Chem. Res. Toxicol. 2008, 21, 1949–1955
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Identification of Cytochrome P450 Enzymes Involved in the Metabolism of the Designer Drugs N-(1-Phenylcyclohexyl)-3-ethoxypropanamine and N-(1-Phenylcyclohexyl)-3-methoxypropanamine Christoph Sauer, Frank T. Peters, Andrea E. Schwaninger, Markus R. Meyer, and Hans H. Maurer* Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland UniVersity, D-66421 Homburg (Saar), Germany ReceiVed April 11, 2008
The involvement of human hepatic cytochrome P450 isoenzymes (P450s) in the metabolism of the designer drugs N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA) and N-(1-phenylcyclohexyl)-3methoxypropanamine (PCMPA) to the common metabolite N-(1-phenylcyclohexyl)-3-hydroxypropanamine (PCHPA) was studied using insect cell microsomes with cDNA-expressed human P450s and human liver microsomes (HLMs). Incubation samples were analyzed by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry. Among the tested isoenzymes, P450 2B6, P450 2C19, P450 2D6, and P450 3A4 catalyzed PCEPA O-deethylation, and P450 2B6, P450 2C19, and P450 2D6 catalyzed PCMPA O-demethylation. According to the relative activity factor approach, these enzymes accounted for 22, 3, 30, and 45% of the net clearance for PCEPA and 51, 8, and 40% of the net clearance for PCMPA, respectively. At 1 µM PCEPA, the chemical inhibitors 4-(4-chlorobenzyl)pyridine for P450 2B6 and quinidine for P450 2D6 reduced metabolite formation in pooled HLMs by 37 and 73%, respectively, and at 10 µM PCEPA, they reduced metabolite formation by 57 and 26%, respectively. At 1 µM PCMPA, 4-(4-chlorobenzyl)pyridine and quinidine reduced metabolite formation in pooled HLMs by 25 and 39%, respectively, and at 10 µM PCMPA, they reduced metabolite formation by 62 and 27%, respectively. The experiments with the MAB inhibitory to P450 3A4 and the chemical inhibitor ketoconazole for P450 3A4 showed no inhibitory effect concerning PCEPA O-dealkylation. Experiments with HLMs from P450 2D6 poor metabolizers showed a reduction of metabolite formation as compared to pooled HLM of 73 and 25% (1 µM and 10 µM PCEPA) and 40 and 38% (1 µM and 10 µM PCMPA), respectively. In conclusion, the main metabolic step was catalyzed by different P450s. Introduction More than 90% of oxidative metabolic reactions of xenobiotics are catalyzed by enzymes of the P450 family (1). Before a drug can be marketed, the involvement of particular P450 enzymes in its biotransformation is usually thoroughly investigated to assess the risk of increased side effects in poor metabolizer subjects (2) and of drug-drug or drug-food interactions (3). However, such data are typically acquired for substances intended for therapeutic use but not for drugs of the illicit market. In the late 1990s, a considerable number of new synthetic drugs from various drug classes were seized in the German federal state of Hesse and surrounding federal states. One of these substances was N-(1-phenylcyclohexyl)propanamine (PCPR),1 a phencyclidine (PCP)-derived compound. After a short time, further members of this new class of PCP-derived * To whom correspondence should be addressed. Tel: +49-68411626050. Fax: +49-6841-1626051. E-mail:
[email protected]. 1 Abbreviations: PCPR, N-(1-phenylcyclohexyl)propanamine; PCP, phencyclidine; PCMPA, N-(1-phenylcyclohexyl)-3-methoxypropanamine; PCMEA, N-(1-phenylcyclohexyl)-2-methoxyethanamine; PCEEA, N-(1-phenylcyclohexyl)-2-ethoxyethanamine; PCEPA, N-(1-phenylcyclohexyl)-3-ethoxypropanamine; PCHPA, N-(1-phenylcyclohexyl)-3-hydroxypropanamine; CBP, 4-(4chlorobenzyl)pyridine; ICM, insect cell microsomes; pHLM, pooled human liver microsomes; PMD6 HLM, single donor human liver microsomes from donors with poor metabolizer genotype for CYP2D6; MAB, monoclonal antibody; IS, internal standard; RAF, relative activity factor; TR, turnover rates; PS, probe substrate; HP, Hewlett-Packard; AT, Agilent Technologies; APCI, atmospheric pressure chemical ionization; SIM, selected ion monitoring.
designer drugs appeared on the illicit drug market, namely, N(1-phenylcyclohexyl)-3-methoxy-propanamine (PCMPA), N-(1phenylcyclohexyl)-2-methoxyethanamine (PCMEA), and N-(1phenylcyclohexyl)-2-ethoxyethanamine (PCEEA). The seized preparations contained either one compound alone or in mixture with other designer drugs (4). In expectance of its appearance on the illicit drug market, a further homologue, namely, N-(1phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), was synthesized as a reference substance for scientific purposes. No information is available on the pharmacological properties of these compounds. However, because of structural similarities, they might be assumed to be similar to those of PCP or ketamine, which both act as antagonists at N-methyl-D-aspartate receptors and have psychotomimetic as well as anesthetic properties (5). Furthermore, it has been reported that (1phenylcyclohexyl)amine, a known metabolite of PCP and of the above-mentioned PCP-derived compounds (6-9), produced a long-lasting dose-dependent effect on the efflux of dopamine in the rat (10). For PCEPA, PCPR, PCMEA, and PCEEA studies on the metabolism and toxicological detection have been described (6, 8, 9). PCEPA and PCMPA are mainly metabolized by N-dealkylation, O-dealkylation followed by oxidation to the corresponding acid, hydroxylation of the cyclohexyl ring at different positions, aromatic hydroxylation, and finally combinations of those. O-Dealkylation of PCEPA and PCMPA leads to
10.1021/tx8001302 CCC: $40.75 2008 American Chemical Society Published on Web 09/09/2008
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the same O-dealkyl metabolite [N-(1-phenylcyclohexyl)-3hydroxypropanamine, PCHPA] (7). So far, no data are available on the P450-mediated metabolism of PCEPA and PCMPA. Therefore, the aim of the presented study was to examine the involvement of human P450 isoenzymes in the main metabolic step to clarify whether a higher risk of increased toxic side effects in poor metabolizer subjects and of drug-drug or drug-food interactions can be expected (2, 3).
Materials and Methods Materials. Hydrochlorides of PCEPA, PCMPA, and PCEEA were provided by the Hessian State Criminal Office (Wiesbaden, Germany) for research purposes. PCHPA was biotechnologically synthesized as described previously (11). NADP+ was obtained from Biomol (Hamburg, Germany), isocitrate and isocitrate dehydrogenase were from Sigma (Taufkirchen, Germany), 4-(4-chlorobenzyl)pyridine (CBP) was obtained by ABCR (Karlsruhe, Germany), and ketoconazole (Janssen, Beerse, Belgium) and all other chemicals and reagents were from Merck (Darmstadt, Germany). The monoclonal antibody (MAB) inhibitory to P450 3A4 (10 mg/mL MAB protein) and a P450 3A4 nonbinding MAB inhibitory to P450 2C8 (10 mg/mL MAB protein) as well as the following microsomes were from Gentest and delivered by NatuTec (Frankfurt/Main, Germany): baculovirus-infected insect cell microsomes (ICM, Supersomes) containing 1 nmol/mL human cDNAexpressed P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 3A4, or 2 nmol/mL P450 2E1, P450 3A5, wild-type baculovirus-infected ICM (control Supersomes), pooled human liver microsomes (pHLM 20 mg microsomal protein/ mL, 400 pmol total P450/mg protein), and single donor human liver microsomes (20 mg microsomal protein/mL) from donors with poor metabolizer genotype for P450 2D6 (PMD6 HLM). After delivery, the microsomes and the MAB were thawed at 37 °C, aliquoted, shock-frozen in liquid nitrogen, and stored at -80 °C until use. Microsomal Incubations. Incubation mixtures (final volume, 50 µL) consisted of 90 mM phosphate buffer (pH 7.4), 5 mM Mg2+, 5 mM isocitrate, 1.2 mM NADP+, 0.5 U/mL isocitrate dehydrogenase, 200 U/mL superoxide dismutase, and substrate at 37 °C. The substrate was added after dilution of a 5000 µM aqueous stock solution for PCEPA and a 7000 µM aqueous stock solution for PCMPA in the above-mentioned phosphate buffer. Reactions were started by the addition of ice-cold microsomes and terminated with 50 µL of acetonitrile. After the addition of 5 µL of internal standard (IS) solution (100 µM PCEEA in water for PCEPA and 100 µM PCEPA in water for PCMPA), the samples were centrifuged. In the initial screening experiments, the supernatants were diluted with 1 mL of water and worked-up by solid-phase extraction and acetylation as previously described for urine samples by Sauer et al. (8). Aliquots (2 µL) of the derivatized extracts were analyzed by GC/MS as described below. In all other experiments, the supernatants were directly transferred to autosampler vials, and 5-30 µL was analyzed by LC/MS as described below. Initial Screening Studies. Incubations were performed with 250 µM PCEPA or PCMPA and 50 pmol/mL P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1, P450 3A4, or P450 3A5 for 30 min. For incubations with P450 2A6 or P450 2C9, phosphate buffer was replaced with 45 or 90 mM Tris buffer, respectively, according to the Gentest manual. Kinetic Studies. Kinetic constants of PCHPA formation were derived from incubations with the following PCEPA concentrations, incubation times, and protein concentrations: 10, 25, 50, 100, 200, 300, 400, 500, 700, 1000, and 1500 µM PCEPA with 8 pmol P450 2B6/mL for 10 min; 10, 25, 50, 100, 200, 300, 400, 500, 700, 1000, and 1500 µM PCEPA with 15 pmol P450 2C19/mL for 10 min; 0.1, 0.2, 0.3, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 300, 500, 700, 1000, 1500, and 2000 µM PCEPA with 15 pmol P450 2D6/mL for 25 min; 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 1000, and 1500 µM PCEPA with 15 pmol P450 3A4/mL for 10 min; and 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 1000, and 1500 µM PCEPA
Sauer et al. with 0.2 mg HLM protein/mL for 10 min. For the determination of the kinetic constants of PCHPA formation by incubations with PCMPA, used were the following PCMPA concentrations, incubation times, and protein concentrations: 7, 17.5, 35, 70, 140, 210, 280, 350, 490, 700, 1050, 1400, and 1750 µM PCMPA with 10 pmol P450 2B6/mL for 15 min; 7, 17.5, 35, 70, 140, 210, 280, 350, 490, 700, 1050, 1400, and 1750 µM PCMPA with 20 pmol P450 2C19/mL for 15 min; 0.5, 1, 2, 3.5, 5, 7, 17.5, 35, 70, 140, 280, 350, 490, 700, 1050, 1400, and 1750 µM PCMPA with 15 pmol P450 2D6/mL for 25 min; and 7, 17.5, 35, 70, 140, 280, 350, 490, 700, 1050, 1400, and 1750 µM PCMPA with 0.3 mg HLM protein/mL for 15 min. Enzyme kinetic constants were estimated by nonlinear regression using GraphPad Prism 3.02 software (San Diego, CA). The Michaelis-Menten equation (eq 1) was used to calculate apparent Km and Vmax values for single-enzyme systems and HLM.
V)
Vmax × [S] Km + [S]
(1)
Eadie-Hofstee plots were used to check for biphasic kinetics (12). If the Eadie-Hofstee plot indicated biphasic kinetics, eq 1 and the alternative eq 2 for a two-enzyme model (12) were applied to the respective data. For eq 2, CLint,2 represents the intrinsic clearance or Vmax/Km of the low-affinity component (12). If eq 2 was found to fit the data significantly better (F test, P < 0.05), biphasic kinetics were assumed.
V)
Vmax,1 × [S] + CLint,2 × [S] Km,1 + [S]
(2)
Calculation of Relative Activity Factors (RAFs), Contributions, and Percentages of Net Clearance. The RAF approach (13-15) was used to account for differences in functional levels of redox partners between the two enzyme sources. The turnover rates (TRs) of P450 2B6 [probe substrate (PS) 7-ethoxy-4trifluoromethylcoumarin], P450 2C19 (PS S-mephenytoin), P450 2D6 (PS bufuralol), and P450 3A4 (PS testosterone) in ICM and HLM were taken from the supplier’s data sheets. The RAFs were calculated according to eq 3.
RAFenzyme )
TRPS in HLM (pmol/min/mg protein) TRPS in ICM (pmol/min/mg protein)
(3)
Vmax values for O-dealkylation of PCEPA/PCMPA obtained from incubations with cDNA-expressed P450s were then multiplied with the corresponding RAF leading to a value, which is defined as “contribution”:
contributionenzyme ) RAF × Vmax of PCHPA formation in ICM (4) From these corrected activities (contributions), the percentages of net clearance by a particular P450 can be calculated according to eq 5, where clearance equals contribution/Km:
clearanceenzyme (%) )
clearanceenzyme
∑ clearancesenzymes
× 100
(5)
Chemical Inhibition Studies. The effect of 3 µM quinidine, 0.1 µM CBP, and 0.08 µM ketoconazole (only for PCEPA) on PCHPA formation was assessed in incubations containing 0.2 mg HLM protein/mL for 10 min for PCEPA and 0.3 mg HLM protein/mL for 15 min for PCMPA at a concentration of 1 and 10 µM of the respective drug (n ) 6 each). Control incubations contained none of these chemical inhibitors (n ) 6 each). The significance of inhibition was tested by one-tailed unpaired t test using GraphPad Prism 3.02 software. Inhibition Studies with MAB. The effect of MAB inhibitory to P450 3A4 on PCHPA formation was assessed in incubations containing 0.2 mg HLM protein/mL and 0.2 mg MAB protein/mL
P450s Responsible for PCEPA and PCMPA Metabolism for 10 min for PCEPA at a concentration of 1 and 10 µM of the respective drug (n ) 6 each). Control incubations contained a P450 3A4 nonbinding MAB (n ) 6 each) also at a concentration of 0.2 mg HLM protein/mL and 0.2 mg MAB protein/mL. Inhibitory effectiveness and selectivity for both the MAB inhibitory to P450 3A4 and the P450 3A4 nonbinding MAB are reported in the corresponding Gentest manuals. Both MABs were added to the HLM preincubated for 15 min on ice according to the NatuTec manual. HLM with MABs were added to the incubation mixture. The significance of inhibition was tested by one-tailed unpaired t test using GraphPad Prism 3.02 software. Studies for Comparison of pHLM with PMD6 HLM. Incubations were carried out with pHLM, PMD6 HLM (0.2 mg protein/ mL, n ) 6 each) for 10 min (PCEPA), and pHLM and PMD6 HLM (0.3 mg protein/mL, n ) 6 each) for 15 min (PCMPA) at concentrations of 1 and 10 µM, respectively. The significance of differences in metabolite formation was tested by one-tailed unpaired t test using GraphPad Prism 3.02 software. GC/MS Apparatus for Identification of Metabolites. The extracts were analyzed using a Hewlett-Packard (HP, Agilent, Waldbronn, Germany) 5890 Series II gas chromatograph combined with an HP 5989B MS Engine mass spectrometer and an HP MS ChemStation (DOS series) with HP G1034C software version C03.00. The GC conditions were as follows: splitless injection mode; column, HP-1 capillary (12 m × 0.2 mm i.d.), cross-linked methyl silicone, 330 nm film thickness; injection port temperature, 280 °C; carrier gas, helium; flow rate, 1 mL/min; column temperature, programmed from 100-310 °C at 30°/min, initial time 3 min, final time 8 min. The MS conditions were as follows: full-scan mode, m/z 50-800 u; EI mode, ionization energy, 70 eV; ion source temperature, 220 °C; capillary direct interface, heated at 260 °C. LC/MS Conditions and Quantification of Metabolite. PCEPA, PCMPA, PCEEA, and PCHPA were analyzed using an Agilent Technologies (AT, Waldbronn, Germany) AT 1100 series LC-MSD, SL version, with an atmospheric pressure chemical ionization (APCI) interface and an LC-MSD ChemStation using the A.08.03 software. LC Conditions. Separation was achieved on an Alltech MixedMode/Cation exchange column (150 mm × 4.6 mm i.d.) with an isocratic mobile phase consisting of 50 mM ammonium formate buffer, pH 3.5, and acetonitrile at a flow rate of 1.0 mL-1. The analytes were quantified by positive APCI-MS in the selected ion monitoring (SIM) mode. APCI-MS Conditions. The APCI inlet conditions were applied as follows: drying gas, nitrogen (7000 mL/min, 300 °C); nebulizer gas, nitrogen (25 psi, 172.3 kPa); capillary voltage, 4000 V; drying gas temperature set at 300 °C; vaporizer temperature set at 400 °C; corona current was 5.0 µA; positive SIM mode, m/z 262 for PCEPA, m/z 248 for PCEEA and PCMPA, and m/z 234 for PCHPA; and fragmentor voltage, 50 V. Metabolite Quantification. Calibration curves were constructed plotting peak area ratios (PCHPA vs IS) of spiked calibrators vs their concentrations (0.05, 0.1, 0.25, 0.5, 1.0, 10.0, 30.0, 50.0, and 100.0 µM). Quantification was carried out using a weighted (1/x2) linear regression model.
Results Initial Screening Studies. PCHPA was the only metabolite detected in any of the incubation experiments with human liver microsomes or with ICM. Among the 10 P450s tested, only P450 2B6, P450 2C19, P450 2D6, and P450 3A4 were markedly capable of catalyzing the O-dealkylation of PCEPA. In the case of PCMPA, only P450 2B6, P450 2C19, and P450 2D6 showed relevant metabolite formation. In incubations of the other P450s, only very little (P450 3A4 in case of PCMPA) or no formation of PCHPA was detectable. LC/MS Procedure. The applied LC/MS conditions provided separation of PCHPA, PCEPA, PCMPA, and PCEEA. The chosen target ions were selective for the analytes under these
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conditions as proven with blank samples (control microsomes without substrate and standard) and zero samples (control microsomes without substrate but with standard). The method showed good linearity in a range of 0.05-100 µM PCHPA (R2 ) 1.000). Matrix effect studies comparing the peak areas of PCHPA in neat standard solutions with those in spiked incubation mixtures containing the same concentrations of PCHPA gave no indication of ion suppression. Kinetic Studies. The Michaelis-Menten plots for PCEPA O-deethylation are depicted in Figure 1, and those for PCMPA O-demethylation are depicted in Figure 2. Duration and protein contents of all incubations in these studies were within the linear range of metabolite formation (data not shown). P450 2B6 (Figures 1 and 2, upper left), P450 2C19 (Figures 1 and 2, upper right), P450 3A4 (Figure 1, middle right), and pHLM (Figure 2, lower right) showed typical hyperbolic metabolite formation profiles allowing use of eq 1 for estimation of the kinetic constants. The resulting Km and Vmax values are listed in Table 1 for PCEPA O-deethylation and in Table 2 for PCMPA O-demethylation. Visual inspection of the data for P450 2D6 (Figure 1, middle left, and Figure 2, lower left) and pHLM (Figure 1, lower middle) and Michaelis-Menten plots gave evidence of biphasic kinetics, which led to significantly better results for eq 2 (F test, p < 0.05). The corresponding Eadie-Hofstee plot clearly confirmed this (data not shown). Hence, the P450 2D6 and pHLM kinetic parameters were estimated by fitting the data into eq 2 for a biphasic kinetic. The second enzyme component is represented by CLint,2 × [S]. Km,1 and Vmax,1 data are also reported in Tables 1 and 2. Calculation of RAFs, Contributions, and Percentages of Net Clearance. The supplier-provided TRs of the specific PSs in the used batches of ICM and pHLM, respectively, were as follows: 1486 and 40 pmol/min/mg protein for 7-hydroxy4-trifluoromethylcoumarin formation (P450 2B6); 5814 (ICM lot used in PCEPA experiments), 2857 (ICM lot used in PCMPA experiments), and 31 pmol/min/mg protein for 4′-hydroxymephenytoin formation (P450 2C19); 4500 and 69 pmol/min/mg protein for 1′-hydroxybufuralol formation (P450 2D6); and 16667 and 6400 pmol/min/mg for 6β-testosterone formation (P450 3A4). The protein content of each lot ICM has been taken into account and has been converted from activity [pmol/(min × pmol enzyme)] to activity [pmol/(min × mg enzyme)]. The RAFs, contribution and intrinsic clearance data, and percentages of net clearance calculated from these and the above-mentioned kinetic data are reported in Tables 1 and 2. Inhibition Studies with Chemical Inhibitors or MAB and Comparative Studies of pHLM with PMD6 HLM. The results of the experiments with the chemical inhibitors CBP for P450 2B6 and quinidine for P450 2D6 and the results of the studies comparing PCHPA formation in pHLM with those in PMD6 HLM are presented in Table 3 for both PCEPA and PCMPA. All observed inhibition effects were statistically significant (p < 0.05). In the case of the experiments with the MAB inhibitory to P450 3A4 and the chemical inhibitor ketoconazole for P450 3A4, no inhibitory effect could be observed concerning PCEPA O-dealkylation.
Discussion The initial screening studies with the 10 most abundant human hepatic P450s were performed for identification of the main metabolite of PCEPA and PCMPA in microsomes and to clarify their role in metabolism of PCEPA and PCMPA. According to the supplier’s advice, the chosen incubation conditions were applicable for checking the general involvement of particular
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Sauer et al.
Figure 1. Michaelis-Menten plots for PCEPA O-deethylation catalyzed by P450 2B6 (upper left), P450 2C19 (upper right), P450 2D6 (middle left), P450 3A4 (middle right), and pHLM (lower middle). Data points represent means (solid squares) and ranges (error bars) of duplicate measurements. The curves for P450 2B6 (upper left), P450 2C19 (upper right), and P450 3A4 (middle right) were calculated by nonlinear regression according to eq 1. The curves for P450 2D6 (middle left) and pHLM (lower middle) were calculated according to eq 2 (two-enzyme model).
P450 enzymes. Both drugs are extensively metabolized in vivo (7, 8), so it first had to be clarified which of the metabolites were formed in incubations with HLM and ICM. Therefore, the supernatants of the initial screening experiments were analyzed by GC/MS, which allowed identification of formed metabolites by library search as described by Sauer et al. (7, 8). Except PCHPA, no further metabolite of PCEPA and PCMPA was detected in any of the incubations with human liver microsomes or with ICM. Therefore, we assumed the Odealkylation of PCEPA and PCMPA to be the main metabolic step and the initial reaction for formation of further metabolites of these designer drugs. Because of the very low activity of P450 3A4 with respect to PCMPA O-dealkylation, only the P450s involved in the O-dealkylation of both drugs, namely, P450 2B6, P450 2C19, and P450 2D6, were characterized by their kinetic profiles. LC/MS analysis was chosen for these experiments, because it allowed direct injection of the supernatants without further workup. Less than 20% of substrate was metabolized in all incubations with exception of the lowest substrate concentrations. The kinetic data for P450 2B6, P450 2C19, and P450 3A4 followed the expected classical hyperbolic Michaelis-Menten plots (Figures 1 and 2). In contrast, P450
2D6 revealed a biphasic kinetic profile. P450 2D6 turned out to have the highest affinity toward both PCEPA and PCMPA with apparent Km,1 values markedly lower than the Km values of P450 2B6, P450 2C19, and 3A4 (Tables 1 and 2), whereas the capacity of P450 2B6, P450 2C19, and P450 3A4 was considerably higher than those of P450 2D6. No data of plasma levels of PCEPA and PCMPA in humans are available. Considering the plasma levels of the derived compound PCP to estimate the approximate plasma level of PCEPA and PCMPA after a common drug user’s dose, the expected plasma levels should be approximately 1 µM or lower (16, 17). However, with respect to expected plasma concentrations of 1 µM or lower, P450 2D6 should play a major role in PCEPA and PCMPA metabolism because of the higher affinity to PCEPA and PCMPA as compared with P450 2B6, P450 2C19, and P450 3A4. The RAF approach (14, 18, 19) is an accepted strategy to correct recombinant P450 formation rates for native human liver enzyme activity. According to the results of the RAF approach, P450 2D6 accounted for 30 (PCEPA) and 40% (PCMPA) of the net clearance. Because these P450s are expressed polymorphically in humans, further studies were performed to confirm their role in O-dealkylation of PCEPA
P450s Responsible for PCEPA and PCMPA Metabolism
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Figure 2. Michaelis-Menten plots for PCMPA O-demethylation catalyzed by P450 2B6 (upper left), P450 2C19 (upper right), P450 2D6 (lower left), and pHLM (lower right). Data points represent means (solid squares) and ranges (error bars) of duplicate measurements. The curves for P450 2B6 (upper left), P450 2C19 (upper right), and HLM (lower right) were calculated by nonlinear regression according to eq 1. The curve for P450 2D6 (lower left) was calculated according to eq 2 (two-enzyme model).
Table 1. Kinetic Data of PCEPA O-Deethylation by P450 2B6, P450 2C19, P450 2D6, P450 3A4, and HLMa Km (best fit value ( standard error) Vmax (best fit value ( standard error) RAF contribution (Vmax × RAF) clearance (contribution/Km) percentage of net clearance
P450 2B6
P450 2C19
P450 2D6 (1)
220 ( 14 220 ( 4.3 0.027 5.923 0.027 22
59 ( 10.3 41 ( 1.4b 0.005 0.218 0.004 3
Km,1, 1.0 ( 0.14 Vmax,1, 2.4 ( 0.07c 0.015 0.037 0.037 30
b
P450 2D6 (2) c
P450 3A4
pHLM (1)
242 ( 23 34.9 ( 1.09b 0.384 13.4 0.055 45
Km,1, 108.4 ( 14.9 Vmax,1, 1.1 ( 0.07c
b
2.5 × 10-5 ,0.1
pHLM (2) c
3.8 × 10-3d
a Units are as follows: Km in µM and Vmax and contribution in pmol/min/pmol P450 (P450 2B6, P450 2C19, P450 2D6, and P450 3A4) or pmol/min/ mg protein (pHLM). b Kinetic data estimated according to eq 1. c Kinetic data estimated according to eq 2. d Kinetic data estimated according to eq 2 without RAF.
Table 2. Kinetic Data of PCMPA O-Demethylation by P450 2B6, P450 2C19, P450 2D6, and HLMa P450 2B6 Km (best fit value ( standard error) Vmax (best fit value ( standard error) RAF contribution (Vmax × RAF) clearance (contribution/Km) percentage of net clearance
248 ( 12.9 59 ( 0.98b 0.027 1.593 0.006 51
b
P450 2C19
P450 2D6 (1)
147 ( 8.8 15 ( 0.23b 0.011 0.163 0.001 8
Km,1, 3.6 ( 0.8 Vmax,1, 1.2 ( 0.06c 0.015 0.018 0.005 40
b
P450 2D6 (2)
pHLM 690 ( 41.7b 1.4 ( 0.04b
c
1.6 × 10-5
