S-CT and S-DCT are also unlikely to cause clinically important drug interactions via CYP inhibition. Previous studies have evaluated the properties of biotransformation of racemic citalopram (CT) to monodesmethylcitalopram (DCT) in vitro by human liver microsomes and by heterologously expressed individual human cytochromes The present study evaluated the biotransformation of the individual R-and S-isomers of CT to DCT, and of R-and S-DCT to didesmethylcitalopram (DDCT), by human liver microsomes, and by microsomes containing pure human cytochromes expressed by cDNAtransfected human lymphoblastoid cells. The inhibitory actions of Rand S-CT, -DCT, and -DDCT versus index reactions representing activity of specific human cytochromes were also evaluated. Materials and Methods In Vitro Procedures. Liver samples from individual human donors with no known liver disease were provided by the International Institute for the Advancement of Medicine, Exton, PA, the Liver Tissue Procurement and Distribution System, University of Minnesota, Minneapolis, MN, and the National Disease Research Interchange, Philadelphia, PA. All samples were of the CYP2D6 and CYP2C19 normal metabolizer phenotype based on prior in vitro phenotyping studies. Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 M potassium phosphate buffer containing 20% glycerol and stored at Ϫ80°C until use. Microsomes containing individual human cytochromes expressed by cDNA-transfected human lymphoblastoid cells (Gentest, Woburn, MA) were similarly stored at Ϫ80°C until use. Pure samples of R-and S-CT, -DCT, and -DDCT were provided by H. Lundbeck A/S (Copenhagen-Valby, Denmark) through Forest Laboratories (New York, NY). Other chemical reagents and drug entities were purchased from commercial sources or kindly provided by their pharmaceutical manufacturers