Reactive Metabolites in Early Drug Development: Predictive In vitro Tools

Abstract

Drug metabolism can result in the formation of highly reactive metabolites that are known to play a role in toxicity resulting in a significant proportion of attrition during drug development and clinical use. Thus, the earlier such reactivity was detected, the better. This review summarizes our multi-year project, together with pertinent literature, to examine a battery of in vitro tests capable of detecting the formation of reactive metabolites. Principal prerequisites for such tests were delineated: chemicals known/not known to cause tissue injury and produce reactive metabolites, activation system (mainly human-derived), small- and large-molecular targets (small-molecular trappers, peptides, proteins), analytical techniques (mass spectrometry), and cellular toxicity biomarkers. The current status of in vitro tools to detect reactive intermediates is the following: 1. Small-molecular trapping agents such glutathione or cyanide detect the production of reactive species with high sensitivity by proper MS technique. However, it seems that also putative "negatives" give rise to corresponding adducts. 2. Results from peptide and dG (DNA targeting) trapper studies are generally in line with those of small-molecular trappers, although also important differences exist. These two trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct studies could be fully interpreted only in conjunction with toxicity biomarker (such as the Nrf2 pathway) information from whole cells or tissues. However, while there are tools to characterize the chemical liability and there are correlation between individual/integrated endpoints and toxicity, there are still severe gaps in understanding the mechanisms behind the link between reactive metabolites and adverse effects.

Drug Metabolism – From In Vitro to In Vivo, From Simple to Complex

“…Therefore, we started already during the EUROCYP project a collaboration with the Department of Chemistry at University of Oulu, which over the years resulted in a large number of publications, some MSc and PhD degrees, founding of an SME to serve pharmaceutical companies in metabolism and pharmacokinetics needs and participation in national research programmes and also in the EU COST and framework programmes together with a number of national and international collaborators. Some examples of outcome of these projects have been described in review articles from the last 15 years .…”

Metabolic activation and drug‐induced liver injury: in vitro approaches for the safety risk assessment of new drugs

“…Recombinant enzymes, cell preparations and subcellular liver fractions (hepatic microsomes, S9 mix, tissue homogenates) are used for the in vitro production of reactive metabolites (Orhan and Vermeulen, 2011;Pelkonen et al, 2015). Several studies have supported the utility of hepatocytes for bioactivation assays (Bauman et al, 2009;Gómez-Lechón et al, 2010;Thompson et al, 2012).…”

“…However, the cellular environment is complex, and the detection of the formation of reactive intermediates in cultured cells is technically demanding. In contrast, hepatic microsomes, which can be found readily available from human and non-human sources, are relatively cheap, robust and easy to use and are, therefore, more frequently used for the generation and early screening of reactive metabolites (Pelkonen et al, 2015;Prakash et al, 2008).…”

“…Trapped metabolites are subjected to LC/MS/MS and/or nuclear magnetic resonance spectroscopic analyses for structural identification purposes. Recent advances, particularly in MS technologies, offer researchers new opportunities in this area (Huang et al, 2015;Kalgutkar and Didiuk, 2009;Pelkonen et al, 2015).…”

“…The pharmaceutical industry is committed to market safer drugs with fewer side effects, and thus to reduce the bioactivation risk early in drug discovery may help reduce compound attrition and provide safer drug therapies (Dalvie et al , ). Early preclinical approaches have been implemented to both identify bioactivable molecules by using metabolic competent systems capable of generating reactive metabolites and detect the formation of reactive intermediates for the purpose of eliminating or minimizing them by structural modifications made to problematic lead compounds based on the structural alerts concept (Kalgutkar and Didiuk, ; Liu et al , ; Obach et al , ; Pelkonen et al , ). Several cell‐based assays have been developed as in vitro tools for drug metabolism and hepatotoxicity studies that may be utilized throughout the discovery and development phases for decision making during drug selection and safety risk assessment (Donato et al , ; Godoy et al , ; Gómez‐Lechón et al , ).…”