Jaroslav Květina

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.

Caco-2 cells, biopharmaceutics classification system (BCS) and biowaiver.

Almost all orally administered drugs are absorbed across the intestinal mucosa. The Caco-2 monolayers are used as an in vitro model to predict drug absorption in humans and to explore mechanism of drug absorption. The Caco-2 cells are derived from a human colon adenocarcinoma and spontaneously differentiate to form confluent monolayer of polarized cells structurally and functionally resembling the small intestinal epithelium. For studying drug permeability, Caco-2 cells are seeded onto the Transwell inserts with semipermeable membrane and grown to late confluence (21 days). After determination of cell viability, the integrity of monolayer is checked by phenol red permeability and by 14C-mannitol permeability. The transport from apical to basolateral (AP-BL) and basolateral to apical (BL-AP) is studied by adding the diluted drug on the apical or basolateral side and withdrawing the samples from the opposite compartment, respectively, for HPLC analysis or liquid scintillation spectrometry. Ca2+ -free transport medium is used to determine paracellular component of the drug transport. On the basis of permeability and solubility, drugs can be categorized into four classes of Biopharmaceutics Classification System (BCS). For certain drugs, the BCS-based biowaiver approach can be used which enables to reduce in vivo bioequivalence studies.

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.

Determination of enzyme (angiotensin convertase) inhibitors based on enzymatic reaction followed by HPLC

For determination of levels of plasmatic inhibitor of ACE (angiotensin convertase) a simple method was used based on a combination of enzymatic reaction followed by an HPLC determination of its product. The inhibitor (e.g. enalaprilat) was at first separated from the biological material by deproteination (methanol). Then, an aliquot of the sample was added to the reaction mixture containing a commercial ACE enzyme, its specific substrate FAPGG (N-(3-[2-furyl]acryloyl)-Phe-Gly-Gly) and buffer (Tris--HCl, pH 7.5). Degree of inhibition of the conversion of this substrate to FAP (desGlyGlyFAPGG) by the inhibitor present in the sample is related to its amount by a simple dose-response relationship. The amount of the FAP was determined by an HPLC on a RP-18 column with an acetonitril--nonylamine buffer (pH 2.4, adjusted with phosphoric acid) as a mobile phase with detection at 305 nm. Alternatively, the activity of the endogenous ACE present in the plasma was measured. The substrate FAPGG was added to the plasmatic sample containing both the inhibitor and endogenous ACE (as the sample was not deproteinized in this case) and the reaction product was determined as above. Inhibitor concentration has been obtained from a dose--response curve expressing the interaction with inhibitor with an ACE enzyme.

Pharmacokinetics and plasma protein binding of two platinum cytostatics CHIP and CBDCA in rats.

SummaryPlasma protein binding and pharmacokinetic parameters of CHIP (cis-dichloro-trans-dihydroxy-bisisopropylamine platinum IV) and CBDCA (cis-diammine-1,1-cyclobutane dicarboxylate platinum II) were investigated in male Wistar rats. The plasma clearance of total and non-protein-bound platinum was determined and compared with that of 99mTc-DTPA. for binding experiments, a novel, simple, and quick method based on adsorption of non-protein-bound platinum species to charcoal was used. The clearance of total platinum after CHIP and CBDCA administration was markedly lower than the glomerular filtration rate (determined as the clearance of 99mTc-DTPA). The renal clearance of non-proteinbound platinum corresponded to 168% and 50% of the glomerular filtration rate for CHIP and CBDCA, respectively. These studies suggested that CHIP was excreted by the rat kidney.

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

Preclinical electrogastrography in experimental pigs

Preclinical electrogastrography in experimental pigs Surface electrogastrography (EGG) is a non-invasive means of recording gastric myoelectric activity or slow waves from cutaneous leads placed over the stomach. This paper provides a comprehensive review of preclinical EGG. Our group recently set up and worked out the methods for EGG in experimental pigs. We gained our initial experience in the use of EGG in assessment of porcine gastric myoelectric activity after volume challenge and after intragastric administration of itopride and erythromycin. The mean dominant frequency in pigs is comparable with that found in humans. EGG in experimental pigs is feasible. Experimental EGG is an important basis for further preclinical projects in pharmacology and toxicology.

Plasma protein binding-lipophilicity relationships: interspecies comparison of some organic acids

Relationships between plasma protein binding of 11 organic acids (benzoic and phenylacetic acid derivatives) and their lipophilicity were studied in man, rabbits, rats and mice. For description, the relationship fu = 1/(1 + aDb) was developed, where fu is the fraction of the unbound drug in plasma, D is the partition coefficient octanol/water and a and b are parameters. While the value of the parameter a is widely different in interspecies comparison, the value of the parameter b is very close in all species studied and is approximately equal to 1. The model used allows the simple calculation of the extent of plasma binding of structurally similar drugs from their lipophilicity, or conversion of the extent of plasma binding from one species to another.

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.

The effect of general anaesthesia on gastric myoelectric activity in experimental pigs

BackgroundSurface electrogastrography (EGG) is a non-invasive method for clinical assessment of gastric myoelectrical activity. Different forms of general anaesthesia might have various effects on porcine EGG. The aim of this study was to evaluate the impact of different anaesthetic agents on EGG in experimental pigs.MethodsFour 15-minute EGG intervals were recorded and analysed. A baseline EGG recording was started 20 minutes after intramuscular injection of ketamine and azaperone (periods A and B). Four different regimens of general anaesthesia followed immediately after the baseline EGG (5 pigs in each experimental group): thiopental, isoflurane, nitrous oxide and isoflurane plus nitrous oxide. EGG recordings followed for the next 30 minutes under general anaesthesia (periods C and D). The dominant frequencies of slow waves were compared between the baseline intervals A and B and periods C and D under general anaesthesia.ResultsThe mean dominant frequency was within the normal range (2.3 – 3.5 cycles per minute) in all animals in all regimens. Thiopental general anaesthesia did not influence any change of the dominant frequency of slow waves. Nitrous oxide general anaesthesia increased the dominant frequency of slow waves in a statistically significant manner (baseline: 2.93 ± 0.53 and 3.01 ± 0.53; under general anaesthesia: 3.25 ± 0.34 and 3.29 ± 0.38 cycles per minute; p < 0.001, p = 0.003, p < 0.001, p < 0.001). Nitrous oxide together with isoflurane induced a statistically significant decrease of dominant frequency in the last 15-minute interval (2.66 ± 0.55 cycles per minute) compared to the baseline recording (2.81 ± 0.49; p = 0.030).ConclusionsAll changes of porcine gastric myoelectric activity assessed by the dominant frequency of slow waves during EGG remained within the normal range although some of them achieved statistical significance. Thus all tested agents used for general anaesthesia can be recommended in preclinical studies with porcine models focused on gastric myoelectric activity without any risk of compromising the results. Thiopental seems to be the most suitable as it did not cause any changes at all.