WRIB 2021
Sept. 27 - Oct. 1, 2021
WE’RE EXHIBITING at WRIB
Sept. 27 - Oct. 1, 2021; Disney Spring Orlando,
MEET US at booth #5 and enter a chance to win 3 years Disney+ subscription & movie package goodies
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don't miss our 3 new poster presentations
Using Anti-Drug-Antibody Screening Assay to Resolve Selectivity Issues in Toxicokinetic Ligand-Binding Assay
Presented by Olga Malykhina, Senior Research Scientist
The selectivity parameter for regulatory Toxicokinetic (TK) quantitative ligand-binding assays (LBAs) is required to meet the accuracy requirements for at least 80% of the individual selectivity matrix lots. Due to the complexity of biological matrices, often this parameter requires extensive method optimization to achieve the required level of accuracy. Individual selectivity lots, spiked with the drug at the level of High-Quality Control (HQC) and Lower Limit of Quantitation (LLOQ) concentrations, are back-calculated from a regressed calibration curve which is prepared in pooled matrix consisting of multiple individual lots. If the pooled matrix contains individual lots that contain interfering factors, such as pre-existing/cross-reacting antibodies or soluble drug targets, then the back-calculated concentration of selectivity samples might be inaccurately represented. Therefore, a routine screening in an anti-drug antibody (ADA) assay of individual matrix lots can be used to assess the presence of those interfering lots due to the presence of pre-existing/cross-reacting antibodies and exclude them from the TK pooled matrix. In this case study, the testing of 20 treatment-naive serum individuals in an ADA screening assay revealed the presence of 2 biological outliers, while the rest of the individuals had a similarly low signal in the ADA screening assay. Using a pooled serum of these lots excluding the ADA biological outliers for the calibration curve preparation resolved the TK selectivity over-recovery issue observed at the LLOQ level when the pooled serum was prepared using random individual lots.
Comparing Cytokine Data to In-Life Parameters on Non-human Primates in Nonclinical Toxicology Studies
Presented by Vivienne Bunker, Study Director, Research Scientist
Authors: V. Bunker, C. Do, J. Forget, and T. Rogers. Altasciences, Seattle, WA.
Cytokines are important immunoregulatory proteins that have gained attention in safety assessment associated with innate or adaptive immune responses. Interpreting cytokine data comes with challenges due to the variable nature of their stimuli and responses. Contributing factors to the variability in cytokine expression include species-specific reactions, individual variations, dose-response relationships, and unanticipated immunotoxicity. For these reasons, cytokine measurements should not be used as standalone biomarkers for immunotoxicity assessment. However, in conjunction with additional parameters such as clinical observations, body weights, and clinical pathology data, cytokine interpretation can be used to provide more definitive assessments in nonclinical safety studies. In several case studies, cytokines were evaluated for a dose response relationship. Multiplex platforms such as Luminex or MSD® were used to determine cytokine levels in non-human primates including IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12/IL-23p40, MCP-1, IFN-γ, and TNF-α. In several instances, measurable levels of IL-6 or IL-12 correlated with clinical observations of bruising, injury, or abnormal feces, not necessarily considered test article-related. Elevated TNF- α and IL-6, pro-inflammatory cytokines, were detected in animals observed as dehydrated with elevated BUN, creatine, and decreased electrolytes. In cases of test article-related effects, animals becoming moribund also had elevated TNF-α and IL-6 levels. Increased levels of MCP-1, a monocyte chemotactic factor, were observed in one study with an animal with petechial bruising, and another study with a cohort with test-article associated renal failure, characterized by hypoproteinemia, azotemia, and hyperkalemia. In conclusion, cytokines are useful markers when assessing potential toxicity when evaluated with other measurements. Clinical observations, body weights, and clinical pathology parameters should also be considered in addition to the test article-related effects.
Establishment of a Historical Database for Normal Cynomolgus Macaque Spermatozoa Evaluation in Male Reproductive Toxicology Studies.
Presented by Anthony Celori Associate Scientist/Study Director
Authors: A. Celori, L. Baydak, P. Ampo, S. Kaur, J. Hynek, S. McGrath, N. Reed, L. Lawrence, and E. Leung. Altasciences, Seattle, WA. Sponsor: T. Rogers
ICH guidance S5(R2) recommends the assessment of sperm count, motility, and morphology to help confirm or characterize effects of pharmaceuticals on male reproduction, with the cynomolgus macaque commonly used as a test system. However, S5(R2) highlights that non-human primates lack historical background data and may differ kinetically from humans and that animal numbers used are too low for detection of risk. Additionally, studies using cynomolgus macaques are typically analyzed via the WHO Laboratory Manual for the Examination and Processing of Human Semen (currently Fifth Ed., 2010), as few publications exist specifically describing the normal cynomolgus macaque sperm. To assist and improve the assessment of cynomolgus macaque sperm for toxicology studies, historical sperm counts/concentration, motility, and morphology assessments were collated from over 400 non-human primates between 2007 and 2019. All animals were either facility stock or had been dosed with a control/sham article. Sperm was collected via direct penile electrostimulation, with ejaculate volume recorded and motility and sperm count assessed using the TOX IVOS or TOX IVOS II computerized assisted sperm analysis (CASA) systems. Morphology was evaluated using light microscopy. Potential differences due to age and origin were also evaluated.
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