1. The molecular and functional characterization of transport proteins is emerging rapidly and significant numbers of drugs have been shown to be substrates or inhibitors. The purpose of this review is to highlight the in vivo preclinical and clinical evidence that supports a role for transport proteins in attenuating the absorption, distribution and excretion (ADE) of drugs. 2. For absorption, a clear role has emerged for P-glycoprotein in limiting permeability across the gastrointestinal tract. As a result, a wide variety of drugs suffer from incomplete, variable and non-linear absorption. Similarly, at the blood-brain barrier a range of drugs has limited brain penetration due to P-glycoprotein-mediated efflux, which can limit therapeutic effectiveness of CNS agents. In the liver, transport proteins are present on the sinusoidal membrane that can be the rate-limiting step in hepatic clearance for some drugs. Mechanistic studies clearly suggest a key role and broad substrate specificity for the OATP family of sinusoidal transporters. Mainly ATP-dependent transport proteins such as P-glycoprotein and MRP2 govern active biliary excretion. 3. Drug-drug interactions have been demonstrated involving inhibition or induction of transport proteins. Clinically significant interactions in the gastrointestinal tract and kidney have been observed with inhibitors such as ketoconazole, erythromycin, verapamil, quinidine, probenecid and cimetidine. Clinically significant inhibition at the blood-brain barrier is more difficult to demonstrate, relying on pharmacodynamic and toxicodynamic changes, but an example is quinidine increasing loperamide-induced central effects in humans. 4. This review highlights the emerging role of transport proteins in ADE of drugs and suggests these need to be considered, in drug discovery and development, with respect to variability in drug disposition and response.

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.

1. The importance of CYP3A enzymes in drug metabolism and toxicology has yielded a wealth of information on the structure, function and regulation of this subfamily and recent research emphasis has been placed on the human forms, namely CYP3A4, CYP3A5, CYP3A7 and CYP3A43. 2. The current review will focus on the receptor-dependency of CYP3A regulation and includes consideration of the regulatory roles of the glucocorticoid (GR), pregnane X (PXR) and constitutive androstane (CAR) receptors. 3. Emphasis has been placed on the topics of expression and substrate specificity, assessment of induction, species differences in induction, CYP3A promoter sequences and regulation of gene expression, structural and functional aspects of receptor-mediated, CYP3A gene activation, receptor variants and interindividual variation in human CYP3A expression, the latter encompassing environmental, physiological and genetic aspects. 4. An outline of future research needs will be discussed in the context of receptor-mediated molecular mechanisms of CYP3A gene regulation and the impact on interindividual variations in CYP3A expression. 5. Taken collectively, this review highlights the importance of understanding the molecular mechanisms of CYP3A induction as a means of rationalizing human responses to many clinically used drugs, in addition to providing a mechanistically coherent platform to understand and predict interindividual variations in response and drug-drug interactions.

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Drug-induced liver injury is the most common cause of market withdrawal of pharmaceuticals, and thus, there is considerable need for better prediction models for DILI early in drug discovery. We present a study involving 223 marketed drugs (51% associated with clinical hepatotoxicity; 49% non-hepatotoxic) to assess the concordance of in vitro bioactivation data with clinical hepatotoxicity and have used these data to develop a decision tree to help reduce late-stage candidate attrition. Data to assess P450 metabolism-dependent inhibition (MDI) for all common drug-metabolizing P450 enzymes were generated for 179 of these compounds, GSH adduct data generated for 190 compounds, covalent binding data obtained for 53 compounds, and clinical dose data obtained for all compounds. Individual data for all 223 compounds are presented here and interrogated to determine what level of an alert to consider termination of a compound. The analysis showed that 76% of drugs with a daily dose of <100 mg were non-hepatotoxic (p < 0.0001). Drugs with a daily dose of ≥100 mg or with GSH adduct formation, marked P450 MDI, or covalent binding ≥200 pmol eq/mg protein tended to be hepatotoxic (∼ 65% in each case). Combining dose with each bioactivation assay increased this association significantly (80-100%, p < 0.0001). These analyses were then used to develop the decision tree and the tree tested using 196 of the compounds with sufficient data (49% hepatotoxic; 51% non-hepatotoxic). The results of these outcome analyses demonstrated the utility of the tree in selectively terminating hepatotoxic compounds early; 45% of the hepatotoxic compounds evaluated using the tree were recommended for termination before candidate selection, whereas only 10% of the non-hepatotoxic compounds were recommended for termination. An independent set of 10 GSK compounds with known clinical hepatotoxicity status were also assessed using the tree, with similar results.