František Perlík

High-performance liquid chromatographic determination of tramadol and its O-desmethylated metabolite in blood plasma Application to a bioequivalence study in humans

Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the above-mentioned tramadol metabolite and N1,N1-dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-microm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v/v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v/v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection (lambda(ex) 202 nm/lambda(em) 296 nm for tramadol and its metabolite, lambda(ex) 264 nm/lambda(em) 344 nm for N1,N1-dimethylsulfanilamide) was used. The validated analytical method was applied to pharmacokinetic studies of tramadol in human volunteers.

Human Urinary Metabolomic Profile of PPARα Induced Fatty Acid β-Oxidation

Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with increased fatty acid catabolism and is commonly targeted for the treatment of hyperlipidemia. To identify latent, endogenous biomarkers of PPARalpha activation and hence increased fatty acid beta-oxidation, healthy human volunteers were given fenofibrate orally for 2 weeks and their urine was profiled by UPLC-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid (>5-fold) and acetylcarnitine (>20-fold), observations that are consistent with known targets of PPARalpha including pantothenate kinase and genes encoding proteins involved in the transport and synthesis of acylcarnitines. It was also concluded that serum cholesterol (-12.7%), triglycerides (-25.6%), uric acid (-34.7%), together with urinary propylcarnitine (>10-fold), isobutyrylcarnitine (>2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine (>5-fold) were all reduced by day 14. Specificity of these biomarkers as indicators of PPARalpha activation was demonstrated using the Ppara-null mouse. Urinary pantothenic acid and acylcarnitines may prove useful indicators of PPARalpha-induced fatty acid beta-oxidation in humans. This study illustrates the utility of a pharmacometabolomic approach to understand drug effects on lipid metabolism in both human populations and in inbred mouse models.

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography-mass spectrometry

A GC-MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography-electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-betaDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1-20 microg/mL (R(2) > or =0.998). Intra-day accuracies ranged between 97.2-104.9%, 96.1-103.2%, and 97.3-102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2-105.7%, 99.1-105.2%, and 96.5-101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.

Tramadol efficacy in patients with postoperative pain in relation to CYP2D6 and MDR1 polymorphisms.

OBJECTIVES The aim of our study was to evaluate impact of CYP2D6 and MDR1 polymorphisms on the analgesic efficacy of tramadol in patients after a knee arthroscopy. BACKGROUND Pharmacokinetics of tramadol and its metabolites is stereoselective and displays high interindividual variability correlating with polymorphic CYP2D6 in the population. Available data provide controversial results regarding the analgesic efficacy of tramadol in subjects with different CYP2D6 genotypes. METHODS Pain intensity was assessed using visual analogue scale at 2 and 24 hours after the knee arthroscopy in 156 patients. Polymorphisms CYP2D6*3,*4,*5,*6, and gene duplication and C3435T in MDR1 gene were analyzed by PCR - RFLP. RESULTS Mean VAS2h value in the whole study group was 44.0 ± 16.5 mm. Mean pain difference, was lowest in the UM group and highest in the PM group. The pain difference varied significantly among the CYP2D6 subgroups (F = 4.29; p = 0.006) with significant differences between homEM vs hetEM, homEM vs PM, and UM vs PM subgroups. There were no significant differences among MDR1 subgroups with regards of pain difference. Mean tramadol consumption was 2.47 ± 1.17 mg/kg during the 24 h period. There were no significant differences in the drug consumption, reporting of adverse reactions, need for rescue analgesic medication or verbal description of pain among the CYP2D6 or MDR1 genotype subgroups. CONCLUSION CYP2D6 plays a significant role in tramadol analgesic efficacy. The non-opioid analgesia in PMs was associated with better subjective pain relief in patients after a knee arthroscopy (Tab. 3, Ref. 18).

Pharmacokinetics of tramadol is affected by MDR1 polymorphism C3435T

Dear Professor Dahlqvist, We have read with interest the articles by Pedersen et al. and Wang et al. who highlighted the importance of functional polymorphisms of CYP2D6 on the pharmacokinetics of tramadol and its major metabolite O-demethyltramadol in recent issues of the European Journal of Clinical Pharmacology [1, 2]. The pharmacology of tramadol is unusually complex, having at least 11 unconjugated metabolites and 12 conjugated compounds [3]. There are three major metabolic pathways, CYP2D6, CYP3A, and CYP2B6, forming Oand N-demethylated metabolites. Tramadol is believed to undergo first-pass metabolism, reducing its bioavailability to approximately 80% after oral administration. The CYP2D6-dependent pharmacokinetics of tramadol is usually also reflected in increased bioavailability in poor metabolizers (PM) compared with extensive metabolizers (EM). Surprisingly, Pedersen et al. did not observe any significant difference between bioavailability of tramadol in EMs and PMs, while large interindividual variability was noted [1]. It is recognized that the bioavailabity of some drugs can be substantially affected by active transporters expressed in the gut lumen, like P-glycoprotein. We recently conducted a study in order to uncover MDR1 genotype-dependent variations in pharmacokinetic parameters of tramadol and O-demethyltramadol. Twenty-one healthy young volunteers selected from our database participated in the study after providing informed consent. Presence of CYP2D6*3, *4, *5, *6 alleles and gene duplications was analyzed using PCRand RFLPbased methods. MDR1 polymorphisms C3435T and G2677T/A were also detected. Three groups of seven CYP2D6 EM, heterozygous EM, and PM subjects were investigated. Four and nine subjects were homozygous carriers of C3435 and T3435 alleles, respectively. Each volunteer was administered a 100-mg sustainedrelease tramadol tablet (Tramal Retard 100 mg, Zentiva Praha a.s.), and plasma concentrations of (R,S)-(±)-tramadol (TMD) and (±)-O-demethyltramadol (M1) were analyzed by HPLC at baseline and at 2.5, 4, 8, 12, and 24 h post-dose. The average Cmax and AUC0–24 values of TMD increased slightly in groups with increasing numbers of 3435T alleles of MDR1 irrespective of CYP2D6 status. The mean (SD) Cmax values of TMD were 495.4 (91.1), 529.3 (161.7), and 600.2 (179.9) nmol/l in 3435CC, 3435CT, and 3435TT groups, respectively. Corresponding values for AUC0–24 in the respective groups were 7,393.9 (2,299.1), 7,710.1 (3,304.7), and 8,478.8 (3,771.0) nmol·h/l. The differences, however, did not reach the level of statistical significance. Interestingly, a similar trend was not observed for M1, for which production was more dependent on relative numbers of CYP2D6 extensive metabolizers. Detailed analysis focused on comparison of subjects according to mixed CYP2D6 and MDR1 genotypes. Figure 1 shows pharmacokinetics profiles of TMD and Eur J Clin Pharmacol (2007) 63:419–421 DOI 10.1007/s00228-006-0255-3

Pupillometry in healthy volunteers as a biomarker of tramadol efficacy.

What is known and Objective:  The opioid effect of tramadol, which can be detected by pupillary response, is predominantly mediated by the O‐demethylated metabolite, formed via CYP2D6. This study was designed to evaluate the effects of tramadol using different parameters of pupillometry as biomarkers.

CYP2D6 polymorphism, tramadol pharmacokinetics and pupillary response

Dear Professor Dahlqvist, We read with interest the article by Fliegert et al. published online on May 20 that describes pupillometry as an evaluation tool for pharmacodynamic profiling [1]. We have previously studied the pharmacokinetics of (R,S)-(±)tramadol and (±)-O-demethyltramadol (M1) in relationship to drug-induced miosis, as measured by infrared pupillometry in 21 young healthy volunteers comprising three equally sized groups of CYP2D6 EMs, heterozygous EMs, and PMs [2]. Our data differ from those of Fliegert et al. in that both pharmacokinetics and pharmacodynamics of tramadol are genotype-dependent in the groups of heterozygous and homozygous EMs (Fig. 1). We have analysed our data in relation to genotype in order to uncover correlations between pharmacokinetic parameters of tramadol and M1 with pupillary response. As shown in Fig. 1, the plasma levels of the parent compound in heterozygous EMs are, at all sampling intervals, lower and the production of M1 is delayed, leading to a shift to the right of the M1 plasma concentration–time curve in comparison with homozygous EM subjects. Also, pupillary response differed considerably between homozygous and heterozygous EMs. The mean maximal effect in homozygous EMs occurred at 4 h post dose, in heterozygous EMs at 12 h. In contrast to Fliegert et al., we also observed a small miotic action of the drug in the PM group using static pupillometry. Significant negative correlations (Spearman’s test) between both tramadol Cmax and AUC0–24 vs Emax (rs= −0.39 and −0.51, respectively; p<0.05) and AUC0–24 vs area under the effect–time curve (AUD0–12) (rs=−0.41; p<0.05) were observed. Higher and positive correlations between both the M1 Cmax and AUC0–24 vs Emax (rs=0.59 and 0.55, respectively; p<0.01) and vs AUD0–12 (rs=0.55 and 0.52, respectively; p<0.01) were observed. The correlations of pharmacokinetic parameters of M1 vs pupillary effect were thus somewhat stronger than the respective values for the parent compound, but we found the strongest correlation of metabolic ratio (concentration of tramadol/concentration of M1) at all sampling intervals (2.5–24 h post dose) vs. the effects (rs range 0.85–0.89; p<0.01). This presumably means that the parent compound itself possesses a minor miotic action, which is observable in healthy volunteers. We have observed a longer time to maximal miosis in heterozygous subjects than in homozygous ones. Fliegert et al. reported that the time to maximal effect was 4–10 h for the mixed homozygous and heterozygous EMs and speculated that it was be due to delayed transfer of M1 through the blood–brain barrier. Based on our data, the pharmacokinetic differences between homozygotes and heterozygotes could be the reason for this observation. In our opinion, it is necessary to consider heterozygous and homozygous EMs as two separate groups when assessing the pharmacokinetic and/or pharmacodynamic parameters of tramadol. Moreover, in reality, there exists no subject with a mixed homozygous and heterozygous EM genotype, and heterozygous and homozygous EM subjects in European populations represent groups that account for approximately 40% and 50% of persons, respectively. O. Slanař (*) . J. R. Idle . F. Perlik Clinical Pharmacology Unit, Institute of Pharmacology, First Faculty of Medicine, Charles University, Na Bojisti 1, Praha 2, 120 00, Czech Republic e-mail: oslan@lf1.cuni.cz Tel.: +420-2-24964135 Fax: +420-2-24964133

Genetic Polymorphisms of CYP2C8 in the Czech Republic

AIM CYP2C8 represents 7% of the hepatic cytochrome system and metabolizes around 5% of drugs in phase I processes. It also plays a significant role in metabolism of endogenous compounds. More than 20 single-nucleotide polymorphisms (SNPs) have been noted, mainly in exons 3, 5, and 8. The most studied SNPs may lead to decreased enzyme activity and may have impact on drug metabolism. Variant alleles are called CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), and CYP2C8*4(I264M). Our aim was to investigate the frequency of major functional SNPs among the Czech population. MATERIAL AND METHODS DNA was isolated from whole blood of 161 healthy, young, and unrelated subjects (94 men and 67 women, aged from 23 to 28 years). The genotypes of polymorphic positions CYP2C8*2, CYP2C8*3 (G416A, A1196G), and CYP2C8*4 were determined by polymerase chain reaction-restriction fragment length polymorphism. RESULTS AND CONCLUSION Observed allele frequencies were 10.9%, 5.9%, and 0.3% for the alleles CYP2C8*3, CYP2C8*4, and CYP2C8*2, respectively. Both CYP2C8*3 (G416A, A1196G) alleles have been found in complete linkage disequilibrium. The allele distribution complies well with Hardy-Weinberg equilibrium. Allele frequencies of functionally important CYP2C8 variants in the Czech population are similar to that of other Caucasian populations.

Enantiomeric determination of tramadol and O-desmethyltramadol in human plasma by fast liquid chromatographic technique coupled with mass spectrometric detection.

A rapid and sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for enantiomeric determination of tramadol and its primary phase metabolite O-desmethyltramadol in human plasma has been developed. Tramadol hydrochloride-(13)C, d(3), was used as an isotopic labeled internal standard for quantification. The method involves a simple solid phase extraction. The analytes and internal standard were separated on Lux Cellulose-2 packed with cellulose tris(3-chloro-4-methylphenylcarbamate) using isocratic elution with hexane/isopropanol/diethylamine (90:10:0.1, v/v/v) at a flow rate of 1.3 mL/min. The APCI positive ionization mass spectrometry was used with multiple reaction monitoring of the transitions at m/z 264.2-->58.2 for tramadol, m/z 250.1-->58.2 for O-desmethyltramadol and m/z 268.2-->58.2 for internal standard. Linearity was achieved between 1-800 ng/mL and 1-400 ng/mL (R(2) > or = 0.999) for each enantiomer of tramadol and O-desmethyltramadol, respectively. Intra-day accuracies ranged among 98.2-102.8%, 97.1-109.1% and 97.4-102.9% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged among 95.5-104.1%, 99.2-104.7%, and 94.2-105.6% at the lower, intermediate, and high concentration for all analytes, respectively. This assay was successfully used to determine the concentration of enantiomers of tramadol and O-desmethyltramadol in a pharmacogenetic study.