Redox potential and metabolic stability. development of high throughput assays for early compound profiling

Abstract

In recent decades, the pharmaceutical drug discovery and development process has been hampered by identifying efficacious and safe drugs. Among absorption problems, interference with ion channels (hERG), extensive hepatic metabolism leading to inadequate metabolic profiles of discovery compounds, has been cited as one of the most common problems associated with failures in early drug discovery and development. Inadequate metabolic profiles of discovery compounds, namely low metabolic stability, an increased risk of CYP450 dependent inhibition and a number of clinically significant drug-drug interactions are largely due to oxidative degradation (phase-I metabolism). This degradation is usually enzymatically catalyzed by the most important members of the mono-oxygenases, the CYP450 superfamily. The most important and common member of the CYP450 superfamily, CYP3A4, is responsible for the metabolic degradation of over 60% of known drugs [1, 2]. Additionally, the relatively large active cavity site of CYP3A4, resulting in a large diversity of possible substrates, makes this isoform especially important in the evaluation of metabolic and safety profiles of drugs and metabolites. Furthermore, the large cavity site allows the optimal orientation of the drug molecule resulting in a successful attack of the chemically most liable positions of CYP3A4 substrates. Unlike for other CYP450 isoforms, structure-property relationships for CYP3A4 have so far not been feasible as various functions and mechanisms of components of the CYP3A4 active site are still not identified. Besides the electronic orientation of liable structural moieties within the cavity site is an additional factor influencing the CYP3A4 induced catalysis. Therefore improved knowledge of the ionization potentials could be an important factor in a better understanding of CYP450 catalysis. The ionization potential of a compound can be described as the compound’s redox potential. Guengerich and Lewis, for example, have proven the correlation between ionization and redox potentials [1, 3, 4]. In the current study we could show that the redox potentials of discovery compounds are an important factor to be considered in the description of rat or human clearance and thus with the metabolic stability. Standard early metabolic stability determinations are biological based mainly focusing on the degradation of drug by microsomal or hepatic cell preparations. Usually these assays only deliver information on the rate of metabolism by the determination of the drug disappearance. Without doubt, there is a need for a better understanding of metabolic processes. A larger focus on structural aspects of drugs candidates could probably improve understanding of metabolic degradation processes and structure-effect relationships. Two novel approaches for early metabolic stability profiling of drug candidates have been developed and investigated in the current PhD-thesis. The first approach is based on redox chemistry. Ideally, the optimal redoxchemical indicator should exhibit reversible two-electron transfer behaviour to best simulate the two-electron transfer process occurring in the CYP450 catalytic cycle. After intensive investigations, p-chloranil has been identified as a suitable component for a redoxchemical based assay which meets the required criteria off reversible twoelectron transfer behaviour. The second approach is electrochemical based. Several research groups have already worked on electrochemical approaches and tried to establish EC/LC/MS as screening/profiling tool for metabolic stability. Quite a few disadvantages, e.g. nonphysiological experimental conditions and low throughput have prevented EC/LC/MS from routine use so far. An external collaboration with Gatlik (Gatlik Ltd., Basel/CH) gave rise to the Electroactive Pharmaceutical Screening System (EPSS), a novel HT-cyclic voltammetric screening/profiling system which allows electrochemical determinations under more physiological conditions in the 96-well format. For the first time, a larger quantity of compounds can be measured per day. Obtained oxidation potentials well correlate with the microsomal rat clearance so that EPSS can be regarded as an attractive screening/profiling method compound ranking/selection based on the found relationship between redox potential and metabolic stability. The aim of the PhD-thesis was the development of fast and easy profiling systems, allowing improvement of the understanding of metabolic processes at the structural/compound level. Thus, compounds/compound classes with high probability to be metabolically instable with focus on phase-I metabolism processes can be identified. Based on the previously reported hypothesis and the obtained results of the PhD study, we therefore propose EPSS as a promising and attractive screening or profiling tool for early metabolic stability determinations in early drug discovery, as first information on the compound’s metabolic stability can be easily obtained without the use of biological materials.