Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept

Ensuring confidence in predictions: A scheme to assess the scientific validity of in silico models

The use of in silico tools within the drug development process to predict a wide range of properties including absorption, distribution, metabolism, elimination and toxicity has become increasingly important due to changes in legislation and both ethical and economic drivers to reduce animal testing. Whilst in silico tools have been used for decades there remains reluctance to accept predictions based on these methods particularly in regulatory settings. This apprehension arises in part due to lack of confidence in the reliability, robustness and applicability of the models. To address this issue we propose a scheme for the verification of in silico models that enables end users and modellers to assess the scientific validity of models in accordance with the principles of good computer modelling practice. We report here the implementation of the scheme within the Innovative Medicines Initiative project “eTOX” (electronic toxicity) and its application to the in silico models developed within the frame of this project

New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop

© 2020 The Authors The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a ‘Blue Sky Workshop’ on new ideas for non-animal approaches to predict repeated-dose systemic toxicity. The aim of the Workshop was to formulate strategic ideas to improve and increase the applicability, implementation and acceptance of modern non-animal methods to determine systemic toxicity. The Workshop concluded that good progress is being made to assess repeated dose toxicity without animals taking advantage of existing knowledge in toxicology, thresholds of toxicological concern, adverse outcome pathways and read-across workflows. These approaches can be supported by New Approach Methodologies (NAMs) utilising modern molecular technologies and computational methods. Recommendations from the Workshop were based around the needs for better chemical safety assessment: how to strengthen the evidence base for decision making; to develop, standardise and harmonise NAMs for human toxicity; and the improvement in the applicability and acceptance of novel techniques. “Disruptive thinking” is required to reconsider chemical legislation, validation of NAMs and the opportunities to move away from reliance on animal tests. Case study practices and data sharing, ensuring reproducibility of NAMs, were viewed as crucial to the improvement of non-animal test approaches for systemic toxicity.U.S.Environmental Protection Agency (EPA); Agency for Science, Technology and Research ( A*STAR), Singapor

GATA2 Mediates Thyrotropin-Releasing Hormone-Induced Transcriptional Activation of the Thyrotropin β Gene

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.Toxicolog

DataSheet1_Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.docx

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.</p

DataSheet5_Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.docx

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.</p