Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to aneugens after single-dose applications

Detection of clastogenic compounds in the peripheral blood micronucleus test (MNT) in rats is a well-established methodology. However, the results obtained on the induction of micronuclei by aneugens in rat peripheral blood are controversial. Our aim was a comparative evaluation of the peripheral blood flow cytometry MNT in Wistar Han rat and CD1 mouse exposed to three aneugens (vinblastine, vincristine and colchicine) after single-dose applications. In addition, the same compounds were tested in the rat bone marrow MNT. The treatment with vinblastine (0.25, 0.5, 1, mg/kg), vincristine (0.025, 0.05, 0.1 mg/kg) or colchicine (0.7, 1, 1.3 mg/kg) induced no statistically significant increase in MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) in rat peripheral blood. In rat bone marrow, a clear statistically significant increase in MN-PCE was found with vincristine and vinblastine. However, colchicine showed a clear increase in MN-PCE frequency without reaching statistically significant level only at 1 mg/kg. The positive effect in the bone marrow MNT shows that the target organ was exposed to the appropriate concentration levels of the respective aneugens. In mouse, the peripheral blood flow cytometry analysis after the treatment with vinblastine, vincristine and colchicine showed clear statistically significant increase in MN-PCE with all three compounds. The experiments with splenectomized rats treated with vincristine and colchicine were performed and statistically significant increases in MN-PCE were found with 0.05, 0.1, 0.15 mg/kg of vincristine and 0.7 and 1 mg/kg of colchicine. Our results demonstrate that micronucleated cells induced by aneugens are removed from rat peripheral blood by the spleen due to the large size of micronuclei. Based on our data, it is concluded that the flow cytometry peripheral blood MNT after single-dose applications is an appropriate test system for evaluating the genotoxic effects of aneugens in mice. However, in rats peripheral blood MNT aneugen detection might require multiple-dose applications to overwhelm the spleen effec

Loss of phosphatase activity in myotubularin-related protein 2 is associated with Charcot-Marie-Tooth disease type 4B1

Mutations in the gene encoding myotubularin-related protein 2 (MTMR2) are responsible for autosomal recessive Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a severe hereditary motor and sensory neuropathy characterized by focally folded myelin sheaths and demyelination. MTMR2 belongs to the myotubularin family, which is characterized by the presence of a phosphatase domain. Myotubularin (MTM), the archetype member of this family, is mutated in X-linked myotubular myopathy. Although MTMR2 and MTM are closely related, they are likely to have different functions. Recent studies revealed that MTM dephosphorylates specifically phosphatidylinositol 3-phosphate. Here we analyze the biochemical properties of the mouse Mtmr2 protein, which shares 97% amino acid identity with human MTMR2. We show that phosphatidylinositol-3-phosphate is also a substrate for Mtmr2, but, unlike myotubularin, Mtmr2 dephosphorylates phosphatidylinositol 3,5-bisphosphate with high efficiency and peak activity at neutral pH. We demonstrate that the known disease-associated MTMR2 mutations lead to dramatically reduced phosphatase activity, suggesting that the MTMR2 phosphatase activity is crucial for the proper function of peripheral nerves in CMT4B1. Expression analysis of Mtmr2 suggests particularly high levels in neurons. Thus, the demyelinating neuropathy CMT4B1 might be triggered by the malfunction of neural membrane recycling, membrane trafficking, and/or endocytic or exocytotic processes, combined with altered axon-Schwann cell interactions. Furthermore, the different biochemical properties of MTM and MTMR2 offer a potential explanation for the different human diseases caused by mutations in their respective gene

Bacterial Mutagenicity Screening in the pharmaceutical industry

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed

Automatic Analysis of the Micronucleus Test in Primary Human Lymphocytes Using Image Analysis

The in vitro micronucleus test (MNT) is a well established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and specific parameter. Various automated systems to replace the tedious and time consuming manual slide analysis procedure have been described. Flow cytrometric approaches have been discussed elsewhere. The ROBIAS image analysis system for both automatic cytotoxicity assessment and highly sensitive micronucleus detection in primary human lymphocytes was developed at Novartis, where the assay is used as to confirm positive results obtained in the MNT in TK6 cells which serves as the primary screening system for genotoxicity profiling in early drug development. The comparison of manual with automatic analysis results showed a high degree of concordance for 27 independent experiments conducted for profiling of 12 compounds. For concentration series of Cyclophosphamide (CP) and Carbendazim (MBC), a very good correlation between automatic and manual analysis could be established, both for the relative division index used as cytotoxicity parameter, and for MN scoring in mono- and bi-nucleated cells. Generally, false positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyze 24 slides within 65 hours by fully automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS supporting various assays in genetic toxicology and other biomedical areas

IN VITRO PRIMARY HUMAN LYMPHOCYTE FLOW CYTOMETRY BASED MICRONUCLEUS ASSAY: SIMULTANEOUS ASSESSMENT OF CELL PROLIFERATION, APOPTOSIS AND MN FREQUENCY.

In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug developmen

AN ANALYSIS OF THE CONSCIOUS DOG TELEMETRY MODEL AND ITS ABILITY TO DETECT AND PREDICT CARDIOVASCULAR CHANGE IN PHASE I

It is widely accepted that more needs to be done to bring new safe and efficacious drugs to the market. Cardiovascular safety detected both in early drug discovery as well as in the clinic, is a major contributor to the high failure rate of new drugs. The growth of translational safety, combined with the considered use of well understood and characterised animal models, as per regulatory guidelines, offers a promising approach to improve the probability of success for new drugs. Nonclinical and phase I clinical cardiovascular data with corresponding plasma exposure, have been shared for 114 small molecules between 7 pharmaceutical companies. The aim was to (a) investigate the concordance between nonclinical and phase I outcome and (b) propose an objective framework for assessing animal models used in the drug discovery process. Accounting for the lack of clinical positives and the heterogeneous data as a consequence of data sharing, the conclusions that can be drawn were limited. Nonetheless, the data indicate that the ability to detect and predict compounds that affect the QT interval was good and within a similar exposure range in animals and man. The ability to detect and predict compounds that affect haemodynamic parameters was severely limited but was reasonable for true negatives (e.g. those without haemodynamic liability). The placement of these data within a 2 dimensional framework confirmed this finding. In conclusion, improved concordance and hence probability of success, may be achieved by refinements to both the animal and clinical study designs

Differentiating Electrophysiological Effects and Cardiac Safety of Drugs Based on the Electrocardiogram: A Blinded Validation

Background: The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the properties of ventricular action potentials that can be modulated by drugs via specific ion channels. However, the correlation of ECG ventricular waveforms with underlying ion actions is not well established and has been extensively debated. We conducted a blinded in vitro assessment of the ionic mechanisms for drug-induced ECG changes. Methods and Results: 14 cardiac and non-cardiac drugs with known effects on cardiac ion channels were selected by the study sponsor, and were tested in the rabbit left ventricular (LV) wedge preparation with recording of the ECG and contractility. The investigators who performed the experiments and analyzed the data were blinded to names, concentrations and molecular weights of the drugs. The compounds were prepared by the sponsor and sent to the investigators as 56 stock solutions. The effect of IKr, IKs, ICa,L, INa blocker and IKATP opener on QRS, QT and Tp-e, were evaluated. Disclosure of the names and concentrations after completion of the study revealed that there were highly correlated ECG changes with underlying ionic mechanisms and proarrhythmic potential of drugs that respectively target IKr, IKs, ICa,L, INa and IKATP. Among ECG parameters, Tp-e was more useful in differentiating drugs’ actions. Conclusions: Specific electrophysiological action and the consequent proarrhythmic potential of a drug can be accurately determined by analysis of drug-induced changes in ECG in the rabbit LV wedge preparation. Change in Tp-e provides the most relevant information