Transporters and Drug-Metabolizing Enzymes in Drug Toxicity. Albert P. Li
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) are most abundant in the liver [22] and are responsible for the uptake of many endogenous compounds, as well as statins, methotrexate, fexofenadine, bosentan, and rifampicin, among other drugs. OATP1B1 and OAT1B3 are also involved in bilirubin and bile acid uptake; however, the NTCP is primarily responsible for bile acid uptake. The OATP transporters are associated with various drug–drug interactions; for example, OATP1B1 and OATP1B3 are inhibited by atazanavir and ritonavir, cyclosporine, and gemfibrozil [23], and OATP1B1 is inhibited by the tyrosine kinase inhibitors pazopanib and nilotinib [24]. Drugs such as cyclosporin and rifampin that inhibit both OATP1B1 and OATP1B3 disrupt bilirubin uptake and can cause hyperbilirubinemia [25, 26]. Rotor type hyperbilirubinemia occurs when genetic mutations render both OATP1B1 and OATP1B3 deficient, thereby inhibiting bilirubin uptake [27].
Figure 2.4 Hepatic proteins involved in drug, bilirubin, and bile acid transport. Drugs are removed from the blood by influx transporters on the basolateral membrane. These influx transporters include organic anion‐transporting polypeptides (OATP), organic anion transporters (OAT), and organic cation transporters (OCT), all of which belong to the solute carrier superfamily. OATP1B1 and OAT1B3 also are involved in bilirubin and bile acid uptake; however, the sodium taurocholate co‐transporting polypeptide (NTCP) is primarily responsible for transporting bile acids into the hepatocyte from the sinusoidal space. The bile salt export pump (BSEP) transports bile salts from the hepatocyte into the biliary canaliculi, while the multidrug resistance‐associated protein 2 (MRP2) transports sulphated bile salts as well as drug metabolites into the biliary canaliculi. Multidrug resistance protein 3 (MDR3) and ATPase Phospholipid Transporting 8B1 (APT8B1) translocate phospholipids that, together with bile acids, form mixed micelles which protect the biliary tree from the detergent‐like effects of bile salts. Located on the basolateral membrane, multidrug resistance associated proteins MRP3 (ABCC3) and MRP4 (ABCC4) return drug metabolites and bile acids to the liver sinusoids. If cholestatic conditions exist, MRP3 and MRP4 are upregulated to protect hepatocytes from toxic bile acid accumulation. Drug metabolites are exported into the biliary canaliculi by multidrug resistance protein 1 (MDR1), breast cancer resistance protein (BCRP), multidrug and toxin extrusion protein 1 (MATE1), and multidrug resistance‐associated protein 2 (MRP2). ATP8B1, ATPase Phospholipid Transporting 8B1; BCRP, breast cancer resistance protein (BCRP); BSEP, bile salt export pump; MATE, multidrug and toxin extrusion protein; MDR, multidrug resistance protein; MRP, multidrug resistance‐associated protein; NTCP, sodium taurocholate co‐transporting polypeptide; OAT, organic anion transporter; OATP, organic anion‐transporting polypeptide; OCT, organic cation transporter.
Efflux transporters are located either on the basolateral membrane or the canalicular membrane and belong to the ATP‐binding cassette (ABC) transporter superfamily. Multidrug resistance‐associated proteins MRP3 (ABCC3) and MRP4 (ABCC4) are located on the basolateral membrane and return drug metabolites and bile acids from liver cells to the sinusoid blood. Under cholestatic conditions, MRP3 and MRP4 are upregulated in order to protect hepatocytes from bile acid accumulation [28, 29].
Transporters on the canalicular membrane are responsible for the export of drug metabolites and bile components. Drug metabolites are exported into the biliary canaliculi by multidrug resistance protein 1 (MDR1), breast cancer resistance protein (BCRP), multidrug and toxin extrusion protein 1 (MATE1) and multidrug resistance‐associated protein 2 (MRP2). In addition to drug metabolites, MRP2 also transports sulphated bile salts and conjugated bilirubin into the biliary canaliculi. The BSEP transports bile salts from the hepatocyte into the biliary canaliculi. Multidrug resistance protein 3 (MDR3) and ATPase Phospholipid Transporting 8B1 (APT8B1) translocate phospholipids that, together with bile acids, form mixed micelles which protect the biliary tree from the detergent‐like effects of bile salts.
Efficient export of bile salts and acids is essential, as their accumulation can be toxic for hepatocytes due to their detergent‐like effects. Drug interactions or genetic defects that impair hepatic transporters involved in bile salt or phospholipid secretion can result in intrahepatic cholestasis (Table 2.1). For example, mutations in ABCB3, which encodes the phospholipid transporter MDR3, can cause progressive familial intrahepatic cholestasis 3, a condition that usually presents as cholestasis in early childhood and may progress to end‐stage liver disease [31]. MDR3 is inhibited by azole antifungals, including ketoconazole, itraconazole, and posaconazole [33]. Loss‐of‐function mutations in ABCB11, the gene encoding the BSEP, cause progressive familial intrahepatic cholestasis 2 [31] and drugs that inhibit the BSEP, such as bosentan, cyclosporin A, and troglitazone, can induce cholestatic DILI [34, 35].
Table 2.1 Genetic diseases related to hepatic transporters.
| Gene/protein | Function | Related disease |
|---|