Advertisement
Research ArticleArticle

Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s

Dermot F. McGinnity, Alison J. Parker, Matthew Soars and Robert J. Riley
Drug Metabolism and Disposition November 2000, 28 (11) 1327-1334;

Abstract

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 inEscherichia 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 (CLint) values were obtained for substrates in both pooled human liver microsomes (HLM; 1–380 μl · min−1mg−1) and recombinant CYPs (0.03–7 μl · min−1pmol−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 CLint and HLM CLint(r2 = 0.8, P < .001) for the substrates indicating that recombinant CYPs may be used to predict HLM CLint 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.

Much interest is currently focused on the early identification of the drug-metabolizing enzymes responsible for the biotransformations commonly encountered in drug development (Becquemont et al., 1998). Such information may help identify the key organs for clearance and explain or even predict the observed variability in pharmacokinetics with some substrates and prioritize drug-drug interaction studies. Because most (∼60%) marketed compounds are cleared metabolically by cytochrome P450 (CYP)2 enzymes, the major activity in this area has focused on this family of enzymes (Bertz and Granneman, 1997).

Traditionally, human liver microsomes (HLM) have been the in vitro tool for these studies and have provided both qualitative, e.g., identifying which CYP isoform(s) metabolize the compound of interest (Pichard et al., 1990; Andersson et al., 1993; Otton et al., 1990; Jacqz-Aigrain et al., 1993; Doecke et al., 1991; Wester et al., 2000; Yasumori et al., 1993; Kroemer et al., 1993) and quantitative information, e.g., predicted CLint (Houston, 1994; Rodrigues, 1994; Carlile et al., 1999). Identifying the enzymology of metabolism by human CYPs has proved somewhat labor- and time-intensive, requiring comparative kinetics across a bank of characterized HLM, chemical, and/or antibody inhibition followed by the use of recombinant CYP isoforms (Rodrigues, 1999). The routine access to recombinant CYPs has facilitated direct identification of the isoform(s) responsible for the oxidative metabolism of the drug of interest, although their use in vitro has generally been to support HLM data (Aoyama et al., 1990;Tassaneeyakul et al., 1992; Kroemer et al., 1993; Rodrigues et al., 1994; Yamazaki et al., 1997; Von Moltke et al., 1998; Rodrigues, 1999).

With the advent of combinatorial chemistry and parallel synthesis techniques, there is an expectation to achieve both higher throughput and faster turnaround times in many biological assays. There is an increasing emphasis within drug metabolism in the pharmaceutical industry to develop enhanced throughput frontline in vitro models, including those to determine both the extent and route of the metabolism of new chemical entities (NCEs) and to screen for inducers and inhibitors of drug-metabolizing enzymes (Ayrton et al., 1998; Moody et al., 1999).

The ability to predict directly the human enzymology using enhanced throughput methods would represent a major breakthrough in this technology (Becquemont et al., 1998; Roy et al., 1999) in a similar manner to that adopted for CYP inhibition assays (Crespi et al., 1998;Moody et al., 1999). This laboratory has demonstrated that the five major human hepatic CYPs expressed in Escherichia coli(CYP1A2, -2C9, -2C19, -2D6, and -3A4) are faithful surrogates for their human liver counterparts with respect to their kinetic profiles and inhibition properties (McGinnity et al., 1999; Moody et al., 1999). In this study, the application of recombinant enzymes as a first line approach for identifying the CYP(s) responsible for metabolizing NCEs has been proposed. A fully automated assay has been developed using the major drug-metabolizing human hepatic cytochrome P450s (CYP1A2, -2C9, -2C19, -2D6, and -3A4) coexpressed functionally in E. coliwith human NADPH-P450 reductase, to predict the CYP isoform(s) involved in the oxidative metabolism of NCEs.

Materials and Methods

Chemicals.

All chemicals and reagents used were of the highest available commercial grade. Diltiazem, testosterone, dextromethorphan, (±)-propranolol, (±)-metoprolol, diazepam, tolbutamide, ibuprofen, and β-nicotinamide adenine dinucleotide phosphate, reduced form (β-NADPH) were purchased from Sigma Chemical Co. (Poole, UK). (±)-Verapamil was purchased from Aldrich Chemical Co. Ltd. (Gillingham, UK). Omeprazole was synthesized at AstraZeneca R&D Charnwood (Loughborough, UK).

Source of Cytochrome P450.

The LINK consortium, a collaboration between UK-based academia and industry, provided stocks of transformed cells with human CYP1A2, CYP2C9, CYP2D6, and CYP3A4 individually coexpressed with human NADPH-P450 reductase in E. coli as described previously (McGinnity et al., 1999). All experiments with CYP1A2, CYP2C9, CYP2D6, and CYP3A4 utilized the E. coli membrane source. All transformed cells were stored as glycerol stocks at −80°C. Expression of the recombinant proteins and preparation of the respective E. coli membranes were carried out as described previously (McGinnity et al., 1999).

Microsomes prepared from insect cells infected with a baculovirus containing the cDNA for human CYP2C19 and rabbit NADPH-P450 reductase were purchased from PanVera Corp. (Madison, WI). All experiments with CYP2C19 utilized this enzyme source. Pooled HLM were purchased from IIAM (Leicester, UK) and In Vitro Technologies (Baltimore, MD). Table1 displays the CYP isoform characterization of the individual HLM pools as determined by the commercial supplier.

View this table:
Table 1

CYP isoform characterization of individual HLM pools

Cytochrome P450 contents were estimated spectrally by the method ofOmura and Sato (1964). Protein concentrations were measured using the Randox Laboratories Ltd. (Crumlin, UK) protein kit based on pyrogallol red complexing with protein in an acid environment containing molybdate ions (Watanabe et al., 1986), using bovine serum albumin as a standard.

Probe Substrates.

Ten commercially available drugs were selected as probe substrates to establish the suitability of this approach. The compounds were selected from the literature as marketed drugs for which the relative CYP-dependent metabolic formation was known and their metabolism by the five CYPs was adequately represented: tolbutamide (Back et al., 1988;Bourrie et al., 1996; Jung et al., 1997; Wester et al., 2000); diazepam (Ono et al., 1996); metoprolol (Otton et al., 1988; Mautz et al., 1995); ibuprofen (Hamman et al., 1997); propranolol (Otton et al., 1990; Yoshimoto et al., 1995); dextromethorphan (Dayer et al., 1989;Broly et al., 1990; Jacqz-Aigrain et al., 1993; Kerry et al., 1994; Von Moltke et al., 1998); omeprazole (Andersson et al., 1993; Kobayashi et al., 1994; Yamazaki et al., 1997); diltiazem (Pichard et al., 1990;Sutton et al., 1997); testosterone (Waxman et al., 1988; Wang et al., 1997); and verapamil (Kroemer et al., 1993; Tracy et al., 1999).

Automated CYP CLint Determination.

CYP CLint determination assays were fully automated and performed by a robotic sample processor (RSP) (Genesis RSP 150; Tecan, Reading, UK). Assays performed by the RSP were done using a program created by the user and not by a default program supplied with the hardware.3 The primary stock of all probe substrates was prepared manually in dimethyl sulfoxide or acetonitrile at 100-fold final incubation concentration. The final concentration of organic solvent in the incubation was 1% v/v. At this concentration dimethyl sulfoxide has been shown to reduce the activities of CYP2C9/19 (Chauret et al., 1998; Hickman et al., 1998), although this effect appears to be substrate-dependent. All substrates were incubated at 3 μM except tolbutamide (CLint calculated by determiningVmax and Km), ibuprofen (10 μM), and testosterone (10 μM). The RSP was programmed to add chilled quenching solvent (100 μl of acetonitrile) to 96-well refrigerated blocks, and compound stocks were then prediluted in 100 mM potassium phosphate buffer, pH 7.4. E. coli membranes and microsomes prepared from baculovirus coexpressing individual CYPs and NADPH-reductase were added to incubation tubes (100 pmol of CYP · ml−1 final concentration) located in a 96-well heated block (37°C). A subaliquot was removed to produce a 0-min time point, and the assay was initiated via addition of NADPH (1 mM final concentration). Aliquots (50 μl) were removed at 5, 10, 15, and 20 min and quenched in acetonitrile. Samples were subsequently removed from the RSP, frozen for 1 h at −20°C, and then centrifuged at 3500 rpm for 20 min. The supernatants were removed and transferred into HPLC vials using the RSP.

Automated Human Liver Microsome CLint Determination.

HLM were diluted in 100 mM potassium phosphate buffer, pH 7.4 (1 mg · ml−1 final). Probe substrates were incubated at identical concentrations as the CYP CLint assay, and incubations were carried out on the RSP as described above. Reactions were again initiated by addition of NADPH (1 mM final concentration), and several aliquots were taken over 45 min.

HPLC Methods.

Aliquots (20 μl) were analyzed by HPLC-UV or HPLC-fluorescence for either parent loss or metabolite appearance using a model 1100 Chemstation (Hewlett-Packard, Palo Alto, CA) and a Hewlett-Packard 1046A fluorescence detector. A symmetry shield RP8 3.9- × 50-mm cartridge (Waters, Watford, UK) and a mobile phase of 0.025% (w/v) ammonium acetate (solvent 1A) and acetonitrile (solvent 1B) was used for the chromatography of most analytes. Testosterone required a mobile phase of 0.025% ammonium acetate:methanol (95:5, v/v) (solvent 2A) and acetonitrile:methanol (95:5, v/v) (solvent 2B). The flow rate for all methods was 1.5 ml · min−1. Diazepam, metoprolol, propranolol, omeprazole, diltiazem, and verapamil eluted using a 5-min linear gradient from 80% solvent 1A to 20% solvent 1A, tolbutamide 99% to 65% over 5 min, dextromethorphan 80% to 20% over 3.5 min, and ibuprofen 85% to 20% over 5 min. Testosterone was eluted using a linear gradient from 85% solvent 2A to 75% solvent 2A over 12 min, 75% to 20% over 3 min followed by isocratic conditions (20:80) for 2 min. UV detection was performed for omeprazole (302 nm), diltiazem (237 nm), ibuprofen (222 nm), and testosterone (254 nm) and for metabolites of diazepam (229 nm) and tolbutamide (230 nm). Fluorometric detection was performed for metoprolol (Excitation 222 nm and Emission 320 nm), dextromethorphan (270, 312 nm), propranolol (205, 340 nm) and verapamil (280 nm, 310 nm).

Metabolite Identification.

HLM or recombinant CYPs were diluted in 100 mM potassium phosphate buffer, pH 7.4 (1 mg · ml−1 or 100 pmol · ml−1, respectively). Probe substrates were incubated at 30 μM, reactions were initiated by addition of NADPH (1 mM), and aliquots were quenched in 1:1 (v/v) methanol at 0 and 45 min. Aliquots (20 μl) were analyzed by liquid chromatography-mass spectrometry using the Hewlett-Packard 1100 Chemstation with a symmetry shield RP8 3.9- × 50-mm cartridge and a mobile phase of 0.025% (w/v) ammonium acetate (solvent 3A) and methanol (solvent 3B). Analytes were eluted using a gradient of 95% solvent 3A to 10% solvent 3A over 7 min. Metabolites were detected using a TSQ 7000 mass spectrophotometer (Finnigan MAT, San Diego, CA) with an atmospheric pressure chemical ionization ion source and a triple quadrupole mass analyzer in full scan mode. The molecular ion (either M + H+ or M − H+ depending on the orifice polarity) was detected for each metabolite.

Data Analysis.

Throughout this study, several approaches were adopted for quantifying intrinsic clearance:

Metabolite appearance—low turnover compounds.

CLint=Vmax/km (tolbutamide) V=Vmax×SKm+S if S ≪ Km (≤10%)V=Vmax×SKm soVS=VmaxKm=CLint (diazepam)

Parent loss.

Because dose/C0 gives a term for the volume of the incubation (expressed in ml · pmol of CYP−1) and the elimination rate constantk = 0.693/T1/2, an equation expressing CLint in terms ofT1/2 of parent loss can be derived:Clint=Volume×0.693T1/2 (majority) The contribution of individual CYP to HLM CLint was estimated as follows:⇒CYP Clint×%content of CYP isoform in HLM×Avg.total CYP in HLM (320 pmol/mg) Table 2 shows the average percentage content of the five major isoforms in human hepatic microsomes.

View this table:
Table 2

Estimated levels of the five major isoforms in HLM pools

All individual data represent means from at least duplicate determinations.

Results

Marker Substrates.

CLint values were obtained for each of the prototypic substrates, tolbutamide, diazepam, metoprolol, ibuprofen, propranolol, dextromethorphan, omeprazole, diltiazem, testosterone, and verapamil in three individual preparations of pooled HLM. Table3 compares the CLint for the three batches of HLM together with the mean and values obtained from the literature. For substrates with significant CYP3A4 metabolism, diltiazem, testosterone, and verapamil, CLint was significantly higher in batch 1 compared with batches 2 and 3. The coefficient of variation was generally <15% for CLint determined from one pool of HLM.

View this table:
Table 3

CLint of marker substrates in different HLM pools and literature values

Substrates were incubated with recombinant CYP1A2, -2C9, -2C19, -2D6, and -3A4, respectively, using the RSP as described underMaterials and Methods. Figure 1displays the loss of propranolol against time by the five different CYP isoforms used in the automated assay and shows significant metabolism by CYP1A2, -2C19, and -2D6. Table 4 shows the CYP CLint of individual CYP isoforms to oxidative metabolism for each marker substrate. The range of CYP CLint determined was 0.03 to 7 μl · min−1 pmol of P450−1. The percentage contributions of individual CYPs toward oxidative metabolism of a compound in HLM were estimated, and Table5 compares our values with common literature assignments. Tolbutamide (CLintdetermined byVmax/Km) was metabolized by both CYP2C9 (70%) and CYP2C19 (30%), diazepam (10 μM) by CYP2C19 (100%), ibuprofen (10 μM) by CYP2C9 (90%) and CYP2C19 (10%), and omeprazole (3 μM) by CYP2C19 (68%) and CYP3A4 (32%), respectively. Metoprolol (3 μM) and dextromethorphan (3 μM) are primarily CYP2D6 substrates and propranolol (3 μM) was metabolized by CYP2D6 (59%), CYP1A2 (26%), and CYP2C19 (15%). Diltiazem (3 μM), testosterone (10 μM), and verapamil (3 μM) were predominantly metabolized by CYP3A4.

Figure 1
Figure 1

CYP1A2, -2C9, -2C19, -2D6, and -3A4-dependent clearance of propranolol.

The determination of the CYP-dependent propranolol metabolism using the automated assay is as described under Materials and Methods. Aliquots were taken at 0, 5, 10, 15, and 20 min, and the amount of propranolol remaining in the incubation media is reflected by the peak area after HPLC-fluorescence detection. The data represent propranolol clearance by E. coli membranes expressing CYP1A2 (▪), CYP2C9 (●), CYP2D6 (♦), CYP3A4 (▾), and baculovirus expressing CYP2C19 (▴), as described underMaterials and Methods. The solid lines indicate linear regression of the data.

View this table:
Table 4

Determination of CYP Clint of individual human CYPs to oxidative metabolism for marker substrates

View this table:
Table 5

Mean percentage contribution of individual CYPs to oxidative metabolism

For each compound, the sum of the CLint(μl · min−1mg−1) from the five individual isoforms was compared with the respective CLint derived from the mean of three separate HLM pools (Table 6). Figure2 shows the correlation (r2 = 0.8, P < .001) between the CYP CLint and HLM CLint. Compounds with a HLM CLint of <8 μl · min−1mg−1 may be described as low clearance, 8 to 65 μl · min−1mg−1 as intermediate, and >65 μl · min−1mg−1 as high clearance.

View this table:
Table 6

Determination of CYP Clint for marker and AR-C compounds

Figure 2
Figure 2

Comparison of summed CYP CLintwith HLM CLint for several probe substrates.

Both the summed CYP and HLM CLint determinations were carried out as described under Materials and Methods. The data points represent the mean CLint determinations, and the error bars reflect the standard deviation from the mean as shown in Table 6. The dotted boxes illustrate HLM CLints of <8 μl · min−1mg−1 (low clearance) and >65 μl · min−1mg−1 (high clearance). The solid line depicts a linear regression analysis of the data (r2 = 0.8, P < .001) with the equation logCYP CLint = 0.91 × logHLM CLint + 0.3. The dashed line indicates line of unity.

Predictions of HLM CLint from the sum of individual CYP CLint were excellent for tolbutamide (CYP CLint = 1.3 μl · min−1mg−1 versus HLM CLint = 0.7 μl · min−1mg−1), diazepam (2 ± 1 versus 3 ± 1), and metoprolol (7 ± 0 versus 6 ± 1). All predictions of HLM CLint from the individually summed CYP CLint except for those derived for propranolol (CYP CLint = 55 ± 15 μl · min−1mg−1 versus HLM CLint = 15 ± 0 μl · min−1mg−1) and omeprazole (131 ± 25 versus 34 ± 14) were within 3-fold. The summed CYP CLint of these two compounds significantly overestimated their CLint determined in HLM.

To investigate the relationship between the CLintof propranolol with increasing HLM protein concentration, CLint was determined at 0.4, 1, and 2 mg · ml−1 of HLM. Figure 3shows the relationship between increasing microsomal protein and decreasing CLint of propranolol.

Figure 3
Figure 3

Determination of propranolol CLint at different concentrations of HLM.

The HLM CLint determinations were carried out as described under Materials and Methods. The histograms reflect a mean CLint, and the error bars give the standard deviation from the mean. Experiments were carried out in duplicate a minimum of three times.

The metabolite profile for the CYP-dependent clearance of dextromethorphan observed by HPLC-mass spectrometry was as predicted from Von Moltke et al. (1998), where microsomes containing individual CYPs expressed by a human lymphoblastoid expression system were used (Fig. 4). Dextromethorphan was chosen for this detailed analysis, because four of the five major human CYPs (CYP2C9, -2C19, -2D6, and -3A4) have been implicated in its metabolism. Dextromethorphan was incubated at 30 μM with all five isoforms, and as expected, two metabolites, dextrorphan and 3-methoxymorphinan, were observed, based on their m/z values and distinguished using standards. Based on UV response, 88% of the metabolites formed from dextromethorphan (m/z = 272) were dextrorphan (m/z = 258) and 12% were 3-methoxymorphinan (m/z = 258), which compares well with Von Moltke et al. (1998) (98 and 2%, respectively). The isoform responsible for dextrorphan formation was primarily CYP2D6 (92% versus 97%; as determined from Von Moltke et al., 1998) with minor contributions from CYP2C9, -2C19, and -3A4 (<3% in both laboratories). The isoforms responsible for 3-methoxymorphinan formation were CYP2C9 (43% versus 55%), CYP3A4 (42% versus 20%), CYP2C19 (8% versus 16%), and CYP2D6 (7% versus 9%). In addition, it was also determined that CYP1A2 metabolized propranolol (m/z = 260) to the expectedN-deisopropylation product (m/z = 218) (Yoshimoto et al., 1995) and CYP2D6 metabolized propranolol to the expected hydroxylated product (m/z = 276), although the regiochemistry of hydroxylation was not investigated. Several other markers also generated the product profile as expected from the literature (data not shown).

Figure 4
Figure 4

Metabolite profile for the CYP-dependent clearance of dextromethorphan.

The identification of CYP-dependent dextromethorphan metabolite formation is as described under Materials and Methods. The data from this laboratory, where dextromethorphan was incubated at 30 μM, are compared with that of Von Moltke et al. (1998) where the data reflect CLint.

Discussion

To generate confidence that recombinant CYPs may be used to predict HLM CLint data, the in vitro kinetics for commonly used CYP probes were determined previously in this laboratory in both pooled HLM and CYP coexpressed with NADPH-reductase in E. coli cells (McGinnity et al., 1999). The kinetic parameters (including CLint) of these recombinant enzymes were similar to their human liver counterparts for the enzyme substrate pairs that were directly comparable, and thus they would appear to be faithful surrogates. Indeed, Eddershaw and Dickins (1999) reported an excellent comparison between the rates of metabolism of several compounds determined from HLM and microsomes containing a mixture of the major recombinant CYPs. However, this “artificial HLM” approach gives little information as to the enzymology of metabolism.

To demonstrate the potential for predicting both the extent and route of oxidative metabolic clearance for NCEs by recombinant human CYPs, several marketed drugs were selected in which metabolism via CYP pathways was well established. Of the marketed drugs that are primarily cleared by human hepatic CYP-mediated metabolism, the vast majority were metabolized by one or more of the five isoforms, CYP1A2, -2C9, -2C19, -2D6, and -3A4 (Bertz and Granneman, 1997), and, for that reason, only these isoforms were employed in this initial study. There are limited available data on the relative levels of the five major isoforms in human hepatic microsomes (Table 2), and we have relied heavily on the seminal study by Shimada et al. (1994), which is widely cited for this purpose. The marker compounds tolbutamide, diazepam, metoprolol, ibuprofen, propranolol, dextromethorphan, omeprazole, diltiazem, testosterone, and verapamil were chosen so that metabolism by each of the five CYPs was adequately represented. The choice of DMSO as a solvent was based on its value for compounds with relatively low solubility (often encountered in early drug discovery programs) and its implementation as the solvent of choice in many liquid banks. Any inhibitory effects should not affect the comparison between recombinant CYPs and HLM.

There is a reasonable agreement between the CLintof the probe substrates determined in HLM to available literature values (Table 3), although the comparison is somewhat compromised due to the large spread of the literature data. There is agreement as to whether a compound demonstrates a low, intermediate, or high CLint. The limitations of such an interlaboratory comparison and the inherent variability of such an exercise are well established (Boobis et al., 1998). Literature CLint values have been obtained from a variety of sources, includingVmax/Kmcalculations, microsomes (prepared from individual as well as pooled livers), and hepatocytes [data converted to μl · min−1mg−1 assuming 2.67 × 106 cells/mg of microsomal protein (Carlile et al., 1999)]. Variability will also result from the fact that isoform levels and activities may vary significantly between the different metabolizing sources (Boobis et al., 1998). However, the agreement is excellent where a direct comparison between two laboratories determining HLM CLint for several compounds can be made (Obach, 1999).

Without exception, our data and the prevailing literature assign the same isoform to be the predominant CYP responsible for the metabolism of each marker compound. Diazepam at low micromolar concentrations was metabolized by CYP2C19, which agrees with Jung et al. (1997), Yasumori et al. (1993), and Andersson et al. (1994). Indeed, detailed HLM kinetics of diazepam metabolism (not shown) suggests the involvement of multiple CYPs (e.g., CYP2C9/18, -2B6, and -3A4), but the data indicate that the high affinity component of diazepam N-demethylation in vivo may be CYP2C19. Metoprolol and dextromethorphan are primarily CYP2D6 substrates (Otton et al., 1988; Dayer et al., 1989;Jacqz-Aigrain et al., 1993; Kerry et al., 1994; Von Moltke et al., 1998). Diltiazem, testosterone, and verapamil are predominantly metabolized by CYP3A4 (Waxman et al., 1988; Pichard et al., 1990;Kroemer et al., 1993; Sutton et al., 1997; Tracy et al., 1999).

In addition, for propranolol, omeprazole, tolbutamide, and ibuprofen, there was excellent concordance between our data and the literature on the relative contribution of several isoforms in the metabolism of the respective compounds. Tolbutamide is metabolized by both CYP2C9 (70%) and CYP2C19 (30%), which agrees with Inoue et al. (1997), Wester et al. (2000), Venkatakrishnan et al. (1998), and Lasker et al. (1998). Similarly, ibuprofen is metabolized by CYP2C9 (90%) and CYP2C19 (10%) (Leemann et al., 1993; Hamman et al., 1997) and omeprazole by CYP2C19 (68%) and CYP3A4 (32%) (Andersson et al., 1993; Karam et al., 1996;Yamazaki et al., 1997; Lasker et al., 1998). Indeed, when recombinant CYP2C19 and CYP3A4 were mixed at a ratio similar to that found in HLM, the metabolism of omeprazole resembled that of HLM (Yamazaki et al., 1997). The assignment of CYP2D6 (59%)-, CYP1A2 (26%)-, and CYP2C19 (15%)-dependent metabolism for propranolol agrees with several sources (Lennard et al., 1984; Otton et al., 1990; Yoshimoto et al., 1995), which implicate these three isoforms. In addition, the appropriate metabolites from each isoform were identified by mass spectrometry analysis.

A method based on the rate of enzyme activity (relative activity factor) of recombinant CYPs and HLM has proven useful in assigning the contribution of individual CYPs to several biotransformations (Rodrigues, 1999; Roy et al., 1999). Recently, it has been suggested that a complementary approach using the ratio ofintrinsic clearance as a relative activity factor may be more predictive, where the kinetics for recombinant CYPs and HLM are equivalent (Nakajima et al., 1999). The correlation observed in this study between the sum of CLint from the different CYP isoforms and HLM CLint for the compounds tested confirms this concept. This study has additionally provided a more thorough evaluation of these recombinant proteins expressed inE. coli.

The summed CYP CLint correctly predicted a low HLM CLint (<8 μl · min−1mg−1) for tolbutamide, diazepam, and metoprolol; an intermediate HLM CLint (8–65 μl · min−1mg−1) for ibuprofen, propranolol, dextromethorphan, diltiazem, and testosterone; and a high HLM CLint (>65 μl · min−1mg−1) for verapamil. However, the summed CYP CLint of omeprazole and propranolol did overpredict somewhat HLM CLint. One possible explanation for this is an increase in “futile” binding with increased protein concentration for some compounds. For propranolol there is 50% free at 0.4 mg · ml−1 and 25% at 2 mg · ml−1 (Obach, 1997), which results in a 2-fold decrease of propranolol CLint (Fig. 4). Typical assay conditions used 0.2 to 0.4 mg of protein/ml−1 of CYPs (exact amount depended on the CYP expression level, because all incubations contain 100 pmol of CYP/ml−1) and 1 mg · ml−1 HLM. The HLM CLint of propranolol at 0.4 mg · ml−1 was determined to be 22 ± 4 μl · min−1mg−1, which compares more favorably with the summed CYP CLint at the same protein level (55 ± 15 μl · min−1mg−1).

Generally, lower protein levels in the recombinant CYP assay may allow a more accurate reflection of unbound CLint and provide a greater dynamic CLint range when discriminating between large numbers of compounds. There is likely to be no significant differences between the extent of futile binding for HLM and recombinant CYPs at the same total protein concentration (Venkatakrishnan et al., 2000). Differential protein binding between in vitro matrices for predicting in vivo Clmet is currently under investigation.

In our experience, an accurate determination of a wide range of CLint is achieved at an incubation concentration for recombinant CYP of 100 pmol of CYP/ml−1, which may be subsequently optimized. The molar ratio of NADPH-P450 reductase to recombinant CYP has been manipulated for the E. coli expression constructs to produce optimal reaction kinetics for probe substrates (McGinnity et al., 1999). For example, optimal CYP2C19-mediated diazepam N-demethylation can be achieved, in the absence of cytochrome b5, by increasing the molar ratio of NADPH-P450 reductase:CYP2C19 to approximately 20:1 (McGinnity et al., 1999). Indeed, to optimize CYP expression systems, further elucidation of the role and importance of ancillary electron transporters such as b5in the metabolism of xenobiotics is required (Yamazaki et al., 1999).

A correlation between HLM and CYP CLint allows compounds to be ranked with respect to metabolic stability, should expedite knowledge of the pharmacophore of individual CYP isoforms, and may facilitate more rational compound synthesis to achieve greater metabolic stability. Furthermore, an underprediction of HLM CLint from the five major human hepatic isoforms should prompt an investigation into possible metabolism by the more minor human hepatic CYPs, i.e., CYP2A6, -2B6, -2C8, or -2E1 (Houston, 1994).

This automated assay is being used early in drug discovery at AstraZeneca R&D Charnwood, a strategy distinct from the comprehensive isoform profiling of a drug later in the development process by other groups (Machinist et al., 1998; Fischer et al., 1999; Nakajima et al., 1999; Roy et al., 1999). The early identification of the major CYP isoforms involved in the metabolism of a drug candidate is useful for several purposes, including understanding ligand-enzyme structure-activity relationships, expanding the database for substrates of the polymorphic isoforms, assessing the potential intersubject variability, and predicting the drug-drug interactions and, ultimately, the direction of clinical trials.

These data indicate that recombinant CYPs may be used to predict HLM CLint. Furthermore, it may prove feasible to scale human CLint data to the fractional metabolic clearance encountered clinically (Iwatsubo et al., 1997;Obach, 1999). Therefore, although very much in its infancy, data in this report demonstrate that E. coli-expressed CYPs may be useful as an early approach for the prediction of the enzymology of human CYP metabolism. Further efforts to examine the differential nonspecific binding between the separate in vitro models and the effects on CLint are underway.

Footnotes

  • Send reprint requests to: Dr. Rob Riley, Department of Physical & Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, Leicestershire LE11 5RH, UK. E-mail: Rob.Riley{at}astrazeneca.com

  • ↵1 Current address: Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.

  • ↵3 Copies of the program are available from the corresponding author upon request.

  • Abbreviations used are::
    CYP
    cytochrome P450
    HLM
    human liver microsomes
    CLint
    intrinsic clearance
    NCE
    new chemical entity
    RSP
    robotic sample processor
    • Received May 24, 2000.
    • Accepted August 7, 2000.

References

  1. ↵
    1. Andersson T,
    2. Miners JO,
    3. Veronese ME,
    4. Birkett DJ
    (1994) Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms. Br J Clin Pharmacol 38:131–137.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Andersson T,
    2. Miners JO,
    3. Veronese ME,
    4. Tassaneeyakul W,
    5. Tassaneeyakul W,
    6. Meyer UA,
    7. Birkett DJ
    (1993) Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism. Br J Clin Pharmacol 36:521–530.
    OpenUrlPubMed
  3. ↵
    1. Aoyama T,
    2. Korzekwa K,
    3. Nagata K,
    4. Gillette J,
    5. Gelboin HV,
    6. Gonzalez FJ
    (1990) Estradiol metabolism by complementary deoxyribonucleic acid-expressed human cytochrome P450s. Endocrinology 126:3101–3106.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Ayrton J,
    2. Plumb R,
    3. Leavens WJ,
    4. Mallett D,
    5. Dickins M,
    6. Dear GJ
    (1998) Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates. Rapid Commun Mass Spectrom 12:217–224.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Back DJ,
    2. Tjia JF,
    3. Karbwang J,
    4. Colbert J
    (1988) In vitro inhibition studies of tolbutamide hydroxylase activity of human liver microsomes by azoles, sulphonamides and quinolines. Br J Clin Pharmacol 26:23–29.
    OpenUrlPubMed
  6. ↵
    1. Becquemont L,
    2. Le Bot MA,
    3. Riche C,
    4. Funck-Brentano C,
    5. Jaillon P,
    6. Beaune P
    (1998) Use of heterologously expressed human cytochrome P450 1A2 to predict tacrine-fluvoxamine drug interaction in man. Pharmacogenetics 8:101–108.
    OpenUrlPubMed
  7. ↵
    1. Belloc C,
    2. Baird S,
    3. Cosme J,
    4. Lecoeur S,
    5. Gautier JC,
    6. Challine D,
    7. de Waziers I,
    8. Flinois JP,
    9. Beaune PH
    (1996) Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies. Toxicology 106:207–219.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Bertz RJ,
    2. Granneman GR
    (1997) Use of in vitro and in vivo data to estimate the likelihood of metabolic pharmacokinetic interactions. Clin Pharmacokinet 32:210–258.
    OpenUrlPubMed
  9. ↵
    1. Boobis AR,
    2. Mckillop D,
    3. Robinson DT,
    4. Adams DA,
    5. McCormick DJ
    (1998) Interlaboratory comparison of the assessment of P450 activities in human hepatic microsomal samples. Xenobiotica 28:493–506.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Bourrie M,
    2. Meunier V,
    3. Berger Y,
    4. Fabre G
    (1996) Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp Ther 277:321–332.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Broly F,
    2. Libersa C,
    3. Lhermitte M,
    4. Dupuis B
    (1990) Inhibitory studies of mexiletine and dextromethorphan oxidation in human liver microsomes. Biochem Pharmacol 39:1045–1053.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Carlile DJ,
    2. Hakooz N,
    3. Bayliss MK,
    4. Houston JB
    (1999) Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans. Br J Clin Pharmacol 47:625–635.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Chauret N,
    2. Gauthier A,
    3. Nicoll-Griffith DA
    (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab Dispos 26:1–4.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Crespi CL,
    2. Miller VP,
    3. Penman BW
    (1998) High throughput screening for inhibition of cytochrome P450 metabolism. Med Chem Res 8:457–471.
    OpenUrl
  15. ↵
    1. Dayer P,
    2. Leemann T,
    3. Striberni R
    (1989) Dextromethorphan O-demethylation in liver microsomes as a prototype reaction to monitor cytochrome P-450 db1 activity. Clin Pharmacol Ther 45:34–40.
    OpenUrlPubMed
  16. ↵
    1. Doecke CJ,
    2. Veronese ME,
    3. Pond SM,
    4. Miners JO,
    5. Birkett DJ,
    6. Sansom LN,
    7. McManus ME
    (1991) Relationship between phenytoin and tolbutamide hydroxylations in human liver microsomes. Br J Clin Pharmacol 31:125–130.
    OpenUrlPubMed
  17. ↵
    1. Eddershaw PJ,
    2. Dickins M
    (1999) Advances in in vitro drug metabolism screening. Pharm Sci Technol Today 2:13–19.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Fischer V,
    2. Johanson L,
    3. Heitz F,
    4. Tullman R,
    5. Graham E,
    6. Baldeck JP,
    7. Robinson WT
    (1999) The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin: Effect on human cytochrome P-450 and implications for metabolic drug interactions. Drug Metab Dispos 27:410–416.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Guengerich FP,
    2. Turvy CG
    (1991) Comparison of levels of several human microsomal cytochrome P-450 enzymes and epoxide hydrolase in normal and disease states using immunochemical analysis of surgical liver samples. J Pharmacol Exp Ther 256:1189–1194.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Hamman MA,
    2. Thompson GA,
    3. Hall SD
    (1997) Regioselective and stereoselective metabolism of ibuprofen by human cytochrome P450 2C. Biochem Pharmacol 54:33–41.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Hickman D,
    2. Wang J-P,
    3. Wang Y,
    4. Unadkat JD
    (1998) Evaluation of the selectivity of in vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos 26:207–215.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Houston JB
    (1994) Relevance of in vitro kinetic parameters to in vivo metabolism of xenobiotics. Toxicol in Vitro 8:507–512.
    OpenUrlPubMed
  23. ↵
    1. Imaoka S,
    2. Yamada T,
    3. Jiroi T,
    4. Hayashi K,
    5. Sakaki T,
    6. Yabusaki Y,
    7. Funae Y
    (1996) Multiple forms of human P450 expressed in Saccharomyces cerevisiae: Systematic characterization and comparison with those of the rat. Biochem Pharmacol 51:1041–1050.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Inoue K,
    2. Yamazaki H,
    3. Imiya K,
    4. Akasaka S,
    5. Guengerich FP,
    6. Shimada T
    (1997) Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S-mephenytoin 4′-hydroxylation activities in livers of Japanese and Caucasian populations. Pharmacogenetics 7:103–113.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Iwatsubo T,
    2. Hirota N,
    3. Ooie T,
    4. Suzuki H,
    5. Shimada N,
    6. Chiba K,
    7. Ishizaki T,
    8. Green CE,
    9. Tyson CA,
    10. Sugiyama Y
    (1997) Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol Ther 73:5147–5171.
    OpenUrl
  26. ↵
    1. Jacqz-Aigrain E,
    2. Funck-Brentano C,
    3. Cresteil T
    (1993) CYP2D6- and CYP3A-dependent metabolism of dextromethorphan in humans. Pharmacogenetics 3:197–204.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Jung F,
    2. Richardson TH,
    3. Raucy JL,
    4. Johnson EF
    (1997) 0 Diazepam metabolism by cDNA-expressed human 2C P450s: Identification of P4502C18 and P4502C19 as low Km diazepam N-demethylases. Drug Metab Dispos 25:133–139.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Karam WG,
    2. Goldstein JA,
    3. Lasker JM,
    4. Ghanayem BI
    (1996) 0 Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes. Drug Metab Dispos 24:1081–1087.
    OpenUrlAbstract
  29. ↵
    1. Kerry NL,
    2. Somogyi AA,
    3. Bochner F,
    4. Mikus G
    (1994) 0 The role of CYP2D6 in primary and secondary oxidative metabolism of dextromethorphan: In vitro studies using human liver microsomes. Br J Clin Pharmacol 38:243–248.
    OpenUrlPubMed
  30. ↵
    1. Kobayashi K,
    2. Chiba K,
    3. Tani M,
    4. Kuroiwa Y,
    5. Ishizaki T
    (1994) Development and preliminary application of a high-performance liquid chromatographic assay for omeprazole metabolism in human liver microsomes. J Pharm Biomed Anal 12:839–844.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Kroemer HK,
    2. Gautier JC,
    3. Beaune P,
    4. Henderson C,
    5. Wolf CR,
    6. Eichelbaum M
    (1993) Identification of P450 enzymes involved in metabolism of verapamil in humans. Naunyn-Schmiedeberg's Arch Pharmacol 348:332–337.
    OpenUrlPubMed
  32. ↵
    1. Lasker JM,
    2. Wester MR,
    3. Aramsombatdee E,
    4. Raucy JL
    (1998) 0 Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:16–28.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Leemann TD,
    2. Transon C,
    3. Bonnabry P,
    4. Dayer P
    (1993) A major role for cytochrome P450TB (CYP2C subfamily) in the actions of non-steroidal anti-inflammatory drugs. Drugs Exp Clin Res 19:189–195.
    OpenUrlPubMed
  34. ↵
    1. Lennard MS,
    2. Jackson PR,
    3. Freestone S,
    4. Tucker GT,
    5. Ramsay LE,
    6. Woods HF
    (1984) The relationship between debrisoquine oxidation phenotype and the pharmacokinetics and pharmacodynamics of propranolol. Br J Clin Pharmacol 17:679–685.
    OpenUrlPubMed
  35. ↵
    1. Machinist JM,
    2. Mayer MD,
    3. Roberts EM,
    4. Surber BW,
    5. Rodrigues AD
    (1998) Identification of the human liver cytochrome P450 enzymes involved in the in vitro metabolism of a novel 5-lipoxygenase inhibitor. Drug Metab Dispos 26:970–976.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Mautz DS,
    2. Nelson WL,
    3. Shen DD
    (1995) Regioselective and stereoselective oxidation of metoprolol and bufuralol catalyzed by microsomes containing cDNA-expressed human P4502D6. Drug Metab Dispos 23:513–517.
    OpenUrlAbstract
  37. ↵
    1. McGinnity DF,
    2. Griffin SJ,
    3. Moody GC,
    4. Voice M,
    5. Hanlon S,
    6. Friedberg T,
    7. Riley RJ
    (1999) Rapid characterization of the major drug-metabolizing human hepatic cytochrome P-450 enzymes expressed in Escherichia coli. Drug Metab Dispos 27:1017–1023.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Moody GC,
    2. Griffin SJ,
    3. Mather AN,
    4. McGinnity DF,
    5. Riley RJ
    (1999) Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: Assessment of human CYP inhibition potential. Xenobiotica 29:53–75.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Nakajima M,
    2. Nakamura S,
    3. Tokudome S,
    4. Shimada N,
    5. Yamazaki H,
    6. Yokoi T
    (1999) Azelastine N-demethylation by cytochrome P-450 (CYP)3A4, CYP2D6, and CYP1A2 in human liver microsomes: evaluation of approach to predict the contribution of multiple CYPs. Drug Metab Dispos 27:1381–1391.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Obach RS
    (1997) Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol. Drug Metab Dispos 25:1359–1369.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Obach RS
    (1999) Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab Dispos 27:1350–1359.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Omura T,
    2. Sato R
    (1964) 0 The carbon monoxide-binding pigment of liver microsomes I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378.
    OpenUrlFREE Full Text
  43. ↵
    1. Ono S,
    2. Hatanaka T,
    3. Miyazawa S,
    4. Tsutsui M,
    5. Aoyama T,
    6. Gonzalez FJ,
    7. Satoh T
    (1996) Human liver microsomal diazepam metabolism using cDNA-expressed cytochrome P450s: Role of CYP2B6, 2C19 and the 3A subfamily. Xenobiotica 26:1155–1166.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Otton SV,
    2. Crewe HK,
    3. Lennard MS,
    4. Tucker GT,
    5. Woods HF
    (1988) Use of quinidine inhibition to define the role of the sparteine/debrisoquine cytochrome P450 in metoprolol oxidation by human liver microsomes. J Pharmacol Exp Ther 247:242–247.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Otton SV,
    2. Gillam EMJ,
    3. Lennard MS,
    4. Tucker GT,
    5. Woods HF
    (1990) Propranolol oxidation by human liver microsomes: The use of cumene hydroperoxide to probe isoenzyme specificity and regio- and stereoselectivity. Br J Clin Pharmacol 30:751–760.
    OpenUrlPubMed
  46. ↵
    1. Pichard L,
    2. Gillet G,
    3. Fabre I,
    4. Dalet-Beluche I,
    5. Bonfils G,
    6. Thenot JP,
    7. Maurel P
    (1990) Identification of the rabbit and human cytochromes P-450IIIA as the major enzymes involved in the N-demethylation of diltiazem. Drug Metab Dispos 18:711–719.
    OpenUrlAbstract
  47. ↵
    1. Rodrigues AD
    (1994) Use of in vitro human metabolism studies in drug development: An industrial perspective. Biochem Pharmacol 48:2147–2156.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Rodrigues AD
    (1999) Integrated cytochrome P450 reaction phenotyping: Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol 57:465–480.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Rodrigues AD,
    2. Kukulka MJ,
    3. Surber BW,
    4. Thomas SB,
    5. Uchic JT,
    6. Rotert GA,
    7. Michel G,
    8. Thome-Kromer B,
    9. Machinist JM
    (1994) Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C]dextromethorphan. Anal Biochem 219:309–320.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Roy P,
    2. Yu LJ,
    3. Crespi CL,
    4. Waxman DJ
    (1999) Development of a substrate-activity based approach to identify the major human liver P-450 catalyts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab Dispos 27:655–666.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Mimura M,
    4. Inui Y,
    5. Guengerich FP
    (1994) 0 Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Sutton D,
    2. Butler AM,
    3. Nadin L,
    4. Murray M
    (1997) Role of CYP3A4 in human hepatic diltiazem N-demethylation: Inhibition of CYP3A4 activity by oxidized diltiazem metabolites. J Pharmacol Exp Ther 282:294–300.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Tassaneeyakul W,
    2. Mohamed Z,
    3. Birkett DJ,
    4. McManus ME,
    5. Veronese ME,
    6. Tukey RH,
    7. Quattrochi LC,
    8. Gonzalez FJ,
    9. Miners JO
    (1992) Caffeine as a probe for human cytochromes P450: Validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques. Pharmacogenetics 2:173–183.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Tracy TS,
    2. Korzekwa KR,
    3. Gonzalez FJ,
    4. Wainer IW
    (1999) Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil. Br J Clin Pharmacol 47:545–552.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Venkatakrishnan K,
    2. Von Moltke LL,
    3. Greenblatt DJ
    (1998) Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: Application of the relative activity factor approach. J Pharm Sci 87:845–853.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Venkatakrishnan K,
    2. Von Moltke LL,
    3. Obach RS,
    4. Greenblatt DJ
    (2000) Microsomal binding of amitriptyline: Effect on estimation of enzyme kinetic parameters in vitro. J Pharmacol Exp Ther 293:343–350.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Von Moltke LL,
    2. Greenblatt DJ,
    3. Grassi JM,
    4. Granda BW,
    5. Venkatakrishnan K,
    6. Schmider J,
    7. Harmatz JS,
    8. Shader RI
    (1998) Multiple human cytochromes contribute to biotransformation of dextromethorphan in vitro: Role of CYP2C9, CYP2C19, CYP2D6, and CYP3A. J Pharm Pharmacol 50:997–1004.
    OpenUrlPubMed
  58. ↵
    1. Wang RW,
    2. Newton DJ,
    3. Scheri TD,
    4. Lu AYH
    (1997) Human cytochrome P450 3A4-catalyzed testosterone 6β-hydroxylation and erythromycin N-demethylation: Competition during catalysis. Drug Metab Dispos 25:502–507.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Watanabe N,
    2. Kamei S,
    3. Ohkubo A,
    4. Yamanaka M,
    5. Ohsawa S,
    6. Makino K,
    7. Tokuda K
    (1986) Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer. Clin Chem 32:1551–1554.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    1. Waxman DJ,
    2. Attisano C,
    3. Guengerich FP,
    4. Lapenson DP
    (1988) Human liver microsomal steroid metabolism: Identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme. Arch Biochem Biophys 263:424–436.
    OpenUrlCrossRefPubMed
  61. ↵
    1. Wester MR,
    2. Lasker JM,
    3. Johnson EF,
    4. Raucy JL
    (2000) CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes. Drug Metab Dispos 28:354–359.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Yamazaki H,
    2. Inoue K,
    3. Shaw PM,
    4. Checovich WJ,
    5. Guengerich FP,
    6. Shimada T
    (1997) Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: Effects of contents of these two forms in individual human samples. J Pharmacol Exp Ther 283:434–442.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Yamazaki H,
    2. Nakajima M,
    3. Nakamura M,
    4. Asahi S,
    5. Shimada N,
    6. Gillam EMJ,
    7. Guengerich FP,
    8. Shimada T,
    9. Yokoi T
    (1999) Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b5 in bacterial membranes. Drug Metab Dispos 27:999–1004.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Yasumori T,
    2. Nagata K,
    3. Yang SK,
    4. Chen LS,
    5. Murayama N,
    6. Yamazoe Y,
    7. Kato R
    (1993) Cytochrome P450-mediated metabolism of diazepam in humans and rats: Involvement of human CYP2C in N-demethylation in a substrate concentration-dependent manner. Pharmacogenetics 3:291–301.
    OpenUrlCrossRefPubMed
  65. ↵
    1. Yoshimoto K,
    2. Echizen H,
    3. Chiba K,
    4. Tani M,
    5. Ishizaki T
    (1995) Identification of human CYP isoforms involved in the metabolism of propranolol enantiomers-N-desisopropylation is mediated mainly by CYP1A2. Br J Clin Pharmacol 39:421–431.
    OpenUrlPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s

Dermot F. McGinnity, Alison J. Parker, Matthew Soars and Robert J. Riley
Drug Metabolism and Disposition November 1, 2000, 28 (11) 1327-1334;
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement