The recent literature has seen a flurry of papers describing the issue of, and potential approaches to minimising the incidence of, drug-induced liver injury (DILI) in the clinic. It is well recognised that attrition throughout the development pipeline is often driven by toxicity/adverse drug reations (ADRs), leading to 16 withdrawals and 56 black box warning labels out of the 548 NCEs approved in the US from 1975-1999. Although the mechanisms of DILI are complex, the formation of reactive metabolites has been clearly highlighted (although many publications do stress the lack of correlation between reactive meabolite formation and the degree of toxicity observed, and indeed the potential for ADRs in the absence of metabolic activation). A 2009 publication by Nakayama discussed the use of covalent binding of metabolites to human hepatocyte proteins as an in vitro surrogate marker for DILI as well as the impact of mitochondrial toxicity, cellular toxicity, biliary efflux inhibition and reactive metabolite formation in general. A more recent paper from AstraZeneca highlights the use of an in vitro hepatic liability panel providing a 3 tiered approach to discharge risk :
- An in vitro cell viability screen in THLE cells (+/- P450 transfection) (See a key reference describing a related screen from Pfizer).
- A HepG2 ‘Crabtree effect’ toxicity assay – to detect mitochondrial poisons (in galactose medium cultured cells – see presentation from AstraZeneca).
- Biliary transport inhibition via a membrane vesicle assay in insect cells overexpressing specific transporters. Quantification of inhibition of ATP-dependent biliary transport (e.g. inhibition of bile salt transporter [BSEP]) correlates with DILI – see Greer (2009)).
The latest Nature Reviews|Drug Discovery contains an extensive review article which summarises much of this material as well as additional background information, in particular reviewing the pros & cons of reactive metabolite screening using GSH/CN trapping assays (and highlighting an interesting new hard & soft nucleophilic trapping agent containing both Cys & Lys residues). A number of decision trees and methodologies are reviewed, including data arising from Merck’s extensive use of early covalent binding studies (which require commitment to the preparation of radio-labelled drug substance and are generally viewed as definitive go/no go experiments) and which, with no correlation between the incidence of liver toxicity and the observed levels of covalent binding, actually questioned the whole approach. The review finishes somewhat philosophically by challenging a number of existing opinions/dogma and emphasises the need to continue informatics approaches to help to identify potentially harmful protein adducts (e.g. use of the Target Protein Database which is a repository for all publicly available information relating to known covalent adducts of mammalian proteins and chemically-reactive metabolites of xenobiotic agents, including drugs).