ABSTRACT
AZD9708 is a new chemical entity with selective and long-acting ß2-agonistic properties currently being evaluated by AstraZeneca for potential use in treatment of respiratory diseases by the inhaled route. As part of the toxicological characterisation of this compound, an increased incidence of micronucleated immature erythrocytes (MIEs) was seen in the bone marrow of rats following single intravenous doses near the maximum tolerated. This effect was seen in the absence of in vitro genotoxicity in bacterial and mammalian cells and no consistent evidence of in vivo DNA damage in the the bone marrow or liver using the comet assay was observed. Because of the lack of signals for mutagenic potential, combined with the observation that MIE frequencies appeared to be increased in only some of the rats and the clearest response was seen at the intermediate dose, it was hypothesised that the effect was secondary to ß2-adrenergic receptor overstimulation. Because it appears that this has not been previously described for ß2-agonists and because pharmacodynamic/pharmacokinetic factors may influence the response, studies using repeated dosing were performed to investigate whether this would lead to compound-induced tachyphylaxis with tolerance induction and decreased responses indicated by ß2-effect biomarkers. A series of experiments confirmed that a sequence of five escalating daily doses leading to systemic exposure corresponding to that after a single dose led to symptomatic tolerance, declining or diminished effects on plasma biomarkers of ß2-effects (plasma glucose and potassium) and elimination of the micronucleus response. This suggests that the increased MIE frequencies after single doses of AZD9708 are secondary to physiological overstimulation of ß2-adrenergic receptors, not a consequence of genotoxicity.
Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Benzothiazoles/pharmacology , Bone Marrow/drug effects , Micronucleus Tests/methods , beta-Alanine/analogs & derivatives , Animals , Blood Glucose/analysis , Blood Glucose/drug effects , Comet Assay , DNA Damage/drug effects , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Liver/drug effects , Male , Mutagens/toxicity , Potassium/blood , Rats , Rats, Wistar , Receptors, Adrenergic/metabolism , beta-Alanine/pharmacologyABSTRACT
An improved protocol for the radiolabel DNA-binding assay, which gives a high yield of highly pure DNA has been developed by use of mouse lymphoma cells. The critical difference from previously published methods is the use of enzymatic degradation of proteins in the later DNA purification steps rather than during the homogenisation procedure. Different DNA-purification methodologies were first compared and the protocol of choice was optimized later on; both steps were performed with [(35)S]-labelled amino acids for labelling of cellular protein, which enabled both the quantification of cellular protein contaminating the DNA sample and the distinction between cellular and enzyme-derived protein. The assay was later evaluated and shown to give reproducible results based on the data obtained with benzo[a]pyrene (B[a]P) and doxorubicin in two different laboratories. In addition, two further reference compounds, dopamine and diazepam and one proprietary AstraZeneca compound were also tested in mouse lymphoma cells in one laboratory. The two compounds B[a]P and doxorubicin were identified as suitable positive controls for routine testing in the presence and absence of S9, respectively. Exposing 90-100×10(6) cells to (14)C-labelled compound with a molar radioactivity of 2MBq/µmol, yields approximately 500µg DNA with <3% total protein contamination, of which approximately 7% is of cellular origin (<0.2%). The detection level is approximately 2adducts/10(8) dNTP.
Subject(s)
DNA Adducts/biosynthesis , DNA/isolation & purification , DNA/metabolism , Mutagenicity Tests/methods , Mutagens/metabolism , Animals , Biotransformation , Cell Line, Tumor , Isotope Labeling , Leukemia L5178 , Mice , Nucleic Acid Denaturation , Protein Binding , Research DesignABSTRACT
OBJECTIVE: To investigate the possible mechanisms underlying the liver enzyme elevations seen during clinical studies of long-term treatment (>35 days) with ximelagatran, and investigate the usefulness of pre-clinical in vitro systems to predict drug-induced liver effects. METHODS: Ximelagatran and its metabolites were tested for effects on cell viability, mitochondrial function, formation of reactive metabolites and reactive oxygen species, protein binding, and induction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) gene expression or nuclear orphan receptors. Experimental systems included fresh and cryopreserved hepatocytes, human hepatoma cell lines (HepG2 and HuH-7) and subcellular human liver fractions. RESULTS: Loss of cell viability was only seen in HepG2 cells at ximelagatran concentrations 100 microM and in cryopreserved human hepatocytes at 300 microM, while HuH-7 cells were not affected by 24 h exposure at up to 300 microM ximelagatran. Calcium homeostasis was not affected in HepG2 cells exposed to ximelagatran up to 300 microM for 15 min. There was no evidence for the formation of reactive metabolites when cell systems were exposed to ximelagatran. ALT and AST expression in human hepatoma cell lines were also unchanged by ximelagatran. Mitochondrial functions such as respiration, opening of the transition pore, mitochondrial membrane depolarization and beta-oxidation were not affected by ximelagatran or its metabolites. CONCLUSION: Ximelagatran at concentrations considerably higher than that found in plasma following therapeutic dosing had little or no effect on cellular functions studied in vitro. The in vitro studies therefore did not elucidate the mechanism by which ximelagatran induces liver effects in humans, possibly because of limitations in the experimental systems not reflecting characteristics of the human hepatocyte, restricted exposure time, or because the primary mechanism for the observed clinical liver effects is not on the parenchymal liver cell.