Science Pool

Cardiotoxicity is one of the most reported adverse effects that leads to pre-clinical and clinical drug failure. To tackle this, the International Conference of Harmonization (ICH) S7B guideline in 2005, proposed a non-clinical assessment of new drug entities using in vitro electrophysiology studies (typically hERG ion channel) and in vivo telemetry in animal models. Although these are very sensitive approaches, they may have also led to unwarranted drug attrition of many potentially valuable therapeutics due to the low specificity nature of the assays.

More recently, the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative was proposed by experts in the field and was established to move safety pharmacology towards in silico and in vitro approaches utilising new and emerging technologies such as stem-cell-derived cardiomyocytes. Building on this new safety paradigm, colleagues from Evotec and Cyprotex have worked collaboratively to develop a non-clinical model using cutting-edge techniques with improved predictive power to de-risk cardiotoxicity in early drug discovery. We have presented the output of this work in a research article titled “In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes” published in a recent edition of Expert Opinion on Drug Metabolism and Toxicology.

In this article, we describe the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro model system together with three high-throughput technologies incorporating structural assays (high-content imaging, HCI), functional assays (Ca²⁺ transience, CaT) and high-throughput RNA-sequencing (ScreenSeq) for the pre-clinical risk assessment of novel compounds. The transcriptional responses of hiPSC-CMs to 24 h treatment with 33 cardiotoxicants (12 structural cardiotoxicants, 14 functional cardiotoxicants, 7 structural/functional cardiotoxicants) and 9 non-cardiotoxicants of mixed therapeutic indications were investigated and compound-induced differential gene expression (DEG) was calculated in comparison with vehicle treated controls. Likewise, the hiPSC-CMs responses to six structural readouts (cell count, cellular ATP, mitochondrial mass, mitochondrial membrane potential, calcium content, DNA structure and nuclear size) and four functional readouts (amplitude, frequency, peak width and decay time) were analysed. In summary, hiPSC-CMs recapitulated expected structural and functional toxicity mechanisms, validating their use as in vitro model system to detect and characterize modes of toxicity. ScreenSeq identified several molecular mechanisms of toxicity such as alterations in cardiac pathways, genotoxicity, ER stress and mitochondrial toxicity. Together, HCI, CaT and ScreenSeq provided the best cardiotoxicity prediction metrics (10x C_max: 100% specificity, 82% sensitivity, 86% accuracy; 25x C_max: 89% specificity, 91% sensi-tivity, 90% accuracy).

This study not only provides invaluable cardiotoxic mechanistic information of the drugs tested, but it also demonstrates the potential of this mechanism-driven risk assessment approach in predicting drug-induced cardiotoxicity in hiPSC-CMs.

Read the paper.

Read more about our Cardiotox Screen assay consisting of both a functional assay (examining the mechanical function of the cardiomyocytes) and a structural assay (assessing morphological changes and loss of viability).

In addition, discover more about our transcriptomics offerings here.

Find out more:

Contact Us

Rapid turnaround is essential for ADME-Tox services, especially during drug discovery. However, turnaround time for the assay isn’t the only consideration. The time required to prepare the quotation may delay the scheduling of the assay and, in turn, the study start date.

The new Cyprotex e-Store provides a solution to this. Registered users of the e-Store are able to view instant pricing for a comprehensive range of ADME-Tox services. As well as having access to detailed protocols and the latest editions of our educational guides, users also benefit from special offers only available through the e-Store.

Online ordering is simple. Previous orders can be tracked through the e-Store and it is easy to re-order your favourite assays. All of this information can be accessed outside business hours. A range of payment options are available on the e-Store including credit card, existing purchase order or invoice. These features allow services to be ordered and scheduled the same day so saving you time.

Despite this automated approach, you are still supported by a dedicated scientific study manager and account manager who are on-hand to guide you at all stages of your study.

Want to learn more?

Explore the Cyprotex e-Store

The industry gold-standard in vitro cell based test system for studying Breast Cancer Resistance Protein (BCRP) or P-glycoprotein (P-gp) transporters is the polarized cell monolayer system using either Caco-2 cells or MDCK-MDR1 cells (Elsby et al., 2022), grown on semi permeable membrane inserts to form a brush border membrane barrier separating two experimental compartments (apical and basolateral) of equivalent pH (7.4). Apparent permeability (P_app, cm/s x10^-6) of the test article is determined in the apical to basolateral (A-B) and the basolateral to apical (B-A) direction and from this an efflux ratio is calculated (B-A P_app/A-B P_app). An efflux ratio greater than 2 which is reduced by greater than 50% with a corresponding reduction in B-A P_app in the presence of a reference inhibitor (Atkinson et al., 2022) indicates the test article is a substrate of the efflux transporter being investigated. The bidirectional P_app values and subsequent efflux ratios of the known P-gp substrate quinidine, determined in polarized MDCK-MDR1 cell monolayers across a concentration range is summarized in Table 1 (left) and demonstrates that in the MDCK-MDR1 polarized monolayer test system quinidine has been determined to be a substrate of the P-gp transporter.

Table 1: Mean P_app values and corresponding efflux ratios of quinidine (left) and N-methyl quinidine (right) across MDCK-MDR1 monolayer cells in the presence and absence of reference inhibitor cyclosporin A

Despite polarized monolayers being the industry gold standard test system for studying P-gp and BCRP interactions, there are limitations and risk of inconclusive results and false negatives if the test article being studied exhibits low P_app indicating inherently low passive membrane permeability. This is due to the physico-chemical characteristics of the test article limiting cellular entry required to access the efflux transporter on the apical membrane. This is true for the quinidine oxidative metabolite, N-methyl quinidine (NMQ). The presence of the methyl group results in lower lipophilicity and therefore passive permeability without diminishing its ability to bind P-gp. As summarized in Table 1 (right) bidirectional P_app values for the more polar NMQ are very low and although efflux ratios determined are around two, a 50% reduction in efflux ratio with corresponding decrease in B-A P_app is not observed in the presence of inhibitor therefore the result is inconclusive at best, or at worst could be interpreted as not a substrate of the P-gp transporter.

Where assessment of a test article in polarized monolayer cells indicates a compound has inherently low passive permeability, membrane vesicles have the membrane orientated “inside out”, this results in the intracellular binding site of the transporter being positioned on the outside of the vesicle (outwardly facing), therefore compounds do not need to cross a membrane in order to interact with the transporter. As such for efflux transporters such as P-gp or BCRP, substrates will be actively taken up into the vesicle and, due to poor permeability, will remain inside the vesicle for quantification. As demonstrated in Table 2, using P-gp membrane vesicles as an in vitro test system for P-gp substrate identification, NMQ is clearly demonstrated to be, and subsequently correctly identified as, a substrate of the P-gp transporter.

Table 2: Mean uptake rate and corresponding uptake ratios of N-methyl quinidine using P-gp expressing membrane vesicles in the presence of ATP or AMP in the absence and presence of the reference inhibitor cyclosporin A. Figure: Mean uptake rate of N-methyl quinidine into P-gp expressing membrane vesicles in the presence of ATP or AMP in the absence and presence of the reference inhibitor (I) cyclosporin A

Furthermore, if data from P-gp or BCRP substrate identification studies in polarized cell monolayers indicate a compound has inherently low passive permeability then any P-gp or BCRP inhibition data generated in the same in vitro test system should also be scrutinized and a follow up inhibition study in membrane vesicles considered, particularly if no inhibition was observed in the cells.

Whilst membrane vesicles have their advantages over polarized cell monolayers as a test system to identify P-gp or BCRP substrates that are poorly permeable, it is not advisable to utilize membrane vesicles as a first-choice test system. This is because for lipophilic test articles such as quinidine, sufficient passive permeability allows the substrate to move freely across the membrane and would not be trapped within the vesicle lumen, therefore there is a risk of inconclusive results and false negatives.

In summary, polarized cell monolayers are the gold standard for studying P-gp and BCRP interactions as indicated in regulatory guidances and as such should be the first choice of test system for identifying P-gp or BCRP substrates and inhibitors. However, if results indicate the compound has inherently low passive permeability then P-gp or BCRP expressing membrane vesicles should be considered as an alternative follow up in vitro test system to mitigate against the clinical implications of false negatives.

References:

Atkinson, H., Mahon-Smith, K. and Elsby, R. (2022) ‘Drug permeability and transporter assessment: Polarized Cell Lines’, The ADME Encyclopedia, pp. 401–412. doi:10.1007/978-3-030-84860-6_142.

Elsby, R. et al. (2022) Studying the right transporter at the right time: An in vitro strategy for assessing drug-drug interaction risk during drug discovery and development. Expert Opin Drug Metab Toxicol 18(10):. 619–655. doi:10.1080/17425255.2022.2132932.