Milan Nobilis

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.

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.

Comparative biotransformation and disposition studies of nabumetone in humans and minipigs using high-performance liquid chromatography with ultraviolet, fluorescence and mass spectrometric detection

The disposition of the non-steroidal anti-inflammatory drug (NSAID) nabumetone after a single oral dose administration of nabumetone tablets to humans and minipigs was investigated. Nabumetone is a prodrug, which is metabolized in the organism to the principal pharmacodynamically active metabolite -- 6-methoxy-2-naphthylacetic acid (6-MNA), and some other minor metabolites (carbonyl group reduction products, O-desmethylation products and their conjugates with glucuronic and sulphuric acids). Standards of the above-mentioned metabolites were prepared using simple synthetic procedures and their structures were confirmed by NMR and mass spectrometry. A simple HPLC method for the simultaneous determination of nabumetone, 6-MNA and the other metabolites was developed, validated and used for xenobiochemical and pharmacokinetic studies in humans and minipigs and for distribution studies in minipigs. Naproxen was chosen as the internal standard (I.S.), both UV (for higher concentrations) and fluorescence detection (for very low concentrations) were used. The identity of the nabumetone metabolites in biological samples was confirmed using HPLC-MS experiments. Pharmacokinetics of nabumetone, 6-MNA and 6-HNA (6-hydroxy-2-naphthylacetic acid) in human and minipig plasma was evaluated and compared. The concentration levels of nabumetone metabolites in urine, bile and synovial fluid were also evaluated.

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

Role of carbonyl reducing enzymes in the phase I biotransformation of the non-steroidal anti-inflammatory drug nabumetone in vitro

1. Nabumetone is a clinically used non-steroidal anti-inflammatory drug, its biotransformation includes major active metabolite 6-methoxy-2-naphtylacetic acid and another three phase I as well as corresponding phase II metabolites which are regarded as inactive. One important biotransformation pathway is carbonyl reduction, which leads to the phase I metabolite, reduced nabumetone. 2. The aim of this study is the determination of the role of a particular human liver subcellular fraction in the nabumetone reduction and the identification of participating carbonyl reducing enzymes along with their stereospecificities. 3. Both subcellular fractions take part in the carbonyl reduction of nabumetone and the reduction is at least in vitro the main biotransformation pathway. The activities of eight cytosolic carbonyl reducing enzymes – CBR1, CBR3, AKR1B1, AKR1B10, AKR1C1-4 – toward nabumetone were tested. Except for CBR3, all tested reductases transform nabumetone to its reduced metabolite. AKR1C4 and AKR1C3 have the highest intrinsic clearances. 4. The stereospecificity of the majority of the tested enzymes is shifted to the production of an (+)-enantiomer of reduced nabumetone; only AKR1C1 and AKR1C4 produce predominantly an (−)-enantiomer. This project provides for the first time evidence that seven specific carbonyl reducing enzymes participate in nabumetone metabolism.

Antifungal 3,5-disubstituted furanones: From 5-acyloxymethyl to 5-alkylidene derivatives

5-Acetoxymethyl-3-(4-bromophenyl)-2,5-dihydrofuran-2-one previously described as highly antifungally active was found to provide the corresponding 5-methylene derivative via an unusual DMSO-promoted elimination of the ester group at C5 under antifungal assay conditions. Since the latter possessed nearly the same antifungal effect as that originally reported for the former, the 5-acetoxymethyl furanone just served as a precursor of the actual antifungally active species. A few series of compounds with alkyloxy, aryloxy and alkylidene substituents at C5 of the parent furanone structure were therefore prepared and evaluated. In line with the ease of elimination of the substituent from C5, low activities of the 5-alkoxy compounds were observed. On the other hand, their 5-aryloxymethyl congeners were found to be capable of liberating the antifungally active 5-methylene furanone into the testing medium. The antifungal effect of the 5-alkylidene derivatives was highly sensitive to substitution of the alkylidene moiety; a substituent in the allylic position was necessary for a compound to retain high activity. Parallel evaluation of cytostatic activity showed moderate activities of the antifungally active derivatives against HeLa S3 and CCRF-CEM lines. Cell cycle analysis of CCRF-CEM cells following the treatment with 5-methylene-3-(4-bromophenyl)-2,5-dihydrofuran-2-one revealed that this compound is a necrotic agent.

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

Chromatographic characterization of in vitro metabolites of 5-[2-(N,N-dimethylamino)ethoxy]-7-oxo-7H-benzo-[c]fluorene

Detection reactions and RF values in thin-layer chromatography on silica gel were studied for the antineoplastic drug Ih (benfluron) and related substances. On incubation of Ih with homogenate fractions of mammalian livers the N-oxide Ii and 5-[2-(N,N-dimethylamino)ethoxy]-7-hydroxy-7 H-benzo[c]fluorene (IIh) were established as products, and 5-[2-(N-methylamino)ethoxy]-7-oxo-7 H-benzo[c]fluorene (Ig), 5-[2-(N-methylamino)ethoxy]-7-hydroxy-7 H-benzo[c]fluorene (IIg) and a phenolic product of benfluron (IV) were tentatively identified.

LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT

AbstractThe iron chelator, 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone (Bp4eT), was identified as a lead compound of the 2-benzoylpyridine thiosemicarbazone series, which were designed as potential anti-cancer agents. This ligand has been shown to possess potent anti-proliferative activity with a highly selective mechanism of action. However, further progress in the development of this compound requires data regarding its metabolism in mammals. The aim of this study was to identify the main in vitro and in vivo phase I metabolites of Bp4eT using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two metabolites were detected after incubation of this drug with rat and human liver microsomal fractions. Based on LC-MSn analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N3-ethyl-N1-[phenyl(pyridin-2-yl)methylene]formamidrazone, with both resulting from the oxidation of the thiocarbonyl group. The identity of these metabolites was further shown by LC-MS/MS analysis of these latter compounds which were prepared by oxidation of Bp4eT with hydrogen peroxide and their structures confirmed by nuclear magnetic resonance and infrared spectra. Both the semicarbazone and the amidrazone metabolites were detected in plasma, urine, and feces after i.v. administration of Bp4eT to rats. In addition, another metabolite that could correspond to hydroxylated amidrazone was found in vivo. Thus, oxidative pathways play a major role in the phase I metabolism of this promising anti-tumor agent. The outcomes of this study will be further utilized for: (1) the development and validation of the analytical method for the quantification of Bp4eT and its metabolites in biological materials; (2) to design pharmacokinetic experiments; and to (3) evaluate the potential contribution of the individual metabolites to the pharmacodynamics/toxico-dynamics of this novel anti-proliferative agent. Figurea LC-MS chromatogram of the analysis of the sample from in vivo experiment, b proposed metabolic pathway of Bp4eT and c MS/MS fragmentation of the parent compound and metabolites M1-M3

Study of the biotransformation of a potential benzo[c]fluorene antineoplastic using high-performance liquid chromatography with high-speed-scanning ultraviolet detection

As the sum of benfluron metabolites found was only a part of the total amount applied, a search for undiscovered metabolites was undertaken in the extracts from isolated rat hepatocytes and in the bile and perfusate in the experiments with an isolated perfused rat liver. To identify the metabolites, high-performance liquid chromatography with UV spectral analysis was used, as benfluron derivatives exhibit characteristic absorption spectra. Administration of known metabolites to experimental animals and selective induction of certain metabolic pathways led to the finding of new metabolites and of the respective conjugates. Fast atom bombardment-mass spectrometry analysis was used to identify the newly found metabolites and conjugates.