Systematic Development and Verification of a Physiologically Based Pharmacokinetic Model of Rivaroxaban.

Rivaroxaban is indicated for stroke prevention in nonvalvular atrial fibrillation (AF). Its elimination is mediated by both hepatic metabolism and renal excretion. Consequently, its clearance is susceptible to both intrinsic (pathophysiological) and extrinsic (concomitant drugs) variabilities that in turn implicate bleeding risks. Upon systematic model verification, physiologically based pharmacokinetic (PBPK) models are qualified for the quantitative rationalization of complex drug-drug-disease interactions (DDDIs). Hence, this study aimed to develop and verify a PBPK model of rivaroxaban systematically. Key parameters required to define rivaroxaban's disposition were either obtained from in vivo data or generated via in vitro metabolism and transport kinetic assays. Our developed PBPK model successfully predicted rivaroxaban's clinical pharmacokinetic parameters within predefined success metrics. Consideration of basolateral organic anion transporter 3 (OAT3)-mediated proximal tubular uptake in tandem with apical P-glycoprotein (P-gp)-mediated efflux facilitated mechanistic characterization of the renal elimination of rivaroxaban in both healthy and renal impaired patients. Retrospective drug-drug interaction (DDI) simulations, incorporating in vitro metabolic inhibitory parameters, accurately recapitulated clinically observed attenuation of rivaroxaban's hepatic clearance due to enzyme-mediated DDIs with CYP3A4/2J2 inhibitors (verapamil and ketoconazole). Notably, transporter-mediated DDI simulations between rivaroxaban and the P-gp inhibitor ketoconazole yielded minimal increases in rivaroxaban's systemic exposure when P-gp-mediated efflux was solely inhibited, but were successfully characterized when concomitant basolateral uptake inhibition was incorporated in the simulation. In conclusion, our developed PBPK model of rivaroxaban is systematically verified for prospective interrogation and management of untested yet clinically relevant DDDIs pertinent to AF management using rivaroxaban. SIGNIFICANCE STATEMENT: Rivaroxaban is susceptible to DDDIs comprising renal impairment and P-gp and CYP3A4/2J2 inhibition. Here, systematic construction and verification of a PBPK model of rivaroxaban, with the inclusion of a mechanistic kidney component, provided insight into the previously arcane role of OAT3-mediated basolateral uptake in influencing both clinically observed renal elimination of rivaroxaban and differential extents of transporter-mediated DDIs. The verified model holds potential for investigating clinically relevant DDDIs involving rivaroxaban and designing dosing adjustments to optimize its pharmacotherapy in atrial fibrillation.

Rat Organic Anion Transport Protein 1A1 Interacts Directly With Organic Anion Transport Protein 1A4 Facilitating Its Maturation and Trafficking to the Hepatocyte Plasma Membrane.

Organic anion transport proteins (OATPs) on the basolateral surface of hepatocytes mediate uptake of a number of drugs and endogenous compounds. Previous studies showed that rat OATP1A1 (rOATP1A1) has a postsynaptic density protein, drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) consensus binding motif at its C-terminus and binds to PDZ domain containing 1 (PDZK1), which is required for its cell-surface localization. PDZK1 associates with rOATP1A1-containing endocytic vesicles within cells, mediating recruitment of motor proteins required for microtubule-based trafficking to the plasma membrane. rOATP1A4 also traffics to the plasma membrane, although it lacks a PDZ binding consensus sequence. The current study was designed to test the hypothesis that trafficking of rOATP1A4 to the plasma membrane requires its direct interaction with rOATP1A1 resulting in a complex that traffics through the cell in common subcellular vesicles in which the cytosolic tail of rOATP1A1 is bound to PDZK1. We found that 74% of rOATP1A4-containing rat liver endocytic vesicles (n = 12,044) also contained rOATP1A1. Studies in transfected HEK293 cells showed surface localization of rOATP1A1 only when coexpressed with PDZK1 whereas rOATP1A4 required coexpression with rOATP1A1 and PDZK1. Studies in stably transfected HeLa cells that constitutively expressed PDZK1 showed that coexpression of rOATP1A4 with rOATP1A1 resulted in more rapid appearance of rOATP1A4 on the plasma membrane and faster maturation to its fully glycosylated form. Similar results were observed on immunofluorescence analysis of single cells. Immunoprecipitation of rat liver or transfected HeLa cell lysates with rOATP1A1 antibody specifically co-immunoprecipitated rOATP1A4 as determined by western blotting. Conclusion: These studies indicate that optimal rOATP1A4 trafficking to the cell surface is dependent upon coexpression and interaction with rOATP1A1. As rOATP1A1 binds to the chaperone protein, PDZK1, rOATP1A4 functionally hitchhikes through the cell with this complex.

The SUMO-Specific Protease Senp2 Regulates SUMOylation, Expression and Function of Human Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3) plays a vital role in removing a broad array of anionic drugs from kidney, thereby avoiding their possibly toxic side effects in the body. We earlier demonstrated that OAT3 is subjected to a specific type of post-translational modification called SUMOylation. SUMOylation is a dynamic event, where de-SUMOylation is catalyzed by a class of SUMO-specific proteases. In the present investigation, we assessed the role of SUMO-specific protease Senp2 in OAT3 SUMOylation, expression and function. We report here that overexpression of Senp2 in COS-7 cells led to a reduced OAT3 SUMOylation, which correlated well with a decreased OAT3 expression and transport activity. Such phenomenon was not observed in cells overexpressing an inactive mutant of Senp2. Furthermore, transfection of cells with Senp2-specific siRNA to knockdown the endogenous Senp2 resulted in an increased OAT3 SUMOylation, which correlated well with an enhanced OAT3 expression and transport activity. Coimmunoprecipitation experiments showed that Senp2 directly interacted with OAT3 in the kidneys of rats. Together these results provided first demonstration that Senp2 is a significant regulator for OAT3-mediated organic anion/drug transport.

Evidence for the Validity of Pyridoxic Acid (PDA) as a Plasma-Based Endogenous Probe for OAT1 and OAT3 Function in Healthy Subjects.

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of organic anion transporter (OAT) 1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single-dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 hour after receiving PROB (40 and 1000 mg orally) on days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased the area under the plasma concentration-time curve (AUC) of PDA by 3.1-fold (dosed alone; P < 0.05), and 3.2-fold (coadministered with FSM; P < 0.01), compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold (P > 0.05) and 2.1-fold (P < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC, whereas those of HVA are smaller (3.1-3.2 and 1.8-2.1 vs. 3.3, respectively). PDA and HVA renal clearance (CL R) values were decreased by PROB to smaller extents compared with FSM (0.35-0.37 and 0.67-0.73 vs. 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and that its plasma exposure responds in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CL R values between subjects is more favorable relative to HVA.

AG490, a JAK2-specific inhibitor, downregulates the expression and activity of organic anion transporter-3.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of AG490, a specific inhibitor of the Janus tyrosine kinase 2 (JAK2), in hOAT3 transport activity in the kidney COS-7 cells. AG490 induced a time- and concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter. The inhibitory effect of AG490 correlated with a reduced expression of hOAT3 at the cell surface. Our lab previously demonstrated that Nedd4-2, a ubiquitin ligase, down regulates OAT expression and transport activity by enhancing OAT ubiquitination, which leads to an internalization of OAT from cell surface to intracellular compartments and subsequent degradation. In the current study, we showed that treatment of hOAT3-expressing cells with AG490 resulted in an enhanced hOAT3 ubiquitination and degradation, which was accompanied by a strengthened association of Nedd4-2 with hOAT3 and a reduction in Nedd4-2 phosphorylation. SiRNA knockdown of endogenous Nedd4-2 abrogated the effects of AG490 on hOAT3. In summary, our study demonstrated that AG490 regulates hOAT3 expression and transport activity through the modulation of Nedd4-2.

The activity of organic anion transporter-3: Role of dexamethasone.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion, from the blood to urine, of clinically important drugs, such as anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of dexamethasone in hOAT3 transport activity in the kidney HEK293 cells. Cis-inhibition study showed that dexamethasone exhibited a concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter, with IC50 value of 49.91 µM. Dixon plot analysis revealed that inhibition by dexamethasone was competitive with a Ki = 47.08 µM. In contrast to the cis-inhibition effect of dexamethasone, prolonged incubation (6 h) of hOAT3-expressing cells with dexamethasone resulted in an upregulation of hOAT3 expression and transport activity, kinetically revealed as an increase in the maximum transport velocity Vmax without meaningful alteration in substrate-binding affinity Km. Such upregulation was abrogated by GSK650394, a specific inhibitor for serum- and glucocorticoid-inducible kinases (sgk). Dexamethasone also enhanced sgk1 phosphorylation. Our study demonstrated that dexamethasone exhibits dual effects on hOAT3: it is a competitive inhibitor for hOAT3-mediated transport, and interestingly, when entering the cells, it stimulates hOAT3 expression and transport activity through sgk1.

Prebiotic prevents impaired kidney and renal Oat3 functions in obese rats.

Obesity is health issue worldwide, which can lead to kidney dysfunction. Prebiotics are non-digestible foods that have beneficial effects on health. This study aimed to investigate the effects of xylooligosaccharide (XOS) on renal function, renal organic anion transporter 3 (Oat3) and the mechanisms involved. High-fat diet was provided for 12 weeks in male Wistar rats. After that, the rats were divided into normal diet (ND); normal diet treated with XOS (NDX); high-fat diet (HF) and high-fat diet treated with XOS (HFX). XOS was given daily at a dose of 1000 mg for 12 weeks. At week 24, HF rats showed a significant increase in obesity and insulin resistance associated with podocyte injury, increased microalbuminuria, decreased creatinine clearance and impaired Oat3 function. These alterations were improved by XOS supplementation. Renal MDA level and the expression of AT1R, NOX4, p67phox, 4-HNE, phosphorylated PKCα and ERK1/2 were significantly decreased after XOS treatment. In addition, Nrf2-Keap1 pathway, SOD2 and GCLC expression as well as renal apoptosis were also significantly reduced by XOS. These data suggest that XOS could indirectly restore renal function and Oat3 function via the reduction of oxidative stress and apoptosis through the modulating of AT1R-PKCα-NOXs activation in obese insulin-resistant rats. These attenuations were instigated by the improvement of obesity, hyperlipidemia and insulin resistance.

The N-terminal region of organic anion transporting polypeptide 1B3 (OATP1B3) plays an essential role in regulating its plasma membrane trafficking.

Organic anion transporting polypeptide 1B3 (OATP1B3) is a major influx transporter mediating the hepatic uptake of various endogenous substrates as well as clinically important drugs such as statins and anticancer drugs. However, molecular mechanisms controlling the membrane trafficking of OATP1B3 have been largely unknown. Several reports recently indicated the presence of a distinct, cancer-type OATP1B3 variant lacking the N-terminal 28 amino acids compared to OATP1B3 expressed in non-malignant hepatocytes. Interestingly, the cancer-type OATP1B3 variant is located predominantly in the cytoplasm, implicating the involvement of the N-terminal region of OATP1B3 in its membrane trafficking. In the current study, we set out to experimentally validate the importance of the N-terminal region of OATP1B3 and to identify responsible sequence motif(s) in that region. A number of truncation or point mutants of OATP1B3 were transiently expressed in HEK293T, HCT-8 or MDCK II cells and their expression in cytoplasmic and surface membrane fractions were analyzed by immunoblotting. Our results indicated that the N-terminal sequence of OATP1B3, in particular, at the amino acid positions between 12 and 28, may be indispensable in its membrane trafficking. Moreover, our results using a fusion construct indicated that the first 50 amino acids of OATP1B3 are sufficient for its membrane localization. The importance of the N-terminal region in membranous localization was shared among the other OATP1B subfamily members, OATP1B1 and rat Oatp1b2. Our efforts to identify the responsible amino acid(s) or structure motif(s) in the N-terminal region did not pinpoint individual amino acids or motifs with putative secondary structures. Our current findings however demonstrate that the N-terminal region is important for the membrane localization of the OATP1B subfamily members and should facilitate future investigations of the mechanisms involved in the regulation and membrane trafficking of these important transporter proteins.

Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.

Loss of SLCO1B3 drives taxane resistance in prostate cancer.

BACKGROUND: Both taxanes, docetaxel and cabazitaxel, are effective treatments for metastatic castration-resistant prostate cancer (mCRPC). However, resistance to taxanes is common. Our objective was to investigate mechanisms of taxane resistance in prostate cancer. METHODS: Two docetaxel-resistant patient-derived xenografts (PDXs) of CRPC were established (PC339-DOC and PC346C-DOC) in male athymic nude mice by frequent intraperitoneal administrations of docetaxel. Next-generation sequencing was performed on PDX tissue pre- and post-docetaxel resistance and gene expression profiles were compared. [(14)C]-docetaxel and [(14)C]-cabazitaxel uptake assays in vitro and cytotoxicity assays were performed to validate direct involvement of transporter genes in taxane sensitivity. RESULTS: Organic anion-transporting polypeptide (SLCO1B3), an influx transporter of docetaxel, was significantly downregulated in PC346C-DOC tumours. In accordance with this finding, intratumoural concentrations of docetaxel and cabazitaxel were significantly decreased in PC346C-DOC as compared with levels in chemotherapy-naive PC346C tumours. In addition, silencing of SLCO1B3 in chemo-naive PC346C resulted in a two-fold decrease in intracellular concentrations of both taxanes. Overexpression of SLCO1B3 showed higher sensitivity to docetaxel and cabazitaxel. CONCLUSIONS: The SLCO1B3 determines intracellular concentrations of docetaxel and cabazitaxel and consequently influences taxane efficacy. Loss of the drug transporter SLCO1B3 may drive taxane resistance in prostate cancer.

Modulation of ALDH5A1 and SLC22A7 by microRNA hsa-miR-29a-3p in human liver cells.

Observed variations in drug responses among patients may result from differences in heritable genetic traits or from alterations in the epigenetic regulation of drug metabolizing enzymes and transporters (DMETs). MicroRNAs (miRNAs), a group of small non-coding RNAs, provide an epigenetic mechanism for fine-tuning the expression of targeted DMET genes by regulating the efficiency of protein translation and by decreasing mRNA stability via enhanced degradation. In the current study we systematically screened 374 important genes encoding DMETs for potential response elements to hsa-miR-29a-3p, a highly abundant miRNA in human liver. RNA electrophoresis mobility shift assays displayed direct interactions between hsa-miR-29a-3p and its cognate targets within the mRNA transcripts for the ABCC6, SLC22A7 and ALDH5A1 genes. The expression of luciferase reporter genes containing the 3'-UTRs of SLC22A7 or ALDH5A1 and the expression of endogenous SLC22A7 and ALDH5A1 were each suppressed by transfection with hsa-miR-29a-3p mimics. Importantly, chemically-induced up-regulation of hsa-miR-29a-3p correlated inversely with the expression of SLC22A7 and ALDH5A1. However, our studies failed to detect suppressive effects of hsa-miR-29a-3p on ABCC6 expression, which might be explained by the notion that the interaction of hsa-miR-29a-3p and ABCC6 mRNA was unable to recruit ribonucleoproteins to form a RNA-induced silencing complex.

Quercetin does not alter the oral bioavailability of Atorvastatin in rats.

The study was undertaken to evaluate the effect of Quercetin on the pharmacokinetics of Atorvastatin Calcium. In-vivo Pharmacokinetic studies were performed on rats in a single dose study and multiple dose study. Rats were treated with Quercetin (10 mg/kg) and Atorvastatin Calcium (20 mg/kg) orally and blood samples were collected at (0) pretreatment and 0.5, 1, 1.5, 2, 2.5, 3, 4, 8, 12, 24 hours post treatment. Plasma concentrations of Atorvastatin were estimated by HPLC method. Quercetin treatment did not significantly alter the pharmacokinetic parameters of atorvastatin like AUC(0-24), AUC(0-α) , T(max), C(max) and T(½) in both single dose and multiple dose studies of Atorvastatin Calcium. Quercetin does not alter the oral bioavailability of Atorvastatin Calcium in rats.

Atenolol Renal Secretion Is Mediated by Human Organic Cation Transporter 2 and Multidrug and Toxin Extrusion Proteins.

Atenolol is a ß-blocker widely used in the treatment of hypertension. Atenolol is cleared predominantly by the kidney by both glomerular filtration and active secretion, but the molecular mechanisms involved in its renal secretion are unclear. Using a panel of human embryonic kidney cell lines stably expressing human organic cation transporter (hOCT) 1-3, human organic anion transporter (hOAT) 1, hOAT3, human multidrug and toxin extrusion protein (hMATE) 1, and hMATE2-K, we found that atenolol interacted with both organic cation and anion transporters. However, it is transported by hOCT1, hOCT2, hMATE1, and hMATE2-K, but not by hOCT3, hOAT1, and hOAT3. A detailed kinetic analysis coupled with absolute quantification of membrane transporter proteins by liquid chromatography-tandem mass spectrometry revealed that atenolol is an excellent substrate for the renal transporters hOCT2, hMATE1, and hMATE2-K. The Km values for hOCT2, hMATE1, and hMATE2-K are 280 ± 4, 32 ± 5, and 76 ± 14 µM, respectively, and the calculated turnover numbers are 2.76, 0.41, and 2.20 s(-1), respectively. To demonstrate unidirectional transepithelial transport of atenolol, we developed and functionally validated a hOCT2/hMATE1 double-transfected Madin-Darby canine kidney cell culture model. Transwell studies showed that atenolol transport in the basal (B)-to-apical (A) direction is 27-fold higher than in the A-to-B direction, whereas its B-to-A/A-to-B transport ratio was only 2 in the vector-transfected control cells. The overall permeability of atenolol in the B-to-A direction in hOCT2/hMATE1 cells was 44-fold higher than in control cells. Together, our data support that atenolol tubular secretion is mediated through the hOCT2/hMATEs secretion pathway and suggest a significant role of organic cation transporters in the disposition of an important antihypertensive drug.

Modulation of OCT3 expression by stress, and antidepressant-like activity of decynium-22 in an animal model of depression.

The organic cation transporter-3 (OCT3) is a glucocorticoid-sensitive uptake mechanism that has been shown to regulate the bioavailability of monoamines in brain regions that are implicated in the pathophysiology of depression. In the present study, the relative impacts of acute stress alone and acute stress with a history of repeated stress (chronic+acute) were evaluated in two strains of rats: the stress-vulnerable Wistar-Kyoto (WKY) strain and the somewhat more stress-resilient Long-Evans (LE) strain. OCT3 mRNA was significantly upregulated in the hippocampus of LE rats 2h after exposure to acute restraint stress, but not in acutely-restrained rats with a history of repeated social defeat stress. WKY rats exhibited a very different pattern. OCT3 mRNA was unaffected by acute restraint stress alone but was robustly upregulated after repeated+acute stress. There was also a corresponding increase in cytosolic OCT3 protein following repeated+acute stress in WKY rats 3h after presentation of the acute stressor. These results are consistent with the hypothesis that altered expression of the OCT3 may play a role in stress coping, and strain differences in regulation of this expression may contribute to differences in physiological and behavioral responses to stress. Furthermore, the OCT3 inhibitor, decynium 22 (1 and 10µg/kg, i.p.) reduced immobility of WKY rats, but not that of LE rats, in the forced swim test, suggesting that blockade of the OCT3 has antidepressant-like effects. Since WKY rats also appear to be resistant to the behavioral effects of traditional antidepressants, this also suggests that OCT3 antagonism may be an alternative therapeutic strategy for the treatment of depression in individuals who do not respond to conventional antidepressants.

Generation of Bayesian prediction models for OATP-mediated drug-drug interactions based on inhibition screen of OATP1B1, OATP1B1∗15 and OATP1B3.

Human organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are important hepatic uptake transporters. Early assessment of OATP1B1/1B3-mediated drug-drug interactions (DDIs) is therefore important for successful drug development. A promising approach for early screening and prediction of DDIs is computational modeling. In this study we aimed to generate a rapid, single Bayesian prediction model for OATP1B1, OATP1B1∗15 and OATP1B3 inhibition. Besides our previously generated HEK-OATP1B1 and HEK-OATP1B1∗15 cells, we now generated and characterized HEK-OATP1B3 cells. Using these cell lines we investigated the inhibitory potential of 640 FDA-approved drugs from a commercial library (10µM) on the uptake of [(3)H]-estradiol-17ß-d-glucuronide (1µM) by OATP1B1, OATP1B1∗15, and OATP1B3. Using a cut-off of ⩾60% inhibition, 8% and 7% of the 640 drugs were potent OATP1B1 and OATP1B1∗15 inhibitors, respectively. Only 1% of the tested drugs significantly inhibited OATP1B3, which was not sufficient for Bayesian modeling. Modeling of OATP1B1 and OATP1B1∗15 inhibition revealed that presence of conjugated systems and (hetero)cycles with acceptor/donor atoms in- or outside the ring enhance the probability of a molecule binding these transporters. The overall performance of the model for OATP1B1 and OATP1B1∗15 was ⩾80%, including evaluation with a true external test set. Our Bayesian classification model thus represents a fast, inexpensive and robust means of assessing potential binding of new chemical entities to OATP1B1 and OATP1B1∗15. As such, this model may be used to rank compounds early in the drug development process, helping to avoid adverse effects in a later stage due to inhibition of OATP1B1 and/or OATP1B1∗15.

The effect of organic anion-transporting polypeptides 1B1, 1B3 and 2B1 on the antitumor activity of flavopiridol in breast cancer cells.

The contribution of organic anion transporting polypeptides (OATPs) to the cellular uptake of flavopiridol was investigated in OATP1B1-, OATP1B3- and OATP2B1-expressing Chinese hamster ovary (CHO) cells. Uptake of flavopiridol into these cells showed typical Michaelis-Menten kinetics with much higher transport capacity for OATP1B3 compared to OATP1B1 and OATP2B1 (Vmax/Km, 33.9 vs. 8.84 and 2.41 µl/mg/min, respectively). The predominant role of OATPs was further supported by a dramatic inhibition of flavopiridol uptake in the presence of the OATP substrate rifampicin. Uptake of flavopiridol by OATPs also seems to be an important determinant in breast cancer cells. The much higher mRNA level for OATP1B1 found in wild-type compared to ZR-75-1 OATP1B1 knockdown cells correlated with higher flavopiridol initial uptake leading to 4.6-fold decreased IC50 values in the cytotoxicity assay (IC50, 1.45 vs. 6.64 µM). Cell cycle profile also showed a clear incidence for a stronger cell cycle arrest in the G2/M phase for ZR-75-1 wild-type cells compared to OATP1B1 knockdown cells, further indicating an active uptake via OATP1B1. In conclusion, our results revealed OATP1B1, OATP1B3 and OATP2B1 as uptake transporters for flavopiridol in cancer cells, which may also apply in patients during cancer therapy.

Human OATP1B1, OATP1B3 and OATP1A2 can mediate the in vivo uptake and clearance of docetaxel.

Organic anion transporting polypeptides (human: OATPs and mouse: Oatps) are uptake transporters with important roles in drug pharmacokinetics and toxicity. We aimed to study the in vivo impact of mouse and human OATP1A/1B transporters on docetaxel plasma clearance and liver and intestinal uptake. Docetaxel was administered to Oatp1a/1b knockout and liver-specific humanized OATP1B1, OATP1B3 and OATP1A2 transgenic mice. Experiments were conducted with a low polysorbate 80 (2.8%) formulation, as 8% polysorbate somewhat inhibited docetaxel plasma clearance after intravenous administration. After intravenous administration (10 mg/kg), Oatp1a/1b knockout mice had an approximately threefold higher plasma area under the curve (AUC). Impaired liver uptake was evident from the significantly reduced (approximately threefold) liver-to-plasma AUC ratios. Absence of mouse Oatp1a/1b transporters did not affect the intestinal absorption of orally administered docetaxel (10 mg/kg), while the systemic exposure of docetaxel was again substantially increased owing to impaired liver uptake. Most importantly, liver-specific expression of each of the human OATP1B1, OATP1B3 and OATP1A2 transporters provided a nearly complete rescue of the increased plasma levels of docetaxel in Oatp1a/1b-null mice after intravenous administration. Our data show that one or more of the mouse Oatp1a/1b transporters and each of the human OATP1A/1B transporters can mediate docetaxel uptake in vivo. This might be clinically relevant for OATP1A/1B-mediated tumor uptake of docetaxel and for docetaxel clearance in patients in whom the transport activity of OATP1A/1B transporters is reduced owing to genetic variation or pharmacological inhibition, leading to potentially altered toxicity and therapeutic efficacy of this drug.

Novel mechanism of impaired function of organic anion-transporting polypeptide 1B3 in human hepatocytes: post-translational regulation of OATP1B3 by protein kinase C activation.

The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [(3)H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [(3)H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti-phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.

Effect of selective cyclooxygenase-2 inhibitor lumiracoxib on phenolsulfonphthalein disposition in rats.

Selective cyclooxygenase-2 inhibitor lumiracoxib was shown to have the strong inhibitory potencies on the renal organic anion transporter (OAT)1 and also on OAT3 from drug transport experiments. The purpose of this study was to examine the effect of lumiracoxib on disposition of phenolsulfonphthalein (PSP) - which is mainly excreted into urine via OATs - from in vivo experiments. After the intravenous injection of PSP and lumiracoxib into rats, pharmacokinetic analysis was performed. After the intravenous injection of PSP as a bolus, its plasma concentration decreased time-dependently. Until 60 min after the injection, 51.1% of the dose was recovered into urine. The simultaneous administration of lumiracoxib increased the plasma levels of PSP and reduced its urinary recovery to 23.6% of the dose. The pharmacokinetic analysis using a two-compartment model exhibited that lumiracoxib affected the parameters implying the elimination of PSP. The present study demonstrates that lumiracoxib interfered with urinary excretion of PSP in rats.