In Vitro Screening of 50 Highly Prescribed Drugs for Thiol Adduct Formation—Comparison of Potential for Drug-Induced Toxicity and Extent of Adduct Formation

Abstract

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

Quantifying the Metabolic Activation of Nevirapine in Patients by Integrated Applications of NMR and Mass Spectrometries

“…However, as only one mercapturate and GSH adduct [NVP-3-mercapturate (NVP-M2) and NVP-3-GSH (NVP-G2), respectively] was formed by HLM (Table 1), either the purely oxidative reactions of unsupplemented HLM fail to reproduce metabolic activation of NVP in vivo or HLM do not trap the reactive metabolite(s) with GSH or NAC via both the addition reactions that operate in vivo. A coincubation of HLM and GSH or a GSH derivative is now a standard preparation used in screens for reactive metabolites that are soft electrophiles (Gan et al, 2009), but it frequently yields more conjugates than can be accounted for in vivo (Bu et al, 2007;Rousu et al, 2009). NVP is an exception and emphasizes the need for whole animal studies.…”

“…Considerable efforts have been made to develop screening systems for reactive drug metabolites based on subcellular liver fractions (Bauman et al, 2009;Gan et al, 2009) or isolated hepatocytes (Bauman et al, 2009). What is currently lacking is a noninvasive, generic, and quantitative method for assessing bioactivation of drugs in experimental animals, human volunteers, and patients.…”

Evaluation of the Potential for Drug-Induced Liver Injury Based on in Vitro Covalent Binding to Human Liver Proteins

“…To detect reactive metabolites as adducts with nucleophilic trapping agents, many in vitro trapping assays featuring a hepatic microsomal enzyme system (one of the most commonly used techniques is the GSH trapping screen) are available for application during the early stages of drug discovery. However, with use of these assays false-negative and false-positive results cannot be avoided (Gan et al, 2009). Therefore, trapping assays may not be adequate for judgment of whether a compound should advance to clinical development, and a higherprecision approach is required.…”

Novel Metabolic Bioactivation Mechanism for a Series of Anti-Inflammatory Agents (2,5-Diaminothiophene Derivatives) Mediated by Cytochrome P450 Enzymes

“…In addition, efflux transporters efficiently pump out xenobiotics and protect cells and tissues from potential toxicities. A trend was recently observed between the body burden of total daily reactive metabolite formation and the occurrence of drug-induced toxicity from a study comparing reactive metabolite formation with a series of 12 marketed drugs (Gan et al, 2009). Drugs were associated with toxicity when FIG.…”

“…As a result, reactive metabolites have become an important issue in modern drug discovery and development. Whether or not protein modification by reactive metabolites results in toxicity depends on many factors, including the function and consequence of the modified targets (covalently bound enzymes/receptors), dose and absorption of drugs, pharmacokinetic profile of the parent drug, fraction of reactive metabolites formed, and detoxification capacity for the reactive species, individual variability, and susceptibility (Obach et al, 2008;Bauman et al, 2009;Gan et al, 2009;Nakayama et al, 2009). P450s are the major metabolizing enzymes that transform lipophilic xenobiotics to hydrophilic metabolites for direct elimination or further conjugation and therefore play an important role in the detoxification of drugs.…”