No abstract

The evaluation of toxicity in preclinical species is important for identifying potential safety liabilities of experimental medicines. Toxicology studies provide translational insight into potential adverse clinical findings, but data interpretation may be limited due to our understanding of cross-species biological differences. With the recent technological advances in sequencing and analyzing omics data, gene expression data can be used to predict cross species biological differences and improve experimental design and toxicology data interpretation. However, interpreting the translational significance of toxicogenomics analyses can pose a challenge due to the lack of comprehensive preclinical gene expression datasets. In this work, we performed RNA-sequencing across four preclinical species/strains widely used for safety assessment (CD1 mouse, Sprague Dawley rat, Beagle dog, and Cynomolgus monkey) in ∼50 relevant tissues/organs to establish a comprehensive preclinical gene expression body atlas for both males and females. In addition, we performed a meta-analysis across the large dataset to highlight species and tissue differences that may be relevant for drug safety analyses. Further, we made these databases available to the scientific community. This multi-species, tissue-, and sex-specific transcriptomic database should serve as a valuable resource to enable informed safety decision-making not only during drug development, but also in a variety of disciplines that use these preclinical species.

Mitochondrial dysfunction has been frequently implicated in some drug‐induced toxicities. Various methods for the detection of mitochondrial toxicity are available such as an oxygen consumption assay to measure mitochondrial basal respiration, assays to measure direct effects of drugs on the five complexes in the mitochondrial electron transport chain (ETC), and assays to detect changes in mitochondrial DNA (mtDNA) copy number or changes in mitochondrial membrane potential. The Seahorse technology platform has emerged as a new technique in assessing mitochondrial respiration in cells, where basal, ATP‐linked, maximal and non‐mitochondrial respiration can be measured in the same experiment. Spare respiratory capacity (SRC) is calculated as the difference between maximal and basal respiration. In this study, Seahorse XF Cell Mito Stress Test was evaluated in the PC‐3 cell line using tool compounds known to directly inhibit the complex or complexes in the ETC. Cells were acutely treated with amiodarone (5–80 μM), simvastatin (5–75 μM), tamoxifen (0.625–20 μM), troglitazone (2.5–50 μM), penicillin (12.5–100 μM, negative control), and vehicle (0.1% DMSO). Exposure of these tool compounds revealed an immediate and pronounced decrease in SRC with a ranking of (greatest to lowest inhibition) troglitazone (IC50< 2.5 μM)> amiodarone (IC50<5 μM) >tamoxifen (IC50 = 17.7 μM) >simvastatin (IC50 = 20.7μM).Tool compounds known to cause mitochondrial toxicity through an indirect mechanism were also explored in the Seahorse platform. 2′, 3′‐dideoxycytidine (ddC), an inhibitor of mtDNA synthesis, and chloramphenicol (CAM), an inhibitor of mitochondrial protein synthesis, were tested. A 3‐day pre‐treatment of ddC (0.12–30 μM) and CAM (0.04–10 μM) showed a decrease of SRC in PC‐3 cells with IC50=0.39 μM and 2.7 μM respectively. The decrease of SRC was further supported by an in‐cell ELISA assay where a set of proteins in the ETC complexes was measured. A decrease of mitochondrial encoded COX‐1 to nuclear encoded SDH‐A protein ratio was observed after a 5‐day treatment of ddC and CAM in PC‐3 cells with IC50=0.016 μM and <0.1 μM respectively. In the dose ranges tested, the 3 or 5 day pre‐treatment of ddC and CAM showed minimal cytotoxicity in the PC‐3 cells. In conclusion, the Seahorse XF Cell Mito Stress Test is a sensitive assay to evaluate mitochondrial dysfunction using a set of tool compounds, and this new method combined with traditional methods will be helpful to better understand the mechanism of mitochondrial toxicity in the development of new drugs.Support or Funding InformationThe design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.