Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells.

In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non-genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by testing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect genotoxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage.

The development of RAD51C, Cystatin A, p53 and Nrf2 luciferase-reporter assays in metabolically competent HepG2 cells for the assessment of mechanism-based genotoxicity and of oxidative stress in the early research phase of drug development.

Four different mechanism-based high-throughput luciferase-reporter assays were developed in human HepG2 cells, which contain phase I and II metabolic activity and a functionally active p53 protein. The promoter regions of RAD51C and Cystatin A, as well as the responsive element of the p53 protein, were selected for the generation of the genotoxicity reporter assays. Moreover, a luciferase-based reporter assay was generated that measures the activation of the Nrf2 oxidative stress pathway. Validation with respect to the ECVAM compound list [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108] resulted in an overall sensitivity of the HepG2 genotoxicity reporter assays for genotoxicity of 85% (17/20). The specificity and predictivity were high with 81% (34/42) and 82% (51/62), respectively. Various compounds had a positive score although metabolic activation was needed. The HepG2 reporter data were also compared with the available data on bacterial mutagenicity (Ames test), in vitro clastogenicity and in vivo clastogenicity for an additional set of 192 compounds. The predictivity for mutagenicity results was 74% (sensitivity, 61%, 30/49; specificity, 80%, 77/96) and for in vitro clastogenicity 59% (sensitivity, 45%, 35/78; specificity 83%, 38/46). The correlation between results from the HepG2 genotoxicity reporter assays and in vivo clastogenicity was much higher with 77% (sensitivity, 74%, 28/38; specificity 81%, 26/32). Results from the Nrf2 reporter assay showed that a large number of genotoxic compounds activated the Nrf2 oxidative stress pathway. In conclusion, four high-throughput mechanism-based reporter assays in the HepG2 cell line were developed, which can be applied for screening in the early research phase of drug development. The use of these assays in combination with the previously validated Vitotox and RadarScreen assays will certainly reduce the attrition rate due to genotoxicity in the developmental phase of drug development.

DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing.

BACKGROUND: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. METHODS: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. FINDINGS: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.

High-throughput screening for analysis of in vitro toxicity.

The influence of combinatorial chemistry and high-throughput screening (HTS) technologies in the pharmaceutical industry during the last 10 years has been enormous. However, the attrition rate of drugs in the clinic due to toxicity during this period still remained 40-50%. The need for reduced toxicity failure led to the development of early toxicity screening assays. This chapter describes the state of the art for assays in the area of genotoxicity, cytotoxicity, carcinogenicity, induction of specific enzymes from phase I and II metabolism, competition assays for enzymes of phase I and II metabolism, embryotoxicity as well as endocrine disruption and reprotoxicity. With respect to genotoxicity, the full Ames, Ames II, Vitotox, GreenScreen GC, RadarScreen, and non-genotoxic carcinogenicity assays are discussed. For cytotoxicity, cellular proliferation, calcein uptake, oxygen consumption, mitochondrial activity, radical formation, glutathione depletion as well as apoptosis are described. For high-content screening (HCS), the possibilities for analysis of cytotoxicity, micronuclei, centrosome formation and phospholipidosis are examined. For embryotoxicity, endocrine disruption and reprotoxicity alternative assays are reviewed for fast track analysis by means of nuclear receptors and membrane receptors. Moreover, solutions for analyzing enzyme induction by activation of nuclear receptors, like AhR, CAR, PXR, PPAR, FXR, LXR, TR and RAR are given.

Evaluation of the Vitotox and RadarScreen assays for the rapid assessment of genotoxicity in the early research phase of drug development.

The Vitotox and RadarScreen assays were evaluated as early screens for mutagenicity and clastogenicity, respectively. The Vitotox assay is a bacterial reporter assay in Salmonella typhimurium based on the SOS-response, and it contains a luciferase gene under control of the recN promoter. The RadarScreen assay is a RAD54 promoter-linked beta-galactosidase reporter assay in yeast. The expression of this beta-galactosidase can easily be quantified by use of the substrate d-luciferin-o-beta-galactopyranoside, which is converted into galactose and luciferin that can be measured luminometrically. Recently, an ECVAM workgroup defined a list of 20 genotoxic and 42 non-genotoxic compounds [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108.] that can be used for the validation and/or optimization of in vitro genotoxicity assays. In the present study, this compound set was used for the validation of the assays. Moreover, an additional set of 192 compounds was used to broaden this validation study. The compounds of this additional set can be classified as non-genotoxins and genotoxins and consists of both in-house and reference compounds. In case of the ECVAM compound list, the results from the Vitotox and RadarScreen assays were compared to the genotoxic/non-genotoxic classification of the compounds in this list. In case of the additionally tested compounds, the results of the Vitotox and RadarScreen assays were compared, respectively, with bacterial mutagenicity (Ames) results or in vitro clastogenicity data obtained in-house or from the literature. The validation with respect to the ECVAM compound list resulted in a sensitivity for both the Vitotox and RadarScreen assay of 70% (14/20). If both assays were combined the sensitivity increased to 85% (17/20). Both tests also gave a low number of false positive results. The specificity of the Vitotox and RadarScreen assays was 93% (39/42) and 83% (35/42), respectively. This resulted in a predictivity of the Vitotox and RadarScreen assay of 85% (53/62) and 79% (49/62), respectively. In case both tests were combined the specificity and the predictivity of the Vitotox and RadarScreen assay turned out to be 81% (34/42) and 82% (51/62), respectively. The results from the additional list of 192 compounds confirmed the results found with the ECVAM compound list. The results from the Vitotox assay showed a high correlation with Ames test of 91% (132/145). Subsequently, the RadarScreen assay had a correlation with in vitro clastogenicity of 76% (93/123). The specificity of the Vitotox assay was 94% (90/96) for Ames test results and that of the RadarScreen assay was 74% (34/46) for clastogenicity. Moreover, the sensitivities of the Vitotox and RadarScreen assays were 86% (42/49) and 77% (59/77), respectively. Implementation of the Vitotox and RadarScreen assays in the early research phase of drug development can lead to fast de-selection for genotoxicity. It is expected that this application will reduce the number of compounds that have a positive score in the regulatory Ames and clastogenicity tests. Moreover, problems with a complete compound class can be foreseen at an early time point in the research phase, which gives more time for issue resolution than late detection of these problems with the regulatory tests.

Uniform procedure of (1)H NMR analysis of rat urine and toxicometabonomics Part II: comparison of NMR profiles for classification of hepatotoxicity.

A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition is proposed for early detection of toxicity of investigational compounds in rats. The method is applied to detect toxicity upon administration of 13 toxic reference compounds and one nontoxic control compound (mianserine) in rats. The toxic compounds are expected to induce necrosis (bromobenzene, paracetamol, carbon tetrachloride, iproniazid, isoniazid, thioacetamide), cholestasis (alpha-naphthylisothiocyanate (ANIT), chlorpromazine, ethinylestradiol, methyltestosterone, ibuprofen), or steatosis (phenobarbital, tetracycline). Animals were treated daily for 2 or 4 days except for paracetamol and bromobenzene (1 and 2 days) and carbon tetrachloride (1 day only). Urine was collected 24 h after the first and second treatment. The animals were sacrificed 24 h after the last treatment, and NMR data were compared with liver histopathology as well as blood and urine biochemistry. Pathology and biochemistry showed marked toxicity in the liver at high doses of bromobenzene, paracetamol, carbon tetrachloride, ANIT, and ibuprofen. Thioacetamide and chlorpromazine showed less extensive changes, while the influences of iproniazid, isoniazid, phenobarbital, ethinylestradiol, and tetracycline on the toxic parameters were marginal or for methyltestosterone and mianserine negligible. NMR spectroscopy revealed significant changes upon dosing in 88 NMR biomarker signals preselected with the Procrustus Rotation method on principal component discriminant analysis (PCDA) plots. Further evaluation of the specific changes led to the identification of biomarker patterns for the specific types of liver toxicity. Comparison of our rat NMR PCDA data with histopathological changes reported in humans and/or rats suggests that rat NMR urinalysis can be used to predict hepatotoxicity.

Sensitivity of (1)H NMR analysis of rat urine in relation to toxicometabonomics. Part I: dose-dependent toxic effects of bromobenzene and paracetamol.

(1)H nuclear magnetic resonance (NMR) spectroscopy of rat urine in combination with pattern recognition analysis was evaluated for early noninvasive detection of toxicity of investigational chemical entities. Bromobenzene (B) and paracetamol (P) were administered at five single oral dosages between 2 and 500 mg/kg and between 6 and 1800 mg/kg, respectively. The sensitivity of the proposed method to detect changes in the NMR spectra 24 and 48 h after single dosing was compared with histopathology and biochemical parameters in plasma and urine. Both B and P applied at the highest dosages induced liver necrosis and markedly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) plasma levels. At dosages of 125 mg/kg B and 450 mg/kg P, liver necrosis and changes in AST and ALT were less pronounced, while at lower dose levels these effects could not be detected. Changes in kidney pathology or standard urine biochemistry were not observed at any of these dosages. Evaluation of the total NMR dataset showed 80 signals to be sensitive for B and P dosing. Principal component analysis on the reduced dataset revealed that NMR spectra were significantly different at dosages above 8 mg/kg (B) and 110 mg/kg (P) at both sampling times. This implies a 4- to 16-fold increased sensitivity of NMR versus histopathology and clinical chemistry in recognizing early events of liver toxicity.

Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™.

In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5mM of glucose. The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10(-3)M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16×10(-5)M, and only 19 (38%) of these compounds appeared to be toxic in both assays. In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72 h. With AB more toxic compounds were identified, whereas MitoXpress was more sensitive for a few compounds. A species specific difference was clearly found with digoxin, a human specific potassium channel inhibitor. Thus both assays are valuable identifiers of early toxicity with discrimination in time, compounds and species.