Development of a platform for drug testing and pharmacological safety assessment using in vitro models based on human induced pluripotent stem cells

Abstract

The pharmaceutical industry is one of the areas with the greatest investment in research and development worldwide, being strategic for the technological development of a country. It is estimated that between 2007 and 2011 the investment to launch a new drug was US$ 4.2 billion according to a report published in 2017 by the Brazilian Center for Management and Strategic Studies of the Ministry of Science, Technology and Innovation. Part of this cost is justified by following regulatory norms and considering drug costs that are discontinued in reason of side effects. In order to minimize these costs, methods have been developed to identify the harmful effect of compounds in the early stages of development. One of these effects is the pro-arrhythmic potential that is currently studied, according to guidelines of the International Council of Harmonization (ICH), through evaluation of the potassium flux of hERG channel and prolongation of the QT interval in animals and humans. Despite these efforts, false positives or false negatives are eventually obtained, resulting in millions of dollars of loss. In 2013 the CiPA initiative (Comprehensive in Vitro Proarrhythmia Assay) was created to define trials to improve accuracy in identifying the proarrhythmic potential of drugs from the evaluation of ion currents, in silico assays and evaluation of membrane potential and intracellular calcium handling in cardiomyocytes derived from stem cells. In recent years, several groups have contributed to the establishment of the CiPA, but challenges remain to be overcome, especially those related to the evaluation of intracellular calcium and electrical stimulation at low frequencies. In this context, the objective of this project is to develop a platform to evaluate the proarrhythmic risk of drugs through analysis of the intracellular calcium cycle. To this end, a validated system will be used to record and analyze intracellular calcium transients from cell cultures under electrical stimulation using a high throughput fluorescence reader. Preliminary results in cultures of cells treated with verapamil suggested the potential of the system to identify drug-induced alterations. The effects induced by six compounds classified by the CiPA according to their proarrhythmic potential (high, intermediate and low) on the activity of cardiomyocytes derived from stem cells in culture will be evaluated from variations in the parameters of the calcium transient profile. The results will be compared with studies that evaluated dose-dependent side effects generated by the drugs tested and will be used to validate the system as a platform for drug evaluation. It is expected to observe more intense variations in the transient profile parameters of cells treated with drugs with greater proarrhythmic potential. The project has an impact on current methods for assessing the proarrhythmic effect of drugs as an alternative method to study the intracellular calcium handling using cell cultures electrically stimulated at low frequencies. In this way, the developed platform could contribute with the pharmaceutical industry reducing costs of the development of new drugs by providing a new method to determine its proarrhythmic risk. (AU)