The world's oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)-100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals.

ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug–transporter interactions.

Lipophilic (logP > 1) and amphiphilic drugs (also known as cationic amphiphilic drugs) with ionizable amines (pKa > 6) can accumulate in lysosomes, a process known as lysosomal trapping. This process contributes to presystemic extraction by lysosome-rich organs (such as liver and lung), which, together with the binding of lipophilic amines to phospholipids, contributes to the large volume of distribution characteristic of numerous cardiovascular and central nervous system drugs. Accumulation of lipophilic amines in lysosomes has been implicated as a cause of phospholipidosis. Furthermore, elevated levels of lipophilic amines in lysosomes can lead to high organ-to-blood ratios of drugs that can be mistaken for active drug transport. In the present study, we describe an in vitro fluorescence-based method (using the lysosome-specific probe LysoTracker Red) to identify lysosomotropic agents in immortalized hepatocytes (Fa2N-4 cells). A diverse set of compounds with various physicochemical properties were tested, such as acids, bases, and zwitterions. In addition, the partitioning of the nonlysosomotropic atorvastatin (an anion) and the lysosomotropics propranolol and imipramine (cations) were quantified in Fa2N-4 cells in the presence or absence of various lysosomotropic or nonlysosomotropic agents and inhibitors of lysosomal sequestration (NH4Cl, nigericin, and monensin). Cellular partitioning of propranolol and imipramine was markedly reduced (by at least 40%) by NH4Cl, nigericin, or monensin. Lysosomotropic drugs also inhibited the partitioning of propranolol by at least 50%, with imipramine partitioning affected to a lesser degree. This study demonstrates the usefulness of immortalized hepatocytes (Fa2N-4 cells) for determining the lysosomal sequestration of lipophilic amines.

Purpose: K i values were determined for prototypical inhibitors of ABC transporters P-gp and BCRP with an experimental design incorporating multiple substrate and inhibitor concentrations in vesicles. The EMA’s Guideline on the Investigation of Drug Interactions (2012) recommends K i values for evaluating transporter inhibition. The use of IC 50 values is recommended only when K i determinations are not possible. Although K i values have been reported for inhibitors of various transporters, the methodology differs between labs and oftentimes relies on evaluation of a single probe substrate concentration combined with mathematical extrapolation. These experiments were conducted to compare IC 50 and K i values determined in transporterexpressing membrane vesicles. Methods: Transporter-expressing membrane vesicles were preincubated with a prototypical inhibitor for 15 min at 37±2°C and then a mixture of probe substrate with or without ATP was added and incubated for an additional time point. Terminated reactions were transferred to a filter plate and washed. The filtered vesicles were lysed to extract the probe substrate by either adding internal standard (P-gp) or scintillation fluid (BCRP). Experimental conditions are described in Table 1. Results: Experimentally determined K i and IC 50 values are summarized in Table 2. P-gp and BCRP K i values were approximately 2-fold higher than the IC 50 values. Using experimentally determined K m and IC 50 values (K m data not shown), theoretical K i values were determined using the Cheng-Prusoff equation. Theoretical K i values for P-gp and BCRP were 0.4 and 0.09 µM, respectively. Compared with the theoretical K i , the experimentally determined P-gp and BCRP K i values were 7- and 2-fold higher, respectively. Conclusions: These results suggest that using experimentally determined K i values as opposed to the IC 50 values would not have changed the prediction of inhibitory potential. However, if theoretical K i values were extrapolated mathematically, inhibition would have been over-predicted in the case of P-gp. It is possible to determine K i values for P-gp and BCRP in transporter-expressing vesicles; however, the experiments are timeconsuming and expensive compared to IC 50 experiments and may not provide additional value in terms of predicting inhibitory potential. • P-gp: The experimentally determined K i and IC 50 values for verapamil (1.2 and 2.9 µM, respectively) differed by ~2fold. The estimated and experimentally determined K i values (0.4 and 2.9, respectively) differed by ~7-fold. The experimentally determined IC 50 and K i values provided similar predictions of inhibitor potential (within 2-fold). The estimated K i over-predicted inhibitory potential. • BCRP: The experimentally determined IC 50 , low affinity K i and high affinity K i values for Ko143 (0.090, 0.15 and 0.074 µM, respectively) differed by < 2-fold. The estimated low and high affinity K i values (0.08 and 0.06 µM, respectively) and experimentally determined low and high affinity K i values (0.15 and 0.074 µM, respectively) also differed by < 2-fold. The experimentally determined IC 50 and K i values provided similar predictions of inhibitor potential (within 2-fold). The estimated and experimentally determined K i values also provided similar predictions (within 2-fold). Although the difference between the low and high affinity K i values was only 2-fold, the high affinity K i revealed more potent inhibition and the type of inhibition became competitive. The biphasic nature of ES3 kinetics should be considered when determining K i values.

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.