PHARMACOGENETICS and GENOMICS Tramadol As a New Probe for Cytochrome P450 2D6 Phenotyping: a Population Study

PHARMACOGENETICS AND GENOMICS Tramadol as a new probe for cytochrome P450 2D6 phenotyping: A population study

Background and Objective: Polymorphic cytochrome P450 (CYP) 2D6 activity has been shown to be a determinant of the pharmacokinetics and pharmacodynamics of tramadol via hepatic phase I O-demethylation of (؉)-tramadol to (؉)-O-desmethyltramadol. Our objective was to investigate whether tramadol can be used as a probe for CYP2D6 phenotyping by determining the concordance between the 8-hour tramadol and 12-hour sparteine metabolic urinary ratios. Methods: Sparteine phenotyping test was carried out in 278 healthy, white subjects. At a minimum of 2 weeks later, each subject took 50 mg tramadol hydrochloride followed by 8-hour urine collection, and a venous blood sample was drawn from 276 subjects. Urine and plasma concentrations of (؉/؊)-tramadol and ,؉/؊)-O-desmethyltramadol were determined. CYP2D6 genotyping was performed with regard to *3, *4) *6, and *9 alleles. Results: There were 28 poor metabolizers of sparteine (10.1% [confidence interval, 6.8%-14.2%]). Very low recoveries of (؉)-M1 were found in poor metabolizers (0.53% [range, 0.1%-1.1%]) compared with extensive metabolizers (8.7% [range, 1.7%-23.2%]). A bimodal distribution of the metabolic ratio of (؊)-M1/(؉)-M1 was found. The visual antimode was 2.0. This new phenotype test had only 1 misclassified subject compared with sparteine phenotyping (sensitivity and negative predictive value, 100%; specificity, 99.6%; positive predictive value, 96.6%). Of the 28 sparteine poor metabolizers, 26 were found to be genotypically poor metabolizers with regard to the inactivating mutations *3, *4, and *6. Conclusion: Fifty milligrams of tramadol is an alternative CYP2D6 phenotype probe by use of the 8-hour urinary ratio of (؊)-M1/(؉)-M1. The poor metabolizers have a metabolic ratio of 2.0 or higher. (Clin Pharmacol Ther 2005;77:458-67.)

Rasmus Steen Pedersen, MSc, Per Damkier, MD, PhD, and Kim Brosen, MD, DMSc Odense, Denmark

Cytochrome P450 (CYP) 2D6 phenotyping is a valu- polymorphism in 1977,1 a number of substrates have able tool in drug metabolism research. Since the been proposed and used for CYP2D6 phenotyping discovery of the debrisoquin (INN, debrisoquine) —bufuralol,2 dextromethorphan,3 metoprolol,4 and sparteine.5 Sparteine has become a classical and gold- standard CYP2D6 probe.6,7 Sparteine is metabolized From the Institute of Public Health, Faculty of Health Sciences, by CYP2D6 to the 2 oxidative metabolites 2,3- Clinical Pharmacology, University of Southern Denmark. didehydrosparteine and 5,6-didehydrosparteine.8 The Received for publication Aug 27, 2004; accepted Jan 14, 2005. Reprint requests: Rasmus Steen Pedersen, MSc, Institute of Public population is divided into 2 general phenotypes— Health, Faculty of Health Sciences, Clinical Pharmacology, Uni- extensive metabolizers (EMs) and poor metabolizers versity of Southern Denmark, Winslowparken 19, DK-5000 (PMs)—according to the activity of CYP2D6 defined Odense C, Denmark. by a metabolic ratio of the parent substrate to the E-mail: [email protected] metabolites. CYP2D6 is encoded by the functional al- 0009-9236/$30.00 Copyright © 2005 by the American Society for Clinical Pharmacology leles in EMs, and the absence of the active enzyme in 9 and Therapeutics. PMs is caused by mutations and deletions. About 7% doi:10.1016/j.clpt.2005.01.014 of the Danish white population are PMs.10

458 CLINICAL PHARMACOLOGY & THERAPEUTICS 2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 459

Fig 1. Major metabolic pathways for tramadol in vivo.

Tramadol is a racemic mixture of the 2 enantiomers (Ͻ24 hours) administration (6011 patients) is quite (ϩ)-tramadol hydrochloride and (Ϫ)-tramadol hydro- similar, quantitatively and qualitatively, to that of long- chloride: (1RS,2RS)-2-[(dimethylamino)methyl]-1-(3- term administration.16 Rare cases of seizures and ana- methoxyphenyl)-cyclohexanol hydrochloride (Fig 1). phylactic reactions have been reported, with an in- Tramadol is a synthetic 4-phenyl-piperidine analog of creased risk at tramadol doses above the recommended codeine and thus an aminocyclohexanol derivative.11 It dose. A British study evaluated the risk of seizures in is a centrally acting synthetic analgesic with a wide- 10,916 patients receiving tramadol. Eleven definite spread clinical use. It has an analgesic efficacy similar cases of idiopathic seizures were identified. With ex- to weak opioids and is devoid of some of the side posure to tramadol alone, the risk of idiopathic incident effects seen with other opioids of comparable effi- seizures was found not to be increased.17 Tramadol has cacy.12,13 Tramadol produces analgesia in short-term been prescribed in Europe for close to 3 decades. More and long-term pain states by synergistically combining than 5 million patients in the United States and more weak opioid and monoaminergically mediated mecha- than 40 million patients worldwide have received tra- nisms.14,15 Safety data for tramadol have been summa- madol.18 Therefore phenotyping with 50 mg tramadol rized by Cossmann et al.16 It is a well-tolerated anal- is considered safe and can be conducted worldwide, gesic. Information from phase II to IV clinical studies because tramadol is registered in more than 100 coun- and postmarketing surveillance studies, covering safety tries. It has been licensed for children aged over 1 year data from more than 21,000 patients, were considered. in several European countries since 1977, and several The most frequent adverse events were nausea (6.1%), studies have shown that it is safe in children. Murthy et dizziness (4.6%), drowsiness (2.4%), tiredness/fatigue al19 showed that none of the pharmacokinetic parame- (2.3%), sweating (1.9%), vomiting (1.7%), and dry ters were significantly different from adults. Payne et mouth (1.6%). The profile for single-dose or short-term al20 concluded that tramadol at 3 mg/kg in children has CLINICAL PHARMACOLOGY & THERAPEUTICS 460 Pedersen, Damkier, and Brosen JUNE 2005

no clinical respiratory depressant effect and behavior ducted as an open, 2-phase, crossover trial. The mini- and recovery are unaffected. Kaabachi et al21 also con- mum washout time between the 2 phases was 2 weeks. cluded that tramadol is safe and efficient in children. Subjects. The study included 278 healthy, white vol- According to Food and Drug Administration (FDA) unteers. The 155 men and 123 women, aged 19 to 40 guidelines, tramadol should not be used in pregnant, years, were primarily Danish students at the University delivering, and nursing women because no adequate of Southern Denmark (Odense, Denmark). None of the and well-controlled studies have been conducted in volunteers had a daily intake of alcohol or drugs (ex- these populations. The FDA also states that healthy cept oral contraceptives), and none of the volunteers elderly subjects aged 65 to 75 years have plasma con- were pregnant or breastfeeding. centrations and elimination half-lives comparable to Phenotyping. In phase A of the trial, the CYP2D6 those observed in healthy subjects aged less than 65 phenotype was determined. Oral intake of 100 mg years. sparteine sulfate pentahydrate (supplied by the Central The in vivo metabolism of tramadol is complex, with Pharmacy, Odense University Hospital, Odense, Den- 23 metabolites having been identified: 11 phase I metab- mark) was followed by urine collection for 12 hours. olites and 12 phase II conjugates.22 The major metabolic The urine volume was recorded, and two 10-mL ali- pathways are O-demethylation to O-desmethyltramadol quots were stored at Ϫ20°C until analysis. The amounts (M1) and N-demethylation to N-desmethyltramadol (M2) of excreted sparteine, 2,3-didehydrosparteine, and 5,6- (Fig 1).23 The O-demethylation of tramadol is mediated didehydrosparteine were determined by gas chromatog- by CYP2D6 both in vitro and in vivo.24-27 Paar et al26 raphy.6 The design of phase B was identical to a pilot suggested that tramadol metabolism via CYP2D6 is study that separated EMs and PMs completely.28 The stereospecific. The fact that the O-demethylation of volunteers were given 50 mg tramadol hydrochloride as (ϩ)-tramadol to (ϩ)-M1 is catalyzed by CYP2D6 has a single oral dose (Nobligan; Grünenthal, Germany; been verified.28,29 Tramadol is also directly excreted in distribution in Denmark by Meda, Allerod, Denmark). the urine.28 CYP2D6 activity has also been shown to be Urine was collected for 8 hours. The urine volume was a determinant of the pharmacodynamics of tramadol. recorded, and two 10-mL aliquots were stored at Laugesen et al29 concluded that paroxetine, a very Ϫ20°C until analysis. The concentrations of (ϩ)- potent CYP2D6 inhibitor, inhibits the metabolism of tramadol, (Ϫ)-tramadol, (ϩ)-M1, and (Ϫ)-M1 in urine tramadol to M1 and reduces the hypoalgesic effect of and plasma were analyzed in duplicate by an HPLC tramadol in human experimental pain models, particu- method.28 The HPLC method for urine samples was larly in opioid-sensitive tests. validated and published with a range of linearity from Because sparteine is no longer marketed as a drug, it 0.1 to 3.0 ␮mol/L and a maximum coefficient of vari- is becoming increasingly difficult to obtain commercial ation of 5.7% for precision and reproducibility (n ϭ formulations and reference substances. Therefore there 10). Samples with higher concentrations were diluted is an interest for new validated probe drugs for in vivo with millipore water before analysis. The two 10-mL phenotyping of CYP2D6. The metabolism of total tra- aliquots of venous blood were obtained with ethyl- madol is well described, but the direct relationship enediaminetetraacetic acid as anticoagulant from each between the CYP2D6 phenotype and the stereospecific volunteer approximately 2 hours after medication dur- tramadol O-demethylation is to some extent still un- ing phase B. The blood sample was centrifuged for 10 known. No other population study has determined the minutes at 2000g, and the plasma was isolated and relationship between sparteine oxidation and the stored at Ϫ20°C until analysis. The HPLC method for O-demethylation of the individual isomers of tramadol. plasma samples was validated and published with a The main purpose of this study was to investigate range of linearity from 0.01 to 1.55 ␮mol/L and a whether tramadol can be used as a CYP2D6 probe. maximum coefficient of variation of 6.0% for precision and reproducibility (n ϭ 10). The remainder of the blood sample was stored at Ϫ20°C until deoxyribonu- METHODS cleic acid (DNA) isolation. The protocol was approved by the Danish Medicines Genotyping. The DNA from peripheral leukocytes Agency and the Regional Ethical Committee of Vejle was isolated by use of the PUREGENE genomic DNA and Funen Counties. The study was carried out in purification kit from Gentra Systems (Minneapolis, accordance with Good Clinical Practice guidelines. Minn), according to the manufacturer’s guidelines. The Written informed consent to participate in the study first step in genotyping was a polymerase chain reaction was obtained from the volunteers. The study was con- with primers specific for the active CYP2D6 gene en- CLINICAL PHARMACOLOGY & THERAPEUTICS 2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 461

Table I. Location and sequence of oligonucleotide CYP2D6 primers and probes Primers/probes Sequence 5Ј¡3Ј

Primers CYP1 (long PCR) att tcc cag gaa tcc CYP4 (long PCR) ccg gcc ctg aca ctc ctt ct CYP2D6*3 sense tcc tga ccc agc tgg atg a CYP2D6*3 antisense ctt ctc cat ctc tgc cag gaa CYP2D6*4 sense cga ccc ctt acc cgc atc CYP2D6*4 antisense ctc acg gct ttg tcc aag aga CYP2D6*6 sense ctt ggg cct ggg caa ga CYP2D6*6 antisense agg gca gca caa agg ca CYP2D6*9 sense gag acc tga ctg agg cct tcc t CYP2D6*9 antisense tca ttc ctc ctg gga cgc t Probes CYP2D6*3 wtMGB FAM–tca tcc tgt gct cag tt–NFQ CYP2D6*3 VIC–tca tcc gtg ctc agt t–NFQ mutMGB CYP2D6*4 wt FAM–acc ccc agg acg ccc ctt t–tamra CYP2D6*4 mut JOE–acc ccc aag acg ccc ctt tc–tamra CYP2D6*6 wtMGB FAM–cgg tca ccc act gc–NFQ CYP2D6*6 VIC–cgg tca ccc ctg c–NFQ mutMGB CYP2D6*9 wtMGB FAM–tct cac ctt ctc cat ct–NFQ CYP2D6*9 VIC–tct cac ctc cat ctc–NFQ mutMGB PCR, Polymerase chain reaction.

compassing the region with the 4 selected single- 0- to 12-hour urinary excretion nucleotide polymorphisms. TaqMan technology (Ap- MR ϭ of sparteine plera, Norwalk, Conn) was used to genotype the 3 sparteine 0- to 12-hour urinary excretion of known inactivation mutations CYP2D6*3, CYP2D6*4, 2, 3-didehydrosparteine and and CYP2D6*6 and the low-activity allele CYP2D6*9. 5, 6-didehydrosparteine The CYP2D6*3 allele is derived from a deletion of EMs were defined as having an MRsparteine value of A2549 in the fifth exon, which causes a frame shift, and no in vivo enzyme activity is observed.30,31 CYP2D6*4 less than 20, and PMs were defined as having an 10 is the most common inactivating allele. It has a single MRsparteine value of 20 or greater. Ͼ Two empiric metabolic ratios describing tramadol base change, G1934 A, in intron 3, causing a splicing defect.32,33 In the CYP2D6*6 allele, a deletion of O-demethylation were calculated as follows:

T1795 in exon 3 results in a frame shift and generates a premature stop codon.34-36 The decreased enzyme 0- to 8-hour urinary excretion of (ϩ)-tramadol MR ϭ activity of CYP2 D6*9 is a result of a 3-base 1 0- to 8-hour urinary excretion of (ϩ)-Ml 37,38 (A2613G2614A2615) deletion in exon 5. Table I shows the primers and probes, which were designed by 0- to 8-hour urinary excretion of (–)-Ml MR ϭ use of Primer Express software (Applied Biosystems, 2 0- to 8-hour urinary excretion of (ϩ)-Ml Foster City, Calif). The real-time analysis was per- formed on the ABI PRISM 7700 Sequence Detection The mean and range of metabolic ratios were calcu- System equipped with the allelic discrimination module lated for each CYP2D6 genotype. (software version 1.7; Applied Biosystems). The concordance between the gold standard of Data analysis. Urinary recoveries (percent of dose) sparteine toward the genotype test and the tramadol of all drugs and metabolites were calculated. metabolic ratio were determined by calculating the

The metabolic ratio of sparteine oxidation (MRsparteine) sensitivity, specificity, positive predictive value, and was calculated as follows: negative predictive value as follows: CLINICAL PHARMACOLOGY & THERAPEUTICS 462 Pedersen, Damkier, and Brosen JUNE 2005

No. of true positives Positive predictive value ϭ No. of true positives ϩ No. of false positives No. of true negatives Negative predictive value ϭ No. of true negatives ϩ No. of false negatives

RESULTS Four subjects had moderate nausea 3 to 5 hours after tramadol medication. This adverse event lasted for a few hours and had no practical consequence. Unfortu- nately, genotyping of only 276 of 278 volunteers was performed because of difficulties in drawing blood samples from 2 subjects. Fig 2 shows the classic clear-cut bimodal distribution

of MRsparteine in the population. The empiric antimode of 20 separated the 2 phenotypes completely, and no other subgroups appeared. Of the 278 individuals, 28 (17 women and 11 men) were phenotyped as PMs (10.1% [confidence interval, 6.8%-14.2%]). The mean

MRsparteine in the 28 PMs of sparteine (PMsparteine) was 68.8 (range, 20.1-250.2), and the mean MRsparteine in the 250 EMs of sparteine (EMsparteine) was 0.6 (range, 0.1-7.0).

Fig 3 shows the distribution of MR1 in the popula- Fig 2. Frequency distribution of MR (N ϭ 278). Solid sparteine tion. Apparently, there was no bimodality, although the bars, Sparteine extensive metabolizers. Open bars, Sparteine PMsparteine in general had high values of MR1. The 2 poor metabolizers. phenotypes overlapped, and in best case, 4 false- ϭ positive subjects were misclassified. The mean MR1 in Table II. Relationship between MR2 (N 278) and genotype (N ϭ 276) with that of sparteine phenotype the 28 PMsparteine was 58.9 (range, 12.7-344.5), and the mean MR1 in the 250 EMsparteine was 2.5 (range, 0.4- PM EM Total sparteine sparteine 23.4). Ն Fig 4 shows the distribution of MR in the popula- PM if MR2 2.0 2 PMMR2 28 1 29 tion. The distribution of MR2 was bimodal, and an EMMR2 0 249 249 empiric antimode of 2.0 could be placed. This antimode Total 28 250 278 separated the 2 phenotypes completely, and only 1 PM if genotype is subject was misclassified. This misclassified subject composed had an MRsparteine of 5.0 and an MR2 of 3.0. This of *3, *4, subject was heterozygous for *4 by this genotype test, and *6 alleles which apparently indicates an EM. By use of the anti- PMgenotype 26 0 26 mode MR2 of 2.0 or greater as a new PM phenotype EMgenotype 2 248 250 Total 28 248 276 test, the sensitivity and negative predictive value were 100%. Because of the false-positive, misclassified sub- PM, Poor metabolizer; EM, extensive metabolizer; MR2, 0- to 8-hour urinary tramadol metabolic ratio: (Ϫ)-M1/(ϩ)-M1. ject, the specificity was 99.6% and the positive predic- tive value was 96.6%. The mean MR2 in the 28 PMsparteine was 4.7 (range, 2.3-9.5), and the mean MR2 No. of true positives in the 250 EMsparteine was 0.7 (range, 0.4-3.0). Sensitivity ϭ No. of true positives ϩ No. of false negatives Of the 28 PMsparteine, 26 were genotypically PMs with regard to the inactivating mutations *3, *4, and *6. No. of true negatives About half of the study population carries functional Specificity ϭ ϩ No. of false positives No. of true negatives alleles. The mean MRsparteine for the apparent wild type CLINICAL PHARMACOLOGY & THERAPEUTICS 2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 463

ϭ Fig 3. Frequency distribution of MR1 (N 278). Solid bars, ϭ Fig 4. Frequency distribution of MR2 (N 278). Solid bars, Sparteine extensive metabolizers. Open bars, Sparteine poor Sparteine extensive metabolizers. Open bars, Sparteine poor metabolizers. metabolizers.

(CYP2D6*1/*1) was lower (0.4) than for other EMs difference between PMsparteine and EMsparteine in mean (0.6-2.0). CYP2D6*4 was by far the most common urinary recoveries for (ϩ)-M1 and (Ϫ)-M1 were larger. mutated allele compared with *3, *6, and *9. One The mean urinary recoveries in the PMsparteine were ϩ PMsparteine was genotyped as CYP2D6*1/*4. This indi- 0.53% (range, 0.1%-1.1%) for ( )-M1 and 2.2% Ϫ vidual had MRsparteine of 84.7, MR1 of 344.5, and MR2 (range, 0.7%-4.5%) for ( )-M1, as compared with the of 9.5. Another PMsparteine was genotyped as following values for EMsparteine: 8.7% (range, 1.7%- ϩ CYP2D6*6/*9 with MRsparteine of 23.1, MR1 of 17.1, 23.2%) for ( )-M1 and 5.6% (range, 1.3%-14.6%) for Ϫ and MR2 of 2.3. As a result of these 2 false-negative ( )-M1. There was a marked interindividual variability genotypes, the genotype test had a sensitivity of 92.9%, for each compound, and there were overlaps for (ϩ)- a specificity of 100%, a positive predictive value of tramadol, (Ϫ)-tramadol, and (Ϫ)-M1 between the phe- 100%, and a negative predictive value of 99.2%. Table notypes. Only the amount of (ϩ)-M1 was significantly II summarizes the relationship between the new phe- different between the phenotypes. The coefficient of notype test and the genotype test with that of the gold variation was high for all groups (34%-60%). standard sparteine. Table III shows the mean and range Unfortunately, the data obtained from the plasma of the metabolic ratios for the genotypes. samples were not sufficiently informative. The plasma The mean urinary recoveries (Table IV)inthe samples from 19 of 28 PMs had very low concentra- ϩ ϩ PMsparteine were higher for ( )-tramadol, 20.2% (range, tions of ( )-O-desmethyltramadol, resulting in unac- 7.4%-42.5%), and (Ϫ)-tramadol, 17.2% (range, 5.0%- ceptable deviations or concentrations of (ϩ)-O- 41.1%), compared with the following values for desmethyltramadol below the limit of quantification (5 ϩ 28 EMsparteine:( )-tramadol, 15.4% (range, 4.0%-39.8%), nmol/L). Therefore it was not possible to calculate Ϫ and ( )-tramadol, 13.2% (range, 2.8%-39.9%). The MR1 and MR2. CLINICAL PHARMACOLOGY & THERAPEUTICS 464 Pedersen, Damkier, and Brosen JUNE 2005

Table III. Mean metabolic ratios for genotypes (N ϭ 276) Sparteine Tramadol

Genotype MRsparteine (range) MR1 (range) MR2 (range) CYP2D6*1/*1 (n ϭ 139) 0.4 (0.1-3.4) 1.7 (0.4-8.2) 0.6 (0.4-1.4) CYP2D6*1/*4 (n ϭ 81) 2.0 (0.2-84.7) 7.5 (0.8-344.5) 0.9 (0.5-9.5) CYP2D6*1/*9 (n ϭ 12) 0.7 (0.2-3.9) 3.2 (1.2-10.9) 0.8 (0.5-1.9) CYP2D6*1/*3 (n ϭ 6) 0.6 (0.4-0.7) 1.8 (1.1-2.2) 0.7 (0.6-0.8) CYP2D6*1/*6 (n ϭ 6) 1.2 (0.5-2.8) 4.6 (1.4-10.8) 0.9 (0.5-1.9) CYP2D6*9/*9 (n ϭ 4) 1.2 (0.8-2.0) 5.0 (3.1-6.8) 0.7 (0.5-1.0) CYP2D6*6/*9 (n ϭ 1) 23.1 17.1 2.3 CYP2D6*4/*4 (n ϭ 19) 64.9 (21.6-141.1) 50.1 (12.7-139.3) 4.7 (3.1-7.4) CYP2D6*4/*6 (n ϭ 4) 48.7 (20.1-66.1) 46.4 (29.4-67.0) 4.7 (3.6-6.5) CYP2D6*3/*3 (n ϭ 2) 156.3 (62.4-250.2) 66.2 (52.8-79.5) 4.4 (4.3-4.5) CYP2D6*3/*4 (n ϭ 2) 71.4 (53.0-89.8) 33.6 (29.0-38.1) 3.7 (2.9-4.5)

MRsparteine, Zero- to twelve-hour urinary sparteine metabolic ratio of sparteine over 2,3- and 5,6-didehydrosparteine; MR1, 0- to 8-hour urinary tramadol metabolic ϩ ϩ Ϫ ϩ ratio of ( )-tramadol over ( )-M1; MR2, 0- to 8-hour urinary tramadol metabolic ratio of ( )-M1 over ( )-M1.

Table IV. Urinary recoveries (percent of dose) of tramadol and M1 in relation to sparteine oxidation polymorphism ϭ ϭ EMsparteine (n 250) PMsparteine (n 28) [mean (range) (CV)] [mean (range) (CV)]

(ϩ)-Tramadol 15.4 (4.0-39.8) (36) 20.2 (7.4-42.5) (34) (Ϫ)-Tramadol 13.2 (2.8-39.9) (38) 17.2 (5.0-41.1) (38) (ϩ)-M1 8.7 (1.7-23.2) (42) 0.53 (0.1-1.1) (60) (Ϫ)-M1 5.6 (1.3-14.6) (41) 2.2 (0.7-4.5) (42) CV, Coefficient of variation.

DISCUSSION for both isomers of tramadol and lower for both isomers

Phenotyping with tramadol should not be carried out of M1 (Table IV). In EMsparteine, the amounts of in pregnant, delivering, and nursing women. Apart (ϩ)-M1 were higher than those of (Ϫ)-M1. This sup- from that, phenotyping with 50 mg tramadol hydro- ports the finding that the maximum metabolizing ve- Ϫ chloride is considered to be safe in healthy volunteers locity (Vmax) for the O-demethylation of ( )-tramadol ϩ aged from 1 to 75 years. Only 4 incidents of minor is higher than Vmax for the O-demethylation of ( )- adverse effects were reported in the total population of tramadol.24 Very low recovery of (ϩ)-M1 in the

278 subjects, because the dose of 50 mg is the smallest PMsparteine confirms that CYP2D6 is stereoselective and tablet available and a subtherapeutic dose. The pheno- CYP2D6 is responsible for the O-demethylation of type test is easily conducted by collecting urine for only (ϩ)-tramadol in particular. The clinical significance of 8 hours. the very low amounts of (ϩ)-M1 is described in the ϩ As expected, MRsparteine was bimodally distributed, literature. Studies have shown that ( )-M1 is respon- separating the phenotypes clearly. In this study 10.1% sible for the hypoalgesic effect of tramadol.29,40 How- of subjects were PMs, which was similar to most other ever, in contrast to sparteine, the distribution of the white populations.39 Because the confidence interval is metabolic ratio of the parent substrate (ϩ)-tramadol ϩ 6.8% to 14.2%, there is no statistical reason to assume over the metabolite ( )-M1 (MR1) was not bimodal. that the population is different from earlier findings in Even the best antimode resulted in the finding that 4 10 the Danish population (7.3%). As shown in Figs 3 EMsparteine were misclassified as tramadol PMs accord- Ϫ and 4, the PMsparteine had high MR1 and MR2, confirm- ing to MR1. Surprisingly, the metabolic ratio of ( )- ϩ ing that CYP2D6 is highly involved in the M1/( )-M1 (MR2) showed better distribution, separat- O-demethylation of (ϩ)-tramadol. As a result, the me- ing the 2 phenotypes at the metabolic ratio of 2.0. The dian urinary recoveries in the PMsparteine were higher specificity and positive predictive value were also CLINICAL PHARMACOLOGY & THERAPEUTICS 2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 465

higher by use of MR2. Only 1 EMsparteine was misclas- detect all mutations resulting in PMs. Therefore ϭ sified according to MR2. some of the homozygote genotypes (*1/*1 [n 139], As a result of the low dose of tramadol hydrochloride *4/*4 [n ϭ 19], *3/*3 [n ϭ 2], and *9/*9 [n ϭ 4]) (50 mg) given, plasma concentrations of (ϩ)-M1 were may carry another mutation on 1 allele. One common too low or undetectable in most PMs. Unfortunately, it mutation in white populations is *5, which is a was therefore not possible on the basis of the study to deletion of an entire functional CYP2D6 gene.41 evaluate whether plasma concentrations of (ϩ/Ϫ)- However, it should be kept in mind that this issue has tramadol and (ϩ/Ϫ)-M1 have the potential to be a no influence on the genotype classification of EMs measure of CYP2D6 activity. To evaluate the plasma and PMs. A better genotype test for white popula- concentrations, a higher dose or a more sensitive ana- tions would detect *5 and *10, because both muta- lytic method was needed. Neither option was chosen. tions are present with considerable frequencies.42 The dose was kept as low as possible to avoid possible Still, without the detection of *5 and *10, only 2 adverse effects. Increasing the sensitivity of the method subjects were misclassified and a sensitivity of was unfortunately not possible without new analytic 92.9%, specificity of 100%, positive predictive value instrumentation, followed by a new method, validation, of 100%, and negative predictive value of 99.2% and publication. Because the plasma concentrations of were achieved. (ϩ/Ϫ)-tramadol and (ϩ/Ϫ)-M1 of samples were not In conclusion, 50 mg tramadol hydrochloride fol- the primary endpoint in the study, the lack of plasma lowed by 8-hour urine collection provides an alter- data was not decisive. native CYP2D6 probe to sparteine. The metabolic Sparteine is an excellent probe with a clear-cut bi- ratio of (Ϫ)-M1/(ϩ)-M1 with an antimode of 2.0 modal distribution. MR2 does not have the same obvi- separated the 2 phenotypes, with the exception of 1 ous antimode. There could be several reasons for this. misclassified subject. The ratio is only a picture of the first 8 hours after We gratefully thank The Alfred Benzon Foundation, The Lund- medication. The decision to use 8-hour urine collection beck Foundation, and The Danish Medical Research Council for was based on the results from a pilot study that clearly financial support. 28 None of the authors have any conflicts of interest. separated PMs and EMs by both MR1 and MR2. What will happen after 8 hours is not clearly described, but this is currently under investigation in our laboratory. References Second, M1 is not a terminal metabolite, and the 1. Mahgoub A, Idle JR, Dring LG, Lancaster R, Smith RL. amount of M1 is dependent on further subsequent met- Polymorphic hydroxylation of debrisoquine in man. Lan- abolic steps. At least 4 M1 metabolites have been cet 1977;2:584-6. described (M5, M13, M20, and M32).22 A third 2. Dayer P, Gasser R, Gut J, Kronbach T, Robertz GM, Eich- reason lies in the alternative N-demethylation. The elbaum M, et al. Characterization of a common genetic N-demethylation is also stereoselective, but in contrast defect of cytochrome P-450 function (debrisoquinine- to O-demethylation, (ϩ)-tramadol is N-demethylated sparteine type polymorphism). Biomed Biophys Res Com- mun 1984;125:374-80. considerably faster than the (Ϫ)-isomer.24 CYP2D6 is 25 3. 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