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HARMACOGENETICS ANDENOMICS

ramadol as a new probe for cytochrome450 2D6 phenotyping: A population study

Background and Objective: Polymorphic cytochrome P450 (CYP) 2D6 activity has been shown to be adeterminant of the pharmacokinetics and pharmacodynamics of tramadol via hepatic phase IO-demethylation of (�)-tramadol to (�)-O-desmethyltramadol. Our objective was to investigate whethertramadol can be used as a probe for CYP2D6 phenotyping by determining the concordance between the8-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 weekslater, each subject took 50 mg tramadol hydrochloride followed by 8-hour urine collection, and a venousblood 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 lowrecoveries of (�)-M1 were found in poor metabolizers (0.53% [range, 0.1%-1.1%]) compared with extensivemetabolizers (8.7% [range, 1.7%-23.2%]). A bimodal distribution of the metabolic ratio of (�)-M1/(�)-M1was found. The visual antimode was 2.0. This new phenotype test had only 1 misclassified subject comparedwith sparteine phenotyping (sensitivity and negative predictive value, 100%; specificity, 99.6%; positivepredictive value, 96.6%). Of the 28 sparteine poor metabolizers, 26 were found to be genotypically poormetabolizers 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-hoururinary ratio of (�)-M1/(�)-M1. The poor metabolizers have a metabolic ratio of 2.0 or higher. (ClinPharmacol Ther 2005;77:458-67.)

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

Odense, Denmark

pb—ssbdpe(bmlP

Cytochrome P450 (CYP) 2D6 phenotyping is a valu-ble tool in drug metabolism research. Since theiscovery of the debrisoquin (INN, debrisoquine)

rom the Institute of Public Health, Faculty of Health Sciences,Clinical Pharmacology, University of Southern Denmark.

eceived for publication Aug 27, 2004; accepted Jan 14, 2005.eprint requests: Rasmus Steen Pedersen, MSc, Institute of PublicHealth, Faculty of Health Sciences, Clinical Pharmacology, Uni-versity of Southern Denmark, Winslowparken 19, DK-5000Odense C, Denmark.

-mail: rpedersen@health.sdu.dk009-9236/$30.00opyright © 2005 by the American Society for Clinical Pharmacologyand Therapeutics.

ooi:10.1016/j.clpt.2005.01.014

58

olymorphism in 1977,1 a number of substrates haveeen proposed and used for CYP2D6 phenotyping

bufuralol,2 dextromethorphan,3 metoprolol,4 andparteine.5 Sparteine has become a classical and gold-tandard CYP2D6 probe.6,7 Sparteine is metabolizedy CYP2D6 to the 2 oxidative metabolites 2,3-idehydrosparteine and 5,6-didehydrosparteine.8 Theopulation is divided into 2 general phenotypes—xtensive metabolizers (EMs) and poor metabolizersPMs)—according to the activity of CYP2D6 definedy a metabolic ratio of the parent substrate to theetabolites. CYP2D6 is encoded by the functional al-

eles in EMs, and the absence of the active enzyme inMs is caused by mutations and deletions.9 About 7%

f the Danish white population are PMs.10

(cmTcistecawnrgadTd(m

(stpcd1cpsbttmibtisat

pathway

CLINICAL PHARMACOLOGY & THERAPEUTICS2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 459

Tramadol is a racemic mixture of the 2 enantiomers�)-tramadol hydrochloride and (�)-tramadol hydro-hloride: (1RS, 2RS)-2-[(dimethylamino)methyl]-1-(3-ethoxyphenyl)-cyclohexanol hydrochloride (Fig 1).ramadol is a synthetic 4-phenyl-piperidine analog ofodeine and thus an aminocyclohexanol derivative.11 Its a centrally acting synthetic analgesic with a wide-pread clinical use. It has an analgesic efficacy similaro weak opioids and is devoid of some of the sideffects seen with other opioids of comparable effi-acy.12,13 Tramadol produces analgesia in short-termnd long-term pain states by synergistically combiningeak opioid and monoaminergically mediated mecha-isms.14,15 Safety data for tramadol have been summa-ized by Cossmann et al.16 It is a well-tolerated anal-esic. Information from phase II to IV clinical studiesnd postmarketing surveillance studies, covering safetyata from more than 21,000 patients, were considered.he most frequent adverse events were nausea (6.1%),izziness (4.6%), drowsiness (2.4%), tiredness/fatigue2.3%), sweating (1.9%), vomiting (1.7%), and dry

Fig 1. Major metabolic

outh (1.6%). The profile for single-dose or short-term a

�24 hours) administration (6011 patients) is quiteimilar, quantitatively and qualitatively, to that of long-erm administration.16 Rare cases of seizures and ana-hylactic reactions have been reported, with an in-reased risk at tramadol doses above the recommendedose. A British study evaluated the risk of seizures in0,916 patients receiving tramadol. Eleven definiteases of idiopathic seizures were identified. With ex-osure to tramadol alone, the risk of idiopathic incidenteizures was found not to be increased.17 Tramadol haseen prescribed in Europe for close to 3 decades. Morehan 5 million patients in the United States and morehan 40 million patients worldwide have received tra-adol.18 Therefore phenotyping with 50 mg tramadol

s considered safe and can be conducted worldwide,ecause tramadol is registered in more than 100 coun-ries. It has been licensed for children aged over 1 yearn several European countries since 1977, and severaltudies have shown that it is safe in children. Murthy etl19 showed that none of the pharmacokinetic parame-ers were significantly different from adults. Payne et

s for tramadol in vivo.

l20 concluded that tramadol at 3 mg/kg in children has

nacAgdatecty

2op((bss(btaLpttl

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CLINICAL PHARMACOLOGY & THERAPEUTICS460 Pedersen, Damkier, and Brosen JUNE 2005

o clinical respiratory depressant effect and behaviornd recovery are unaffected. Kaabachi et al21 also con-luded that tramadol is safe and efficient in children.ccording to Food and Drug Administration (FDA)uidelines, tramadol should not be used in pregnant,elivering, and nursing women because no adequatend well-controlled studies have been conducted inhese populations. The FDA also states that healthylderly subjects aged 65 to 75 years have plasma con-entrations and elimination half-lives comparable tohose observed in healthy subjects aged less than 65ears.The in vivo metabolism of tramadol is complex, with

3 metabolites having been identified: 11 phase I metab-lites and 12 phase II conjugates.22 The major metabolicathways are O-demethylation to O-desmethyltramadolM1) and N-demethylation to N-desmethyltramadol (M2)Fig 1).23 The O-demethylation of tramadol is mediatedy CYP2D6 both in vitro and in vivo.24-27 Paar et al26

uggested that tramadol metabolism via CYP2D6 istereospecific. The fact that the O-demethylation of�)-tramadol to (�)-M1 is catalyzed by CYP2D6 haseen verified.28,29 Tramadol is also directly excreted inhe urine.28 CYP2D6 activity has also been shown to be

determinant of the pharmacodynamics of tramadol.augesen et al29 concluded that paroxetine, a veryotent CYP2D6 inhibitor, inhibits the metabolism oframadol to M1 and reduces the hypoalgesic effect oframadol in human experimental pain models, particu-arly in opioid-sensitive tests.

Because sparteine is no longer marketed as a drug, its becoming increasingly difficult to obtain commercialormulations and reference substances. Therefore theres an interest for new validated probe drugs for in vivohenotyping of CYP2D6. The metabolism of total tra-adol is well described, but the direct relationship

etween the CYP2D6 phenotype and the stereospecificramadol O-demethylation is to some extent still un-nown. No other population study has determined theelationship between sparteine oxidation and the-demethylation of the individual isomers of tramadol.he main purpose of this study was to investigatehether tramadol can be used as a CYP2D6 probe.

ETHODSThe protocol was approved by the Danish Medicines

gency and the Regional Ethical Committee of Vejlend Funen Counties. The study was carried out inccordance with Good Clinical Practice guidelines.ritten informed consent to participate in the study

as obtained from the volunteers. The study was con- w

ucted as an open, 2-phase, crossover trial. The mini-um washout time between the 2 phases was 2 weeks.Subjects. The study included 278 healthy, white vol-

nteers. The 155 men and 123 women, aged 19 to 40ears, were primarily Danish students at the Universityf Southern Denmark (Odense, Denmark). None of theolunteers had a daily intake of alcohol or drugs (ex-ept oral contraceptives), and none of the volunteersere pregnant or breastfeeding.Phenotyping. In phase A of the trial, the CYP2D6

henotype was determined. Oral intake of 100 mgparteine sulfate pentahydrate (supplied by the Centralharmacy, Odense University Hospital, Odense, Den-ark) was followed by urine collection for 12 hours.he urine volume was recorded, and two 10-mL ali-uots were stored at �20°C until analysis. The amountsf excreted sparteine, 2,3-didehydrosparteine, and 5,6-idehydrosparteine were determined by gas chromatog-aphy.6 The design of phase B was identical to a pilottudy that separated EMs and PMs completely.28 Theolunteers were given 50 mg tramadol hydrochloride assingle oral dose (Nobligan; Grünenthal, Germany;

istribution in Denmark by Meda, Allerod, Denmark).rine was collected for 8 hours. The urine volume was

ecorded, and two 10-mL aliquots were stored at20°C until analysis. The concentrations of (�)-

ramadol, (�)-tramadol, (�)-M1, and (�)-M1 in urinend plasma were analyzed in duplicate by an HPLCethod.28 The HPLC method for urine samples was

alidated and published with a range of linearity from.1 to 3.0 �mol/L and a maximum coefficient of vari-tion of 5.7% for precision and reproducibility (n �0). Samples with higher concentrations were dilutedith millipore water before analysis. The two 10-mL

liquots of venous blood were obtained with ethyl-nediaminetetraacetic acid as anticoagulant from eacholunteer approximately 2 hours after medication dur-ng phase B. The blood sample was centrifuged for 10inutes at 2000g, and the plasma was isolated and

tored at �20°C until analysis. The HPLC method forlasma samples was validated and published with aange of linearity from 0.01 to 1.55 �mol/L and aaximum coefficient of variation of 6.0% for precision

nd reproducibility (n � 10). The remainder of thelood sample was stored at �20°C until deoxyribonu-leic acid (DNA) isolation.

Genotyping. The DNA from peripheral leukocytesas isolated by use of the PUREGENE genomic DNAurification kit from Gentra Systems (Minneapolis,inn), according to the manufacturer’s guidelines. The

rst step in genotyping was a polymerase chain reaction

ith primers specific for the active CYP2D6 gene en-

cnpkaTAnibdTaa(suFfS(

o

w

lM

O

l

sms

T

CLINICAL PHARMACOLOGY & THERAPEUTICS2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 461

ompassing the region with the 4 selected single-ucleotide polymorphisms. TaqMan technology (Ap-lera, Norwalk, Conn) was used to genotype the 3nown inactivation mutations CYP2D6*3, CYP2D6*4,nd CYP2D6*6 and the low-activity allele CYP2D6*9.he CYP2D6*3 allele is derived from a deletion of

2549 in the fifth exon, which causes a frame shift, ando in vivo enzyme activity is observed.30,31 CYP2D6*4s the most common inactivating allele. It has a singlease change, G1934�A, in intron 3, causing a splicingefect.32,33 In the CYP2D6*6 allele, a deletion of

1795 in exon 3 results in a frame shift and generatespremature stop codon.34-36 The decreased enzyme

ctivity of CYP2 D6*9 is a result of a 3-baseA2613G2614A2615) deletion in exon 5.37,38 Table Ihows the primers and probes, which were designed byse of Primer Express software (Applied Biosystems,oster City, Calif). The real-time analysis was per-ormed on the ABI PRISM 7700 Sequence Detectionystem equipped with the allelic discrimination modulesoftware version 1.7; Applied Biosystems).

Data analysis. Urinary recoveries (percent of dose)f all drugs and metabolites were calculated.The metabolic ratio of sparteine oxidation (MRsparteine)

able I. Location and sequence of oligonucleotide CY

Primers/probes

PrimersCYP1 (long PCR)CYP4 (long PCR)CYP2D6*3 senseCYP2D6*3 antisenseCYP2D6*4 senseCYP2D6*4 antisenseCYP2D6*6 senseCYP2D6*6 antisenseCYP2D6*9 senseCYP2D6*9 antisense

ProbesCYP2D6*3 wtMGBCYP2D6*3

mutMGBCYP2D6*4 wtCYP2D6*4 mutCYP2D6*6 wtMGBCYP2D6*6

mutMGBCYP2D6*9 wtMGBCYP2D6*9

mutMGB

PCR, Polymerase chain reaction.

as calculated as follows: n

MRsparteine �

0- to 12-hour urinary excretion

of sparteine0- to 12-hour urinary excretion of

2, 3-didehydrosparteine and

5, 6-didehydrosparteine

EMs were defined as having an MRsparteine value ofess than 20, and PMs were defined as having an

Rsparteine value of 20 or greater.10

Two empiric metabolic ratios describing tramadol-demethylation were calculated as follows:

MR1 �0- to 8-hour urinary excretion of (�)-tramadol

0- to 8-hour urinary excretion of (�)-Ml

MR2 �0- to 8-hour urinary excretion of (–)-Ml

0- to 8-hour urinary excretion of (�)-Ml

The mean and range of metabolic ratios were calcu-ated for each CYP2D6 genotype.

The concordance between the gold standard ofparteine toward the genotype test and the tramadoletabolic ratio were determined by calculating the

ensitivity, specificity, positive predictive value, and

rimers and probes

Sequence 5�¡3�

att tcc cag gaa tccccg gcc ctg aca ctc ctt cttcc tga ccc agc tgg atg actt ctc cat ctc tgc cag gaacga ccc ctt acc cgc atcctc acg gct ttg tcc aag agactt ggg cct ggg caa gaagg gca gca caa agg cagag acc tga ctg agg cct tcc ttca ttc ctc ctg gga cgc t

FAM–tca tcc tgt gct cag tt–NFQVIC–tca tcc gtg ctc agt t–NFQ

FAM–acc ccc agg acg ccc ctt t–tamraJOE–acc ccc aag acg ccc ctt tc–tamraFAM–cgg tca ccc act gc–NFQVIC–cgg tca ccc ctg c–NFQ

FAM–tct cac ctt ctc cat ct–NFQVIC–tct cac ctc cat ctc–NFQ

P2D6 p

egative predictive value as follows:

S

S

P

N

R

tfnps

ooo((M6t0

tPpptm2

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Tg

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CLINICAL PHARMACOLOGY & THERAPEUTICS462 Pedersen, Damkier, and Brosen JUNE 2005

ensitivity �No. of true positives

No. of true positives � No. of false negatives

No. of true negatives

ig 2. Frequency distribution of MRsparteine (N � 278). Solidars, Sparteine extensive metabolizers. Open bars, Sparteineoor metabolizers.

able II. Relationship between MR2 (N � 278) andenotype (N � 276) with that of sparteine phenotype

PMsparteine EMsparteine Total

PM if MR2 �2.0PMMR2 28 1 29EMMR2 0 249 249Total 28 250 278

PM if genotype iscomposedof *3, *4,and *6 alleles

PMgenotype 26 0 26EMgenotype 2 248 250Total 28 248 276

PM, Poor metabolizer; EM, extensive metabolizer; MR2, 0- to 8-hour urinaryramadol metabolic ratio: (�)-M1/(�)-M1.

pecificity �No. of false positives � No. of true negatives a

ositive predictive value �No. of true positives

No. of true positives �

No. of false positives

egative predictive value �No. of true negatives

No. of true negatives �

No. of false negatives

ESULTSFour subjects had moderate nausea 3 to 5 hours after

ramadol medication. This adverse event lasted for aew hours and had no practical consequence. Unfortu-ately, genotyping of only 276 of 278 volunteers waserformed because of difficulties in drawing bloodamples from 2 subjects.

Fig 2 shows the classic clear-cut bimodal distributionf MRsparteine in the population. The empiric antimodef 20 separated the 2 phenotypes completely, and nother subgroups appeared. Of the 278 individuals, 2817 women and 11 men) were phenotyped as PMs10.1% [confidence interval, 6.8%-14.2%]). The mean

Rsparteine in the 28 PMs of sparteine (PMsparteine) was8.8 (range, 20.1-250.2), and the mean MRsparteine inhe 250 EMs of sparteine (EMsparteine) was 0.6 (range,.1-7.0).Fig 3 shows the distribution of MR1 in the popula-

ion. Apparently, there was no bimodality, although theMsparteine in general had high values of MR1. The 2henotypes overlapped, and in best case, 4 false-ositive subjects were misclassified. The mean MR1 inhe 28 PMsparteine was 58.9 (range, 12.7-344.5), and the

ean MR1 in the 250 EMsparteine was 2.5 (range, 0.4-3.4).Fig 4 shows the distribution of MR2 in the popula-

ion. The distribution of MR2 was bimodal, and anmpiric antimode of 2.0 could be placed. This antimodeeparated the 2 phenotypes completely, and only 1ubject was misclassified. This misclassified subjectad an MRsparteine of 5.0 and an MR2 of 3.0. Thisubject was heterozygous for *4 by this genotype test,hich apparently indicates an EM. By use of the anti-ode MR2 of 2.0 or greater as a new PM phenotype

est, the sensitivity and negative predictive value were00%. Because of the false-positive, misclassified sub-ect, the specificity was 99.6% and the positive predic-ive value was 96.6%. The mean MR2 in the 28Msparteine was 4.7 (range, 2.3-9.5), and the mean MR2

n the 250 EMsparteine was 0.7 (range, 0.4-3.0).Of the 28 PMsparteine, 26 were genotypically PMs

ith regard to the inactivating mutations *3, *4, and *6.bout half of the study population carries functional

lleles. The mean MRsparteine for the apparent wild type

((mPvoCaga1Inso

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FSm

CLINICAL PHARMACOLOGY & THERAPEUTICS2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 463

CYP2D6*1/*1) was lower (0.4) than for other EMs0.6-2.0). CYP2D6*4 was by far the most commonutated allele compared with *3, *6, and *9. OneMsparteine was genotyped as CYP2D6*1/*4. This indi-idual had MRsparteine of 84.7, MR1 of 344.5, and MR2

f 9.5. Another PMsparteine was genotyped asYP2D6*6/*9 with MRsparteine of 23.1, MR1 of 17.1,nd MR2 of 2.3. As a result of these 2 false-negativeenotypes, the genotype test had a sensitivity of 92.9%,specificity of 100%, a positive predictive value of

00%, and a negative predictive value of 99.2%. TableI summarizes the relationship between the new phe-otype test and the genotype test with that of the goldtandard sparteine. Table III shows the mean and rangef the metabolic ratios for the genotypes.The mean urinary recoveries (Table IV) in the

Msparteine were higher for (�)-tramadol, 20.2% (range,.4%-42.5%), and (�)-tramadol, 17.2% (range, 5.0%-1.1%), compared with the following values forMsparteine: (�)-tramadol, 15.4% (range, 4.0%-39.8%),

ig 3. Frequency distribution of MR1 (N � 278). Solid bars,parteine extensive metabolizers. Open bars, Sparteine pooretabolizers.

nd (�)-tramadol, 13.2% (range, 2.8%-39.9%). The M

ifference between PMsparteine and EMsparteine in meanrinary recoveries for (�)-M1 and (�)-M1 were larger.he mean urinary recoveries in the PMsparteine were.53% (range, 0.1%-1.1%) for (�)-M1 and 2.2%range, 0.7%-4.5%) for (�)-M1, as compared with theollowing values for EMsparteine: 8.7% (range, 1.7%-3.2%) for (�)-M1 and 5.6% (range, 1.3%-14.6%) for�)-M1. There was a marked interindividual variabilityor each compound, and there were overlaps for (�)-ramadol, (�)-tramadol, and (�)-M1 between the phe-otypes. Only the amount of (�)-M1 was significantlyifferent between the phenotypes. The coefficient ofariation was high for all groups (34%-60%).Unfortunately, the data obtained from the plasma

amples were not sufficiently informative. The plasmaamples from 19 of 28 PMs had very low concentra-ions of (�)-O-desmethyltramadol, resulting in unac-eptable deviations or concentrations of (�)-O-esmethyltramadol below the limit of quantification (5mol/L).28 Therefore it was not possible to calculate

ig 4. Frequency distribution of MR2 (N � 278). Solid bars,parteine extensive metabolizers. Open bars, Sparteine pooretabolizers.

R1 and MR2.

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CLINICAL PHARMACOLOGY & THERAPEUTICS464 Pedersen, Damkier, and Brosen JUNE 2005

ISCUSSIONPhenotyping with tramadol should not be carried out

n pregnant, delivering, and nursing women. Apartrom that, phenotyping with 50 mg tramadol hydro-hloride is considered to be safe in healthy volunteersged from 1 to 75 years. Only 4 incidents of minordverse effects were reported in the total population of78 subjects, because the dose of 50 mg is the smallestablet available and a subtherapeutic dose. The pheno-ype test is easily conducted by collecting urine for only

hours.As expected, MRsparteine was bimodally distributed,

eparating the phenotypes clearly. In this study 10.1%f subjects were PMs, which was similar to most otherhite populations.39 Because the confidence interval is.8% to 14.2%, there is no statistical reason to assumehat the population is different from earlier findings inhe Danish population (7.3%).10 As shown in Figs 3nd 4, the PMsparteine had high MR1 and MR2, confirm-ng that CYP2D6 is highly involved in the-demethylation of (�)-tramadol. As a result, the me-

able III. Mean metabolic ratios for genotypes (N �

Genotype

Sparteine

MRsparteine (rang

CYP2D6*1/*1 (n � 139) 0.4 (0.1-3.4)CYP2D6*1/*4 (n � 81) 2.0 (0.2-84.7)CYP2D6*1/*9 (n � 12) 0.7 (0.2-3.9)CYP2D6*1/*3 (n � 6) 0.6 (0.4-0.7)CYP2D6*1/*6 (n � 6) 1.2 (0.5-2.8)CYP2D6*9/*9 (n � 4) 1.2 (0.8-2.0)CYP2D6*6/*9 (n � 1) 23.1CYP2D6*4/*4 (n � 19) 64.9 (21.6-141.CYP2D6*4/*6 (n � 4) 48.7 (20.1-66.1CYP2D6*3/*3 (n � 2) 156.3 (62.4-250.CYP2D6*3/*4 (n � 2) 71.4 (53.0-89.8

MRsparteine, Zero- to twelve-hour urinary sparteine metabolic ratio of sparteatio of (�)-tramadol over (�)-M1; MR2, 0- to 8-hour urinary tramadol meta

able IV. Urinary recoveries (percent of dose) of tramolymorphism

EMsparteine

[mean (ran

(�)-Tramadol 15.4 (4.0-(�)-Tramadol 13.2 (2.8-(�)-M1 8.7 (1.7-(�)-M1 5.6 (1.3-

CV, Coefficient of variation.

ian urinary recoveries in the PMsparteine were higher s

or both isomers of tramadol and lower for both isomersf M1 (Table IV). In EMsparteine, the amounts of�)-M1 were higher than those of (�)-M1. This sup-orts the finding that the maximum metabolizing ve-ocity (Vmax) for the O-demethylation of (�)-tramadols higher than Vmax for the O-demethylation of (�)-ramadol.24 Very low recovery of (�)-M1 in theMsparteine confirms that CYP2D6 is stereoselective andYP2D6 is responsible for the O-demethylation of

�)-tramadol in particular. The clinical significance ofhe very low amounts of (�)-M1 is described in theiterature. Studies have shown that (�)-M1 is respon-ible for the hypoalgesic effect of tramadol.29,40 How-ver, in contrast to sparteine, the distribution of theetabolic ratio of the parent substrate (�)-tramadol

ver the metabolite (�)-M1 (MR1) was not bimodal.ven the best antimode resulted in the finding that 4Msparteine were misclassified as tramadol PMs accord-

ng to MR1. Surprisingly, the metabolic ratio of (�)-1/(�)-M1 (MR2) showed better distribution, separat-

ng the 2 phenotypes at the metabolic ratio of 2.0. The

Tramadol

MR1 (range) MR2 (range)

1.7 (0.4-8.2) 0.6 (0.4-1.4)7.5 (0.8-344.5) 0.9 (0.5-9.5)3.2 (1.2-10.9) 0.8 (0.5-1.9)1.8 (1.1-2.2) 0.7 (0.6-0.8)4.6 (1.4-10.8) 0.9 (0.5-1.9)5.0 (3.1-6.8) 0.7 (0.5-1.0)

17.1 2.350.1 (12.7-139.3) 4.7 (3.1-7.4)46.4 (29.4-67.0) 4.7 (3.6-6.5)66.2 (52.8-79.5) 4.4 (4.3-4.5)33.6 (29.0-38.1) 3.7 (2.9-4.5)

3- and 5,6-didehydrosparteine; MR1, 0- to 8-hour urinary tramadol metabolicof (�)-M1 over (�)-M1.

nd M1 in relation to sparteine oxidation

0))]

PMsparteine (n � 28)[mean (range) (CV)]

) 20.2 (7.4-42.5) (34)) 17.2 (5.0-41.1) (38)) 0.53 (0.1-1.1) (60)) 2.2 (0.7-4.5) (42)

276)

e)

1))2))

adol a

(n � 25ge) (CV

39.8) (3639.9) (3823.2) (4214.6) (41

pecificity and positive predictive value were also

hs

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moTmwswtSaadrNtcnTpat

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lnrsm

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R

CLINICAL PHARMACOLOGY & THERAPEUTICS2005;77(6):458-67 Tramadol as a new probe for CYP2D6 phenotyping 465

igher by use of MR2. Only 1 EMsparteine was misclas-ified according to MR2.

As a result of the low dose of tramadol hydrochloride50 mg) given, plasma concentrations of (�)-M1 wereoo low or undetectable in most PMs. Unfortunately, itas therefore not possible on the basis of the study to

valuate whether plasma concentrations of (�/�)-ramadol and (�/�)-M1 have the potential to be aeasure of CYP2D6 activity. To evaluate the plasma

oncentrations, a higher dose or a more sensitive ana-ytic method was needed. Neither option was chosen.he dose was kept as low as possible to avoid possibledverse effects. Increasing the sensitivity of the methodas unfortunately not possible without new analytic

nstrumentation, followed by a new method, validation,nd publication. Because the plasma concentrations of�/�)-tramadol and (�/�)-M1 of samples were nothe primary endpoint in the study, the lack of plasmaata was not decisive.Sparteine is an excellent probe with a clear-cut bi-odal distribution. MR2 does not have the same obvi-

us antimode. There could be several reasons for this.he ratio is only a picture of the first 8 hours afteredication. The decision to use 8-hour urine collectionas based on the results from a pilot study that clearly

eparated PMs and EMs by both MR1 and MR2.28 Whatill happen after 8 hours is not clearly described, but

his is currently under investigation in our laboratory.econd, M1 is not a terminal metabolite, and themount of M1 is dependent on further subsequent met-bolic steps. At least 4 M1 metabolites have beenescribed (M5, M13, M20, and M32).22 A thirdeason lies in the alternative N-demethylation. The-demethylation is also stereoselective, but in contrast

o O-demethylation, (�)-tramadol is N-demethylatedonsiderably faster than the (�)-isomer.24 CYP2D6 isot involved in the N-demethylation of tramadol.25

herefore the calculated metabolic ratios are not com-letely determined by the CYP2D6 activity, but theylso reflect, to some extent, other enzymes involved inhe tramadol metabolism.

All subjects carrying 2 inactivating mutations (*3,4, or *6) were PMsparteine, but 2 PMsparteine wereisclassified as EMs by the genotype test. One of

hese misclassified subjects had a *6/*9 genotypeith an MRsparteine of 23. This subject was, by def-

nition, a genotype EM but carried an inactive allele,s well as an allele with decreased activity. The otherubject had a *1/*4 genotype with an MRsparteine of5. This subject could carry another inactivatingutation/deletion that was not tested for with this

enotype test. The present genotype test does not

etect all mutations resulting in PMs. Thereforeome of the homozygote genotypes (*1/*1 [n � 139],4/*4 [n � 19], *3/*3 [n � 2], and *9/*9 [n � 4])ay carry another mutation on 1 allele. One commonutation in white populations is *5, which is a

eletion of an entire functional CYP2D6 gene.41

owever, it should be kept in mind that this issue haso influence on the genotype classification of EMsnd PMs. A better genotype test for white popula-ions would detect *5 and *10, because both muta-ions are present with considerable frequencies.42

till, without the detection of *5 and *10, only 2ubjects were misclassified and a sensitivity of2.9%, specificity of 100%, positive predictive valuef 100%, and negative predictive value of 99.2%ere achieved.In conclusion, 50 mg tramadol hydrochloride fol-

owed by 8-hour urine collection provides an alter-ative CYP2D6 probe to sparteine. The metabolicatio of (�)-M1/(�)-M1 with an antimode of 2.0eparated the 2 phenotypes, with the exception of 1isclassified subject.

We gratefully thank The Alfred Benzon Foundation, The Lund-eck Foundation, and The Danish Medical Research Council fornancial support.

None of the authors have any conflicts of interest.

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