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The chapter also provides an overview of the advantages of using such dynamic mechanistic models over static models for clearance predictions to improve IVIVE.Drug transporters are universally acknowledged as important determinants of the absorption, distribution, metabolism, and excretion of both endogenous and exogenous compounds. Altered transporter function, whether due to genetic polymorphism, DDIs, disease, or environmental factors such as dietary constituents, can result in changes in drug efficacy and/or toxicity due to changes in circulating or tissue levels of either drugs or endogenous substrates.Prediction of whether and to what extent the biological fate of a drug is influenced by drug transporters, therefore, requires in vitro test systems that can accurately predict the risk and magnitude of clinical DDIs. While these in vitro assessments appear simple in theory, practitioners recognize that there are multiple factors that can influence experimental outcomes. A better understanding of these variables, including test compound characteristics, test systems, assay formats, and experimental design, will enable clear, actionable steps and translatable outcomes that may avoid unnecessary downstream clinical engagement. This chapter will delineate the role of these variables in improving in vitro assay outcomes.Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation.The cytosolic sulfotransferase (SULT) enzymes are found in human liver, kidney, intestine, and other tissues. These enzymes catalyze the transfer of the -SO3 group from 3'-phospho-adenosyl-5'-phosphosulfate (PAPS) to a nucleophilic hydroxyl or amine group in a drug substrate. SULTs are stable as dimers, with a highly conserved dimerization domain near the C-terminus of the protein. Crystal structures have revealed flexible loop regions in the native proteins, one of which, located near the dimerization domain, is thought to form a gate that changes position once PAPS is bound to the PAPS-binding site and modulates substrate access and enzyme properties. There is also evidence that oxidation and reduction of certain cysteine residues reversibly regulate the binding of the substrate and PAPS or PAP to the enzyme thus modulating sulfonation. Because SULT enzymes have two substrates, the drug and PAPS, it is common to report apparent kinetic constants with either the drug or the PAPS varied while the other is kept at a constant concentration. The kinetics of product formation can follow classic Michaelis-Menten kinetics, typically over a narrow range of substrate concentrations. Over a wide range of substrate concentrations, it is common to observe partial or complete substrate inhibition with SULT enzymes. This chapter describes the function, tissue distribution, structural features, and properties of the human SULT enzymes and presents examples of enzyme kinetics with different substrates.Aldehyde oxidase (AO) has emerged as an important drug metabolizing enzyme over the last decade. Several compounds have failed in the clinic because the clearance or toxicity was underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. PLX51107 Metabolic products from AO-catalyzed oxidation are generally nonreactive and often they have much lower solubility. AO metabolism is not limited to oxidation as AO can also catalyze reduction of oxygen and nitrite. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. AO is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite, and hydroxamic acids. These reductive transformations may cause toxicity due to the formation of reactive metabolites. Moreover, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening.The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 2 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapters 4 and 6 ). Models and equations that can result in these kinetic profiles will be presented and discussed.