Introduction

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Tramadol, (2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol), is a synthetic opioid analgesic of the aminocyclohexanol type that has two chiral centers (Paar et al., 1992). It is a centrally acting analgesic drug with an analgesic efficacy and a potency that ranges between weak opioids and morphine (Poulsen et al., 1996). The biological activity of tramadol was initially attributed to predominantly µ-receptor-mediated opioid analgesia. However, tramadol has only a low affinity for µ-receptors of the central nervous system, being 6000 times lower than that of morphine. One metabolite of tramadol, namely, O-desmethyl-tramadol (metabolite M1²), is known to have a higher affinity for opioid receptors than the parent drug. In contrast to other opioids, the analgesic action of tramadol is only partially inhibited by the opioid antagonist naloxone, which suggests an additional mechanism of action. Indeed, tramadol may act by both opioid and monoaminergic mechanisms, the latter involving inhibition of norepinephrine reuptake and stimulation of serotinin release (Collart et al., 1993a,b; Dayer et al., 1994, 1997; Poulsen et al., 1996).

The metabolism of tramadol has been studied in humans, rats, mice, Syrian hamsters, guinea pigs, rabbits, and dogs (Lintz et al., 1981). Following oral doses of ¹⁴C-labeled tramadol of either 1.06 or 1.25 mg/kg to two human volunteers, around 90% of the administered radioactivity was excreted in the urine. Analysis of 0- to 72-h urine samples revealed some unchanged tramadol (25 or 32% of total urinary radioactivity) and a number of tramadol metabolites. The primary metabolites (Fig. 1) of tramadol, namely, O-desmethyl-tramadol (metabolite M1) and N-desmethyl-tramadol (metabolite M2) may be further metabolized to three additional secondary metabolites, namely, N,N-didesmethyl-tramadol (metabolite M3), N,N,O-tridesmethyl-tramadol (metabolite M4), and N,O-didesmethyl-tramadol (metabolite M5). The urine of the two subjects given ¹⁴C-labeled tramadol contained various amounts of these tramadol metabolites, together with conjugates (glucuronides and sulfates) of metabolites M1, M4, and M5 and some unidentified components.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Some known metabolic pathways of tramadol. Tramadol has been shown to undergo both N- and O-demethylations to various metabolites which can be conjugated with either D-glucuronic acid or sulfate (Lintz et al., 1981).

The O-demethylation of tramadol to metabolite M1 is known to be catalyzed by human hepatic CYP2D6 (Paar et al., 1992, 1997; Poulsen et al., 1996). This cytochrome P-450 (CYP) isoform (Nelson et al., 1996) is expressed polymorphically in humans with some 93% of Caucasians being phenotypically extensive metabolizers and the remainder being poor metabolizers of CYP2D6 probe substrates such as debrisoquine and sparteine (Dahl et al., 1995; Ingelman-Sundberg et al., 1995). A close relationship has been reported between tramadol O-demethylation and sparteine oxidation in human volunteers and tramadol may show a weaker analgesic effect on clinical pain in poor than in extensive metabolizers (Poulsen et al., 1996; Paar et al., 1997). In the study of Lintz et al. (1981), a marked difference was observed between the two subjects examined in the relative amounts of M1 and M2 formed. Possibly one subject may have been CYP2D6 deficient (i.e., a poor metabolizer). In another study where a 50-mg oral dose of tramadol was given to 104 volunteers, mean values for M1 and M2 excretion in 24-h urines were 15 and 4% of the administered dose, respectively (Paar et al., 1997). Unlike tramadol O-demethylation, no correlation was observed between tramadol N-demethylation and sparteine oxidation in extensive metabolizers (Paar et al., 1997).

The in vitro metabolism of both ()- and (+)-tramadol has been investigated in human liver microsomes with a substrate concentration range of 50 to 5000 µM (Paar et al., 1992). For O-demethylation to metabolite M1, the K_m value for both isomers was determined to be 210 µM. While Eadie-Hofstee plots for M1 formation were monophasic, tramadol N-demethylation to metabolite M2 was stated to exhibit biphasic kinetics. Moreover, M1 formation, but not M2 formation, was competitively inhibited by quinidine with a reported K_i value of 15 nM. Other studies have demonstrated that quinidine is a selective inhibitor of human hepatic CYP2D6 (Newton et al., 1995; Clarke, 1998; Pelkonen et al., 1998).

The objective of this study was to obtain more information on the CYP isoforms responsible for cis-tramadol metabolism in human liver. Investigations have been performed with human liver microsomes and cDNA-expressed CYP isoforms. The cis-tramadol metabolites studied were the two primary metabolites, namely, M1 and M2, together with two secondary metabolites, namely, M3 and M5 (Fig. 1).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. NADPH, cytochrome c, Tris, diethyldithiocarbamate, quinidine, sulfaphenazole, and troleandomycin were obtained from Sigma (Poole, Dorset, UK) and furafylline and S-(+)-mephenytoin from Salford Ultrafine Chemicals and Research Ltd. (Manchester, UK). A reaction phenotyping kit (product no. H0500, version 5) containing 16 individual human liver microsomal preparations, characterized for total CYP content and a range of CYP isoform enzyme activities, was purchased from XenoTech LLC (Kansas City, KS) and stored at 80°C. Microsomes from human B-lymphoblastoid cells containing cDNA-expressed human CYP isoforms (GENTEST, Woburn, MA) were obtained from Cambridge Bioscience (Cambridge, UK) and stored at 80°C. The samples of human B-lymphoblastoid cell microsomes comprised control cell microsomes (i.e., no transfected human cytochrome P-450 isoform cDNA but contain native CYP1A1 activity) and cell microsomes containing CYP1A2, CYP2A6 + OR [i.e., CYP2A6 cDNA plus human NADPH-cytochrome P-450 reductase (OR) cDNA], CYP2B6, CYP2C8 + OR, CYP2C9 + OR, CYP2C19, CYP2D6 + OR, CYP2E1 + OR, and CYP3A4 + OR.

Tramadol, Tramadol Metabolites, and Internal Standards. Samples of tramadol, tramadol metabolites, and ¹³C- and deuterium-labeled tramadol and tramadol metabolites [internal standards for liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) analysis] were supplied by Department of Pharmacokinetics and Drug Metabolism, Purdue Pharma L.P. (Ardsley, NY). The compounds (all hydrochlorides) supplied are listed in Table 1.

Human Liver Microsomes. Samples of human liver (surplus to transplant requirements) were transported on ice to TNO BIBRA from other institutions and were stored at 80°C. Washed microsomal fractions were prepared in 0.154 M KCl containing 50 mM Tris-HCl, pH 7.4, as described previously (Lake, 1987). Liver microsomal fractions were assayed for total CYP content (Omura and Sato, 1964) and for protein (Lowry et al., 1951) using bovine serum albumin as standard. Microsomal fractions were diluted to 10 mg of protein/ml and aliquots stored at 80°C. Two separate batches, designated pools I and II, of pooled human liver microsomes were prepared. Each batch was prepared by pooling liver samples from five subjects. Pool I comprised liver samples from males aged 2.5 and 58 years and females aged 14, 59, and 74 years, whereas pool II comprised liver samples from male subjects aged 44 and 50 years and female subjects aged 11, 31, and 55 years. Microsomal NADPH-cytochrome c reductase activity was determined at 37°C in cuvettes containing 0.4 mM NADPH (test cell only), 0.62 mg/ml cytochrome c, 1.2 mM KCN, 20 to 50 µg of microsomal protein and 0.1 M phosphate buffer, pH 7.6, in a final volume of 2.5 ml. The rate of reduction of cytochrome c was monitored at 550 nm using an extinction coefficient of 21 mM¹ cm¹.

Metabolism of Tramadol by Human Liver Microsomes. The NADPH-dependent metabolism of tramadol was studied in incubation mixtures containing 5 to 5000 µM tramadol (added in 5 µl of methanol), 10 mM MgCl₂, 2 mM EDTA, 80 mM phosphate buffer, pH 7.4, and 0.25 to 1.5 mg of microsomal protein in a final volume of 1 ml. After a 5-min preincubation at 37°C in a shaking water bath, the reaction was initiated by the addition of 1 mM NADPH. Incubations were performed for 3 to 30 min at 37°C and were terminated by the addition of 2 ml of ice-cold acetonitrile containing the ¹³C- and deuterium-labeled internal standards for LC-MS-MS analysis (see below). Blank incubations contained all components except NADPH, which was added after the reaction was terminated with acetonitrile. For each tramadol substrate concentration, incubation time, and microsomal protein concentration incubations were conducted in duplicate with either a single or a duplicate blank (no NADPH) tube. Apart from the studies with the CYP1A2 mechanism-based inhibitor furafylline (Newton et al., 1995), where dimethyl sulfoxide (DMSO) was used, methanol was used as the solvent for these investigations to minimize solvent effects on individual CYP isoforms (Chauret et al., 1998; Busby et al., 1999).

Kinetics of Tramadol Metabolism. Kinetic data were analyzed by Michaelis-Menten and Eadie-Hofstee plots. Inspection of Eadie-Hofstee plots revealed that multiple enzymes were involved in the metabolism of tramadol to both tramadol metabolites M1 and M2. Kinetic analysis was performed by inspection of the Eadie-Hofstee plots and considering the data as biphasic kinetics with both high-affinity and low-affinity enzymes. For the high-affinity enzyme, K_m and V_max values were calculated directly from the Eadie-Hofstee plots. Additional calculations were performed to calculate the contribution of the high-affinity enzyme to the low-affinity enzyme kinetic plot. The following equation was used:
where V_max and K_m are the kinetic constants for the high-affinity enzyme and [S] is the substrate concentration. Calculated rates were derived for each substrate concentration used to calculate the low-affinity component. To derive corrected rates for the low-affinity component the following equation was used:
As an alternative approach estimates of K_m and V_max for both the high- and low-affinity enzymes were obtained with the following modified Michaelis-Menten equation (Clarke, 1998):
Inspection of the data revealed that the low-affinity component was not saturated over the range of tramadol substrate concentrations used. The following modified Michaelis-Menten equation (Clarke, 1998) was used:
In this equation CL_int represents the intrinsic clearance (i.e., V_max/K_m) of the low-affinity component.

Tramadol Metabolism Correlation Analysis. Incubation mixtures contained 250 µM tramadol (added in 5 µl of methanol), 10 mM MgCl₂, 2 mM EDTA, 0.4 mg of microsomal protein, and 87 mM phosphate buffer, pH 7.4, in a final volume of 1 ml. After a 5-min preincubation at 37°C in a shaking water bath, the reaction was initiated by addition of 1 mM (final concentration) NADPH. Incubations were conducted in triplicate with a single blank (no NADPH) for each of the 16 preparations of characterized human liver microsomes and were terminated after 5 min by addition of 2 ml of ice-cold acetonitrile containing the ¹³C- and deuterium-labeled tramadol metabolite M1, M2, M3, and M5 internal standards. Incubations were processed for LC-MS-MS analysis as described above.

Tramadol Metabolism by cDNA-Expressed CYP Isoforms. A 100 mM stock solution of tramadol in methanol was diluted to 333 µM with 100 mM phosphate buffer, pH 7.4. Incubation mixtures contained 250 µM tramadol (added as 187.5 µl of 333 µM tramadol), 10 mM MgCl₂, 2 mM EDTA, 1 mM NADPH, and 80 mM phosphate buffer in a final volume of 0.25 ml. After 5-min preincubation in a 37°C water bath, the reaction was initiated by adding 0.25 mg of B-lymphoblastoid cell microsomal protein with gentle mixing. The incubations were conducted in duplicate with a single blank (no microsomes) for each CYP isoform preparation. Reactions were terminated after either 5 or 30 min with 0.5 ml of ice-cold acetonitrile containing the ¹³C- and deuterium-labeled tramadol metabolite M1, M2, M3, and M5 internal standards. Incubations were processed for LC-MS-MS analysis as described above.

Tramadol Metabolism Inhibition Studies. For the mechanism-based inhibitors requiring preincubation with NADPH, incubation mixtures contained 10 mM MgCl₂, 2 mM EDTA, 1 mM NADPH, 85 mM phosphate buffer, pH 7.4, 0.5 mg of pool II microsomal protein, and either 5 to 50 µM furafylline, 5 to 100 µM diethyldithiocarbamate, or 5 to 100 µM troleandomycin in a volume of 0.945 ml. The inhibitors were added in either methanol (5 µl/tube) or DMSO (furafylline only, 5 µl/tube) and the concentrations refer to the final concentrations in a 1-ml incubation. Following a 30-min preincubation at 37°C in a shaking water bath, 250 µM tramadol (added in 5 µl of methanol) and 1 mM NADPH (final concentration 2 mM) were added. Incubations were performed for 5 min at 37°C and were terminated by the addition of 2 ml of ice-cold acetonitrile containing the ¹³C- and deuterium-labeled tramadol metabolite M1, M2, M3, and M5 internal standards. All test incubations were performed in either triplicate (no inhibitor added controls, but with either 5 µl of methanol or 5 µl of DMSO) or duplicate (for each concentration of each inhibitor), with single blank tubes where tramadol was added after the ice-cold acetonitrile. Incubations were processed for LC-MS-MS analysis as described above.

	Results

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Metabolism of Tramadol by Human Liver Microsomes. Tramadol was metabolized by human liver microsomes in the presence of NADPH to all four available tramadol metabolites. While M1 and M2 were major metabolites of tramadol in human liver microsomes, M3 and M5 represented only minor metabolites. Some representative data for tramadol metabolism to metabolites M1, M2, M3, and M5 are shown in Table 2. Under the incubation conditions selected, tramadol metabolism to either metabolite M3 or metabolite M5 was always 3.0% of total tramadol metabolism (i.e., the sum of formation of M1, M2, M3, and M5).

Kinetics of Tramadol Metabolism in Human Liver Microsomes. The kinetics of the NADPH-dependent metabolism of 5 to 5000 µM tramadol to metabolites M1 and M2 was examined with both the pool I and pool II human liver microsomal preparations. Because both metabolites M3 and M5 each constituted 3.0% of total tramadol metabolism in human liver microsomes, it was not considered necessary to investigate the kinetics of formation of these two metabolites. Pooled, rather than individual, liver microsomal preparations were used for these studies, because the aim was to identify average K_m values to select a suitable substrate concentration for the reaction phenotyping studies, rather than to evaluate any interindividual variability in K_m values for M1 and M2 formation. With both liver microsome pools, M1 and M2 were detected at all tramadol substrate concentrations examined (Fig. 2). Figure 2 also demonstrates that while M1 is the major metabolite formed at low tramadol substrate concentrations, M2 formation predominates at high substrate concentrations. In keeping with the higher total CYP content, the pool I liver microsome preparation was more active than the pool II microsomal preparation in catalyzing M1 and M2 formation.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Metabolism of tramadol to tramadol metabolites M1 and M2 in human liver microsomes. The metabolism of 5 to 5000 µM tramadol to M1 and M2 was studied with the pool I (A) and II (B) human liver microsomal preparations. Incubations were performed with 0.5 mg of microsomal protein for 5 min. Results are presented as the mean of a duplicate test and a duplicate blank incubation.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Kinetics of tramadol metabolism to tramadol metabolites M1 and M2 in human liver microsomes. Eadie-Hofstee plots of 5 to 5000 µM tramadol metabolism to M1 (A and C) and M2 (B and D) was studied with the pool I (A and B) and II (C and D) human liver microsomal preparations. For other details see Fig. 2.

Tramadol Metabolism Correlations Analysis. The metabolism of 250 µM tramadol to tramadol metabolites M1 and M2 was examined with a characterized panel of 16 human liver microsomal preparations. Although tramadol metabolism to the secondary metabolites M3 and M5 was not necessarily linear under the incubation conditions selected, the levels of these two minor tramadol metabolites were also quantified in the incubation extracts. Tramadol was metabolized to M1 and M2 by all 16 human liver microsomal preparations examined (Fig. 4A). Compared with M1 and M2 formation, much smaller amounts of the secondary metabolites M3 and M5 were observed in 14 of the 16 human liver microsomal preparations examined (Fig. 4B).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Metabolism of tramadol to tramadol metabolites M1, M2, M3, and M5 in a panel of 16 characterized human liver microsomal preparations. The metabolism of 250 µM tramadol to M1 and M2 (A) and M3 and M5 (B) was studied. Incubations were performed with 0.4 mg of microsomal protein for 5 min. Results are presented as the mean ± S.E. of triplicate test incubations.

Metabolism of Tramadol by cDNA-Expressed CYP Isoforms. The metabolism of 250 µM tramadol by B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, together with microsomes from control cells (which contain native CYP1A1), was studied. Only trace amounts of tramadol metabolites were observed in incubations with the control cell microsomes (data not shown) and with the cDNA-expressed CYP2A6, CYP2C8, and CYP2E1 preparations (Fig. 5). While tramadol metabolism to M1 was greatest with the cDNA-expressed CYP2D6 preparation, M2 formation was greatest with the cDNA-expressed CYP2B6 preparation (Fig. 5A). Tramadol was also metabolized to M1 by the CYP2B6 preparation and to M2 by the CYP2D6 and CYP3A4 preparations. Only low rates of tramadol metabolism to either M1 or M2 were observed with the cDNA-expressed CYP1A2, CYP2C9, and CYP2C19 preparations (Fig. 5A). Low rates of tramadol metabolism to M3 were observed with the CYP2B6 and CYP3A4 preparations, with a low rate of M5 formation being catalyzed by the CYP2D6 preparation (Fig. 5B).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Metabolism of tramadol to tramadol metabolites M1 and M2 by cDNA-expressed human CYP isoforms. The metabolism of 250 µM tramadol by cDNA-expressed CYP isoforms to M1 and M2 (A) and M3 and M5 (B) was studied. Incubations were performed with 0.25 mg of B-lymphoblastoid cell microsomal protein for either 5 min (CYP2B6, CYP2D6, and CYP3A4) or 30 min (all other CYP isoforms). Results are presented as the mean of a duplicate test and a single blank incubation.

Inhibition of Tramadol Metabolism. The effect of some human CYP isoform inhibitors and one CYP isoform substrate (S-mephenytoin) on the metabolism of 250 µM tramadol to metabolites M1 and M2 in human liver microsomes was studied. Because of the low rates of formation of M3 and M5, it was not possible to evaluate the effects of the test compounds on these two tramadol secondary metabolites. For the mechanism-based inhibitors (Newton et al., 1995) furafylline (CYP1A2), diethyldithiocarbamate (CYP2E1), and troleandomycin (CYP3A4), the compounds were preincubated for 30 min at 37°C with liver microsomes and NADPH prior to the addition of tramadol and a further aliquot of NADPH. In the studies with the inhibitors sulfaphenazole (CYP2C9) and quinidine (CYP2D6) and the substrate S-mephenytoin (CYP2C19), the compounds were preincubated with liver microsomes and NADPH for 10 min at 37°C prior to the addition of tramadol.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Inhibition of tramadol metabolism to tramadol metabolites M1 and M2 in human liver microsomes. The effects of the mechanism-based inhibitors (A and B) 5 to 50 µM furafylline (), 5 to 100 µM diethyldithiocarbamate (), and 5 to100 µM troleandomycin (), together with (C and D) 2 to 50 µM sulfaphenazole (), 50 to 500 µM S-mephenytoin (), and 2 to 20 µM quinidine () were evaluated on the metabolism of 250 µM tramadol to M1 (A and C) and M2 (B and D) in human liver microsomes. Incubations were performed with 0.5 mg of pool II microsomal protein for 5 min. Each point represents the mean of a duplicate test and a single blank incubation.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In this study the metabolism of cis-tramadol by human hepatic microsomal fractions and cDNA-expressed human hepatic CYP isoforms has been investigated. The formation of the major tramadol metabolites M1 and M2 was observed in two preparations of pooled liver microsomes and in 16 individual preparations. In addition, two minor secondary metabolites (each 3.0% of total metabolism), namely, M3 and M5 were also observed in the majority of the human liver microsomal preparations examined. Tramadol can thus be metabolized in human liver by either single or multiple O- and N-demethylations (Fig. 1).

For both M1 and M2 formation in human liver microsomes, biphasic Eadie-Hofstee plots were obtained, indicating the participation of more than one CYP isoform in these pathways of tramadol metabolism. The kinetic data were considered as biphasic plots with both high-affinity and low-affinity enzymes. Over a substrate concentration range of 5 to 5000 µM mean K_m values of 116 and 1021 µM were obtained for the high-affinity enzymes responsible for M1 and M2 formation, respectively (Table 3). While the mean K_m value of 116 µM for M1 formation is similar to the value of 210 µM reported by Paar et al. (1992) for metabolism of both ()- and (+)-tramadol to M1, these studies only reported monophasic kinetics. However, in agreement with the present study, Paar et al. (1992) did observe biphasic kinetics for M2 formation although no K_m values were reported. In the present study, the lower K_m value for the high-affinity component of M1 formation compared with M2 formation (i.e., 116 compared with 1021 µM) is in agreement with the observation that in most subjects larger quantities of M1 are formed after oral administration of tramadol (Lintz et al., 1981; Paar et al., 1997).

To identify the CYP isoform responsible for tramadol metabolism, "reaction phenotyping" was performed by correlation analysis with a panel of characterized microsomal preparations, chemical inhibition, and the use of cDNA-expressed human CYP isoforms. A cis-tramadol substrate concentration of 250 µM was selected as being approximately 2 times the K_m for the high-affinity enzyme responsible for M1 formation.

The metabolism of cis-tramadol was examined in a panel of 16 characterized human liver microsomal preparations. Good correlations were obtained between M1 formation and dextromethorphan O-demethylase (r² = 0.676) and between M2 formation and S-mephenytoin N-demethylase (r² = 0.748). These two enzyme activities are considered to be markers for CYP2D6 and CYP2B6, respectively, in human liver microsomes (Heyn et al., 1996; Parkinson, 1996; Clarke, 1998; Pelkonen et al., 1998; Lewis et al., 1999). Some lower correlations between either M1 or M2 formation and enzymatic markers for CYP2C8 and CYP3A4 were also observed (Table 4).

The conclusions of the correlation analysis study are supported by the results of the chemical inhibition studies. Previous studies have demonstrated that furafylline, sulfaphenazole, S-mephenytoin, quinidine, diethyldithiocarbamate, and troleandomycin may be considered either inhibitors or substrates of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4, respectively (Newton et al., 1995; Ono et al., 1996; Parkinson, 1996; Clarke, 1998; Pelkonen et al., 1998; Rodrigues, 1999). Tramadol metabolism to M1 was inhibited to 14 to 23% of control by the CYP2D6 inhibitor quinidine, whereas M2 formation was inhibited to 33 to 44% of control by the CYP3A4 mechanism-based inhibitor troleandomycin (Fig. 6, B and C). In contrast, the inhibitors or substrate of CYP1A2, CYP2C9, CYP2C19, and CYP2E1 studied had no marked effect on tramadol metabolism to either M1 or M2. The effect of a chemical inhibitor of CYP2B6 was not evaluated in this study, although S-mephenytoin, which is metabolized to different products by CYP2B6 and CYP2C19 (Relling et al., 1989; Heyn et al., 1996; Parkinson, 1996; Clarke, 1998; Pelkonen et al., 1998), had no marked effect on either M1 or M2 formation. The lack of a marked effect of S-mephenytoin on CYP2B6-catalyzed cis-tramadol metabolism to M2 in this study is probably attributable to the K_m value for CYP2B6-catalyzed S-mephenytoin N-demethylase. Other studies have demonstrated that while the K_m value for CYP2C19-catalyzed S-mephenytoin 4'-hydroxylase in human liver microsomes is around 60 to 65 µM, the K_m value for CYP2B6-catalyzed S-mephenytoin N-demethylase has been reported to be 805 µM (Relling et al., 1989; Heyn et al., 1996; Pelkonen et al., 1998). Thus, over the 50 to 500 µM concentration range studied, S-mephenytoin would not have been expected to have a marked effect on CYP2B6-catalyzed cis-tramadol metabolism to M2.

cis-Tramadol was metabolized to M1 and M2 by some of the cDNA-expressed human CYP isoforms examined. CYP2D6 was the major CYP isoform catalyzing M1 formation, whereas CYP2B6 and CYP3A4 were the major isoforms catalyzing M2 formation (Fig. 5). In contrast, cDNA-expressed CYP1A2, CYP2C9, and CYP2C19 only catalyzed low rates of M1 and M2 formation. For M2 formation the CYP2B6 preparation exhibited a higher specific activity than the CYP3A4 preparation. However, the levels of CYP3A4 in human liver microsomes have been reported to range from 44 to 250 pmol/mg of protein, whereas those for CYP2B6 have been reported to range from only 1 to 39 pmol/mg of protein (Lecoeur et al., 1994; Shimada et al., 1994; Imaoka et al., 1996; Rodrigues, 1999). Thus, assuming these two CYP isoforms have similar affinities (i.e., K_m values) for tramadol, M2 formation may be predominantly catalyzed by CYP3A4 in human liver microsomes.

This study demonstrates that multiple CYP isoforms are responsible for cis-tramadol metabolism in human liver. Clearly, tramadol metabolism to tramadol metabolite M1 is primarily catalyzed by CYP2D6 and this finding is in agreement with other in vivo and in vitro studies on tramadol metabolism and the effect of quinidine inhibition (Paar et al., 1992, 1997; Dayer et al., 1994; Poulsen et al., 1996). From the correlation analysis and cDNA-expressed CYP isoform data, CYP2B6 may have a minor role in M1 formation in human liver, with very low levels of activity being catalyzed by CYP1A2, CYP2C9, and CYP2C19. While CYP2D6 appears to be the high-affinity enzyme catalyzing M1 formation in human liver microsomes, additional studies would be required to identify the low-affinity enzyme(s) responsible for M1 formation.

Unlike M1, the major CYP isoforms involved in M2 formation appear to be CYP2B6 and CYP3A4. Additional kinetic studies would be required to identify the high-and low-affinity enzymes catalyzing M2 formation in human liver microsomes. From the present data CYP2B6 may constitute a high-affinity enzyme, whereas the high levels of CYP3A4 in human liver (Lecoeur et al., 1994; Shimada et al., 1994; Imaoka et al., 1996; Rodrigues, 1999) would imply that this CYP isoform would also contribute to M2 formation, particularly in subjects with either low or absent levels of CYP2B6 (Mimura et al., 1993; Code et al., 1997; Shimada et al., 1997). Based on the cDNA-expressed CYP isoform data, CYP2D6 may have a minor role in M2 formation, with very low levels of activity being catalyzed by CYP1A2, CYP2C9, and CYP2C19. While some correlation (r² = 0.454) was observed between CYP2C8-catalyzed taxol 6-hydroxylase activity and M2 formation, this finding was not supported by cDNA-expressed CYP isoform studies where only a very low rate of M2 formation was observed with the CYP2C8 preparation.

Some preliminary information was also obtained on the CYP isoforms involved in M3 and M5 formation. However, only tentative conclusions can be drawn from the present data because M3 formation is dependent on the amount of M2 produced and M5 formation is dependent on the production of both M1 and M2. In addition, under the incubation conditions used M3 and M5 formation was not necessarily linear with respect to incubation time and microsomal protein concentration. Based on the observations that CYP2D6 primarily catalyzes tramadol O-demethylation, whereas CYP2B6 and CYP3A4 primarily catalyze N-demethylation, CYP2D6 would be expected to participate in M5 formation from M2. In addition, CYP2B6 and CYP3A4 would be expected to participate in M1 metabolism to M5 and M2 metabolism to M3. Additional studies, including using M1 and M2 as substrates, would be required to fully elucidate the CYP isoforms involved in M3 and M5 formation.

In summary, cis-tramadol can be metabolized to tramadol metabolites M1, M2, M3, and M5 in human liver microsomal preparations. While M1 and M2 constitute major primary metabolites, M3 and M5 are only minor secondary metabolites. By kinetic analysis and the results of the reaction phenotyping studies, tramadol metabolism in human liver is catalyzed by multiple CYP isoforms. While CYP2D6 is primarily responsible for M1 formation, M2 formation is catalyzed by CYP2B6 and CYP3A4.