Development of a non-mammalian, pre-clinical screening tool for the predictive analysis of drug toxicity

The failure to predict drug-induced toxicity reactions is still a major problem contributing to a high attrition rate and tremendous cost in drug development. Xenopus laevis embryos are amenable for the early stage medium to high throughput small molecule screens. We 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. The objective of this study was to assess the use of Xenopus laevis embryos for the prediction of organ-specific toxicity. To do this I used drugs known to generate toxicity reactions in humans. First of all I determined that Xenopus embryos treated with a drug from the age of stage 38 until stage 45, was an appropriate assay for the prediction of drug-induced toxicity. The embryos expressed major drug metabolism enzymes including CYP2E1, CYP2D6, CYP3A4 and glutathione S-transferases, sulphotransferases and glucuronosyltransferases. They also expressed KCNH2, which encodes the α-subunit protein of the potassium ion channel KV11.1 that contributes to heart electrophysiology. For drug-induced liver injury, I used paracetamol treatment. Xenopus laevis embryos treated with paracetamol (0-5 mM) generated predicted paracetamol metabolites, had a dose-dependent depletion of free glutathione and increased expression of microRNA-122 (miR-122) in tissue that did not contain the liver. To investigate drug-induced cardiotoxicity, I treated Xenopus embryos with doxorubicin (0-100 µM) and terfenadine (050 µM). Embryo heart rates increased and decreased with these drugs respectively and arrhythmias occurred with both drug treatments. Embryos treated with doxorubicin had an increasing amount of arrhythmia that correlated with an increasing dose of doxorubicin treatment. Terfenadine treatment induced arrhythmia at a rate that was not concentration dependent. Wholemount in situ hybridisation (WISH) revealed the Xenopus embryos also express miR-208 specifically in the heart, similar to mammalian models. We conclude that Xenopus laevis embryos exhibit some similar characterisations of drug-induced hepatotoxicity and cardiotoxicity observed in mammalian models. These data indicate the Xenopus embryo could be a useful model to assess drug-induced toxicity and aid lead compound prioritisation in early drug development

Paracetamol-induced liver injury modelled in Xenopus laevis embryos

Introduction: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC). Method: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (Dart et al., 2006) microRNA-122 (miR-122) expression (a hepatic marker), (Jaeschke, 2015) free GSH concentration (a marker of oxidative stress) and (Larson et al., 2005) NAC antioxidant intervention. Results: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had an significantly higher amount GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively). Conclusion: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research

In Vivo Assessment of Drug-Induced Hepatotoxicity Using Xenopus Embryos

Failure to predict drug-induced toxicity reactions is a major problem contributing to a high attrition rate and tremendous cost in drug development. Drug screening in X. laevis embryos is high-throughput relative to screening in rodents, potentially making them ideal for this use. Xenopus embryos have been used as a toxicity model in the frog embryo teratogenesis assay on Xenopus (FETAX) for the early stages of drug safety evaluation. We previously developed compound-screening methods using Xenopus embryos and believe they could be used for in vitro drug-induced toxicity safety assessment before expensive preclinical trials in mammals. Specifically, Xenopus embryos could help predict drug-induced hepatotoxicity and consequently aid lead candidate prioritization. Here we present methods, which we have modified for use on Xenopus embryos, to help measure the potential for a drug to induce liver toxicity. One such method examines the release of the liver-specific microRNA (miRNA) miR-122 from the liver into the vasculature as a result of hepatocellular damage, which could be due to drug-induced acute liver injury. Paracetamol, a known hepatotoxin at high doses, can be used as a positive control. We previously showed that some of the phenotypes of mammalian paracetamol overdose are reflected in Xenopus embryos. Consequently, we have also included here a method that measures the concentration of free glutathione (GSH), which is an indicator of paracetamol-induced liver injury. These methods can be used as part of a panel of protocols to help predict the hepatoxicity of a drug at an early stage in drug development

Negative regulation by PD-L1 during drug-specific priming of IL-22-secreting T cells and the influence of PD-1 on effector T cell function

Activation of PD-1 on T cells is thought to inhibit Ag-specific T cell priming and regulate T cell differentiation. Thus, we sought to measure the drug-specific activation of naive T cells after perturbation of PD-L1/2/PD-1 binding and investigate whether PD-1 signaling influences the differentiation of T cells. Priming of naive CD4(+) and CD8(+) T cells against drug Ags was found to be more effective when PD-L1 signaling was blocked. Upon restimulation, T cells proliferated more vigorously and secreted increased levels of IFN-γ, IL-13, and IL-22 but not IL-17. Naive T cells expressed low levels of PD-1; however, a transient increase in PD-1 expression was observed during drug-specific T cell priming. Next, drug-specific responses from in vitro primed T cell clones and clones from hypersensitive patients were measured and correlated with PD-1 expression. All clones were found to secrete IFN-γ, IL-5, and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL-22-secreting clones expressed the skin-homing receptors CCR4, CCR10, and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the Ag-specific proliferative response or the secretion of cytokines/cytolytic molecules. This study shows that PD-L1/PD-1 binding negatively regulates the priming of drug-specific T cells. ELISPOT analysis uncovered an Ag-specific FasL/IL-22-secreting T cell subset with skin-homing properties