Clinical Investigator

Clin Investig (1992) 70: 708-710

ClinicaiPharmacoJogy OriginalArticle

© Springer-Verlag 1992

The metabolism of tramadol by human liver mierosomes W.D. Paar, P. Frankus, and H.J. Dengler Zentrum ffir Innere Medizin-Allgemeine Innere Medizin, Universitfit Bonn

Summary. The metabolism of tramadol was investigated in vitro using microsomal fractions of human liver. The parent compound and its main metabolites were determined by a newly developed high performance liquid chromatography assay. O-demethylation of tramadol was found to be stereoselective. The Vmax of the O-demethylation of ( - ) - t r a m a d o l was 210 pmol. mg - 1. min- 1, whereas (+)-tramadol was O-demethylated with a Vm,x of 125 -1 .min -1. The Km for both enantiomers was determined to be 210 gM. O-demethylation was inhibited competitively by quinidine (ki = 15 nM) and propafenone (ki = 34 nM). N-demethylation was also stereoselective, preferentially metabolizing the (+)-enantiomer. Whereas O-demethylation displayed monophasic MichaelisMenten kinetics, N-demethylation was best described by a two-site model. Competitive inhibition of the O-demethylation both by quinidine and propafenone suggests that O-demethylation is carried out by P-450IID6.

Key words: Tramadol - Drug metabolism - Racemate - Enantiomers - Microsomes - High performance liquid chromatography - Quinidine - Propafenone

Tramadol (T) is used as a centrally acting analgesic. The compound has two chiral centers. The marketed drug is the racemate of the t r a n s isomers. T is metabolized via O-demethylation of metabolite MI and via N-demethylation to metabolite M2. Further demethylation of the primary metabolites results in the formation of three other metabolites [5]. The aim of the present study was to develop an in vitro assay to investigate the metabolism of T with respect to stereoselectivity and to possible inhibition by other drugs. Abbreviations: HPLC=high performance liquid chromatography; k i = inhibitory constant; km = apparent Michaelis-Menten constant; M1 = O-demethylated metabolite of tramadol; M2 = N-demethylated metabolite of tramadol; NADP=nicotinamide-adenine dinucleotide phosphate; T=tramadol; Vm.x= maximum velocity of the reaction

Methods Liver tissue was obtained from tumor-free areas of liver resectates taken on the occasion of partial liver resection because of liver metastases. The patient did not take medicaments prior to the admission to hospital. Preanesthetic medication consisted of midazolam. For the induction of anesthesia, thiopental was used. Relaxation of skeletal muscle was obtained by vecuronium bromide and succinyl chloride. The inhalational anaesthesia was isoflurane. The liver samples were immediately frozen in liquid nitrogen and kept at - 8 0 ° C until assayed. Microsomes were prepared according to a method described by Dayer et al. [1]. The protein content of the microsomal preparations was estimated using the Lowry method, and the cytochrome P-450 content was determined by spectrophotometry. Incubation was carried out in the presence of reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) according to Osikowska-Evers and Eichelbaum [7]. The incubation mixture (final volume 250 lxl) contained: microsomal protein (150 gg, 25-60 pmol cytochrome P-450, 30pl), TR1S buffer (0.05M, pH7.5, 145 gl), MgC12 (0.06 M, 25 ~d), ( + / - ) - r , ( - ) - T or (+)-T (concentration range 50-5000 pM, 25 pl). The reaction was started by the addition of NADPH (0.004 M, 25 gl), which was prepared fresh for each assay. The reaction was terminated after 100 min by the addition of 25% ammonia solution (10 gl). Tramadol and its metabolites were extracted by dichloromethane after alkalization using the ammonia solution. The extraction efficiencies of T and its metabolites from incubation mixtures ranged from 78% to 93%. The isocratic high performance liquid chromatography (HPLC) system employed an analytical column (30 cm. 4 mm internal diameter) filled with Nucleosil RP18. The analytical column was protected by a precolumn (12.5 cm. 3.0 mm internal diameter) containing the same material. Fluorescence detection was used. The precision of the assay was 3.6%, 5.2%, and 4.4% for T, MI, and M2, respectively. The pre-


cision of the incubation procedure was determined by 10-fold incubation of 500 g M T. The coefficient of variation was 10.2% and 9.2% for the quantification of M1 and M2, respectively. Details of the incubation conditions as well as of the HPLC assay will be published elsewhere. The formation of MI and M2 was found to be linear up to an incubation time of 120 min. With respect to the microsomal protein concentration, the metabolite formation was found to be linear in the range of 50-200 gg of microsomal protein/ sample. Results

O-demethylation The kinetics of the O-demethylation of ( + ) - and ( - ) - T by microsomes from human liver was adequately described by a one-site model. The Eadie Hofstee plot is shown in Fig. 1. The apparent Michaelis-Menten constants and maximum reaction velocities for the O-demethylation of tramadol are: ( - ) - t r a m a d o l km=210 g M and vm,x = 210 pmol- mg - 1. min - 1 (+)-tramadol km = 210 g M and Vm,x= 125 pmol" m g - a" min - 1 O-demethylation was inhibited competitively by quinidine and by propafenone. The extent of inhibition was comparable. The ki of quinidine was 15 nM, while the corresponding value for propafenone was 34 nM.

N-demethylation N-demethylation was also found to be stereoselective. In contrast to O-demethylation, the (+)-enantiomer is N-demethylated considerably faster than v (pmol-mg'~min - ' ) 25O



the ( - ) - e n a n t i o m e r . The kinetics of the N-demethylation of both T enantiomers were biphasic, indicating a low affinity/high capacity and a high affinity/low capacity site. There was no inhibition of N-demethylation in the presence of quinidine. Coincubation with propafenone resulted in a minimal inhibition, the ki being greater than 500 gM. Discussion

O-demethylation of T was found to be highly stereoselective. The Vma x of O-demethylation of the ( - ) - e n a n t i o m e r is twofold greater than that of the (+)-enantiomer. This finding of our in vitro investigations suggests differences in the pharmacokinetics of the enantiomers in vivo. O-demethylation of T is inhibited competitively by quinidine and by propafenone. Both drugs are known to be specific inhibitors of the cytochrome P-450IID6 [4, 8]. The absence of this isoenzyme is considered the basis of the most widely studied genetic polymorphism of drug oxidation, affecting the metabolism of sparteine, debrisoquine, and more than 25 other drugs [2, 3, 6]. Because of the competitive inhibition by quinidine and propafenone in vitro, it is likely that O-demethylation of T is carried out by P-450IID6. At present, we are examining whether poor metabolizers of sparteine also exhibit impaired O-demethylation of T. It is difficult to predict whether this expected polymorphism of T has clinical significance. N-demethylation was found to be stereoselective, too, but in contrast to O-demethylation, Ndemethylation preferred the other enantiomer. Ndemethylation was considerably faster after incubation of the (+)-enantiomer as compared with the ( - ) - e n a n t i o m e r or the racemate. The Vmax of the low affinity/high capacity site was in the range of 900 pmol-mg- 1. rain- 1 after incubation of ( - )T, the corresponding value for the ( +)-enantiomer being about 2000 p m o l . m g - 1. m i n - 1. Actually further in vitro investigation is carried out in order to determine the exact kinetic data of both sites of N-demethylation of T. In contrast to O-demethylation, we observed no marked inhibition of Ndemethylation when incubation was carried out in the presence of quinidine.


Acknowledgements. We are thankful to Prof. Dr. A. Hirner and the staff of the Department of Surgery of the University of Bonn for collaboration in regard to the liver biopsies.









v ( I rain-') / S (rnM)

Fig. 1. Eadie Hofstee plot of the O-demethylation of (+)- and (-)-tramadol. + ( + ) Tramadol; • (--) tramadol

References 1. Dayer P, Gasser R, Gut J, Kronbach T, Robertz GM, Eichelbaum M, Meyer UA (1984) Characterization of common

710 genetic defect of cytochrome P-450 function (debrisoquinesparteine type polymorphism) - increased Michaelis constant (Kin) and loss of stereoselectivity of bufuralol 1-hydroxylation in poor metabolizers. Biochem Biophys Res Commun 125:374-380 2. Eichelbaum M, Gross AS (1990) The genetic polymorphism of debrisoquine/sparteine metabolism - clinical aspects. Pharmacol Ther 46: 377-394 3. Eichelbaum M, Spannbrucker N, Dengler HJ (1978) A probably genetic defect of the metabolism of sparteine. In: Gorrod JW (ed) Biological oxidation of nitrogen. Elsevier NorthHolland, Amsterdam, pp 113-118 4. Kroemer HK, Mikus G, Kronbach T, Meyer UA, Eichelbaum M (1989) In vitro characterization of the human cytochrome P-450 involved in polymorphic oxidation of propafenone. Clin Pharmacol Ther 45:28-33 5. Lintz W, Erlacin S, Frankus E, Uragg H (1981) Metabolismus von Tramadol bei Mensch und Tier. Arzneimittelforschung/Drug Res 31 : 1932-1943 6. Meyer U A (1990) Genetic polymorphisms of drug metabolism. Fundam Clin Pharmacol 4:595-615

7. Osikowska-Evers BA, Eichelbaum M (1986) A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver. Life Sci 38 : 1775-1782 8. Otton SV, Crewe HK, Lennard MS, Tucker GT, Woods H F (1988) Use of quinidine inhibition to define the role of the sparteine/debrisoquine cytochrome P450 in metoprolol oxidation by human liver microsomes. J Pharmacol Exp Ther 247: 242-247 Received: April 3, 1992 Returned for revision: April 22, 1992 Accepted: May 25, 1992 Dr. W.D. Paar Zentrum ffir Innere Medizin Allgemeine Innere Medizin Universit/it Bonn Sigmund-Freud-Strasse 25 W-5300 Bonn, F R G

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The metabolism of tramadol by human liver microsomes.

The metabolism of tramadol was investigated in vitro using microsomal fractions of human liver. The parent compound and its main metabolites were dete...
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