The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice.

The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUCinf of 3.67 µg h/mL and an effective half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUCinf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose with a mean AUCinf of 2.10 ± 0.49 µg h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.

The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08µg/mL at 0.25-0.5h post-dose with an AUCinf of 1.74±0.52µgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08µg/mL at 0.25h post-dose with an AUCinf of 1.94±0.22µgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans.

Hepatic drug transporters: the journey so far.

INTRODUCTION: The key role of transporter biology in both the manifestation and treatment of disease is now firmly established. Experiences of sub-optimal drug exposure due to drug-transporter interplay have supported incorporation of studies aimed at understanding the interactions between compounds and drug transporters much earlier in drug discovery. While drug transporters can impact the most pivotal pharmacokinetic parameter with respect to human dose and exposure projections, clearance, at a renal or hepatobiliary level, the latter will form the focus of this perspective. AREAS COVERED: A synopsis of guidelines on which transporters to study together with an overview of the currently available toolkit is presented. A perspective on when to conduct studies with various hepatic transporters is also provided together with structural "alerts" which should prompt early investigation. EXPERT OPINION: Great progress has been made in individual laboratories and via consortia to understand the role of drug transporters in disease, drug disposition, drug-drug interactions and toxicity. A systematic analysis of the value posed by the available approaches and an inter-lab comparison now seems warranted. The emerging ability to use physico-chemical properties to guide future screening cascades promises to revolutionise the efficiency of early drug discovery.

Dissecting the relative contribution of OATP1B1-mediated uptake of xenobiotics into human hepatocytes using siRNA.

1. Organic anion transporting polypeptide 1B1 plays a pivotal role in the disposition of many anionic drugs. Significant overlap in substrate specificity between individual OATP isoforms has hampered the identification of the relative importance of individual isoforms for hepatic uptake of xenobiotics. 2. The present study focused on the use of siRNA technology to decrease OATP1B1 selectively in human hepatocytes. Following delivery of siRNA by the novel lipid, AtuFECT01, mRNA expression of OATP1B1 was reduced by 94%-98% with no significant toxicity. Off-target effects were also shown to be minimal as evidenced by the expression of common drug metabolizing enzymes, transporters, nuclear receptors and associated co-regulators. Uptake of estrone-3-sulfate (5 nM) by OATP1B1 was reduced by 82%-95%. This methodology was subsequently used to assess the relative contribution of OATP1B1 uptake in human hepatocytes for olmesartan (42%-62%), valsartan (28%-81%), rosuvastatin (64%-72%), pitavastatin (84%-98%) and lopinavir (64%-89%). These data are consistent with previous values obtained using a relative activity factor approach. 3. The siRNA approach provides a robust and reproducible method for assessing the relative contribution of OATP1B1 to hepatic uptake of new chemical entities. The technique also has potential utility in facilitating detailed characterization of drug-drug interactions involving hepatic drug transporters.

In vitro-in vivo extrapolation of hepatic clearance involving active uptake: theoretical and experimental aspects.

The importance of hepatic uptake transporters in drug clearance is well recognized. The subject is reviewed with the intention of providing an overview of the concepts in order to link the increasing knowledge of transporter-mediated uptake into established models of hepatic clearance. In order to understand and quantify their impact, models of hepatic elimination that incorporate permeability barriers are required. Models that include both active and passive uptake into hepatocytes are discussed and simulations of the influence of active uptake and passive diffusion on hepatic clearance are presented. The advantages and weaknesses of a number of in vitro assays of hepatic uptake are described, and their ability to predict hepatic clearance is reviewed.

Evaluation of human pharmacokinetics, therapeutic dose and exposure predictions using marketed oral drugs.

In this article approaches to predict human pharmacokinetics (PK) are discussed and the capability of the exemplified methodologies to estimate individual PK parameters and therapeutic dose for a set of marketed oral drugs has been assessed. For a set of 63 drugs where the minimum efficacious concentration (MEC) and human PK were known, the clinical dose was shown to be well predicted or in some cases over-estimated using a simple one-compartment oral PK model. For a subset of these drugs, in vitro potency against the primary human targets was gathered, and compared to the observed MEC. When corrected for plasma protein binding, the MEC of the majority of compounds was < or=3 fold over the respective in vitro target potency value. A series of in vitro and in vivo experiments were conducted to predict the human PK parameters. Metabolic clearance was generally predicted well from human hepatocytes. Interestingly, for this compound set, allometry or glomerular filtration rate (GFR) ratio methods appeared to be applicable for renal CL even where CL(renal) > GFR. For approximately 90% of compounds studied, the predicted CL using in vitro-in vivo (IVIV) extrapolation together with a CL(renal) estimate, where appropriate, was within 2-fold of that observed clinically. Encouragingly volume of distribution at steady state (V(ss)) estimated in preclinical species (rat and dog) when corrected for plasma protein binding, predicted human V(ss) successfully on the majority of occasions--73% of compounds within 2-fold. In this laboratory, absorption estimated from oral rat PK studies was lower than the observed human absorption for most drugs, even when solubility and permeability appeared not to be limiting. Preliminary data indicate absorption in the dog may be more representative of human for compounds absorbed via the transcellular pathway. Using predicted PK and MEC values estimated from in vitro potency assays there was a good correlation between predicted and observed dose. This analysis suggests that for oral therapies, human PK parameters and clinical dose can be estimated from a consideration of data obtained from in vitro screens using human derived material and in vivo animal studies. The benefits and limitations of this holistic approach to PK and dose prediction within the drug discovery process are exemplified and discussed.

Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5.

To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL(int) estimates using CYP3A5 compared with CYP3A4 including midazolam (CL(int) = 3.4 versus 3.3 microl min(-1) pmol(-1)). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL(int) = 10.3 versus 2.4 microl min(-1) pmol(-1)), and ritonavir (CL(int) = 0.76 versus 0.47 microl min(-1) pmol(-1)). The majority of compounds studied showed a greater inhibitory potential (IC(50)) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.

The impact of in vitro binding on in vitro-in vivo extrapolations, projections of metabolic clearance and clinical drug-drug interactions.

This review provides a vista of the current opportunities and remaining challenges in the area of in vitro-in vivo extrapolation, with particular emphasis on drug binding terms in predictive models, which has been the source of much controversy. Although the importance of fu(inc) (fraction unbound in in vitro incubations) has been acknowledged for decades, it is not always applied in practice. This is somewhat disappointing, since although it may be onerous to measure this term for large numbers of compounds, algorithms to estimate the term from logD(7.4) or logP have been detailed in the literature. These are sufficiently robust to negate routine measurement in early drug discovery. Several groups have recently established convincing relationships between unbound in vivo and in vitro metabolic intrinsic clearance (CL(int)). In the authors' laboratory, correlations of this type have been constructed for rat, dog and Man. The use and interpretation of these models within a drug discovery setting is discussed. The quantitative prediction of drug-drug interactions from in vitro cytochrome P450 (CYP) inhibition data remains a challenge. Although extensive literature databases are at last emerging, apparent ad hoc use of terms for in vivo inhibitor concentrations and only occasional consideration of fu(inc) may only have confused matters. The effect of accounting for drug binding on the accuracy of predictions is reviewed. Other themes including the impact of fu(inc) on relative activity factors (RAFs) and how in vitro data quality and inter-laboratory differences can confound quantitative human pharmacokinetic predictions are also developed.

In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance.

The glucuronidation of a number of commonly used hepatic uridine diphosphate glucuronosyltransferase drug substrates has been studied in human tissue microsomes. Prediction of in vivo hepatic drug glucuronidation from liver microsomal data yielded a consistent 10-fold under-prediction. Consideration of protein binding was observed to be pivotal when predicting in vivo glucuronidation for acid substrates. Studies using human intestinal microsomes demonstrated the majority of drugs to be extensively glucuronidated such that the intrinsic clearance (CL(int)) of ethinylestradiol (CL(int) = 1.3 microl/min/mg) was twice that obtained using human liver microsomes (CL(int) = 0.7 microl/min/mg). The potential extrahepatic in vivo glucuronidation was calculated for a range of drug substrates from human microsomal data. These results indicate the contribution of intestinal drug glucuronidation to systemic drug clearance to be much less than either hepatic or renal glucuronidation. Therefore, data obtained with intestinal microsomes may be misleading in the assessment of the contribution of this organ to systemic glucuronidation. The use of hepatocytes to assess metabolic stability for drugs predominantly metabolized by glucuronidation was also investigated. Metabolic clearances for a range of drugs obtained using fresh preparations of human hepatocytes predicted accurately hepatic clearance reported in vivo. The use of cryopreserved hepatocytes as an in vitro tool to predict in vivo metabolism was also assessed with an excellent correlation obtained for a number of extensively glucuronidated drugs (R(2) = 0.80, p < 0.001).

The potential pharmacological and toxicological impact of P450 screening.

Cytochrome P450 (CYP) proteins catalyze the majority of drug biotransformations and play a pivotal role in several homeostatic mechanisms. Perturbation of CYP activity can have profound effects on therapeutic efficacy and in extreme cases may lead to life-threatening toxicities. The trend towards front loading physicochemical properties, and drug metabolism and pharmacokinetics (DMPK), in an attempt to minimize the attrition within drug development, has been a concentrated effort on screens aimed at optimizing interactions with the CYP system. These include improving metabolic stability and minimizing the potential for drug-drug interactions associated with marked reductions (inhibition) or increases (induction) in CYP activity. Increased emphasis on the identification of individual CYPs responsible for drug metabolism is also a common theme, with the desire to provide early alerts to compounds cleared predominantly by polymorphic enzymes or isozymes which demonstrate marked inter-subject variability. These data may provide a rationale for individualized therapy in the near future. Such screens should see the establishment of large databases and better in silico (if not in cerebro) prediction of properties typically associated with successful marketed therapies.

Development of a generalized, quantitative physicochemical model of CYP3A4 inhibition for use in early drug discovery.

PURPOSE: To examine the structure-activity relationships for the inhibition of the activity of recombinant human CYP3A4 and to establish a generalized, quantitative physicochemical model for use in early drug discovery. METHODS: Inhibition of the activity of recombinant human CYP3A4 (erythromycin N-demethylase) by 30 diverse chemicals was studied using enhanced throughput methodology. RESULTS: There was a general, strong correlation between the IC50 value determined against erythromycin N-demethylase activity and lipophilicity (LogD7.4) (r2 = 0.68, p <0.0001). This relationship was strengthened further by subdividing the structures studied into two distinct subpopulations of chemistry within the dataset. These could be identified by the absence (r2 = 0.80, p <0.0001) or presence (r2 = 0.69, p <0.0001) of a sterically uninhindered N-containing heterocycle, more specifically a pyridine, imidazole, of triazole function. The presence of these structural motifs increased the potency of CYP3A4 inhibition by approximately 10-fold for a given lipophilicity (LogD7.4.value). More detailed analyses of AstraZeneca compounds demonstrated that the inhibitory potency of the pyridine structure can be attenuated through direct steric effects or electronic substitution resulting in a modulation of the pKa of the pyridine nitrogen, thereby influencing its ability to interact with the CYP heme. CONCLUSIONS: A generalized, quantitative model is proposed for the inhibition of the major drug metabolizing enzyme, CYP3A4. This model indicates the importance of lipophilicity and rationalizes increased potency arising through additional interactions with the heme iron. These general relationships were shown to be applicable to a selection of compounds of interest to several early research projects.

Cloning and characterization of a canine UDP-glucuronosyltransferase.

UDP-glucuronosyltransferases (UGTs) are a major family of enzymes catalyzing the transfer of glucuronic acid to a range of endogenous compounds and xenobiotics facilitating their elimination in either urine or bile. Although the dog is commonly used in drug metabolism studies, relatively little is known about the expression and activity of UGTs in this species. This report describes the molecular cloning and functional characterization of the first dog UGT, UGT1A6. The cloned protein is composed of 528 amino acids with the variable region demonstrating a 67-72% identity with the variable regions of mouse, rat, and human UGT1A6. The enzyme expressed stably in V79 cells predominantly catalyzed the glucuronidation of simple, planar phenols (e.g., for 1-naphthol, K(m) = 41 microM, V(max) = 0.07 nmol/min/mg protein), a class of compounds extensively glucuronidated by human UGT1A6. Based on sequence homology and common catalytic activity, this dog UGT1A protein appears to be the canine orthologue of human UGT1A6.

Predicting drug pharmacokinetics in humans from in vitro metabolism studies.

The pharmaceutical industry is committed to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. There is an increasing need to develop robust, enhanced-throughput in vitro assays, which accurately extrapolate to humans. The major drug metabolizing human hepatic cytochrome P450s (CYPs; CYP1A2, 2C9, 2C19, 2D6 and 3A4) have been co-expressed functionally in Escherichia coli with human NADPH-cytochrome P450 reductase and validated as surrogates to their counterparts in human liver microsomes (HLM) with respect to their kinetic and inhibition properties. Using these recombinant enzymes, fully automated in vitro assays to assess CYP inhibition and determine the enzymology of drug oxidation have been developed and validated. IC(50) values determined for a series of test compounds in HLM and recombinant CYPs were similar (r(2)=0.9, P<0.001). There was a good correlation between the sum of individual CYP intrinsic clearance (Cl(int)) and HLM Cl(int) (r(2)=0.8, P<0.001) for ten prototypic substrates for which clearance was CYP-dependent. Several in vitro incubation milieu (e.g. CYPs, HLM, human hepatocytes) are routinely used and the level of non-specific binding was investigated with respect to effects on K(m) and K(i) determinations. There were clear correlations between binding and lipophilicity (logD(7.4)) for a selection of bases (r(2)=0.98, P<0.001) and acids (r(2)=0.79, P<0.001) that may allow prediction of this property. Our laboratory has shown that recombinant enzymes are suitable for "frontline" predictive human metabolism studies in early drug discovery.

Evidence for significant differences in microsomal drug glucuronidation by canine and human liver and kidney.

The in vitro glucuronidation of a range of structurally diverse chemicals has been studied in hepatic and renal microsomes from human donors and the beagle dog. These studies were undertaken to improve on the limited knowledge of glucuronidation by the dog and to assess its suitability as a model species for pharmacokinetic studies. In general, the compounds studied were glucuronidated severalfold more rapidly (based on intrinsic clearance estimates) by DLM than by HLM. Intrinsic clearance values for human UGT1A1 and UGT2B7 substrates were an order of magnitude higher in DLM than in HLM (e.g., gemfibrozil: 31 microl/min/mg versus 3.0 microl/min/mg; ketoprofen: 2.4 microl/min/mg versus 0.2 microl/min/mg). There were also drug-specific differences. HLM readily glucuronidated propofol (2.4 microl/min/mg) whereas DLM appeared unable to glucuronidate this drug directly. Regioselective differences in morphine glucuronidation were also apparent. Human kidney microsomes catalyzed the glucuronidation of many xenobiotics, although glucuronidation of the endobiotic bilirubin was not detectable in this tissue. In direct contrast, dog kidney microsomes glucuronidated bilirubin only (no glucuronidation of all other xenobiotics was detected). These preliminary studies indicated significant differences in the glucuronidation of xenobiotics by microsomes from the livers and kidneys of human and dog and should be confirmed using a larger panel of tissues from individual dogs. Early knowledge of the relative rates of in vitro glucuronidation, the UGTs responsible for drug glucuronidation, and their tissue distribution in different species could assist the design and analysis of preclinical pharmacokinetic and safety evaluation studies.

Evaluation of the marmoset as a model species for drug glucuronidation.

1. The in vitro glucuronidation of a wide range of compounds has been studied in microsomes prepared from marmoset liver and kidney. These studies have been undertaken to evaluate the marmoset as a model species for drug glucuronidation and for comparison with conjugation by other species. 2. The compounds studied were glucuronidated by marmoset liver microsomes to varying extents (e.g. naproxen CLint 0.4 microl min(-1) mg(-1), 1-naphthol CLint 43 microl min(-1) mg(-1)) Both marmoset and rat liver microsomes glucuronidated morphine at the 3-position (marmoset CLint 19 microl min(-1) mg(-1), rat CLint 6.3 microl min(-1) mg(-1)) but glucuronidation at the 6-position was below, the level of radiodetection in both species. 3. Interestingly, marmoset liver microsomes were able to catalyse the glucuronidation of the tertiary amine imipramine to a significant extent (0.05 nmol min(-1) mg(-1)). However, no glucuronidation was detected by rat liver microsomes. 4. Conjugation of a range of substrates was detectable by marmoset kidney microsomes in contrast to rat kidney microsomes, which only catalysed the glucurondation of bilirubin and 1-naphthol (CLint 17 microl min(-1) mg(-1) and 18 microl min(-1) mg(-1), respectively). 5. This report and previous work in dog and human tissue microsomes suggest that the marmoset may be an alternative animal model for human drug glucuronidation, especially when the pathway of drug glucuronidation is known to differ between lower laboratory species and man.

Automated definition of the enzymology of drug oxidation by the major human drug metabolizing cytochrome P450s.

A fully automated assay to determine the enzymology of drug oxidation by the major human hepatic cytochrome P450s (CYPs; CYP1A2, -2C9, -2C19, -2D6, and -3A4) coexpressed functionally in Escherichia coli with human NADPH-P450 reductase has been developed and validated. Ten prototypic substrates were chosen for which clearance was primarily CYP-dependent, and the activities of these five major CYPs were represented. A range of intrinsic clearance (CL(int)) values were obtained for substrates in both pooled human liver microsomes (HLM; 1-380 microl. min(-1)mg(-1)) and recombinant CYPs (0.03-7 microl. min(-1)pmol(-1)) and thus the percentage contribution of individual CYPs toward their oxidative metabolism could be estimated. All the assignments were consistent with the available literature data. Tolbutamide was metabolized by CYP2C9 (70%) and CYP2C19 (30%), diazepam by CYP2C19 (100%), ibuprofen by CYP2C9 (90%) and CYP2C19 (10%), and omeprazole by CYP2C19 (68%) and CYP3A4 (32%). Metoprolol and dextromethorphan were primarily CYP2D6 substrates and propranolol was metabolized by CYP2D6 (59%), CYP1A2 (26%), and CYP2C19 (15%). Diltiazem, testosterone, and verapamil were metabolized predominantly by CYP3A4. In addition, the metabolite profile for the CYP-dependent clearance of several markers determined by mass spectroscopy was as predicted from the literature. There was a good correlation between the sum of individual CYP CL(int) and HLM CL(int) (r(2) = 0.8, P <.001) for the substrates indicating that recombinant CYPs may be used to predict HLM CL(int) data. This report demonstrates that recombinant human CYPs may be useful as an approach for the prediction of the enzymology of human CYP metabolism early in the drug discovery process.

Amino acid 305 determines catalytic center accessibility in CYP3A4.

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.

Rapid characterization of the major drug-metabolizing human hepatic cytochrome P-450 enzymes expressed in Escherichia coli.

The major drug-metabolizing human hepatic cytochrome P-450s (CYPs; CYP1A2, 2C9, 2C19, 2D6, and 3A4) coexpressed functionally in Escherichia coli with human NADPH-P-450 reductase have been validated as surrogates to their counterparts in human liver microsomes (HLM) using automated technology. The dealkylation of ethoxyresorufin, dextromethorphan, and erythromycin were all shown to be specific reactions for CYP1A2, CYP2D6, and CYP3A4 that allowed direct comparison with kinetic data for HLM. For CYP2C9 and CYP2C19, the kinetics for the discrete oxidations of naproxen and diazepam were compared to data obtained using established, commercial CYP preparations. Turnover numbers of CYPs expressed in E. coli toward these substrates were generally equal to or even greater than those of the major commercial suppliers [CYP1A2 (ethoxyresorufin), E. coli 0.6 +/- 0.2 min(-1) versus B lymphoblasts 0.4 +/- 0.1 min(-1); CYP2C9 (naproxen), 6.7 +/- 0.9 versus 4.9 min(-1); CYP2C19 (diazepam), 3.7 +/- 0.3 versus 0.2 +/- 0.1 min(-1); CYP2D6 (dextromethorphan), 4.7 +/- 0.1 versus 4.4 +/- 0.1 min(-1); CYP3A4 (erythromycin), 3 +/- 1.2 versus 1.6 min(-1)]. The apparent K(m) values for the specific reactions were also similar (K(m) ranges for expressed CYPs and HLM were: ethoxyresorufin 0.5-1.0 microM, dextromethorphan 1.3-5.9 microM, and erythromycin 18-57 microM), indicating little if any effect of N-terminal modification on the E. coli-expressed CYPs. The data generated for all the probe substrates by HLM and recombinant CYPs also agreed well with literature values. In summary, E. coli-expressed CYPs appear faithful surrogates for the native (HLM) enzyme, and these data suggest that such recombinant enzymes may be suitable for predictive human metabolism studies.

Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential.

1. Fully automated inhibition screens for the major human hepatic cytochrome P450s have been developed and validated. Probe assays were the fluorometric-based ethoxyresorufin O-deethylation for CYP1A2 and radiometric analysis of erythromycin N-demethylation for CYP3A4, dextromethorphan O-demethylation for CYP2D6, naproxen O-demethylation for CYP2C9 and diazepam N-demethylation for CYP2C19. For the radiometric assays > 99.7% of 14C-labelled substrate was routinely extracted from incubations by solid-phase extraction. 2. Furafylline, sulphaphenazole, omeprazole, quinidine and ketoconazole were identified as specific markers for the respective CYP1A2 (IC50 = 6 microM), CYP2C9 (0.7 microM), CYP2C19 (6 microM), CYP2D6 (0.02 microM) and CYP3A4 (0.2 microM) inhibition screens. 3. For the radiometric methods, a two-point IC50 estimate was validated by correlating the IC50 obtained with a full (seven-point) assay (r2 = 0.98, p < 0.001). The two-point IC50 estimate is useful for initial screening, while the full IC50 method provides more definitive quantitation, where required. 4. IC50 determined for a series of test compounds in human liver microsomes and cytochrome P450 cDNA-expressed enzymes were similar (r2 = 0.89, p < 0.001). In particular, the CYP1A2, CYP2D6 and CYP3A4 screens demonstrated the flexibility to accept either enzyme source. As a result of incomplete substrate selectivity, expressed enzymes were utilized for analysis of CYP2C9 and CYP2C19 inhibition. Good agreement was demonstrated between IC50 determined in these assays to IC50 published by other laboratories using a wide range of analytical techniques, which provided confidence in the universality of these inhibition screens. 5. These automated screens for initial assessment of P450 inhibition potential allow rapid determination of IC50. The radiometric assays are flexible, sensitive, robust and free from analytical interference, and they should permit the identification and eradication of inhibitory structural motifs within a series of potential drug candidates.

In vitro analysis of the activity of the major human hepatic CYP enzyme (CYP3A4) using [N-methyl-14C]-erythromycin.

Although the N-demethylation of erythromycin has found widespread use in a noninvasive assay with which to phenotype hepatic CYP3A function, currently, the routine in vitro analysis of erythromycin N-demethylase activity relies on the quantitation of liberated formaldehyde by relatively labor-intensive and insensitive colorimetric or fluorimetric detection. This report describes the development of a rapid, sensitive, and reproducible radioassay for human CYP3A4 using solid-phase extraction (SPE). The kinetics of erythromycin N-demethylation were best described by a one-site Michaelis-Menten model with autoinhibition and the apparent kinetic parameters for pooled human liver microsomes (HLM; Km=88 microM, Vmax=345 pmol/min/mg) and expressed CYP3A4 (Km=33 microM, Vmax=130 pmol/min/ mg) were in good agreement. Erythromycin N-demethylase activity was found to vary 14-fold in HLM and correlated with the rate of testosterone 6beta-hydroxylation (r2=0.92, p < 0.001; N=9). Ketoconazole was a potent inhibitor of the N-demethylation of erythromycin, and the estimated IC50 value (104+/-23 nM) agreed well with that obtained using testosterone as a probe for CYP3A (71+/-4 nM). The addition of this radioassay to those established for human CYP1A2, 2C9, 2D6, and 2E1 and its subsequent automation should enable the routine use of this methodology in the analysis of CYP-dependent reactions.