Lipophilic (logP > 1) and amphiphilic drugs (also known as cationic amphiphilic drugs) with ionizable amines (pK a > 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 (NH 4 Cl, nigericin, and monensin). Cellular partitioning of propranolol and imipramine was markedly reduced (by at least 40%) by NH 4 Cl, 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.

ABSTRACT:UDP-glucuronosyltransferases (UGTs) catalyze phase II biotransformation reactions in which lipophilic substrates are conjugated with glucuronic acid to increase water solubility and enhance excretion. Currently, little information regarding tissue-or genderspecific expression of mouse UGTs is available. Mice are increasingly popular models in biomedical research, and therefore, thorough characterization of murine drug metabolism is desired. The purpose of the present study was to determine both tissueand gender-specific UGT gene expression profiles in mice. RNA from 14 tissues was isolated from male and female C57BL/6 mice and UGT expression was determined by the branched DNA signal amplification assay. UGTs highly expressed in mouse liver include Ugt1a1, Ugt1a5, Ugt1a6, Ugt1a9, Ugt2a3, Ugt2b1, Ugt2b5/37/38, Ugt2b34, Ugt2b35, and Ugt2b36. Several isoforms were expressed in the gastrointestinal (GI) tract, including Ugt1a6, Ugt1a7c, Ugt2a3, Ugt2b34, and Ugt2b35. In kidney, Ugt1a2, Ugt1a7c, Ugt2b5/37/38, Ugt2b35, and Ugt3a1/2 were expressed. UGT expression was also observed in other tissues: lung (Ugt1a6), brain (Ugt2b35), testis and ovary (Ugt1a6 and Ugt2b35), and nasal epithelia (Ugt2a1/2). Male-predominant expression was observed for Ugt2b1 in liver, Ugt2b5/37/38 in kidney, and Ugt1a6 in lung. Female-predominant expression was observed for Ugt1a1 and Ugt1a5 in liver, Ugt1a2 in kidney, Ugt2b35 in brain, and Ugt2a1/2 in nasal epithelia. UDP-glucose pyrophosphorylase was highly expressed in liver, kidney, and GI tract, whereas UDP-glucose dehydrogenase was highly expressed in the GI tract. In conclusion, marked differences in tissue-and gender-specific expression patterns of UGTs exist in mice, potentially influencing drug metabolism and pharmacokinetics.

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Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers, which is consistent with the trend of human NTCP mRNA expression between men and women. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid increased Bsep mRNA expression. In silico analysis indicates that female-predominant mouse and human Ntcp/NTCP expression may be due to GH. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers.

Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporterexpressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics.