by adding the sulfonate moiety, which can easily be removed from the body.
GSH S‐conjugation plays a critical defensive role against oxidative stress. The family of glutathione S‐transferases (GSTs) helps detoxify reactive intermediates formed by other metabolizing enzymes derived from drugs, chemicals, and environmental carcinogens.
Acetylation reactions are mostly driven by two N‐acetyltransferase isoenzymes (NATs), NAT1, and NAT2, in humans. Most drugs in acetylation reactions either are hydrazines or aromatic amines.
Methylation is a minor pathway of xenobiotic biotransformation, which does not dramatically change the solubility of substrates. It is primarily involved in the metabolism of small endogenous compounds and certain drugs.
2.3 Reactive Metabolite Formation and Assessment
The physiological role of drug metabolism is detoxification, i.e. the biotransformation of lipophilic compounds into stable water‐soluble metabolites, which are more readily eliminated from the body. However, drug metabolism can also lead to toxicity by the biotransformation of foreign compounds into metabolites which could be intrinsic to chemical reactivity for macromolecules. Such metabolites typically are unstable and undetectable in plasma, and usually have half‐lives of less than one minute. They can be electron‐deficient electrophiles or free radicals containing an unpaired electron [7], but most are electrophilic in nature [8]. Also, they are reactive with electron‐rich nucleophiles such as biological macromolecules in which the functional side chains of arginine, lysine, histidine, cysteine, aspartic acid, glutamic acid, and tyrosine are potent nucleophiles in the unprotonated state [9].
RMs can irreversibly bind to and modify biological macromolecules such as enzymes, mitochondrial proteins, RNA, and DNA, and some of these mechanisms can eventually cause hepatocellular damage. For example, RMs can lead to an immune response by forming hepatic protein adducts. The overloading of RMs can deplete GSH, and the lack of GSH in the liver results in oxidative stress. They can also directly inhibit the BSEP, which may disrupt liver function and lead to hepatic injury. Free radicals also can bind lipids, initiating a chain reaction leading to lipid peroxidation, oxidative stress, or modification of biological macromolecules.
2.3.1 Metabolism and Reactive Metabolites
Many enzymes can generate and release RMs. Particularly, cytochromes P450, together with the reactivity of their oxygen intermediates, are the main drivers in catalyzing the formation of relatively inactive substrates into diverse chemically reactive species. These P450 enzymes are present in the ER and their hepatic expression is concentrated in the liver’s centrilobular region; this is consistent with the typical histological findings of centrilobular necrosis in many acute DILI cases. Many drugs can form RMs, which are catalyzed by P450 enzymes and cause hepatotoxicity [10]. For example, acetaminophen is primarily metabolized via glucuronidation and sulfonation pathways, and the regular metabolites are rapidly excreted in urine. However, a small proportion of acetaminophen is metabolized by CYP3A4, CYP2E1, and CYP1A2, and undergoes bioactivation to form the RM, N‐acetyl‐p‐benzoquinoneimine (NAPQI). At therapeutic doses, NAPQI can be rapidly detoxified by conjugation to GSH and safely eliminated from the liver. Acetaminophen overdosing, on the other hand, can result in GSH depletion and NAPQI accumulation, eventually leading to oxidative stress reactions, dysfunction of mitochondria, and DNA damage.
Phase II enzymes undoubtedly play an important role in the detoxification of various xenobiotics, but some, including UGTs, GSTs, NATs, and SULTs, also could catalyze the formation of RMs. Hepatotoxicity is considered a class effect of nonsteroidal anti‐inflammatory drugs (NSAIDs) and the carboxylic acid functional groups they contain can undergo bioactivation to form acyl glucuronides by hepatic UGT isoforms. Benoxaprofen was an NSAID withdrawn from market in 1982 following several reports of fatal cholestatic jaundice. It contained a carboxylic acid moiety that can form a reactive acyl glucuronide and results in covalent protein adducts as reported from in vitro incubations with rat liver microsomes and from in vivo studies [11]. In FDA’s guidance for industry on safety testing drug metabolites [12], additional safety assessments are required for products that potentially could form reactive acylglucuronides.
2.3.2 Dose and Reactive Mtabolites
The idiosyncratic nature of DILI suggests that it is independent of dose; however, many DILI cases have occurred with doses of drugs >100 mg per day, and cases for drugs given at doses of 10 mg per day or lower have been reported only rarely [13]. Furthermore, the average daily dose of drugs reported to cause hepatotoxicity was higher compared to drugs not associated with this [13]. Moreover, extensive hepatic metabolism has been associated with a higher risk of hepatic adverse events from oral medications [14]. High daily dose and extensive drug metabolism in the liver are associated with high risk of DILI, supporting the hypothesis that the formation and accumulation of RMs beyond a critical threshold is the precondition triggering the development of liver injury.
The quantitative relationship between daily dose, formation of RMs and risk of DILI in humans was reported by Chen et al. [15]. By considering N = 354 FDA‐approved oral medications, the authors defined an algorithm for assigning a DILI score by factoring the relative contribution of daily dose, logP and RM, which permitted a quantitative assessment of clinical DILI risk:
The three parameters in the DILIscore formula (i.e. daily dose/Cmax, logP, and RM formation) contributed significantly to DILI risk with the order of RM > daily dose > logP. The relationship between calculated DILI scores and DILI risk was validated by three independent datasets retrieved from the literature, and the score model demonstrates its correlation with the severity of clinical outcome by applying to N = 159 clinical cases collected from the NIH’s LiverTox database (https://www.ncbi.nlm.nih.gov/books/NBK547852/).
2.3.3 Structural Alerts for Avoiding Reactive Metabolites
A compound’s potential to form electrophiles and chemically RMs is determined by its chemical structure. Certain functional groups, referred to as structural alerts, are molecular fragments with high chemical reactivity, or which can be transformed into fragments with high chemical reactivity through bioactivation [16]. These structural alerts can be present in the parent compound or in its metabolites.
Structural alerts regularly have been employed for screening potential RM formation from candidate compounds in drug discovery, in order to limit undesirable toxic effects. General avoidance of certain functional groups related to RM formation is considered a normal practice at the lead optimization/candidate selection stage. Sometimes, if the structural alert must be carried into lead optimization, mitigation strategies learned from industry experience may be suggested to attenuate or avoid toxicity, including: (i) use substitutes resistant to metabolism to replace potential structural alerts; (ii) decrease electronic density; and (iii) introduce a structural element to redirect metabolism potential [16].
An increasing concern, however, is that the value of incorporating structural alerts for screening drug molecule‐associated safety hazards may have been overstated. In a study examining the role of RMs, about 50% of the top 200 drugs marketed in the U.S. and ranked by prescription and sales were found to have one or more alerts in their chemical architecture [17]. Meanwhile, the absence of structural alerts cannot serve as a guarantee of drug safety. For example, ximelagatran did not present any alerts in its chemical structure but was recalled as a thrombin inhibitor with idiosyncratic hepatotoxicity [17].
