2.3 lists some typical structural alerts found in the literature. Since the relationships among these alerts in generating RMs and toxicity are largely unclear, most institutes and companies maintain their own libraries of structural alerts. Several review papers have summarized a comprehensive list of alerts. Enoch et al., for example, reported many structural alerts, each with an outline of their associated mechanistic chemistry of adduct formation. These alerts have been incorporated into the Organization for Economic Co‐operation and Development (OECD) (Q)SAR Toolbox, a freely available software tool for analyzing structural alerts in the public domain [18]. Claesson and Minidis summarized 63 alerts with detailed SMART names, SMART pictures and examples of drugs, which could be the basis for an in‐house structural alert application system. [19]
Figure 2.3 Some typical structure alerts for formation of reactive metabolites found in the literature.
2.3.4 Experimental Approaches for Assessing Reactive Metabolites
The structural alerts for formation of RMs sometimes do not imply actual formation of RMs. In this circumstance, experimental evidence is needed to assess whether a drug candidate is bioactivated to RMs. We discuss some selected experimental approaches here, including covalent binding assay, electrophile trapping experiment and time‐dependent inactivation of CYP450.
2.3.4.1 Covalent Binding Assay
The earliest approach for assessing protein covalent binding is using radiolabeled compounds with human liver microsomal or hepatocyte incubations. It is the gold‐standard approach for measuring RM formation and can quantify the extent of RMs covalently binding to proteins. The original experimental protocol for determining covalent binding properties of radioactive drugs was developed by Evans et al. [20]. In their protocol in vitro human liver microsomes are used as the metabolizing enzyme source and incubated with radioactive drug and cofactors for oxidative metabolism. Quantification of covalent binding is measured through counting the radioactivity of the protein pellet.
Although the covalent binding assay is reliable and widely used by pharmaceutical companies, the use of radiolabeled drugs makes running high throughput screening difficult, and it is an expensive option for assessing whether a compound is likely to undergo bioactivation. A more common and economical screening approach is using electrophile trapping experiments that do not require radiolabeled drugs.
2.3.4.2 Electrophile Trapping Experiments
Electrophile trapping is the primary approach for screening RMs in the early phase of drug discovery without involving a radioactive drug. Normally it requires the addition of trapping agents (i.e. GSH) to microsomal incubation followed by liquid chromatography mass spectrometry (LC‐MS) analysis. This approach also helps indicate which metabolite structure is responsible for this reactivity and characterizes the mechanism of covalent binding. It generates the stable trapping adducts and/or conjugates of electrophilic RMs using nucleophilic trapping reagents or in vitro incubation, along with hepatic microsome and cellular, animal, and human studies. The biological nucleophile GSH is the most frequently‐used nucleophile reagent, commonly used for trapping soft electrophiles (e.g. epoxides, quinones, and quinone methides). For certain hard electrophiles with high charge density and high polarization, hard nucleophiles such as cyanide anion (CN−) are preferred because the GSH adducts are unstable and GSHs have limited trapping efficiency.
Following in vitro incubations, various analytical approaches such as LC‐MS, fluorescence or radiochemical detection could be used to analyze the trapped adducts and/or conjugates of RMs. Mass spectrometric methods are the most common approach. Taking advantage of specific fragmentation behavior of the peptide moiety of nucleophilic trapping reagents, stable trapping adducts, and/or conjugates of electrophilic RMs can be detected and characterized. For example, the neutral loss of 129 Da is commonly used by positive ion electrospray‐tandem mass spectrometry to provide a generic endpoint for GSH trapping and measurement. Without using the radiolabeled drugs, this protocol can provide semi‐quantitative estimates of adduct formation for RMs.
2.3.4.3 Time Dependent Inactivation of CYP450 Enzymes
Occasionally the formed RMs (electrophiles) are so highly reactive that they cannot flow out from the active site of the P450 enzymes that catalyzed their formation. Electrophilic intermediates derived from drug molecules are so reactive that they can covalently bind directly to an active site amino acid residue in the CYP enzyme itself. Covalent modification in the active site of a P450 enzyme may lead to the loss of enzyme activity over time via the modification of the P450 apoprotein. Therefore, time dependent inactivation (TDI) of CYP450 enzymes and other drug‐metabolizing enzymes also indicates the generation of RMs during the drug metabolism process. Additionally, drug–drug interactions could be caused by this irreversible P450 inhibition. Covalent modification of P450 enzymes can also result in a neoantigen formation and trigger an autoimmune response in DILI.
The TDI assay is a two‐step assay in which the drug of interest is preincubated with a source of P450 enzymes, such as human liver microsomes. This method can detect two scenarios for TDI: (i) time dependent loss of CYP activity following incubation at a single compound concentration; and (ii) an IC50 shift after preincubation at multiple concentrations. In the first step the tested compound is preincubated at multiple concentrations in the in vitro incubation of human liver microsome in the presence and absence of β‐nicotinamide adenine dinucleotide 2′‐phosphate reduced tetrasodium salt (NADPH) for a preset time period (usually ∼30 minutes). The second step is the measurement of CYP450 activity using a probe CYP substrate. The IC50 values upon preincubation were calculated in the presence and absence of NADPH cofactor, and a decrease in IC50 will suggest the presence of TDI of CYP450 enzymes.
Electrophile trapping experiments are typically used for the stable conjugates of RMs. If a certain amount of formed RMs cannot escape the active site of the P450 enzymes, the trapping assays usually are not able to detect the RMs associated with the inactivation of P450 enzymes. The TDI assay therefore can supplement the electrophile trapping assay in detecting RM formation. Nakayama et al. [21] demonstrated that the combination of TDI assays and GSH trapping assays significantly correlated with the extent of covalent binding assay (r = 0.77, P < 0.0001), but both alone are not correlated, suggesting the combination of these two assays provides an alternative to the covalent binding assay for identifying RM formation by the drug molecules.
2.4 Hepatic Transporters
Hepatic transporter proteins facilitate drug metabolism and elimination by regulating the movement of drugs across hepatocyte cell membranes. They also are essential in maintaining bile acid homeostasis. Drug–drug interactions and genetic variants that impair the function of hepatic drug transporters can result in bile acid accumulation, which can lead to toxicity and DILI.
Hepatic transporters are generally described as influx or efflux transporters (Figure 2.4). Influx transporters located on the basolateral membrane of hepatocytes remove drugs and other compounds from blood in the liver sinusoidal space and transport into hepatocytes. Influx transporters include members of the solute carrier (SLC) superfamily, such as the sodium‐independent organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs), the organic cation transporters (OCTs), and the sodium‐dependent sodium taurocholate cotransporting polypeptide (NTCP) transporter. The OATP transporters OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3), and OATP2B1 (SLCO2B1)
