Metabonomic evaluation of idiosyncrasy-like liver injury in rats cotreated with ranitidine and lipopolysaccharide.

Idiosyncratic liver injury occurs in a small fraction of people on certain drug regimens. The cause of idiosyncratic hepatotoxicity is not known; however, it has been proposed that environmental factors such as concurrent inflammation initiated by bacterial lipopolysaccharide (LPS) increase an individual's susceptibility to drug toxicity. Ranitidine (RAN), a histamine-2 receptor antagonist, causes idiosyncratic liver injury in humans. In a previous report, idiosyncrasy-like liver toxicity was created in rats by cotreating them with LPS and RAN. In the present study, the ability of metabonomic techniques to distinguish animals cotreated with LPS and RAN from those treated with each agent individually was investigated. Rats were treated with LPS or its vehicle and with RAN or its vehicle, and urine was collected for nuclear magnetic resonance (NMR)- and mass spectroscopy-based metabonomic analyses. Blood and liver samples were also collected to compare metabonomic results with clinical chemistry and histopathology. NMR metabonomic analysis indicated changes in the pattern of metabolites consistent with liver damage that occurred only in the LPS/RAN cotreated group. Principal component analysis of urine spectra by either NMR or mass spectroscopy produced a clear separation of the rats treated with LPS/RAN from the other three groups. Clinical chemistry (serum alanine aminotransferase and aspartate aminotransferase activities) and histopathology corroborated these results. These findings support the potential use of a noninvasive metabonomic approach to identify drug candidates with potential to cause idiosyncratic liver toxicity with inflammagen coexposure.

Disposition and pharmacokinetics of L-N6-(1-iminoethyl)lysine-5-tetrazole-amide, a selective iNOS inhibitor, in rats.

The metabolism, pharmacokinetics, tissue distribution, and excretion of L-N6-(1-iminoethyl)lysine-5-tetrazole-amide (L-NIL-TA), a selective inducible NO synthase (iNOS) inhibitor, were investigated in rats. [(14)C]L-NIL-TA is extensively metabolized after either oral or IV administration with a minor amount (<1%) excreted as the prodrug. L-NIL-TA is metabolized via a single hydrolysis pathway to form the active drug, L-N6-(1-iminoethyl)lysine (L-NIL). The oxidative deamination of 2-amino group of L-NIL forms a 2-keto metabolite (M5), which further loses carbon dioxide to yield a carboxylic acid metabolite (M6). Acetylation of L-NIL and M5 resulted in the formations of metabolites M7 and M4, respectively. Complete recovery of the radioactive dose was achieved after either oral (91.2% in urine and 4.66% in feces) and IV (99.3% in urine and 5.11% in feces) administration. L-NIL-TA-related material was extensively distributed to the tissues, with the highest concentration of radioactivity being found in muscle. Maximal concentration of radioactivity was reached between 0.5 and 1 h post-dose in the majority of tissues, with the exception of muscle and skin where the maximal concentrations were achieved at 8 h post-dose.

Collision-induced dissociation of valdecoxib metabolites: a novel rearrangement involving an isoxazole ring.

Valdecoxib is a potent COX-2 inhibitor. During metabolism studies of valdecoxib by liquid chromatography/tandem mass spectrometry, we observed a novel mass spectral rearrangement involving an isoxazole ring for some of the metabolites in the negative ion mode. Accurate mass measurements were performed with quadrupole time-of-flight mass spectrometry to determine the elemental compositions of the fragments. Additionally, two types of stable-isotope labeled analogues were prepared to assist with the assignments of these fragments and possible mechanistic rearrangements resulting from collision-induced dissociation (CID). Detailed analyses of the CID mass spectra suggest that the fragmentation process involves a novel two-step rearrangement. The first step consists of an intramolecular SN2 reaction with a five-membered ring rearrangement to form an intermediate. The second step involves a four-membered ring intramolecular rearrangement followed by a cleavage of the N-O bond on the isoxazole ring to form a unique fragment ion at m/z 196. The same phenomenon was observed for a group of structurally related metabolites that also contain a 5-hydroxymethyl or 5-carboxylic acid moieties. A mechanism for the novel rearrangement involving an isoxazole ring is proposed.

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma.

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.

Involvement of human UGT2B7 and 2B15 in rofecoxib metabolism.

O-Glucuronidation of 5-hydroxyrofecoxib is the major biotransformation pathway of rofecoxib in human, rat, and dog. The glucuronide conjugate is also involved in the reversible metabolism of rofecoxib in rat and human. Atypical bimodal phenomena were observed in their plasma concentration-time curves with a large variability among different human subjects. It is unclear which family members of human UDP-glucuronosyltransferases (UGT) are involved in the formation of the glucuronide. O-Glucuronidation of 5-hydroxyrofecoxib by human liver microsomes and eight cDNA-expressed human UGT isoforms were investigated. Human liver microsomes formed 5-hydroxyrofecoxib glucuronide with apparent V(max) value of 1736 pmol/min/mg of protein and K(m) value of 44.2 microM. Eight individual cDNA-expressed human UGT isozymes (1A1, 1A3, 1A4, 1A6, 1A8, 1A9, 2B7, and 2B15) were evaluated for glucuronidation of 5-hydroxyrofecoxib. Among them UGT2B15 exhibited the highest metabolism rate with apparent V(max) value of 286 pmol/min/mg of protein and K(m) value of 16.1 microM, whereas UGT2B7 showed apparent V(max) value of 47.1 pmol/min/mg of protein and K(m) value of 41.6 microM. These results indicated that human UGT2B15 has the highest level of activity for catalyzing the glucuronidation of 5-hydroxyrofecoxib. Because polymorphisms have been identified in human UGT2B7, 2B15 genes and O-glucuronidation of 5-hydroxyrofecoxib plays a major role in biotransformation of rofecoxib, it is possible that human UGT2B7 and 2B15 polymorphisms for O-glucuronidation of 5-hydroxyrofecoxib are responsible for the high variability in bimodal patterns in human plasma concentration-time curves.

Development and validation of a liquid chromatography-tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma.

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.

Pharmacokinetics and metabolism of a COX-2 inhibitor, valdecoxib, in mice.

The pharmacokinetics and metabolism of valdecoxib, a potent cyclooxygenase-2 selective inhibitor, were investigated in mice. Valdecoxib was extensively metabolized after a single 5 mg/kg oral administration of [(14)C]valdecoxib and elimination of unchanged drug was minor (less than 1%) in male and female mice. The total mean percentage of administered radioactive dose recovered was 99.8% in the male mice and 94.7% in the female mice. Sixteen metabolites were identified in mouse plasma, red blood cells, urine, and feces. The main phase I metabolic pathway of valdecoxib in mice involved the oxidation of the 5-methyl group to form the active hydroxymethyl metabolite M1. M1 was further oxidized to the carboxylic acid metabolite M4, which underwent opening of the isoxazole ring to form M6 and M13. Phase II metabolism included glucuronide, glucoside, and methyl sulfone conjugations. M1 was also conjugated with glucuronic acid and glucose to yield M-G and M1-glucose, respectively. Three novel methylsulfone conjugates M20, M21, and M21-G were detected in blood or urine. Valdecoxib and M1 were the major radioactive components in plasma and red blood cells. The plasma area under the curve from zero to infinity (AUC(0-infinity)) values for valdecoxib and M1 were 3.58 and 0.850 microg. h/ml in males and 2.08 and 1.63 microg. h/ml in females, respectively. The RBC AUC(0-infinity) values for valdecoxib and M1 were 12.1 and 22.6 microg. h/g in males and 6.42 and 35.2 microg. h/g in females, respectively.

Contemporary issues in toxicology the role of metabonomics in toxicology and its evaluation by the COMET project.

The role that metabonomics has in the evaluation of xenobiotic toxicity studies is presented here together with a brief summary of published studies. To provide a comprehensive assessment of this approach, the Consortium for Metabonomic Toxicology (COMET) has been formed between six pharmaceutical companies and Imperial College of Science, Technology and Medicine (IC), London, UK. The objective of this group is to define methodologies and to apply metabonomic data generated using (1)H NMR spectroscopy of urine and blood serum for preclinical toxicological screening of candidate drugs. This is being achieved by generating databases of results for a wide range of model toxins which serve as the raw material for computer-based expert systems for toxicity prediction. The project progress on the generation of comprehensive metabonomic databases and multivariate statistical models for prediction of toxicity, initially for liver and kidney toxicity in the rat and mouse, is reported. Additionally, both the analytical and biological variation which might arise through the use of metabonomics has been evaluated. An evaluation of intersite NMR analytical reproducibility has revealed a high degree of robustness. Second, a detailed comparison has been made of the ability of the six companies to provide consistent urine and serum samples using a study of the toxicity of hydrazine at two doses in the male rat, this study showing a high degree of consistency between samples from the various companies in terms of spectral patterns and biochemical composition. Differences between samples from the various companies were small compared to the biochemical effects of the toxin. A metabonomic model has been constructed for urine from control rats, enabling identification of outlier samples and the metabolic reasons for the deviation. Building on this success, and with the completion of studies on approximately 80 model toxins, first expert systems for prediction of liver and kidney toxicity have been generated.

Use of surface plasmon resonance biosensor technology as a possible alternative to detect differences in binding of enantiomeric drug compounds to immobilized albumins.

The use of biosensors for monitoring real time interactions between biomolecules and drug compounds has a lot of advantages over presently existing detection methods, the major ones being the elimination of radio labels and rapid screening. We can also obtain information about the kinetic parameters and these values may serve as useful indicators towards subtle differences in the binding strength and characteristics of closely related drug compounds and enantiomers. The Biacore 3000 biosensor based on the Surface Plasmon Resonance (SPR) technology was used to assess the albumin protein binding differences between two enantiomers of a drug compound. Normalized responses (NRU) and affinity constants (K(D)) were readily calculated. Statistical parameters like mean normalized responses, %CV values were determined to make the technique robust. The %CV values obtained were within the preset limits of < or = 25% (FDA limits for drug development and method validation protocols) for the binding interactions for majority of the concentrations studied. For example, the %CV values for the normalized responses for the binding of the control drug warfarin to human albumin ranged from 7.9 to 24.3%. The method gave reproducible results, and the results indicated slight differences in binding patterns of the enantiomers to human and rat albumin.

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine.

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry.

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.

Use of optical biosensor technology to study immunological cross-reactivity between different sulfonamide drugs.

Adverse reactions to medications account for a substantial number of hospitalizations and in some cases fatalities. The nature of the many drug-drug interactions caused by the inhibition of drug-metabolizing enzymes can now be predicted and examined with a greater deal of accuracy due to research developments in the understanding of the drug-metabolizing enzymes. However, the more troubling aspects of drug-drug interactions are the idiosyncratic reactions that are unpredictable and quite often life-threatening. These reactions are often caused by a prior sensitization of a person's immune system to a given drug or class of drugs. The following work offers a technique to examine in a medium-throughput system the cross-reactivity of drugs to antibodies in order to predict if structures share the same antigenic potential toward a sensitized individual. Two commercially important sulfonamide drugs, sulfamethazine and furosemide, were taken and their binding to their respective antibodies were tested in the presence of other structurally related sulfonamide drugs. The BIACORE 3000 biosensor was used for the study and the solution-phase equilibrium assay principle was employed. The data obtained help us determine which drugs can react, and to what extent, with sulfamethazine and furosemide, giving rise to possible allergic or hypersensitivity reactions. Though sulfamethazine and furosemide were used in this study; this principle and methodology can be applied to study any drug molecule-antibody pair.