Pharmacometabonomic investigation of dynamic metabolic phenotypes associated with variability in response to galactosamine hepatotoxicity.

Galactosamine (galN) is widely used as an in vivo model of acute liver injury. We have applied an integrative approach, combining histopathology, clinical chemistry, cytokine analysis, and nuclear magnetic resonance (NMR) spectroscopic metabolic profiling of biofluids and tissues, to study variability in response to galactosamine following successive dosing. On re-challenge with galN, primary non-responders displayed galN-induced hepatotoxicity (induced response), whereas primary responders exhibited a less marked response (adaptive response). A systems-level metabonomic approach enabled simultaneous characterization of the xenobiotic and endogenous metabolic perturbations associated with the different response phenotypes. Elevated serum cytokines were identified and correlated with hepatic metabolic profiles to further investigate the inflammatory response to galN. The presence of urinary N-acetylglucosamine (glcNAc) correlated with toxicological outcome and reflected the dynamic shift from a resistant to a sensitive phenotype (induced response). In addition, the urinary level of glcNAc and hepatic level of UDP-N-acetylhexosamines reflected an adaptive response to galN. The unique observation of galN-pyrazines and altered gut microbial metabolites in fecal profiles of non-responders suggested that gut microfloral metabolism was associated with toxic outcome. Pharmacometabonomic modeling of predose urinary and fecal NMR spectroscopic profiles revealed a diverse panel of metabolites that classified the dynamic shift between a resistant and sensitive phenotype. This integrative pharmacometabonomic approach has been demonstrated for a model toxin; however, it is equally applicable to xenobiotic interventions that are associated with wide variation in efficacy or toxicity and, in particular, for prediction of susceptibility to toxicity.

Intra- and interlaboratory reproducibility of ultra performance liquid chromatography-time-of-flight mass spectrometry for urinary metabolic profiling.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a major platform in metabolic profiling but has not yet been comprehensively assessed as to its repeatability and reproducibility across multiple spectrometers and laboratories. Here we report results of a large interlaboratory reproducibility study of ultra performance (UP) LC-MS of human urine. A total of 14 stable isotope labeled standard compounds were spiked into a pooled human urine sample, which was subject to a 2- to 16-fold dilution series and run by UPLC coupled to time-of-flight MS at three different laboratories all using the same platform. In each lab, identical samples were run in two phases, separated by at least 1 week, to assess between-day reproducibility. Overall, platform reproducibility was good with median mass accuracies below 12 ppm, median retention time drifts of less than 0.73 s and coefficients of variation of intensity of less than 18% across laboratories and ionization modes. We found that the intensity response was highly linear within each run, with a median R(2) of 0.95 and 0.93 in positive and negative ionization modes. Between-day reproducibility was also high with a mean R(2) of 0.93 for a linear relationship between the intensities of ions recorded in the two phases across the laboratories and modes. Most importantly, between-lab reproducibility was excellent with median R(2) values of 0.96 and 0.98 for positive and negative ionization modes, respectively, across all pairs of laboratories. Interestingly, the three laboratories observed different amounts of adduct formation, but this did not appear to be related to reproducibility observed in each laboratory. These studies show that UPLC-MS is fit for the purpose of targeted urinary metabolite analysis but that care must be taken to optimize laboratory systems for quantitative detection due to variable adduct formation over many compound classes.

[In silico, in vitro, in omic experimental models and drug safety evaluation]. / La place des méthodes in silico, in vitro, in omic dans l'évaluation de la sécurité des médicaments.

Over the last few decades, toxicology has benefited from scientific, technical, and bioinformatic developments relating to patient safety assessment during clinical and drug marketing studies. Based on this knowledge, new in silico, in vitro, and "omic" experimental models are emerging. Although these models cannot currently replace classic safety evaluations performed on laboratory animals, they allow compounds with unacceptable toxicity to be rejected in the early stages of drug development, thereby reducing the number of laboratory animals needed. In addition, because these models are particularly adapted to mechanistic studies, they can help to improve the relevance of the data obtained, thus enabling better prevention and screening of the adverse effects that may occur in humans. Much progress remains to be done, especially in the field of validation. Nevertheless, current efforts by industrial, academic laboratories, and regulatory agencies should, in coming years, significantly improve preclinical drug safety evaluation thanks to the integration of these new methods into the drug research and development process.

Value of in vitro models for the assessment of drug-induced haematotoxicity.

BACKGROUND: A new antipsychotic compound induced unexpected red cell hypoplasia (reticulocytopenia, red marrow hypoplasia) in rats dosed orally for 7 days. MATERIALS AND METHODS: Since an erythropoietin-mediated pathogenesis was excluded, in vitro tests on rat and human bone marrow cells were performed with measurement of formation of late erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units after incubation with the drug. CFU-E together with growth factors were cultured for 2 days (rat) or 7 days (human) and CFU-GM was cultured for 7 days (rat) or 10 days (human). RESULTS: The drug induced inhibition of erythroid progenitors and myeloid progenitors for both species from 3 x 10(-5) mol/L, with the concentration inhibiting the growth of 50% (IC50) consistent with drug plasma levels measured in rats. CONCLUSION: These cloning assays on rat bone-marrow cells were shown to be adequate models for determining the haematotoxicity of the agent and to be predictive of human toxicity. With only a small amount of compound required, they can be used as screening tools to detect haematotoxic potential of candidate drugs.