Development of non-mammalian, preclinical screening tools for the predictive analysis of cardiotoxicity

Why did we fund this project?

This award aims to help replace the use of mammals in the cardiotoxicity assessment of drugs by introducing a screening stage during early phases of drug development using Xenopus laevis embryos.

Drug-induced cardiotoxicity is a leading cause of attrition during pharmaceutical development and in some instances can result in a drug being withdrawn after market approval. Cardiotoxicity can involve many processes in multiple cell types and impact the heart either functionally, structurally or both. Regulatory pharmacology tests are conducted in mammals such as dogs and monkeys, but drugs will also be screened earlier in development in mice or rats. Current in vitro methods involving tissue or cell-based assays do not fully recapitulate the complexity of the heart. The Xenopus laevis embryo represents an alternative model as it has a fully functioning cardiovascular system approximately four days into development. At this stage the embryos are not considered capable of suffering and so provide a replacement for the use of other animals.

The student will work with Dr Grant Wheeler to investigate drug-induced cardiotoxicity in Xenopus laevis embryos by measuring the heart rate and presence of biomarkers. They will measure cardiotoxicity biomarkers using a method previously developed by Grant where blood from the embryo can be isolated from the tail and the specific organ of interest. The student will also develop skills in qRT-PCR, immunoassays and ELISAs. 

This Studentship was co-awarded with the British Heart Foundation (BHF).

Potential new drugs developed to treat cancer, diabetes or any other disease can cause additional unwanted side effects including cardiotoxicity. The failure to predict such drug induced toxicity during drug development is a major problem contributing to a high attrition rate and tremendous costs. Drug induced cardiotoxicity can affect all components and functions of the cardiovascular system either directly or indirectly and can be functional and or structural (morphological) in nature. In vitro single isolated organ and cell-based assays are therefore limited in what they can test. Even organoid model systems which are currently being developed involving mixtures of human cardiomyocytes, fibroblasts and endothelial cells cannot completely recapitulate a fully functioning cardiovascular system.Cardiotoxicity can involve many processes including degeneration, necrosis leading to inflammatory changes and fibrosis. It can affect multiple cardiac cell types. The hearts ability to contract and function is thus highly dependent on the number, severity and distribution of cells involved. In addition, prolonged changes in haemodynamics, particularly increases in heart rate are associated with subsequent changes in cardiac structure.

We have previously shown Xenopus laevis embryos to be amenable for early stage medium to high throughput small molecule screens. Xenopus tadpoles have a fully functioning cardiovascular system with a three chambered heart and flowing blood. We therefore hypothesise Xenopus embryos can assist in vitro drug-induced toxicity safety assessment in the early phases of drug development before moving on to expensive preclinical trials in mammals. This would lead to a reduction in mammalian animal experiments consistent with the philosophy of the 3Rs. Our studies on nanoparticle toxicity and drug induced liver injury have shown Xenopus to be a potential model for toxicity studies (Marin-Barba et al. 2018, Nanoscale 10(2):690-704; Saide et al. 2019, Toxicology Letters 302: 83-91).

The aim of this project is to further develop Xenopus as a model for the prediction of organ - in this case heart - specific toxicity. In summary the student will investigate drug-induced cardiotoxicity using two drugs with known cardiotoxic effect, Doxorubicin and terfenadine. They will test for cardiotoxicity using two common approaches, measuring heart rate and the release of cardiotoxicity biomarkers into the vasculature/blood. Embryo heart rates will be recorded using movies of live tadpole hearts. To measure blood markers the student will utilise a novel method we have developed (Saide et al. Toxicol. Letts. 2019) where we dissect the specific organ (ie. liver or heart) and tail of the Xenopus tadpole. The tail has an extensive vasculature and so will contain blood born biomarkers that can be readily assessed. Analysis of known microRNA biomarkers for cardiotoxicity will be carried out using qRT-PCR. Analysis for blood protein biomarkers such as the natriuretic peptides, cardiac and skeletal troponins, myosin light chain 3, Creatine Kinase, LDH and fatty acid binding protein 3 will be carried out using immunoassays and ELISAs.