Gut microbiota metabolizes nabumetone in vitro: Consequences for its bioavailability in vivo in the rodents with altered gut microbiome.

1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo, we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo, none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t1/2 in rats with reduced microbiota and germ-free mice.

The Importance of Wireless Capsule Endoscopy for Research into the Intestin al Absorption Window of 5-Aminosalicylic Acid in Experimental Pigs.

BACKGROUND: Absorption windows in particular segments of the small intestine can contribute to the development of orally administered drug formulations and can limit the bioavailability of released compounds. OBJECTIVE: The aim of this study was to evaluate use of wireless capsule enteroscopy regarding the disintegration kinetic process of tablets in the small intestine and its comparison with the levels of the model drug (5- aminosalicylic acid; 5-ASA), and its majority metabolite (N-acetyl-5-aminosalicylic acid; N-acetyl-5-ASA) in blood plasma. METHODS: Tablets were endoscopically introduced into the duodenum and their disintegration was monitored using wireless capsule enteroscopy in anaesthetised pigs. In parallel, blood plasma time profiles of the model drug (5-ASA) released from tablets and its metabolite (N-acetyl-5-ASA) were detected. RESULTS: The disintegration of tablets was evident in the proximal jejunum (until the 90-minute mark) and culminated at the 3rd hour. The maximum plasmatic concentration of 5-ASA was reached at the 3rd hour and in the case of its metabolite (N-acetyl-5-ASA) at the 4th hour. CONCLUSION: The study demonstrated the advantage of combination of wireless capsule enteroscopy and bioanalytical determination of pharmacokinetic parameters in an animal experiment to localise the disintegration site of solid dosage form and following kinetics of intestinal absorption of the released active agent.

In vitro metabolism of fenofibric acid by carbonyl reducing enzymes.

Fenofibric acid is a hypolipidemic drug that is used as an active ingredient per se or is administered in the form of fenofibrate that releases fenofibric acid after absorption. The metabolism of fenofibric acid is mediated primarily by glucuronidation. However, the other part of fenofibric acid is excreted as reduced fenofibric acid. Enzymes responsible for the formation of reduced fenofibric acid as well as their subcellular localization have remained unknown until now. We have found that the predominant site of fenofibric acid reduction is the human liver cytosol, whereas liver microsomes reduced fenofibric acid to a lower extent and exhibited a lower affinity for this drug (Km > 1000 µM). Of nine carbonyl-reducing enzymes (CREs) tested, CBR1 exhibited the greatest activity for fenofibric acid reduction (CLint = 85.975 µl/mg protein/min). CBR1 predominantly formed (-)-enantiomers of reduced fenofibric acid similar to liver cytosol and in accordance with the in vivo data. AKR1C1, AKR1C2, AKR1C3 and AKR1B1 were also identified as reductases of fenofibric acid but are expected to play only a minor role in fenofibric acid metabolism.

Identification of combined conjugation of nabumetone phase I metabolites with glucuronic acid and glycine in minipig biotransformation using coupling high-performance liquid chromatography with electrospray ionization mass spectrometry.

High-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) was applied for the analysis of nabumetone metabolites during the biotransformation in minipigs. In addition to known phase I metabolites, the identification of phase II metabolites was achieved on the basis of their full-scan mass spectra and subsequent MS(n) analysis using both positive-ion and negative-ion ESI mode. Some phase I metabolites are conjugated with both glucuronide acid and glycine, which is quite unusual type of phase II metabolite not presented so far for nabumetone. These metabolites were found in small intestine content, but they were absent in minipigs urine.

Simultaneous determination of the novel thiosemicarbazone anti-cancer agent, Bp4eT, and its main phase I metabolites in plasma: application to a pilot pharmacokinetic study in rats.

Novel thiosemicarbazone metal chelators are extensively studied anti-cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC-MS/MS method for the simultaneous quantification of 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1-E and M1-Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C18 column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid-phase extraction on 96-well plates. This method was validated over the concentration range of 0.18-2.80 µM for Bp4eT, 0.02-0.37 µM for both M1-E and M1-Z, and 0.10-1.60 µM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration-time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti-cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class.

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.

Analytical power of LLE-HPLC-PDA-MS/MS in drug metabolism studies: identification of new nabumetone metabolites.

Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date. In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE-HPLC-PDA and achiral LLE-UHPLC-MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC-MS/MS experiments detected the presence of a structure corresponding to elemental composition C15H16O3, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite. To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE-HPLC-PDA-MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone→3-hydroxy nabumetone→6-MNA).

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.

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

The 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-MS(n) analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N (3)-ethyl-N (1)-[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.

HPLC determination of arginases inhibitor N-(ω)-hydroxy-nor-L-arginine using core-shell particle column and LC-MS/MS identification of principal metabolite in rat plasma.

For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-L-arginine, L-arginine and N-(ω)-ethyl-L-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core-shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH=3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450 nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-L-arginine (i.v. 10 mg kg(-1)). The metabolite was identified as nor-L-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.