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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Amodiaquine Clearance and Its Metabolism toN-Desethylamodiaquine Is Mediated by CYP2C8: A New High Affinity and Turnover Enzyme-Specific Probe Substrate

Xue-Qing Li, Anders Björkman, Tommy B. Andersson, Marianne Ridderström and Collen M. Masimirembwa
Journal of Pharmacology and Experimental Therapeutics February 2002, 300 (2) 399-407; DOI: https://doi.org/10.1124/jpet.300.2.399

Abstract

Amodiaquine (AQ) metabolism to N-desethylamodiaquine (DEAQ) is the principal route of disposition in humans. Using human liver microsomes and two sets of recombinant human cytochrome P450 isoforms (from lymphoblastoids and yeast) we performed studies to identify the CYP isoform(s) involved in the metabolism of AQ. CYP2C8 was the main hepatic isoform that cleared AQ and catalyzed the formation of DEAQ. The extrahepatic P450s, 1A1 and 1B1, also cleared AQ and catalyzed the formation of an unknown metabolite M2. TheKm and Vmaxvalues for AQ N-desethylation were 1.2 μM and 2.6 pmol/min/pmol of CYP2C8 for recombinant CYP2C8, and 2.4 μM and 1462 pmol/min/mg of protein for human liver microsomes (HLMs), respectively. Relative contribution of CYP2C8 in the formation of DEAQ was estimated at 100% using the relative activity factor method. Correlation analyses between AQ metabolism and the activities of eight hepatic P450s were made on 10 different HLM samples. Both the formation of DEAQ and the clearance of AQ showed excellent correlations (r2 = 0.98 and 0.95) with 6α-hydroxylation of paclitaxel, a marker substrate for CYP2C8. The inhibition of DEAQ formation by quercetin was competitive withKi values of 1.96 for CYP2C8 and 1.56 μM for HLMs. Docking of AQ into the active site homology models of the CYP2C isoforms showed favorable interactions with CYP2C8, which supported the likelihood of an N-desethylation reaction. These data show that CYP2C8 is the main hepatic isoform responsible for the metabolism of AQ. The specificity, high affinity, and high turnover make AQ desethylation an excellent marker reaction for CYP2C8 activity.

Amodiaquine (AQ) is a 4-aminoquinoline derivative that has been widely used for treatment of malaria over the past 50 years. It is intrinsically more active than the other 4-aminoquinoline, chloroquine, againstPlasmodium falciparum parasites, which are moderately chloroquine resistant. The drug is therefore increasingly being considered as a replacement for chloroquine as a first line drug in Africa because of widespread chloroquine resistance. Because of major side effects, mainly agranulocytosis, observed during prophylactic use of the drug, AQ is now only recommended for treatment of malaria, for which no serious cases of toxicity have been reported (Laurent et al., 1993).

Upon oral administration, AQ is rapidly absorbed and extensively metabolized such that very little of the parent drug is detected in the plasma. The main metabolite of AQ is N-desethylamodiaquine (DEAQ) with other minor metabolites being 2-hydroxyl-DEAQ andN-bisdesethylAQ (bisDEAQ) (Churchill et al., 1985, 1986;Mount et al., 1986). Whereas the formation of DEAQ is rapid, its elimination is very slow with a terminal half-life of over 100 h (Winstanley et al., 1987; Laurent et al., 1993). AQ and DEAQ both have antimalarial activity, but AQ is 3 times more active (Churchill et al., 1985). However, since AQ is rapidly cleared and the formed DEAQ attains high plasma concentrations for a long time, AQ is considered a prodrug, which is bioactivated to DEAQ.

In vivo pharmacokinetic studies have shown that the primary route of systemic elimination of AQ in humans is via extensive first-pass biotransformation to the active DEAQ (White et al., 1987; Laurent et al., 1993). Jewell et al. (1995) found that DEAQ and bisDEAQ were formed by human liver microsomes and postulated that the liver was the major site of AQ first-pass metabolism. Studies with isolated neutrophils and lymphocytes showed that agranulocytosis caused by AQ might be due to metabolism to a reactive quinoneimine (Naisbitt et al., 1998). All the pharmacokinetic studies on AQ and its major metabolite, DEAQ, show that there is a great interindividual variability in kinetic parameters (Cmax,t1/2, and area under the curve). Such variability probably reflects variability in metabolic capacity and could imply different therapeutic and toxicological responses to AQ.

Besides the report by Jewell et al. (1995) on the possible role of CYP3A4 in AQ metabolism based on inhibition studies with ketoconazole, no detailed study has been performed to identify P450s that metabolize AQ. The aim of this study was therefore to investigate the metabolic clearance of AQ and identify the cytochromes P450 responsible for its metabolism. Our results show that the clearance of AQ and its hepatic metabolism to DEAQ is catalyzed mainly by CYP2C8. Molecular modeling studies also demonstrated the substrate specificity of CYP2C8 for AQ metabolism.

Materials and Methods

Chemicals.

Amodiaquine dihydrochloride and desethylamodiaquine hydrochloride were obtained from Karolinska Institute (Stockholm, Sweden). Quercetin dihydrate was obtained from Aldrich Chemical Co. (Milwaukee, WI). Ketoconazole was purchased from Janssen Biotech (Flander, NJ). NADP and glucose 6-phosphate were obtained from Sigma Chemical Co. (St. Louis, MO). Glucose-6-phosphate dehydrogenase was purchased from ICN Pharmaceuticals Biochemicals Division (Aurora, OH). Paclitaxel and 6α-hydroxypaclitaxel were obtained from Gentest (Woburn, MA). All other reagents used were of analytical or HPLC grade.

Human Liver Microsomes (HLMs) and Recombinant Cytochromes P450 (rCYP).

Ten HLMs were obtained from an in-house bank of liver microsomes maintained at AstraZeneca Research and Development (Mölndal, Sweden) (Äbelö et al., 2000), and the CYP activities (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in individual HLMs were determined using diagnostic marker substrates (C. M. Masimirembwa, M. E. S. Lutz, R. A. Thompson, and T. B. Andersson, submitted for publication). The following marker reactions were used: phenacetin demethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), paclitaxel 6α-hydroxylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), S-mephenytoin 4′-hydroxylation (CYP2C19), bufuralol 1-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1-hydroxylation (CYP3A4). The 10 HLM samples were selected from a bank of 21 HLM samples, ensuring that there was minimal cross P450 activity correlations and that the activities of each P450 covered a wide range. Pooled HLMs were prepared from a pooled set of liver pieces of patients undergoing liver resections. Recombinant human P450 isoforms CYP1A1, 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 were from lymphoblastoid cell lines (Gentest) and yeast (Masimirembwa et al., 1999). P450 isoforms available from lymphoblastoid cell lines only and not yeast were CYP2A6, 1B1, 2B6, 2E1, 3A5, and 4A11, which were obtained from Gentest. The microsomal preparations were stored at −80°C until use.

Incubation Conditions with HLMs and rCYP Isoforms.

The basic incubation medium contained 0.1 mg/ml HLMs, 3.3 mM MgCl2, 1 mM NADP, 3.3 mM glucose 6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, 100 mM potassium phosphate buffer (pH 7.4), and AQ (0.5–67 μM), in a final volume of 200 μl. The mixture was incubated at 35°C for 20 min. The reaction was initiated by the addition of NADP after a preincubation period of 5 min. The incubation was terminated by the addition of 150 μl of ice-cold acetonitrile. The mixture was then centrifuged at 4500g for 20 min, and 20 μl of supernatant was analyzed by HPLC as described below. Incubation conditions used for the 13 different recombinant human P450 isoforms were essentially similar to those used for HLMs, except for the quantity of enzyme used (10 pmol of P450/incubation).

Chromatography and Metabolite Structural Analysis.

The HPLC/UV and LC/MS system consisted of an HP 1100 system (Hewlett-Packard, Palo Alto, CA), and a Finnigan LCQ ion trap mass spectrometer (ThermoFinnigan MAT, San Jose, CA) employing an atmospheric pressure ionization interface. Chromatography was performed on a Symmetry C18 column (3.9 × 150 mm i.d.; 5 μm, Waters, Milford, MA). Compounds were eluted with a linear gradient of acetonitrile (5–40%, v/v, over 7 min) in 5 mM ammonium acetate using formic acid to bring the pH to 3.3, at a flow rate of 1 ml/min. The effluent was split with approximately 0.3 ml/min introduced into the mass spectrometer. Source parameters of mass spectrometer (e.g., spray voltage, temperature, gas flow rates, etc.) were individually optimized for each compound, and the MS/MS spectra were obtained for precursor ions through incidental collision with neutral gas (Helium) molecules in the ion trap. The metabolites of AQ were analyzed on-line, qualitatively by ion trap-based MS, and quantitatively by diode array detector detection set at 342 nm. Instrument control, data acquisition, and data evaluation were performed using Xcalibur software (version 1.2, ThermoFinnigan MAT).

Contribution of CYP2C8 to HLM AQ Metabolism and Estimation of in Vivo Drug Clearance.

The percentage contribution of CYP2C8 to AQN-desethylation was estimated by applying the relative activity factor (RAF) values as proposed by Crespi (1995) using the values of the activities (RAFv). The RAFv of CYP2C8 was determined as the ratio of the activity of paclitaxel 6α-hydroxylation (at the substrate concentration of 100 μM), a specific metabolic reaction mediated by CYP2C8 (Rahman et al., 1994), in HLMs to the activity for the recombinant CYP2C8. Using RAFv, theN-desethylation clearance of AQ by CYP2C8 in HLMs was calculated using equations described previously (Nakajima et al., 1999).

Estimation of in vivo clearance was made from in vitro data according to a venous equilibrium model (well stirred model) using equations described previously (Houston, 1994; Obach et al., 1997). The liver was taken as the main site of drug metabolic clearance. Previous studies have shown that AQ does not bind to human microsomal proteins (Jewell et al., 1995), and DEAQ but not AQ does accumulate in blood, preferentially in lymphocytes at an average blood/plasma distribution ratio of 3.35 (Pussard et al., 1987; Laurent et al., 1993). The clearance we predicted in vitro was compared to the in vivo clearance obtained using plasma concentrations of AQ.

Michaelis-Menten and Inhibition Kinetics.

Linear conditions for the formation of metabolites were established with respect to protein content and incubation time for the HLM and for CYP2C8. The optimum protein concentrations of HLMs and CYP2C8 for kinetic analysis were 0.1 mg/ml and 3 pmol of P450/incubation, respectively. The formation rates of DEAQ were linear at 35°C for incubation times of up to 30 min. The Michaelis-Menten kinetics of AQN-desethylation by HLMs and CYP2C8 were determined using nine substrate concentrations in the range of 0.5 to 67 μM. The substrates were dissolved in water. For inhibition studies, quercetin was dissolved in methanol and added to each microsomal suspension (final solvent content of 1%) while on ice. After a 5-min preincubation, NADP was added to initiate the reaction. Kinetic studies were undertaken to determine the mechanism of inhibition and to calculate the apparent inhibition constant (Ki). AQ N-desethylase activity was measured at six inhibitor concentrations at each of three substrate concentrations (approximately 1/3Km,Km, and 3Km).

Data Analysis.

All data points represent the means of duplicate estimations. Km andVmax values for HLMs and CYP2C8 were determined by nonlinear least-squares regression analysis using GraFit software (version 3.0, Erithacus Software Limited, Middlesex, UK). Correlations between the activities of respective P450 isoform and DEAQ formation or clearance of AQ were determined by least-squares linear regression. P < 0.05 was considered statistically significant.

Docking AQ into CYP2C Isoform Homology Models.

The results from the in vitro experiments described above showed that CYP2C8 was the main enzyme responsible for the metabolism of AQ. In our laboratory, we have generated homology models for human CYP2C8, 2C9, 2C18, and 2C19 (Ridderström et al., 2001) based on the crystal structure of the rabbit CYP2C5 (Williams et al., 2000). To understand the molecular basis of CYP2C8 substrate specificity for AQN-desethylation compared to the closely related CYP2C9, 2C18, and 2C19, AQ was docked into the active site of the homology models of these enzymes. The substrate-binding cavity was defined at various radii (10, 12, or 15Å) from the oxygen atom of the water coordinated to the heme.

The two-dimensional structure of AQ was built in SYBYL 6.7.6 (Tripos Associates Inc., St. Louis, MO). The structure was energy minimized using the MMFF94s force field and MMFF94 atom charges in vacuo conditions. The docking program, GOLD 1.1 (Dr. Gareth Jones, University of Oxford, UK), was used in the docking (Jones et al., 1997) of AQ to homology models of CYP2C8, 2C9, 2C18, and 2C19. The genetic algorithm implemented in GOLD was used to optimize the orientation of the ligand in the active site. During this optimization process the ligand was considered flexible (movement of the flexible bonds), whereas the active site of the enzyme was considered rigid (no movement of the amino acid side chains was allowed). For each experiment 10 dockings were allowed with an early termination if the root mean square distances were within 1.5 Å for the top three solutions.

Results

AQ and DEAQ Identification and Quantitation.

LC/MS and HPLC/UV analysis were used for the qualification and quantitation of the metabolites of AQ after incubation with rCYP isoforms and HLMs. The only metabolite found after AQ incubation with HLMs is DEAQ, and it was characterized by comparing the chromatographic retention time and multistage mass spectra with the reference substance (Fig.1). The limits of quantitation for both AQ and DEAQ were 0.05 μM by UV detection at 342 nm. In our incubation conditions, no detectable bis-desethylated metabolite of AQ was found, which could be due to the low protein concentration (0.1 mg/ml) of HLMs and short incubation time (30 min) we used compared with the high concentration of 2 mg/ml and longer incubation time (60 min) used byJewell et al. (1995).

Figure 1
Figure 1

HPLC chromatograms of metabolites of AQ in human liver microsomes and recombinant CYP1A1, 1B1, and 2C8 after incubation with 10 μM of AQ for 30 min. Peaks: 1 = DEAQ,tR = 4.30 min; 2 = AQ,tR = 4.51 min; 3 = metabolite M2,tR = 6.65 min. AQ was incubated with human liver microsomes (a); AQ was incubated with CYP2C8 (b); AQ was incubated with CYP1A1 (c); AQ was incubated with CYP1B1 (d); and AQ in human liver microsomes without NADPH (e).

Identification of P450s Responsible for AQ and DEAQ Metabolism.

Figure 2 shows the catalytic activities of the 13 recombinant human P450 isoforms with respect to the elimination of AQ (1 μM) and theN-desethylation of AQ (50 μM). In light of the fact that one can get different RAF factors when using different expression systems and that rates of metabolism of a test compound could differ between systems due to differences in cytochrome 450/cytochromeb5 ratios and other physiologic parameters (Venkatakrishnan et al., 2000), we used two sets of recombinant P450s from different expression systems to see if we could arrive at the same conclusions. Figure 2 shows that the qualitative role of the P450s from the different systems in the clearance of AQ and the formation of DEAQ are similar between yeast- and lymphoblastoid-expressed P450s. Figure 2 and Table1 show that there are some quantitative differences with yeast CYP1A1 and CYP2C8 having a greater capacity to clear AQ and to form DEAQ. An unknown metabolite M2 was found to be the major metabolite formed by P450s 1A1 and 1B1 (Fig. 1). Metabolite M2 has the protonated molecular ion atm/z 299 and a long retention time (tR = 6.65 min) compared with AQ (tR = 4.51 min). This is not consistent with the other reported AQ metabolites, AQ quinoneimine, bisDEAQ, or 2-hydroxyDEAQ, and hence represents a new metabolite whose identity has yet to be established.

Figure 2
Figure 2

Amodiaquine clearance (left panel, at 1 μM AQ) andN-desethylation (right panel, at 50 μM AQ) activities of recombinant human P450 isoforms expressing from lymphoblastoid cell lines (lined column) versus yeast (open column).

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Table 1

Michaelis-Menten kinetic parameters of amodiaquineN-desethylation and inhibition Ki values of AQ metabolism by quercetin in HLMs and CYP2C8 expressed in human lymphoblastoid cell lines vs. yeast

The P450 isoforms involved in the metabolism of DEAQ were also studied at 1 μM of the substrate. CYP1A1 completely cleared all the DEAQ, and CYP1B1 also contributes to a significant extent in the clearance of the DEAQ. CYP1A1 and 1B1 are however constitutively extrahepatic. The concentration of DEAQ after incubation with the other 11 P450s and HLMs were similar to the control samples in which there was no NADPH. This result indicated that DEAQ was relatively metabolically stable in HLMs, which is consistent with its long terminal half-life in humans.

Reaction Kinetics Using Liver Microsomes and CYP2C8.

A typical Eadie-Hofstee plot for the formation of DEAQ from AQ shown in Fig.3 exhibits monophasic behavior, suggesting that a single isoform of P450s may be involved in theN-desethylation of AQ in HLMs. Accordingly, a simple Michaelis-Menten kinetic analysis was used to estimate the affinity constant (Km), the maximum enzyme velocity (Vmax), and the intrinsic clearance (CLint), defined asVmax/Km(Table 1). The apparent Km andVmax values for AQ desethylation were 2.4 μM and 1462 pmol/min/mg of protein with HLMs, 1.2 μM and 2.6 pmol/min/pmol with CYP2C8 from lymphoblastoid cell lines (Fig. 3), and 0.9 μM and 3.9 pmol/min/pmol, respectively, with CYP2C8 from yeast. Accordingly, the CLint of AQ for HLMs and CYP2C8 were 608.2 μl/min/mg, and 2.1 or 4.4 μl/min/pmol of CYP2C8 from lymphoblastoid cell lines and yeast, respectively. These data show that AQ is a high clearance drug with high affinity to the associated enzyme(s).

Figure 3
Figure 3

Plot of velocity versus amodiaquine concentration for the formation of desethylamodiaquine in human liver microsomes (left panel) and in CYP2C8 expressed from lymphoblastoid cell lines (right panel). Inset, Eadie-Hofstee plot for the desethylation of amodiaquine in human liver microsomes.

Correlation Study.

Correlations between AQ metabolic stability and the N-desethylation reaction by a panel of 10 HLMs with activities of eight marker substrates for specific P450s were performed at AQ concentration of 1 μM. Figure 4shows an excellent correlation (r2 = 0.98, P < 0.01) between AQ desethylase and CYP2C8 activities (determined as the activity of 6α-hydroxylation of paclitaxel) in 10 different HLMs. A good relationship (r2 = 0.95, P < 0.01) was also observed (Fig. 4) between the clearance of AQ and CYP2C8 activities in those liver microsomes. There were no significant correlations between the desethylation of AQ and catalytic activity for P450s 1A2, 2A6, 2C19, 2D6, 2E1, and 3A4 (r2 < 0.35). Although CYP2C9 showed a fair relationship (r2 = 0.59) between its activity and AQ desethylase activity, it also had some correlation (r2 = 0.64) with the activity of CYP2C8. Since no detectable amount of DEAQ has been found after incubation with CYP2C9, it is unlikely that this enzyme is involved in the metabolism of AQ.

Figure 4
Figure 4

Relationship between amodiaquine metabolism (clearance and desethylation of amodiaquine) and paclitaxel 6α-hydroxylase activities in microsomes obtained from 10 different human livers.

Contribution of CYP2C8 to AQ N-Desethylase Activity in HLMs.

From the P450 identification studies (Fig. 2) of the hepatic isoforms, CYP2C8 had the major role in the clearance of AQ and the formation of its major metabolite, DEAQ. To estimate the relative contribution of this P450 in the hepatic metabolism of AQ, the relative activity factor approach was used. The contribution of CYP2C8 to the AQ N-desethylase activity in pooled HLMs was estimated using the clearance (CL =Vmax/Km) of AQ N-desethylation by recombinant CYP2C8 from lymphoblastoid cell lines (CLCYP2C8, 2.1 μl/min/pmol of CYP2C8) and by pooled HLMs (CLHLM, 608.2 μl/min/mg of protein), respectively (Table 2). The paclitaxel 6α-hydroxylase activity in microsomes from CYP2C8 was 0.35 pmol/min/pmol of CYP2C8, and for pooled HLMs it was 94.2 pmol/min/mg of protein. Thus, RAFv, CYP2C8(lymphoblastoid) was estimated to be 268.8 (pmol of CYP2C8/mg of HLMs). Using the same approach, a RAFv,CYP2C8(yeast) of 151.1 was obtained using yeast-expressed CYP2C8. Table 2 shows that using either the lymphoblastoid- or yeast-expressed CYP2C8, we get similar percentage of contributions of CYP2C8 to the clearance of AQ. The data in Table 2 show that the predicted contribution of CYP2C8 to AQN-desethylase activity was very close to the measured liver microsomal N-desethylation activity for AQ, indicating that CYP2C8 plays a major role in the N-desethylation of AQ.

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Table 2

Contribution of CYP2C8 expressed in human lymphoblastoid cell lines vs. yeast to AQ N-desethylation in HLMs by RAF method

In Vitro-in Vivo Correlation of AQ Clearance.

Using the well stirred model, the clearance of AQ calculated from the in vitrot1/2 (15.6 min, at 1 μM of AQ) predicted an in vivo clearance of 19.1 ml/min/kg. The clearance of AQ calculated fromVmax/Kmin vitro predicted in vivo clearance of 19.3 ml/min/kg.

Inhibition Study.

Quercetin was previously shown to be a potent diagnostic inhibitor for CYP2C8. Figure5 shows that quercetin is a competitive inhibitor of DEAQ formation catalyzed by CYP2C8 from lymphoblastoid cell lines and HLMs, with Ki values of 1.96 and 1.56 μM, respectively. TheKi value for CYP2C8 from yeast was 2.35 μM. Inhibition of AQ N-desethylation by ketoconazole (10 μM) was also observed in HLMs at an AQ concentration of 5 μM. Ketoconazole inhibited the formation of DEAQ by about 60%, whereas the effect by quercetin (10 μM) was 70%. However, ketoconazole has been shown to also inhibit CYP2C8 catalyzed paclitaxel 6α-hydroxylation by more than 50% (Masimirembwa et al., 1999) at 10 μM. The inhibition of AQ N-desethylation caused by ketoconazole could therefore be a reflection of the unspecificity of the inhibitor at high concentration.

Figure 5
Figure 5

Dixon plots for inhibition of CYP2C8 catalyzed amodiaquine desethylation by quercetin in human liver microsomes (left panel) and CYP2C8 expressed from lymphoblastoid cell lines (right panel). Concentrations of substrate (AQ) are shown in each plot. Each point represents the mean of duplicate measurements.

Docking AQ into CYP2C Isoforms.

When AQ was docked into the active site cavities of the four CYP2C isoforms, the docking program, GOLD, found several preferred AQ-CYP2C protein interactions. In the docking query, the program was asked to provide the 10 best solutions. Interpretation of the results was based on the favorability of interactions as deduced by GOLD and the proximity of potential sites of metabolism to oxygen of the water coordinating to the heme. Table3 shows that in all cavity sizes chosen, the greatest number of favorable solutions was found with CYP2C8 and that the cavity with radii of 12 and 15 Å defined the best substrate binding cavity for AQ. AQ docked in the different P450s indicating possible regions for metabolism (Table 3, Fig.6).

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Table 3

Docking of AQ to CYP2C8, 2C9, 2C18, and 2C19

Figure 6
Figure 6

Docking of amodiaquine into CYP2C8, 2C9, 2C18, and 2C19. The heme is magenta-colored and active site amino acids are in green. The docked compound is amodaiquine. GOLD 1.1 was used for the docking.

Discussion

The results of our study show that the clearance of AQ and metabolism to its main metabolite, DEAQ, is catalyzed by CYP2C8. They also show that there is an unidentified metabolite (M2), which is a product of AQ metabolism by extrahepatic CYP1A1 and 1B1. The role of the extrahepatic CYP1A1 and 1B1 in the clearance of AQ or the formation of M2 could have toxicological implications in individuals exposed to inducers (e.g., polyaromatic hydrocarbons) of these extrahepatic P450s if M2 is reactive.

The identification of CYP2C8 as the major enzyme responsible for the hepatic metabolism of AQ was derived from several lines of evidence: 1) of several recombinant cytochrome P450 isoforms tested, CYP2C8 showed a dominant capacity for AQ N-desethylation; 2) CYP2C8 was an efficient catalyst of the reaction, as demonstrated by a turnover of 2.6 pmol/min/pmol of CYP2C8; 3) AQ N-desethylase activity, as well as AQ elimination rate, correlated well with hepatic CYP2C8 activity (r2 = 0.98 and 0.95, respectively); 4) when the activity of CYP2C8 used in this study was adjusted for its content of HLMs, the predicted contribution of CYP2C8 to AQ N-desethylase activity (562.3 μl/min/mg of protein) was similar to that found with HLMs (608.2 μl/min/mg of protein); 5) the inhibitory effects for quercetin were comparable for expressed CYP2C8 and for HLMs with Ki values of 1.96 and 1.56 μM, respectively, which are similar to those obtained using another CYP2C8 substrate, paclitaxel (Rahman et al., 1994); and 6) docking of AQ into CYP2C isoforms showed best solutions with CYP2C8, which also suggested potential metabolism in the proximity of the nitrogen atom on the diethylamino group of AQ. Furthermore, in the correlation study, the amount of the loss of AQ was almost equal to that of the formation of DEAQ, indicating that DEAQ is the dominant metabolite in HLMs and catalyzed by CYP2C8.

The selectivity of AQ-CYP2C8 interaction among the CYP2C isoforms was mainly driven by the geometric structure of the binding cavity. The many solutions of docking into CYP2C8 indicated that the possibility of hydrogen abstractions on the carbons bonded to the nitrogen where AQ isN-desethylated (Table 3 and Fig. 6) and most of the distances from those hydrogens to the oxygen atom were around 4 Å, a distance consistent with that of sites of oxidation of other substrates when docked into active sites of P450s that metabolize them. AQ desethylation is likely to proceed in two steps, a hydrogen abstraction and hydroxylation at the adjacent carbon, forming an unstable carbinolamide that rapidly hydrolyzes to DEAQ and acetaldehyde.

The results from AQ clearance studies using thet1/2 and theVmax/Kmapproach were comparable further indicating that the AQ desethylation is the major hepatic metabolic route for the clearance of the drug. The predicted in vivo clearance value of 19.1 ml/min/kg estimated from in vitro experiments greatly underestimated the observed clearance of 216 ml/min/kg (obtained after intravenous drug administration). There is, however, a wide interindividual variation in the in vivo clearance rates ranging from 78 to 943 ml/min/kg (White et al., 1987). In general, clearances predicted from in vitro data underestimate the observed values for many drugs (Carlile et al., 1999). The possible reasons for this trend have been reviewed by Iwatsubo et al. (1997). The reason for underestimating the in vivo clearance of AQ might have to do with the fact that AQ is probably also metabolized in the blood since CYP1A1 and 1B1 have been found in this fluid (Baron et al., 1998;Nguyen et al., 2000). Our results show that these extrahepatic enzymes extensively metabolize AQ (Fig. 2). Another reason could be that during the preparation of HLMs, the recovery of the specific enzyme metabolizing AQ, CYP2C8, is low and not reflected in the HLM recovery factor we used in estimating hepatic clearance.

Using recombinant P450s from different sources (lymphoblastoid and yeast), similar relative contributions (93 and 109%, respectively) of CYP2C8 to the metabolism of AQ were obtained. SimilarKm andKi (for inhibition by quercetin) values were also obtained, indicating that for AQ metabolism, the enzymes from the two different expression systems are equally predictive of the role of CYP2C8. For the metabolism of other compounds, use of P450s from different expression systems have been shown to result in inconsistent relative contributions of specific P450s (Venkatakrishnan et al., 2000).

Contrary to in vivo studies and the in vitro studies of Jewell et al. (1995) in which two other metabolites, 4-hydroxyDEAQ and bisDEAQ were observed, we did not observe these metabolites with both HLMs and rCYPs. The M2 produced by CYP1A1 and 1B1 has a protonated molecular ion at m/z 299, which is not consistent with these reported metabolites of AQ. This highlights the risk of failing to identify minor and slowly formed metabolites when using low microsomal proteins and short incubation times as used in our study. Another limitation pointed out by Jewell et al. (1995) is that the in vitro assays underestimate the likely bioactivation of AQ to the reactive quinoneimine since HLMs rapidly reduce it.

Our results conclusively show that CYP3A4 is not involved in the metabolism of AQ to DEAQ as had been previously proposed by Jewell et al. (1995) using ketoconazole as a diagnostic inhibitor. That erroneous conclusion is common when a single enzyme identification procedure is used without caution with respect to its shortcomings. In this case (Jewell et al., 1995), ketoconazole is probably only selective for CYP3A in the nanomolar range as we have shown that at 10 μM, it also inhibited CYP2C8 by over 50% (Masimirembwa et al., 1999). In other methods like the correlation analysis, use of HLM samples with P450 activities which cocorrelate can also lead to erroneous P450 identifications. Following the formation of a known metabolite can also be misleading since that might not be the main or only route of elimination. A simultaneous investigation of substrate disappearance is therefore recommended. A multifaceted approach as employed in this study ensures valid P450 identification and better estimations of drug clearance.

Pharmacokinetic studies have shown a large variation in the kinetic parameters of AQ and DEAQ. This variation could have implications in the therapeutic and toxicological response to the drug. In a subject with acute hepatic failure, the concentration of AQ was found to be a quarter that of the DEAQ (Pussard et al., 1987). This was very usual since in most subjects, very low concentrations of AQ itself are detected in blood and urine in the first hours following oral administration. These results are interesting in light of the findings of our study. First, the large interindividual variation in pharmacokinetics is consistent with the up to 38-fold variation among HLMs in metabolizing CYP2C8 substrates (reviewed in Ong et al., 2000). Variability in CYP2C8 either due to coadministered drug inhibitors or coingested dietary inhibitors/inducers, or genetic polymorphism could explain differential susceptibility of individuals to AQ toxicity and maybe explain some of the fatalities reported (Larrey et al., 1986;Rouveix et al., 1989). Regulation of CYP2C8 expression and activity by these factors need to be investigated as they might assist in the optimal use of AQ.

Genetic variants of CYP2C8 have been reported (http://www.imm.ki.se/CYPalleles; Ree et al., 1999). The CYP2C8*2, CYP2C8*3, and CYP2C8*4 variants associated with an increasedKm, decreased activity for paclitaxel 6α-hydroxylation and uncharacterized effects, respectively (Dai, 2001a,b; A. Daly and G. P. Aithal, submitted for publication). Knowledge of frequencies of distribution of CYP2C8 variants in the population likely to be exposed to the CYP2C8 substrate drug amodiaquine will contribute to our understanding of how patients will respond to this drug.

Due to the changing level of knowledge, the number and relative importance of individual P450s is continuously changing. Once it was only 2, then 5 and now over 10 P450s that are thought to be important for drug metabolism. These claims are partly to do with the biased studies with respect to some P450s and the expanding chemical space in the drug discovery process leading to new chemistries preferring previously “minor” P450s. Currently, there are few well characterized CYP2C8 marker substrates and diagnostic inhibitors. Because of CYP2C8 selectivity for AQ desethylation, high affinity, and turnover, AQ could be an excellent in vitro probe drug for CYP2C8 activity. AQ and DEAQ are relatively cheap compared with paclitaxel and its metabolite. Although CYP2C8 is the major hepatic enzyme responsible for both the clearance of AQ and the formation of DEAQ, paclitaxel is mainly metabolized to 6α-OH paclitaxel by CYP2C8 and to other metabolites by a major hepatic enzyme, CYP3A4 (Sonnichsen et al., 1995). These factors make AQ desethylation a relatively better probe.

In conclusion, we have shown that CYP2C8 is the exclusive isoform contributing to the N-desethylation of AQ in HLMs from among a panel of recombinant human hepatic P450 isoforms. This knowledge will give us a better understanding of the basis of the interindividual variability in AQ pharmacokinetics and probably therapeutic and toxicological responses to the drug. AQ desethylation also represents a new high affinity and turnover enzyme-specific marker reaction for assaying CYP2C8 activity.

Footnotes

  • X.-Q.L. is a recipient of the Wenner-Gren Foundation postdoctoral fellowship (Stockholm, Sweden).

  • Abbreviations:
    AQ
    amodiaquine
    DEAQ
    N-desethylamodiaquine
    bisDEAQ
    N-bisdesethylamodiaquine
    P450
    cytochrome P450
    HLM
    human liver microsome
    RAF
    relative activity factor
    LC
    liquid chromatography
    HPLC
    high-performance LC
    MS
    mass spectroscopy
    tR
    retention time
    • Received July 25, 2001.
    • Accepted October 5, 2001.

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Amodiaquine Clearance and Its Metabolism toN-Desethylamodiaquine Is Mediated by CYP2C8: A New High Affinity and Turnover Enzyme-Specific Probe Substrate

Xue-Qing Li, Anders Björkman, Tommy B. Andersson, Marianne Ridderström and Collen M. Masimirembwa
Journal of Pharmacology and Experimental Therapeutics February 1, 2002, 300 (2) 399-407; DOI: https://doi.org/10.1124/jpet.300.2.399
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