drugs is comprised of two reaction phases: biotransformation/detoxification (phase I) and conjugation (phase II). In phase I, reactive or polar groups are introduced into xenobiotics. In phase II, reactions these modified compounds are conjugated to polar compounds, which are easier to eliminate from the body.
2.2.1 Phase I Metabolism
Within phase I metabolism a series of biotransformation reactions, including oxidation, hydroxylation, and other reactions, are triggered. These reactions add functional hydroxyl, carboxyl, amino, or thiol groups to lipophilic drugs and convert them to a more hydrophilic status. This process is primarily mediated by the cytochrome P450 (CYP) enzymes, which account for about 75% of the total metabolism in the liver. CYP is a large family of enzymes containing heme as a cofactor. They function as monooxygenases localized primarily in the membrane of the endoplasmic reticulum (ER) in hepatocytes. In humans approximately 60 genes coding for the various CYP enzymes have been identified. The most frequent human P450 isoforms involved in the metabolism of drug molecules are 3A4/5, 2D6, 2C9, 1A2, 2B6, 2C19, 2C8, 2A6, 2E1, and 2J2 (Figure 2.2) [6]. CYP3A4/5 and 2D6 are the most abundant of all CYP450 enzymes in the human liver and metabolize half of medications, although their expression levels vary widely among individuals.
Figure 2.2 The most frequent human P450 enzymes involved in metabolism of clinically‐used drugs. Numbers represent the fractions of drugs metabolized by the P450 enzyme.
Source: Data from Zanger and Schwab [6].
The expression level of these CYP enzymes controls the rate at which many drugs are metabolized. Usually a drug is metabolized by multiple CYP enzymes. Each of these has a limited capacity to metabolize drugs, and therefore can become overloaded when the drug level in blood is too high. The activities of the CYP enzymes significantly vary among individuals, and their gene expression and enzyme activities are tightly regulated by members of the nuclear receptor (NR) family of ligand‐modulated transcription factors, such as the pregnane X receptor (PXR), farnesoid X receptor, vitamin D receptor, and hepatocyte nuclear factor 4 alpha.
2.2.2 Phase II Metabolism
Phase II metabolism is composed of a series of reactions, including glucuronidation, sulfoconjugation, glutathione (GSH) S‐conjugation, acetylation, and methylation. They introduce glucuronic acid, sulfate, amino acids, or GSH molecules to phase I products and form conjugates, which increase water solubility and decrease pharmacologic activity; and finally, enhance detoxification of the compounds.
Glucuronidation accounts for the conjugation of about 40–70% of marketed drugs in humans, and UDP‐glucuronosyltransferases (UGTs) are the key enzymes metabolizing various exogenous and endogenous compounds.
Sulfoconjugation (or sulfonation), generally described as a detoxification pathway for many xenobiotics, is mediated by a supergene family of enzymes called sulfotransferases (SULTs). The phase I active molecules can form a water‐soluble compound 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