Food Additives as Inhibitors of Intestinal Drug Transporter OATP2B1.

Food additives are compounds that are added to food and beverage to improve the taste, color, preservation, or composition. Generally, food additives are considered safe for human use due to safety evaluations conducted by food safety authorities and high safety margins applied to permitted usage levels. However, the interaction potential of food additives with simultaneously administered medication has not received much attention. Even though many food additives are poorly absorbed into systemic circulation, high concentrations could exist in the intestinal lumen, making intestinal drug transporters, such as the uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1), a possible site of food additive-drug interactions. In the present work, we aimed to characterize the interaction of a selection of 25 food additives including colorants, preservatives, and sweeteners with OATP2B1 in vitro. In human embryonic kidney 293 (HEK293) cells transiently overexpressing OATP2B1 or control, uptake of dibromofluorescein was studied with and without 50 µM food additive at pH 7.4. As OATP2B1 displays substrate- and pH-dependent transport functions and the intraluminal pH varies along the gastrointestinal tract, we performed the studies also at pH 5.5 using estrone sulfate as an OATP2B1 substrate. Food additives that inhibited OATP2B1-mediated substrate transport by ≥50% were subjected to dose-response studies. Six colorants were identified and validated as OATP2B1 inhibitors at pH 5.5, but only three of these were categorized as inhibitors at pH 7.4. One sweetener was validated as an inhibitor under both assay conditions, whereas none of the preservatives exhibited ≥50% inhibition of OATP2B1-mediated transport. Extrapolation of computed inhibitory constants (Ki values) to estimations of intestinal food additive concentrations implies that selected colorants could inhibit intestinal OATP2B1 also in vivo. These results suggest that food additives, especially colorants, could alter the pharmacokinetics of orally administered OATP2B1 substrate drugs, although further in vivo studies are warranted to understand the overall clinical consequences of the findings.

The Impact of Breast Cancer Resistance Protein (BCRP/ABCG2) on Drug Transport Across Caco-2 Cell Monolayers.

Breast cancer resistance protein (BCRP) is expressed on the apical membrane of small intestinal epithelial cells and functions as an efflux pump with broad substrate recognition. Therefore, quantitative evaluation of the contribution of BCRP to the intestinal permeability of new chemical entities is very important in drug research and development. In this study, we assessed the BCRP-mediated efflux of several model drugs in Caco-2 cells using WK-X-34 as a dual inhibitor of P-glycoprotein (P-gp) and BCRP and LY335979 as a selective inhibitor of P-gp. The permeability of daidzein was high with an apparent permeability coefficient for apical-to-basal transport (P AB) of 20.3 × 10-6 cm/s. In addition, its efflux ratio (ER) was 1.55, indicating that the contribution of BCRP to its transport is minimal. Estrone-3-sulfate and ciprofloxacin showed relatively higher ER values (>2.0), whereas their BCRP-related absorptive quotient (AQ BCRP) was 0.21 and 0.3, respectively. These results indicate that BCRP does not play a major role in regulating the permeability of estrone-3-sulfate and ciprofloxacin in Caco-2 cells. Nitrofurantoin showed a P AB of 1.8 × 10-6 cm/s, and its ER was 7.6. However, the AQ BCRP was 0.37, suggesting minimal contribution of BCRP to nitrofurantoin transport in Caco-2 cells. In contrast, topotecan, SN-38, and sulfasalazine had low P AB values (0.81, 1.13, and 0.19 × 10-6 cm/s, respectively), and each AQ BCRP was above 0.6, indicating that BCRP significantly contributes to the transport of these compounds in Caco-2 cells. In conclusion, Caco-2 cells are useful to accurately estimate the contribution of BCRP to intestinal drug absorption. SIGNIFICANCE STATEMENT: We performed an in vitro assessment of the contribution of breast cancer resistance protein (BCRP) to the transport of BCRP and/or P-glycoprotein (P-gp) substrates across Caco-2 cell monolayers using absorptive quotient, which has been proposed to represent the contribution of drug efflux transporters to the net efflux. The present study demonstrates that the combined use of a BCRP/P-gp dual inhibitor and a P-gp selective inhibitor is useful to estimate the impact of BCRP and P-gp on the permeability of tested compounds in Caco-2 cells.

Establishment and Validation of Competitive Counterflow as a Method To Detect Substrates of the Organic Anion Transporting Polypeptide 2B1.

The organic anion transporting polypeptide (OATP) 2B1 is ubiquitously expressed and known to facilitate cellular entry. It is widely accepted that transport proteins play a pivotal role in pharmacokinetics. Consequently, testing for interaction with drug transporters became an important part in the assessment of new molecular entities in order to predict and prevent drug-drug interactions. Recently, competitive counterflow (CCF), an indirect method allowing the identification of substrates, was successfully applied to the organic cation transporter 2. It was the aim of this study to test whether CCF can be used to identify substrates of OATP2B1. A protocol for CCF experiments using estrone 3-sulfate (E1S) as the driven compound in expression-verified MDCKII-OATP2B1 cells was established. The protocol was tested using a substance library, which was prior screened for inhibition of OATP2B1-mediated transport accounting for both E1S-binding sites. In CCF experiments, all previously reported OATP2B1 substrates significantly reduced the amount of E1S in equilibrium, classifying them as substrates. In addition, we identified and verified novel substrates of OATP2B1, namely, astemizole and domperidone. Results of the CCF were complemented with cytotoxicity assays or cell-based reporter gene assays to validate the finding of etoposide and teniposide or hyperforin being substrates of OATP2B1, respectively. Our study indicates that the method of CCF can be used to identify substrates of OATP2B1, irrespective, whether interacting with binding site A or A and B, but is limited by solubility issues or the amount of transporter that is expressed in the used cellular system.

SLCO1B1 genetic variation and hormone therapy in menopausal women.

OBJECTIVE: Response to menopausal hormone therapy (MHT) shows individual variation. SLCO1B1 encodes the OATP1B1 transporter expressed in the liver that transports many endogenous substances, including estrone sulfate, from the blood into hepatocytes. This study evaluated the relationship between genetic variation in SLCO1B1 and response to MHT in women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic, Rochester, MN. METHODS: KEEPS participants were randomized to oral conjugated equine estrogen (n = 33, oCEE), transdermal 17ß-estradiol (n = 33, tE2), or placebo (n = 34) for 48 months. Menopausal symptoms (hot flashes, night sweats, insomnia, palpitations) were self-reported before treatment and at 48 months. Estrone (E1), E2, and sulfated conjugates (E1S, E2S) were measured using high-performance liquid chromatography-tandem mass spectrometry. SLCO1B1 rs4149056 (c.521T>C, p.Val174Ala) was genotyped using a TaqMan assay. RESULTS: After adjusting for treatment, there was a significant association between the SLCO1B1 rs4149056 TT genotype (encoding normal function transporter) and lower E1S, E1S/E1, and E2S (P = 0.032, 0.010, and 0.008, respectively) compared with women who were heterozygous (TC) or homozygous (CC) for the reduced function allele. The interactions between genotype, treatment, and E2S concentration were stronger in women assigned to tE2 (P = 0.013) than the women taking oCEE (P = 0.056). Among women assigned to active treatment, women with the CT genotype showed a significantly greater decrease in night sweats (P = 0.041) than those with the TT genotype. CONCLUSIONS: Individual variation in sulfated estrogens is explained, in part, by genetic variation in SLCO1B1. Bioavailability of sulfated estrogens may contribute to relief of night sweats.

Structural insights into anticancer activity of D-ring modified estrone derivatives using their lipophilicity in estimation of SAR and molecular docking studies.

Many forms of breast carcinoma are hormone-dependent and therefore development of novel aromatase inhibitors is of particular interest. Since brain metastases are frequent in patients with advanced breast carcinoma, one of the goals of modern drug development is the discovery of drugs with specific pharmacokinetic profile. High performance thin layer chromatography (HPTLC) is often used to determine lipophilicity of the molecules based on their retention constant. As a predictive analysis, multiple linear regression method was performed to connect pharmacokinetic-dependent parameters with independent physicochemical properties such are: RM0 , TPSA and Mw of fourteen D-ring modified oestrone derivatives. Additionally, docking studies were performed. Conducted correlation analysis indicates excellent dependence between experimental RM parameter values and calculated values of pharmacokinetic parameters. Results show sufficient intestinal absorption of all the investigated molecules as well as moderate volumes of distribution and strong affinity for binding to plasma proteins. Crossing blood-brain barrier is predicted to be successful for 11 compounds. The created quantitative structure activity relationship model represents an excellent predictive tool and enables determination of pharmacokinetic properties of examined compounds. Docking analysis defined molecules I3 and II3 to be the best candidates; however, compound II3 violates the Lipinski rule. It has been concluded that molecules with hydroxyl group at C-3 more effectively pass through blood-brain barrier while structures with benzyloxy groups have stronger interactions with CYP1A19. Molecules II2 , II4 , II6 , and II7 are regarded as most suitable candidates for further investigation considering their good pharmacokinetic and docking characteristics. Copyright © 2017 John Wiley & Sons, Ltd.

Dioscin protects against ANIT-induced cholestasis via regulating Oatps, Mrp2 and Bsep expression in rats.

Alpha-naphthylisothiocyanate (ANIT) is a toxicant that is widely used in rodents to model human intrahepatic cholestasis. The aim of the study is to investigate whether effects of dioscin on ANIT-induced cholestasis are related to changes in expression of hepatic transporters in rats. Effects of dioscin on cholestasis were examined by histology and biochemical marker levels. The functional changes of hepatic transporters were determined by in vitro, in situ and in vivo. qRT-PCR and western blot were used to assess the expression of hepatic transporters in cholestatic rats. Dioscin administration could ameliorate cholestasis, as evidenced by reduced biochemical markers as well as improved liver pathology. The uptakes of organic anion transporting polypeptide (Oatp) substrates were altered in liver uptake index in vivo, perfused rat liver in situ and isolated rat hepatocytes in vitro in cholestasis rats. qRT-PCR and western blot analysis indicated co-treatment of ANIT with dioscin prevented the adaptive down-regulation of Oatp1a1, 1b2, and prompted the up-regulation of Oatp1a4, multidrug resistance-associated protein (Mrp) 2 and bile salt export pump (Bsep). In addition, concerted effects on Mrp2 and Bsep occurred through up-regulation of small heterodimer partner by activating farnesoid X receptor. Dioscin might prevent impairment of hepatic function by restoring hepatic transporter expression.

Synergistic dual-targeting hydrogel improves targeting and anticancer effect of Taxol in vitro and in vivo.

A synergistic dual-targeting molecular self-assembly hydrogel was designed with estrone (Et) and Arg-Gly-Asp (RGD) peptide. Confocal imaging in vitro and real-time visualization in vivo researches were carried out to evidence enhanced targeted delivery and anticancer effect of Taxol.

Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions.

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC50 ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC50 values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration-IC50 ratios ([C]/IC50 ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC50 ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ.

Estrone-3-sulphate, a potential novel ligand for targeting breast cancers.

The current study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane associated uptake transporters, for detection and diagnosis of hormone dependent breast cancers. E3S, an OATP substrate, is a predominant source of tumour estradiol in post-menopausal patients. To assess the potential of E3S as a ligand, distribution of exogenous E3S was determined at the whole body, tumour and cellular levels in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6 h post injection (p.i) with significant difference (p = 0.04) between the level in MCF-7 (13.9±3.1%ID/g) and MDA-MB-231 (10.4±1.1%ID/g) (%ID/g: percentage of the total injected dose per gram tissue). The highest tumour-to-blood ratios (MCF-7∶7.4±1.2; MDA-MB-231∶9.1±2.1) were observed at 48 p.i., and highest tumour-to-muscle ratios (MCF-7∶10.7±1.5; MDA-MB-231∶3.8±0.7) were observed at 6 h p.i. Analogous to total tumour uptake, ex vivo tumour cell uptake at 2 h p.i. was 6 fold higher in MCF-7 in comparison to MDA-MB-231 tumour cells. Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower (MCF-7: p = 0.01; MDA-MB-231: p = 0.02) tumour uptake in both xenograft models, suggesting the involvement of an active carrier-mediated process. The expression of OATP1A2 was detected in tumour sections from both xenografts, with significantly higher expression (p = 0.002) in the MCF-7 xenografts. Overall, the higher tumour uptake and tumour-to-muscle ratio, alongside the higher expression of OATP1A2, in the MCF-7 xenograft model suggests the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers.

The utility of cold-preserved human hepatocytes in studies on cytochrome P450 induction and hepatic drug transport.

Human hepatocytes that had been cold-preserved in SureTran(TM) matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012). After 5 d of cold preservation, the viability was still more than 70%, and after 8 d it was around 60%. In hepatocytes that had been cold-preserved for 3 d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8 µM rifampicin for 72 h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3 d and thereafter treated with 1 mM phenobarbital for 72 h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3 d, while the activity increased in cells treated with 0.3-25 µM ß-naphthoflavone for 72 h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2 d. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.

Dissecting the relative contribution of OATP1B1-mediated uptake of xenobiotics into human hepatocytes using siRNA.

1. Organic anion transporting polypeptide 1B1 plays a pivotal role in the disposition of many anionic drugs. Significant overlap in substrate specificity between individual OATP isoforms has hampered the identification of the relative importance of individual isoforms for hepatic uptake of xenobiotics. 2. The present study focused on the use of siRNA technology to decrease OATP1B1 selectively in human hepatocytes. Following delivery of siRNA by the novel lipid, AtuFECT01, mRNA expression of OATP1B1 was reduced by 94%-98% with no significant toxicity. Off-target effects were also shown to be minimal as evidenced by the expression of common drug metabolizing enzymes, transporters, nuclear receptors and associated co-regulators. Uptake of estrone-3-sulfate (5 nM) by OATP1B1 was reduced by 82%-95%. This methodology was subsequently used to assess the relative contribution of OATP1B1 uptake in human hepatocytes for olmesartan (42%-62%), valsartan (28%-81%), rosuvastatin (64%-72%), pitavastatin (84%-98%) and lopinavir (64%-89%). These data are consistent with previous values obtained using a relative activity factor approach. 3. The siRNA approach provides a robust and reproducible method for assessing the relative contribution of OATP1B1 to hepatic uptake of new chemical entities. The technique also has potential utility in facilitating detailed characterization of drug-drug interactions involving hepatic drug transporters.

Long-lasting inhibitory effects of saquinavir and ritonavir on OATP1B1-mediated uptake.

Previously, we reported a long-lasting inhibition of transport mediated by organic anion-transporting polypeptides (OATPs) in humans and rats by cyclosporin A (CsA). In the present study, we examined the effects of several other compounds on OATP1B1-mediated transport, with a focus on long-lasting inhibition. Effects of coincubation, preincubation, or preincubation plus coincubation of 12 compounds on uptake of estrone 3-sulfate (E1 S) in OATP1B1-expressing HEK293T cells were examined. The OATP1B1 inhibitors used in the present study inhibited OATP1B1-mediated uptake of E1 S in a concentration-dependent manner. Among them, saquinavir and ritonavir in addition to CsA exhibited long-lasting inhibitory effects on OATP1B1-mediated transport of E1 S at ≥ 5 and 25 µM, respectively, even after they were washed out from the incubation buffer. After preincubation with saquinavir, its inhibitory effect on OATP1B1 remained for at least 6 h, whereas the effect of ritonavir did not remain. Protein expression of OATP1B1 was not altered by preincubation with 25 µM saquinavir or ritonavir. The present study firstly showed that saquinavir and ritonavir as well as CsA have long-lasting inhibitory effects on OATP1B1. But, at plasma unbound concentrations of saquinavir and ritonavir in clinical situations, they may not cause long-lasting inhibition of OATP1B1.

Rapid stimulating effect of the antiarrhythmic agent amiodarone on absorption of organic anion compounds.

In a clinical setting, changes in pharmacokinetics due to drug-drug interactions can often directly affect the therapeutic safety and efficacy of drugs. Recently, interest has been shown in drug-drug interactions in the intestine. It is now recognized that changes in the functions of drug transporters substantially influence the absorption of administered drugs from the intestine. Amiodarone (AMD) is a potent drug used in the treatment of serious supraventricular and ventricular tachyarrhythmias. Despite its potent pharmacological effects, its wide clinical use is precluded by drug-drug interactions. In this study, we characterized the transporter function between AMD and various compounds in human intestinal model Caco-2 cells. AMD significantly and rapidly increased the uptake of [(3)H]estrone-3-sulfate (E-3-S) for 5 min. The apical-to-basal transport of [(3)H]E-3-S was significantly increased by AMD. The AMD-stimulated [(3)H]E-3-S uptake was inhibited by organic anion transporting polypeptide (OATP) substrates. Caco-2 cells treated with AMD showed increased OATP2B1 expression on the cell surface. AMD also increased the absorption of sulfobromophthalein (BSP), which is a typical organic anion compound, and the expression level of Oatp2b1 at the membrane in in vivo experiments. The results indicate that AMD induces OATP2B1/Oatp2b1 expression at the membrane in the intestine and enhances absorption of organic anion compounds.

Classification of inhibitors of hepatic organic anion transporting polypeptides (OATPs): influence of protein expression on drug-drug interactions.

The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug-drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.

Simulating kinetic parameters in transporter mediated permeability across Caco-2 cells. A case study of estrone-3-sulfate.

Substances that compete for the same saturable intestinal transporters may when dosed together lead to altered permeability and hence influence bioavailability. The aim was to simulate kinetic parameters, i.e. K(m) and J(max), for transporter mediated E(1)S permeability across Caco-2 cells by a combined experimental modeling approach. 4 classes of transporters were suggested to be involved in the permeability of E(1)S, i.e. apical influx (T(I)) and efflux (T(III)) as well as basolateral efflux (T(II)) and influx (T(IV)). Efflux ratio of E(1)S was determined to 6.8. E(1)S is suggested to have highest affinity to T(III). T(IV) is however suggested to be rate limiting in exsorptive P(APP) due to lower J(max) of T(IV), compared to T(III). Possible interactions between E(1)S and the excipients erythrosine and Brij35 on these 4 classes of transporters were also studied. From these studies it is suggested that erythrosine does interact with E(1)S on apical efflux transporter T(III) by competitive inhibition. Furthermore interaction between erythrosine and E(1)S is suggested on apical influx transporter (T(I)). Brij35 does not seem to interact with E(1)S on apical transporters. The present model seem to be a valuable tool to simulate kinetic parameters for compounds being substrates to multiple transporters as well as to estimate kinetic parameters for compounds interacting on the same transporters.

pH-Dependent transport of pemetrexed by breast cancer resistance protein.

Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment.

Effects of uremic toxins on transport and metabolism of different biopharmaceutics drug disposition classification system xenobiotics.

Chronic kidney disease (CKD) is recognized to cause pharmacokinetic changes in renally excreted drugs; however, pharmacokinetic changes are also reported for drugs that are nonrenally eliminated. Few studies have investigated how uremic toxins may affect drug transporters and metabolizing enzymes and how these may result in pharmacokinetic/metabolic changes in CKD. Here, we investigated the effects of uremic toxins and human uremic serum on the transport of the prototypical transporter substrate [(3) H]-estrone sulfate and three Biopharmaceutics Drug Disposition Classification System (BDDCS) drugs, propranolol, losartan, and eprosartan. We observed a significant decrease in [(3) H]-estrone sulfate, losartan, and eprosartan uptake with some uremic toxins in both transfected cells and rat hepatocytes. The uptake of losartan was decreased in rat and human hepatocytes (28% and 48%, respectively) in the presence of hemodialysis (HD) serum. Time-course studies of losartan showed a 27%, 65%, and 68% increase in area under the curve (AUC) in the presence of HD serum, rifampin, and sulfaphenazole, respectively. Intracellular losartan AUC decreased significantly in the treatment groups, and the metabolite AUC decreased by 41% and 26% in rifampin- and sulfaphenazole-treated group, respectively. The intracellular AUC of eprosartan increased 190% in the presence of HD serum. These studies indicate that the uremic toxins contained in HD serum play an important role in drug disposition through drug transporters, and that there would be differential effects depending on the BDDCS classification of the drug.

Characterization of organic anion-transporting polypeptide (Oatp) 1a1 and 1a4 null mice reveals altered transport function and urinary metabolomic profiles.

Organic anion-transporting polypeptides (Oatp) 1a1 and 1a4 were deleted by homologous recombination, and mice were characterized for Oatp expression in liver and kidney, transport in isolated hepatocytes, in vivo disposition of substrates, and urinary metabolomic profiles. Oatp1a1 and Oatp1a4 proteins were undetected in liver, and both lines were viable and fertile. Hepatic constitutive messenger RNAs (mRNAs) for Oatp1a4, 1b2, or 2b1 were unchanged in Oatp1a1⁻/⁻ mice, whereas renal Oatp1a4 mRNA decreased approximately 50% (both sexes). In Oatp1a4⁻/⁻ mice, no changes in constitutive mRNAs for other Oatps were observed. Uptake of estradiol-17ß-D-glucuronide and estrone-3-sulfate in primary hepatocytes decreased 95 and 75%, respectively, in Oatp1a1⁻/⁻ mice and by 60 and 30%, respectively, in Oatp1a4⁻/⁻ mice. Taurocholate uptake decreased by 20 and 50% in Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, respectively, whereas digoxin was unaffected. Plasma area under the curve (AUC) for estradiol-17ß-D-glucuronide increased 35 and 55% in male and female Oatp1a1⁻/⁻ mice, respectively, with a concurrent 50% reduction in liver-to-plasma ratios. In contrast, plasma AUC or tissue concentrations of estradiol-17ß-D-glucuronide were unchanged in Oatp1a4⁻/⁻ mice. Plasma AUCs for dibromosulfophthalein increased nearly threefold in male Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, increased by 40% in female Oatp1a4⁻/⁻ mice, and were unchanged in female Oatp1a1⁻/⁻ mice. In both lines, no changes in serum ALT, bilirubin, and cholesterol were noted. NMR analyses showed no generalized increase in urinary excretion of organic anions. However, urinary excretion of taurine decreased by 30-40% and was accompanied by increased excretion of isethionic acid, a taurine metabolite generated by intestinal bacteria, suggesting some perturbations in intestinal bacteria distribution.

Functional analysis of human sodium-phosphate transporter 4 (NPT4/SLC17A3) polymorphisms.

We analyzed the functional properties of five nonsynonymous single nucleotide polymorphisms (SNPs) in the sodium-phosphate transporter NPT4 gene (SLC17A3) using the Xenopus oocyte expression system. NPT4 variants carrying SNP V257F, G279R, or P378L exhibited reduced transport of [(14)C]para-aminohippurate, [(3)H]bumetanide, [(3)H]estrone sulfate, and [(14)C]urate, when each variant clone was expressed in the plasma membrane of oocytes. This study suggests the possibility that the genetic variation of NPT4 contributes to inter-individual differences in disposition of anionic drugs such as diuretics as well as certain endogenous organic anions such as urate.

Ivermectin interacts with human ABCG2.

Ivermectin is an antiparasitic drug frequently administered to humans. It has a limited brain exposure that is attributed to the efflux activity of ABCB1/Abcb1. ABCG2/Abcg2 is also a major transporter present in most pharmacologically important barriers. However, interaction of ivermectin with Abcg2 shows species specificity and in many studies was confounded by the masking effect of ABCB1/Abcb1. In this study using cellular and membrane assays we show that ivermectin displays a high-affinity interaction with human ABCG2 with IC(50) values in the 1-1.5 µM range. This interaction may have implications in human ABCG2-mediated drug-drug interactions of ivermectin.