Zbyněk Svoboda

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.

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.

High-performance liquid chromatographic determination of tiapride and its phase I metabolite in blood plasma using tandem UV photodiode-array and fluorescence detection.

New bioanalytical SPE-HPLC-PDA-FL method for the determination of the neuroleptic drug tiapride and its N-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals. The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 mm × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile-0.01 M phosphate buffer pH=3, flow rate 1 ml min(-1)) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array→fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride and N-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emiss.)=232 nm/334 nm), high concentrations (500-6000 pmol ml(-1)) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 min. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol ml(-1) (10.11 pmol ml(-1)). The recoveries of tiapride ranged from 89.3 to 94.3%, 81.7 to 86.8% for internal standard sulpiride and 90.9 to 91.8% for N-desethyl tiapride.

Cyclosporine A inhibits acetylcholinesterase activity in selected parts of the rat brain

Cyclosporine A (CsA) is the major immunosuppressive drug used for organ and neural transplantation and the therapy of selected autoimmune diseases. We investigated the effect of CsA on the activity of acetylcholinesterase (AChE) in the frontal cortex, hippocampus, septum, and basal ganglia. AChE was determined spectrophotometrically with acetylthiocholine as substrate and 5,5-bis-2-nitrobenzoic acid as chromogen. CsA was administered in single doses of 20 or 45 mg/kg perorally; in the case of the higher dose we also performed a repeated administration of CsA in three consecutive doses separated by 24 h intervals. Both lower and higher doses of CsA decreased AChE activity in the frontal cortex and hippocampus to practically the same extent. On the contrary, AChE activity was more diminished in the case of the higher dose of CsA used in the septum and basal ganglia. Repeated administration of the higher dose of CsA did not lead, with the exception of the hippocampus, to a further decrease in AChE activity in the brain structures observed. These findings contribute to rare evidence concerning the interaction of CsA and the cholinergic system in the brain.

Transintestinal transport mechanisms of 5-aminosalicylic acid (in situ rat intestine perfusion, Caco-2 cells) and Biopharmaceutics Classification System.

The aim of the study was 1) to estimate permeability of 5-aminosalicylic acid (5-ASA), 2) to categorize 5-ASA according to BCS (Biopharmaceutics Classification System), and 3) to contribute to determination of 5-ASA transintestinal transport and biotransformation mechanisms. The in situ rat intestine perfusion was used as an initial method to study 5-ASA transport. The amount of 5-ASA (released from tablet) transferred into portal circulation reached 5.79 ± 0.24%. During this transport, the intestinal formation of 5-ASA main metabolite (N-ac-5-ASA) occurred. N-ac-5-ASA was found in perfusate both from intestinal lumen and from v. portae. In in vitro Caco-2 monolayers, transport of 5-ASA (10-1000 µmol/l) was studied in apical-basolateral and basolateral-apical direction (iso-pH 7.4 conditions). The transport of total 5-ASA (parent drug plus intracellularly formed N-ac-5-ASA) was linear with time, concentration- and direction-dependent. Higher basolateral-apical (secretory) transport was mainly caused by higher transport of the metabolite (suggesting metabolite efflux transport). Transport of 5-ASA (only parent drug) was saturable (transepithelial carrier-mediated) at low doses, dominated by passive, paracellular process in higher doses which was confirmed by increased 5-ASA transport using Ca2+-free transport medium. The estimated low 5-ASA permeability and its low solubility enable to classify 5-ASA as BCS class IV.

The effect of probiotic Escherichia coli strain Nissle 1917 lipopolysaccharide on the 5-aminosalicylic acid transepithelial transport across Caco-2 cell monolayers.

The object of this study was to investigate the effect of probiotic Escherichia coli strain Nissle 1917 (EcN) (i) EcN lipopolysaccharide (EcN LPS) and (ii) bacteria-free supernatant of EcN suspension (EcN supernatant) on in vitro transepithelial transport of mesalazine (5-aminosalicylic acid, 5-ASA), the most commonly prescribed anti-inflammatory drug in inflammatory bowel disease (IBD). Effect of co-administered EcN LPS (100 µg/ml) or EcN supernatant (50 µg/ml) on the 5-ASA transport (300 µmol/l) was studied using the Caco-2 monolayer (a human colon carcinoma cell line) as a model of human intestinal absorption. Permeability characteristics for absorptive and secretory transport of parent drug and its intracellularly-formed metabolite were determined. The quantification of 5-ASA and its main metabolite N-acetyl-5-amino-salicylic acid (N-Ac-5-ASA) was performed by high performance liquid chromatography. Obtained results suggest that neither EcN LPS nor EcN supernatant had effect on the total 5-ASA transport (secretory flux greater than absorptive flux) and on the transport of intracellularly formed N-Ac-5-ASA (preferentially transported in the secretory direction). The percent cumulative transport of the total 5-ASA alone or in combination with EcN LPS or EcN supernatant did not exceed 1%.