Materials and Methods

Chemicals and Reagents.

Bufuralol, 1′-hydroxybufuralol, 4-hydroxydebrisoquine, and dextrorphan were purchased from Ultrafine Chemicals (Manchester, UK). Dextromethorphan, debrisoquine, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, NADP, and EDTA were acquired from Sigma-Aldrich (St. Louis, MO). Hydroxybufuralol was obtained from Sigma/RBI (Natick, MA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). TransfectAMINE Plus reagent was purchased from Invitrogen (Carlsbad, CA). DMEM² and COS-7 cells were obtained from American Type Culture Collection (Manassas, VA). Oligonucleotides were purchased from MWG-Biotech AG (Greensboro, NC). SacI and BsrGI were acquired from New England Biolabs (Beverly, MA). The pcDNA3.1/Hygro (+/−) vector was obtained from Invitrogen. Quantum Prep plasmid miniprep kits were purchased from Bio-Rad (Hercules, CA). Assay plates (96-well) were purchased from Nalge Nunc (Naperville, IL). NADPH-dependent cytochrome P450 reductase and baculovirus-expressed CYP2D6*1 were purchased from Gentest Corp. (Woburn, MA). All other reagents were of analytical grade.

Construction of Expression Plasmids.

A pUC19 plasmid containing the CYP2D6*1 cDNA with a 65-bp long 5′ “leader sequence” optimized for mammalian expression was provided by Gentest Corp. Site-directed mutagenesis to introduce the C → T transition at position 2850 was performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's directions. Oligonucleotides 30-bp long with the desired mismatches located in the center of the sequence were used without gel purification. For restriction fragment exchange, a 645-bp fragment containing C at position 4180 was amplified using primers 5′4213 and 3′4213/4268 (Gaedigk et al., 1999) from a genomic DNA sample genotyped as CYP2D6*2/*2 (Gaedigk et al., 1999) and digested with SacI and BsrGI to yield a 183-bp fragment. This fragment was exchanged with the corresponding fragment released from the intermediate 2850C > T construct. Similarly, a 530-bp fragment carrying T at position 1023 was generated from a genomic DNA sample genotyped as CYP2D6*17/*17 and digested with PflMI and EagI. The resulting 73-bp fragment was exchanged with the fragment released by PflMI andEagI digestion of the CYP2D6*2 construct. Clones containing the desired nucleotide changes were identified by genotyping as described in detail elsewhere (Gaedigk et al., 1999). To confirm the identity of the CYP2D6*17 cDNA, the construct was completely sequenced on a Visible Genetics sequencing system (Visible Genetics Inc., Toronto, Canada) using Cy5 and Cy5.5 dye terminator chemistry (Amersham Biosciences, Piscataway, NJ).

For expression in COS-7 cells, cDNA and “leader sequence” were released from pUC*2 and pUC*17 with XbaI, and the resulting fragments cloned into the XbaI site of the pcDNA3.1/Hygro (+/−) vector. Subsequently, plasmids were purified for transfection experiments with the Quantum Prep plasmid miniprep kit, followed by an ethanol precipitation. DNA concentration and quality were evaluated by spectral photometry and agarose gel analysis.

Transfection of COS-7 Cells and Preparation of Microsomes.

COS-7 cells were plated onto 100-mm dishes (typically 10 dishes per construct) in DMEM supplemented with 10% FBS, 200 U/ml penicillin, and 200 μg/ml streptomycin. With the cells ∼50% confluent, the media was changed to DMEM without penicillin/streptomycin 12 to 24 h prior to transfection. Plasmids were transfected into the COS cells using TransfectAMINE Plus (Invitrogen) according to the manufacturer's guidelines. Optimal cell viability and transfection efficiency were achieved with 6 μg of DNA/dish, 15 μl of TransfectAMINE Plus reagent/dish, and 20 μl of lipid/dish. Cells transfected with vector alone served as a control. Twenty-four hours after transfection, medium was replaced with DMEM containing FBS supplemented with penicillin/streptomycin. Forty-eight hours after transfection, cells were washed once and harvested by scraping in homogenization buffer containing 50 mM Tris-HCl, 150 mM KCl, and 2 mM EDTA. After sonication, microsomes were prepared by differential centrifugation at 9,000g for 20 min and centrifugation of the resulting supernatant at 100,000g for 60 min. The microsomal pellet was resuspended in 250 mM sucrose and stored at −70°C. The CYP2D6*1, CYP2D6*2, and CYP2D6*17 constructs were transfected on three separate occasions into COS-7 cells passaged in succession.

Quantitation of Expressed CYP2D6 Variant Proteins.

Total protein content of microsomes prepared from COS-7 cells was measured using the BCA protein assay kit (Pierce, Rockford, IL). Microsomal CYP2D6 apoprotein content was determined by immunoblot analysis. Microsomal protein (5 μg/lane) was resolved on 4 to 12% Novex polyacrylamide gel electrophoresis gradient gels (Invitrogen) in MOPS buffer and transferred to Hybond-C nitrocellulose (Amersham Biosciences) using a semidry transfer apparatus (Invitrogen). CYP2D6 protein was probed with an anti-CYP2D6 antibody raised against a multivalent antigenic peptide (MAP) antigen prepared from the CYP2D6-derived peptide NH₂-DPAQPPRDLTEAFLA-COOH coupled to the polylysine core using carbodiimide chemistry. MAP antigen (2 mg) was dissolved in 1 ml of phosphate-buffered saline and emulsified with an equal volume of Freund's Complete Adjuvant. After preimmune serum was obtained, 2-kg male, pathogen-free New Zealand White rabbits (Charles River Canada, Montreal, Quebec, Canada) were immunized subcutaneously with emulsified MAP antigen (1 mg per rabbit) distributed over multiple sites. Injections with MAP antigen (1 mg in Freund's Incomplete Adjuvant) divided into six to eight subcutaneous sites were repeated 4, 8, and 16 weeks after the initial immunization. Rabbits were exsanguinated under pentobarbital anesthesia 21 days after the final immunization, and serum recovered and heat-inactivated (56°C for 30 min) prior to storage at −20°C. Immunoblotting with the anti-CYP2D6 antibody was performed under optimized conditions for dilution (1:100,000) and incubation time (1 h, 22°C). A serial dilution of baculovirus-expressed CYP2D6.1 was included on each blot to serve as a “standard curve” for the expressed CYP2D6. The alkaline phosphatase anti-rabbit secondary antibody (Promega, Madison, WI) was diluted 1:10,000, and bands were visualized by incubation with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Blots were scanned with a Kodak 440CF Image Station and subjected to densitometric analysis using Kodak Digital Science 1D software (Eastman Kodak, Rochester, NY). Microsomal CYP2D6 apoprotein content was extrapolated from linear regression of the CYP2D6.1 standards and expressed as picomoles per milligram of microsomal protein.

Baculovirus Expression, Microsome Preparation, and Quantitation of Expressed CYP2D6.

CYP2D6*2 and CYP2D6*17 cDNAs were modified by the addition ofEcoRI linkers and were coexpressed with human NADPH-dependent cytochrome P450 oxidoreductase (OR) in a baculovirus expression system (Sata et al., 2000). Virus bearing active CYP protein was identified by assaying virus-infected Sf9 cell lysates using the fluorometric substrate 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin at a final concentration of 25 μM (Chauret et al., 2001), and the presence of OR was confirmed by measurement of cytochrome creduction. Virus containing both of these activities was amplified, and microsomes were prepared from Trichoplusia ni cells infected with the appropriate virus (Sata et al., 2000).

In Vitro Incubation Conditions.

For proteins expressed in COS-7 cells, dextromethorphanO-demethylation and bufuralol 1′-hydroxylation assays were carried out using 0.5 pmol of enzyme (apoprotein) per incubation. Reactions were performed in round bottom, 96-well plates in a total assay volume of 100 μl containing 50 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl₂, 1 mM EDTA, 5 mM glucose 6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, and 100 mM NADP and supplemented with 6.0 μg of OR. Dextromethorphan and bufuralol concentrations ranged between 0 and 100 μM and 0 and 200 μM, respectively. Reactions were conducted for 60 min at 37 ± 1°C with gentle shaking. Appropriate metabolite standards were included in each assay. Reactions were terminated by the addition of 100 μl of methanol, and samples were centrifuged at 10,000 rpm for 5 min to pellet protein prior to high-performance liquid chromatography analysis.

For baculovirus-expressed enzymes, assay conditions for dextromethorphan O-demethylation and bufuralol 1′-hydroxylase activities were identical to those described for the COS-7-expressed proteins with the exception that the amount of added CYP2D6 variant was based on spectral determination rather than apoprotein content, exogenous reductase was not added, and the incubation time was 30 min. Assays for debrisoquine 4-hydroxylation activity used 2 pmol of enzyme per incubation and were incubated for 1 h using debrisoquine concentrations ranging from 0 to 200 μM.

Analytical Procedures.

All analytic procedures were conducted with a Hewlett Packard HP1100 high-performance liquid chromatography system (Hewlett Packard Instruments, Palo Alto, CA) equipped with a degasser, binary or quaternary pump, autosampler, column heater, and programmable 1100 series variable fluorescence detector. All data were collected and integrated with Hewlett Packard Chemstation V A.0401 software.

The O-demethylation of dextromethorphan to dextrorphan was analyzed using a method modified from that described by Abdel-Rahman et al. (1999). Aliquots (25–75 μl) of assay supernatants were injected onto a Phenomenex Phenyl guard column (4 mm × 3 mm i.d., 5-μm particle size; Phenomenex, Torrance, CA) followed by separation on a Novapak Phenyl column (3.9 mm × 15 cm, 4-μm particle size, 40°C; Waters, Milford, MA) that had been equilibrated with a mobile phase consisting of a 75:25 mixture of buffer (20 mM potassium phosphate and 20 mM hexane sulfonic acid, pH 4.0) and acetonitrile at a flow rate of 1.2 ml/min followed by fluorescence detection with excitation and emission wavelengths of 235 and 310 nm, respectively. Under these conditions, dextromethorphan and dextrorphan had retention times of 4.1 and 15.7 min, respectively. A five-point standard curve was used to quantify the amount of dextrorphan present in sample injections. The limit of quantitation for the assay was 0.75 pmol. The analytical method demonstrated linearity between 1.125 and 112.5 pmol injected, the range of standards evaluated (r² > 0.997). Intraday and interday assay variability for dextrorphan standards over this range of standards varied from 0.6 to 9.3% and 2.2 to 10%, respectively.

Analytical methods to determine bufuralol 1′-hydroxylation and debrisoquine 4-hydroxylation activities were modified from the method described above for quantitation of dextromethorphan and dextrorphan. A Novapak Phenyl column preceded by a Phenomenex Phenyl guard column was used for both methods. For bufuralol 1′-hydroxylation, the mobile phase consisted of a 65:35 mixture of buffer (20 mM potassium phosphate and 20 mM hexane sulfonic acid, pH 4.0) and acetonitrile at a flow rate of 1.0 ml/min and a column temperature of 40°C with fluorescence detection at excitation and emission wavelengths of 252 and 302 nm, respectively. Under these conditions, the retention times of 1′-hydoxybufuralol and bufuralol were 2.1 and 6.5 min, respectively. The analytical method demonstrated linearity over the range of standards evaluated, 0.25 to 5.0 pmol injected (r² > 0.999), and the lower limit of quantitation for the assay was 0.1 pmol. Intraday and interday assay variability for the range of standards was 0.5 to 4.8% and 0.9 to 4.8%, respectively. For debrisoquine and 4-hydroxydebrisoquine, the acetonitrile content of the mobile phase was reduced to 20% at a flow rate of 1.0 ml/min and a column temperature of 40°C with fluorescence detection of column eluants at excitation and emission wavelengths of 208 and 290 nm, respectively. 4-Hydroxydebrisoquine eluted at 2.9 min under these conditions, whereas the parent compound eluted at 6.8 min. The lower limit of quantitation was 3.75 pmol, and the method demonstrated linearity over a 100-fold range between 3.75 and 375 pmol injected (r² > 0.99). Intraday and interday assay variability for 4-hydroxydebrisoquine varied from 0.2 to 21% and 2.8 to 18%, respectively.

Data Analysis.

Kinetic parameters for metabolite formation were estimated from the line of best-fit using least-squares linear regression analysis of Lineweaver-Burk plots (reciprocal substrate concentration versus reciprocal velocity). Data presented for dextromethorphan and bufuralol biotransformation by CYP2D6 variants expressed in COS-7 cells represent the mean ± S.E.M. of three separate transfection experiments. For baculovirus-expressed proteins, experiments with dextromethorphan and bufuralol were conducted in triplicate whereas experiments with debrisoquine were conducted in duplicate. For each expression system, enzyme activity at every substrate concentration studied was determined in duplicate. Statistical comparisons were made using Dunnett's procedure for comparing multiple treatments to a control.

Results

A representative immunoblot of microsomal fractions prepared from COS-7 cells expressing CYP2D6.1, CYP2D6.2, and CYP2D6.17 proteins is presented in Fig. 1. The lane labeled “control” contains microsomes prepared from cells transfected with vector without insert and confirm that CYP2D6 is not constitutively expressed in COS-7 cells. The absolute amount of expressed CYP2D6 apoprotein (in picomoles) from each transfection experiment is presented in Table 1. Although factors such as cell confluency at the time of transfection, concentration of the plasmid preparation, and cell viability at the time of harvest may contribute to some degree of variability between transfections, the data demonstrated that for a given transfection experiment expression of each CYP2D6 allelic product was comparable, suggesting that differences in the stability of the three expressed CYP2D6 variants were minimal.

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Figure 1

Detection by immunoblot analysis of CYP2D6 protein expressed in microsomes isolated from COS-7 cells.

Representative results of expressed CYP2D6.1, CYP2D6.2, and CYP2D6.17, with known quantities of baculovirus-expressed CYP2D6.1 (Gentest Corp.) ranging from 0.1 to 0.5 pmol/lane serving as standards for quantitation.

In contrast to expression in the baculovirus system, insufficient amounts of microsomal protein were available from the COS-7 cell expression experiments to determine P450 holoenzyme levels based on spectral analysis. Therefore, the rate of product formation by COS-7 cell microsomes was expressed relative to immunoreactive apoprotein. Experimentally determined values for the kinetic parametersK_m andV_max describing dextromethorphan and bufuralol biotransformation by CYP2D6.1, CYP2D6.2, and CYP2D6.17 expressed in COS cells are summarized in Table2. Compared with the wild-type protein, the R296C and S486T amino acid substitutions present in CYP2D6.2 had no apparent effect on K_m and a marginal 30% reduction in V_max with dextromethorphan as a substrate and a modest 30% increase inK_m and no apparent effect onV_max with bufuralol as the substrate. The net result of the R296C and S486T substitutions was an approximately 30% reduction in the intrinsic clearance (CL_int;V_max/K_m) for each substrate that did not achieve statistical significance (p = 0.06).

Relative to CYP2D6.2, further addition of the T107I substitution in CYP2D6.17 resulted in an approximately 2-fold increase inK_m and a 33% decrease inV_max for dextromethorphan, but no effect on K_m, and a 45% decrease inV_max with bufuralol as the substrate. The CL_int of dextromethorphan and bufuralol by CYP2D6.17 was significantly less than that observed with CYP2D6.1 being 25% (p < 0.01) and 37% (p < 0.02) of the CYP2D6.1 activity, respectively. The CL_int of each substrate was also significantly lower (p < 0.05) with CYP2D6.17 compared with CYP2D6.2 (Table 2).

In general, when microsomes prepared from baculovirus-transfectedT. ni cells were used, K_mestimates were 2- to 3-fold lower andV_max estimates were 10- to 20-fold higher than those determined using microsomes from COS-7 cells (Fig.2 and Table 3). Consistent changes in the kinetic parameters characterizing dextromethorphan, bufuralol, and debrisoquine biotransformation by CYP2D6.2 relative to CYP2D6.1 were observed for all three substrates, with a 40 to 50% increase in K_m value combined with an approximately 50% increase inV_max such that CL_int values were the same or slightly (10–15%) greater for CYP2D6.2 (Table 3). The reduction in CL_int associated with CYP2D6.17 also appeared to be similar for all three substrates, ranging from 18% of CYP2D6.1 activity for dextromethorphan (p < 0.01) to 22% of CYP2D6.1 activity for bufuralol and debrisoquine (p < 0.05). However, there seemed to be subtle substrate-dependent differences in the effects onK_m andV_max, with a tendency for changes in dextromethorphan and debrisoquine K_mto be greater than changes in V_max, whereas equivalent (2-fold) increases in each parameter were observed when bufuralol was the substrate.

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Figure 2

Kinetics of dextromethorphan O-demethylation, bufuralol 1′-hydroxylation, and debrisoquine 4-hydroxylation in microsomes prepared from insect cells expressing CYP2D6.1 (squares), CYP2D6.2 (circles), and CYP2D6.17 (triangles).

Representative data from a single experiment are presented.

Discussion

In theory, phenotyping individuals for specific drug biotransformation pathways prior to initiation of drug therapy has considerable potential for improving pharmacotherapy, especially for drugs with narrow therapeutic indices. In principle, the phenotype (usually a urinary metabolite ratio) is a surrogate measure of enzyme activity from which drug clearance by the eliminating organ can be inferred. The implicit intent of this process is to tailor initial dose selection according to the individual clearance estimate of the patient such that target therapeutic drug concentrations can be achieved as quickly as possible while minimizing the risk of concentration-dependent toxicity. CYP2D6 pharmacogenetics figure prominently in this process due to the number of clinically useful compounds (tricyclic antidepressants, serotonin selective reuptake inhibitors, and cardiovascular agents) that are dependent upon this polymorphically expressed enzyme for their elimination from the body. In particular, relationships between CYP2D6 genotype and/or phenotype and drug clearance (and as a result, plasma concentrations achieved) have been established for several agents with relatively narrow therapeutic ranges such as nortriptyline (Nordin et al., 1985; Yue et al., 1999), desipramine (Spina et al., 1997), paroxetine (Sindrup et al., 1992; Yoon et al., 2000), and venlafaxine (Lessard et al., 1999), although CYP2D6 phenotype appears to predict toxicity better than it predicts clinical efficacy (Spina et al., 1997; Lessard et al., 1999).

CYP2D6 phenotyping studies indicate that Chinese (Bertilsson et al., 1992) and individuals of black African origin (Woolhouse et al., 1985;Masimirembwa et al., 1996a; Wennerholm et al., 1999) tend to have lower CYP2D6 activity on a population basis, relative to Caucasians. Black African populations are notable for the presence of theCYP2D6*17 allele, which occurs at a frequency as high as 34% in studied populations (Bradford et al., 1998), and for an apparent lack of coregulatory control between debrisoquine and sparteine phenotypes (Woolhouse et al., 1985; Lennard et al., 1992) and debrisoquine and metoprolol phenotypes (Sommers et al., 1989; Simooya et al., 1993; Masimirembwa et al., 1996a). The CYP2D6*17allele, which has been observed to have reduced activity in vitro (Oscarson et al., 1997), is therefore likely to be at least partially responsible for the lower CYP2D6 activity observed in black African populations (Masimirembwa et al., 1996b; Griese et al., 1999;Wennerholm et al., 1999). However, its contribution to the observed discrepancies in phenotype assignment noted above has not been addressed. Therefore, the specific goal of this investigation was to determine whether the CYP2D6.17 protein had differential effects on the biotransformation of model substrates, dextromethorphan and debrisoquine, commonly used as in vivo phenotyping probes in North America.

In essence, the results of these studies are consistent with theCYP2D6*17 allele contributing toward the observation of lower CYP2D6 activity in populations of black African origin relative to white American or European populations. The intrinsic clearance (estimated asV_max/K_m) of all three substrates (dextromethorphan, debrisoquine and bufuralol) catalyzed by CYP2D6.17 was reduced to approximately 15 to 20% of that observed with the CYP2D6.1 and CYP2D6.2 proteins. Furthermore, since CYP2D6.17 affected the intrinsic clearance of all three substrates to a similar extent, it would appear that the CYP2D6*17 allele alone cannot account for the poor correlation between dextromethorphan and debrisoquine metabolic ratios (r_s = 0.52, p > 0.05) observed by Droll et al. (1998) in Ghanaian volunteers. Since sparteine and metoprolol were not specifically investigated in this study, it is not possible to address the effects of CYP2D6.17 on their metabolism nor the contribution of CYPD26.17 to the poor correlations between these two phenotyping probes and debrisoquine in vivo (Woolhouse et al., 1985; Sommers et al., 1989; Lennard et al., 1992; Simooya et al., 1993; Masimirembwa et al., 1996a; Droll et al., 1998).

A novel variant allele with reduced functional activity,CYP2D6*29, was recently discovered at a frequency of 20% in a black Tanzanian population (Wennerholm et al., 2001) compared with only one allele observed in 672 European subjects (Marez et al., 1997). This allele codes for a protein equivalent to CYP2D6.2 with two additional amino acid changes, V136I and V338M. CYP2D6.29 expressed in COS-1 cells and supplemented with exogenous OR had 26% of the catalytic activity of CYP2D6.1 with bufuralol as a substrate whereas debrisoquine biotransformation was less affected (63% of CYP2D6.1 activity) (Wennerholm et al., 2001). Thus, CYP2D6.29 rather than CYP2D6.17 may account for the poor correlation between phenotyping probes observed in individuals of black African origin.

From a technical perspective, estimates ofK_m for dextromethorphan and bufuralol tended to be 2- to 3-fold lower with baculovirus-expressed enzymes compared with the same proteins expressed in COS-7 cells. In addition,V_max values were 5- to 10-fold higher for dextromethorphan and approximately 10-fold higher for bufuralol using the baculovirus-expressed enzymes. A number of factors may contribute to these observed differences including the higher level of protein expression in T. ni cells compared with COS-7 cells and the relative efficiency of coexpressed reductase coupling with the expressed CYP2D6 variants compared with exogenous supplementation, as was the case with COS-7-expressed enzyme. In addition, the CYP2D6.2 variant has been demonstrated to be more labile than CYP2D6.1 when expressed in insect cells, and the amount of holoprotein is reduced to a greater extent than apoprotein (Zanger et al., 2001). In our studies, incubations with baculovirus-expressed enzyme were conducted using equivalent amounts of each variant form based on spectral determination whereas those with COS cell-expressed enzyme were based on immunochemically determined apoprotein content. Thus, it is possible that CYP2D6.17 may demonstrate lability similar to that observed by others with CYP2D6.2 and that the extent of lability may differ between the two expression systems. Regardless, the rank order of dextromethorphan K_m estimates with CYP2D6.1, CYP2D6.2, and CYP2D6.17 proteins determined in either the COS-7 or baculovirus systems in the present study compares favorably with the values reported for morphine formation from codeine using the same enzymes expressed in yeast (Oscarson et al., 1997). In contrast, bufuralol V_max was essentially identical for all three CYP2D6 variants expressed in yeast (Oscarson et al., 1997) whereas a 50% reduction in value was observed for CYP2D6.17 in both the COS-7 and baculovirus expression systems. Nevertheless, both the results of Oscarson et al. (1997) and those presented here confirm that the intrinsic clearance of all substrates tested is reduced with CYP2D6.17 compared with CYP2D6.1. Therefore, since both the mammalian COS-7 and the baculovirus expression systems were able to detect the reduced catalytic activity associated with CYP2D6.17, we conclude that the COS-7 expression system is useful for rapid, initial characterization of the functional consequences of newly identified allelic variants, but systems yielding higher levels of expression are preferred for more accurate estimation of kinetic parameters.

In conclusion, this in vitro study confirms in vivo reports that theCYP2D6*17 allele confers reduced catalytic activity. However, the issue of discrepancies in phenotype assignment when using different model CYP2D6 probes remains unresolved. Population phenotyping studies imply that metabolism and thus clearance of CYP2D6 substrates may be reduced in African Americans relative to Caucasians, and this appears to be the case for nortriptyline. However, clearance of metoprolol appears to be similar (Johnson and Burlew, 1996) and the clearance of propranolol (Sowinski et al., 1996), by both CYP1A2 and CYP2D6 (Johnson et al., 2000), is increased in African Americans compared with Caucasians. Thus, clearer insights into the consequences of existing and yet undiscovered population-specific alleles on a broad range of individual substrates are necessary to optimize pharmacotherapy of CYP2D6 substrates in individuals of black African origin.