Expression Of OATP1A2 Research Articles

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.

Abstract 3929: Estrone-3-sulphate, a potential novel ligand for targeting breast cancers.

Abstract This study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane-associated transport proteins, for detection of hormone dependent breast cancers. E3S, a circulating inactive plasma estrogen, is an OATP substrate and a predominant source of tumour estradiol in post-menopausal patients. It has been reported that some of the OATP isoforms (e.g. OATP1A2, OATP2B1) are over expressed (up to ten times) in breast cancer tissues as compared to surrounding normal tissues. This suggests that the two- three times higher E3S concentration reported in malignant tissues, as compared to the surrounding normal tissues, could be due to higher expression and/or increased functional activity of OATPs detected in breast tumour tissues. We recently demonstrated in vitro that expression of some of the OATPs (i.e. OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, OATP3A1 and OATP4A1) were either exclusive, similar or significantly higher (p<0.001) in breast cancer cells as compared to normalized breast epithelial cells (MCF10A). Thus to assess in vivo, the potential of E3S as a ligand for targeting OATPs in breast cancers, distribution of exogenous E3S (administered intravenously) was determined in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6h post injection (p.i) with significant difference between the level in MCF-7 (13.9 ± 3.1 %ID/g) and MDA-MB-231 (10.4 ± 1.1 %ID/g). 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 6h p.i. Analogous to total tumour uptake, ex vivo tumour cell uptake at 2h p.i. was 6 fold higher in MCF-7 compared to MDA-MB-231 tumour cells. Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower tumour uptake in both xenograft models, suggesting the involvement of an active carrier mediated process. Immunohistochemical analysis detected OATP1A2 in tumour sections from both xenografts, with significantly higher expression 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 suggest the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers. To the best of our knowledge, this is the first report documenting the in vivo distribution of exogenous E3S, in models of hormone dependent and independent breast cancers. Citation Format: Nilasha Banerjee, Humphrey Fonge, Andrew Mikhail, Raymond M. Reilly, Reina Bendayan, Christine Allen. Estrone-3-sulphate, a potential novel ligand for targeting breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2013-3929

Enhanced expression of organic anion transporting polypeptides (OATPs) in androgen receptor-positive prostate cancer cells: Possible role of OATP1A2 in adaptive cell growth under androgen-depleted conditions

The biological mechanisms underlying castration resistance of prostate cancer are not fully understood. In the present study, we examined the role of organic anion transporting polypeptides (OATPs) as importers of dehydroepiandrosterone sulfate (DHEAS) into cells to support growth under androgen-depleted conditions. Cell growth and mRNA expression of OATP genes were studied in human prostate cancer LNCaP and 22Rv1 cells under androgen-depleted conditions. The stimulatory effect of DHEAS on cell growth was investigated in LNCaP cells in which OATP1A2 had been silenced. Growth of both cell lines was stimulated by DHEAS and the effect was attenuated by STX64, an inhibitor of steroid sulfatase which can covert DHEAS to DHEA. OATP1A2 mRNA expression was increased most prominently among various genes tested in LNCaP cells grown in androgen-depleted medium. Similar results were obtained with 22Rv1 cells. Furthermore, the characteristics of [3H]DHEAS uptake by LNCaP cells were consistent with those of OATP-mediated transport. Knockdown of OATP1A2 in LNCaP cells resulted in loss of the DHEAS sensitivity of cell growth. Our results suggest that enhanced OATP1A2 expression is associated with adaptive cell growth of prostate cancer cells under androgen-depleted conditions. Thus, OATP1A2 may be a pharmacological target for prostate cancer treatment.

Hypoxia/Reoxygenation (H/R) Stress Increases Organic Anion Transporting Polypeptide 1a4 (Oatp1a4)‐mediated Transcellular Transport at the Blood‐Brain Barrier (BBB): Relevance to CNS Drug Delivery

Our objective was to investigate effects of H/R stress, a component of ischemic stroke, on Oatp1a4‐mediated transcellular transport at the BBB. Rats (200–250 g) were subjected to normoxia (Nx, 21% O2, 60 min), hypoxia (Hx, 6% O2, 60 min) or H/R (6% O2, 60 min followed by 21% O2, 10 min). In rat brain microvessels, Oatp1a4 expression was increased following H/R but was unchanged by Nx or Hx. Uptake of Oatp1a4 substrates (i.e., taurocholate, atorvastatin) were increased following H/R. Oatp1a4 inhibitors (i.e., fexofenadine, estrone‐3‐sulfate) blocked taurocholate and atorvastatin brain uptake in H/R animals, further implicating Oatp1a4 in BBB transcellular transport of these drugs. Additionally, H/R reduced serum levels of transforming growth factor‐β1 (TGF‐β1) and decreased microvascular expression of TGF‐β receptor activin receptor‐like kinase (ALK)‐5. Pretreatment with SB431542, an ALK5 inhibitor, increased Oatp1a4 functional expression both in H/R and Nx animals. These novel data demonstrate that targeting an endogenous BBB transporter may be a viable approach for delivering drugs to the brain during cerebral ischemia. Our study also identifies a BBB signaling mechanism (i.e., TGF‐β/ALK5 pathway) that can be exploited to control expression of Oatp1a4 during H/R and, therefore, optimize CNS drug delivery. This work was supported by NIH R01 grant DA‐11271 and an AAPS New Investigator Grant.

Impact of testosterone on the expression of organic anion transporting polypeptides (OATP-1A2, OATP-2B1, OATP-3A1) in malignant and non-malignant human breast cells in vitro

ObjectivesPostmenopausal hormone therapy (HT) increases local estrogen formation in breast tissue. The enzymatic substrates depend on transmembrane anion transporting polypeptides (OATPs) to reach intracellular enzymes. The aim of this study was to investigate the effect of testosterone (T) on the expression of OATP-1A2, OATP-2B1, and OATP-3A1 in malignant (MCF-7, BT-474) and non-malignant (HBL-100) breast cells in vitro. Study designCells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3d, and subsequently incubated in the absence or presence of T, anastrozole (A), and T+A (10−6M) for 24h at 37°C. Main outcome measuresOATP expression was determined by immunocytochemical staining. Expression intensity was graded as low, moderate, or strong. Hormone receptor (AR, PR, ESR1, ESR2) expression was investigated by qPCR and Western blotting. Rank variance analysis was performed for statistical analysis (p≤0.05). ResultsOATP-1A2, OATP-2B1, and OATP-3A1 expression was present in all untreated breast cell lines examined, with OATP-1A2 and OATP-3A1 being the predominant ones. There was a trend for a higher baseline expression in untreated HBL-100 and BT-474 in comparison to MCF-7 cells, which was significant for OATP-2B1. T treatment led to decreased OATP-1A2, -2B1, and -3A1 expression in BT-474 and HBL-100 cells, respectively. In contrast, in MCF-7 cells, OATP-2B1 expression was significantly increased. T-induced upregulation of AR and PR protein expression in BT-474 and MCF-7 cells was reduced by A treatment. ConclusionsT may constitute a signal for differential regulation of mammary OATP expression. In non-malignant breast cells T seems to have a beneficial effect by reducing the availability of substrates for the intracellular formation of potent estrogens.

Oral Absorption of Clarithromycin Is Nearly Abolished by Chronic Comedication of Rifampicin in Foals

The delivery of clarithromycin (CRL) to its site of action in bronchial/alveolar epithelial cells (EC), bronchial epithelial lining fluid (ELF), and bronchoalveolar lavage cells (BALC) may be influenced by CYP3A4 and the drug transporters, ATP-binding cassette (ABC) B1 and ABCC2 and organic anion-transporting polypeptides (OATPs), which can be modulated and/or up-regulated via the nuclear pregnane X receptor (PXR) by rifampicin (RIF). Therefore, we evaluated the disposition and pulmonary distribution of CLR (7.5 mg/kg b.i.d., 21 days) and expression of ABCB1, ABCC2, OATP1A2, and OATP2B1 in EC and BALC before and after comedication of RIF (10 mg/kg b.i.d., 11 days) in nine healthy foals (41-61 days, 115-159 kg) in which the genetic homology of drug transporters is close to that of their human analogs. After RIF comedication, relative bioavailability of CLR decreased by more than 90%. Concentrations in plasma (29.8 ± 26.3 versus 462 ± 368 ng/ml), ELF (0.69 ± 0.66 versus 9.49 ± 6.12 μg/ml), and BALC (10.2 ± 10.2 μg/ml 264 ± 375 μg/ml; all P < 0.05) were lowered drastically, whereas levels of the metabolite 14-hydroxyclarithromycin were not elevated despite higher 4β-hydroxycholesterol/cholesterol plasma concentration ratio, a surrogate for CYP3A4 induction. In the presence of CLR, ABCC2 and PXR mRNA contents were significantly and coordinately (r(2) = 0.664, P < 0.001) reduced in BALC after RIF. In EC, mRNA expression of OATP1A2 increased but that of OATP2B1 decreased (both P < 0.05). RIF interrupts oral absorption and decreases CRL plasma levels below the minimal inhibitory concentration for eradication of Rhodococcus equi. Evidence that RIF influences the cellular uptake of CLR in bronchial cells and the PXR expression in BALC in the presence of high CLR concentrations exists.

Inflammatory Pain Signals an Increase in Functional Expression of Organic Anion Transporting Polypeptide 1a4 at the Blood-Brain Barrier

Pain is a dominant symptom associated with inflammatory conditions. Pharmacotherapy with opioids may be limited by poor blood-brain barrier (BBB) permeability. One approach that may improve central nervous system (CNS) delivery is to target endogenous BBB transporters such as organic anion-transporting polypeptide 1a4 (Oatp1a4). It is critical to identify and characterize biological mechanisms that enable peripheral pain/inflammation to "transmit" upstream signals and alter CNS drug transport processes. Our goal was to investigate, in vivo, BBB functional expression of Oatp1a4 in animals subjected to peripheral inflammatory pain. Inflammatory pain was induced in female Sprague-Dawley rats (200-250 g) by subcutaneous injection of 3% λ-carrageenan into the right hind paw; control animals were injected with 0.9% saline. In rat brain microvessels, Oatp1a4 expression was increased during acute pain/inflammation. Uptake of taurocholate and [d-penicillamine(2,5)]-enkephalin, two established Oatp substrates, was increased in animals subjected to peripheral pain, suggesting increased Oatp1a4-mediated transport. Inhibition of inflammatory pain with the anti-inflammatory drug diclofenac attenuated these changes in Oatp1a4 functional expression, suggesting that inflammation in the periphery can modulate BBB transporters. In addition, diclofenac prevented changes in the peripheral signaling cytokine transforming growth factor-β1 (TGF-β1) levels and brain microvascular TGF-β receptor expression induced by inflammatory pain. Pretreatment with the pharmacological TGF-β receptor inhibitor 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) increased Oatp1a4 functional expression in λ-carrageenan-treated animals and saline controls, suggesting that TGF-β signaling is involved in Oatp1a4 regulation at the BBB. Our findings indicate that BBB transporters (i.e., Oatp1a4) can be targeted during drug development to improve CNS delivery of highly promising therapeutics.

Hepatic transporters – Regulation, induction and potential for drug-drug interactions

Event Abstract Back to Event Hepatic transporters – Regulation, induction and potential for drug-drug interactions Gabrielle Hawksworth1* 1 University of Aberdeen, Department of Molecular Toxicology, United Kingdom Organic anion transporting polypeptides (Oatps) are basolateral membrane solute carriers with a broad range of substrates, including bile salts, steroid conjugates, thyroid hormones and numerous drugs. The three predominant Oatps in rat liver are Oatp1a1, Oatp1a4 and Oatp1b2. Nuclear receptors, including the pregnane X receptor (PXR), constitutive androstane receptor (CAR) and farnesoid X receptor (FXR) are implicated in the regulation of both transporters and P450s. In vivo dosing of rats with 150mg/kg dexamethasone for 72h caused a 50 fold induction of Oatp1a4 expression, whilst having no significant effect on the expression of Oatp1a1 or Oatp1b2. Treatment of sandwich cultured hepatocytes with PXR agonists, dexamethasone or pregnenolone-16 α-carbonitrile, induced expression of Oatp1a4 6-8 fold. As was seen in vivo, treatment with PXR agonists did not significantly affect expression of Oatp1a1 or Oatp1b2. The FXR agonists, GW-4064 and chenodeoxycholic acid, caused a dose-dependent decrease in the expression levels of Oatps. The role of PXR in the induction of Oatp1a4 by dexamethasone and PCN was confirmed by transfection of cells with PXRsiRNA prior to treatment with PCN. The ATP-dependent multidrug resistance associated proteins (Mrps/Abccs) are responsible for the efflux of a wide range of substrates, including glutathione, glucuronide and sulphate conjugates, unconjugated organic anions and bile salts. Abccs 1-6 are expressed in rat and human livers, although only Abcc2 is located on the apical membrane. Sandwich culture is essential for promoting bile canalicular reformation in vitro and for the expression and function of the abccs. PXR, CAR and FXR agonists are involved in the induction of abcc2 and abcc3. Abcc2 and 5 are upregulated in response to a toxic insult to the cell, probably via Nrf2 activation, suggesting a role in cell defence. Keywords: Organic anion transporting polypeptides, Oatp1a4, Oatp1a1, Oatp1b2, Abccs Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Invited speaker Topic: Drug transport Citation: Hawksworth G (2010). Hepatic transporters – Regulation, induction and potential for drug-drug interactions. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00128 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Gabrielle Hawksworth, University of Aberdeen, Department of Molecular Toxicology, Aberdeen, United Kingdom, g.m.hawksworth@abdn.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gabrielle Hawksworth Google Gabrielle Hawksworth Google Scholar Gabrielle Hawksworth PubMed Gabrielle Hawksworth Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Different expression patterns of organic anion transporting polypeptides in osteosarcomas, bone metastases and aneurysmal bone cysts

Organic anion transporting polypeptides (OATP) were identified as transmembrane transporters for various endo- and exogenous organic compounds (hormones, prostaglandins, anticancer drugs). OATP expression had been shown in different tissues, but not in bone tumors. Therefore, the expression pattern of all known eleven human OATPs was analyzed by quantitative RT-PCR in 21 human bone tumor specimens (osteosarcomas, bone metastases and benign aneurysmal bone cysts). Transcriptional expression of OATP1A2, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1 and 5A1, but not of OATP1B1, 1B3 and 6A1 was observed in malignant and non-malignant tumor specimens at varying level. Importantly, OATP3A1, 4A1, 2B1 and 1C1 mRNA levels were significantly higher in aneurysmal bone cysts as compared to osteosarcomas. Elevated mRNA levels of OATP2A1, 1A2, and 4C1 in metastases from kidney cancer and of OATP5A1 in prostate cancer suggest that the OATP expression pattern in metastases is comparable to that of the primary tumors. Different to tissue, OATP expression in osteosarcoma cell lines HOS and MG-63, normal human osteoblast outgrowth cells (hOB) and bone marrow stromal cells (BMSC) is limited to OATP3A1 and OATP4A1. High OATP expression levels, particularly in benign bone tumors, suggest an important role of these transporters for providing hormones, their conjugates, prostaglandins and drugs in bone cells. Thereby, they may influence bone resorption and formation.

Targeted Disruption of Murine Organic Anion-Transporting Polypeptide 1b2 (oatp1b2/Slco1b2) Significantly Alters Disposition of Prototypical Drug Substrates Pravastatin and Rifampin

Organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are widely acknowledged as important and rate-limiting to the hepatic uptake of many drugs in clinical use. Accordingly, to better understand the in vivo relevance of OATP1B transporters, targeted disruption of murine Slco1b2 gene was carried out. It is noteworthy that Slco1b2(-/-) mice were fertile, developed normally, and exhibited no overt phenotypic abnormalities. We confirmed the loss of Oatp1b2 expression in liver using real-time polymerase chain reaction, Western Blot analysis, and immunohistochemistry. Expression of Oatp1a4 and Oatp2b1 but not Oatp1a1 was greater in female Slco1b2(-/-) mice, but expression of other non-OATP transporters did not significantly differ between wild-type and Slco1b2(-/-) male mice. Total bilirubin level was elevated by 2-fold in the Slco1b2(-/-) mice despite the fact that liver enzymes ALT and AST were normal. Pharmacological characterization was carried out using two prototypical substrates of human OATP1B1 and -1B3, rifampin and pravastatin. After a single intravenous dose of rifampin (1 mg/kg), a 1.7-fold increase in plasma area under the concentration-time curve (AUC) was observed, whereas the liver-to-plasma ratio was reduced by 5-fold, and nearly 8-fold when assessed at steady-state conditions after 24 h of continuous subcutaneous infusion in Slco1b2(-/-) mice. Likewise, continuous subcutaneous infusion at low (8 microg/h) or high (32 microg/h) dose rates of pravastatin resulted in a 4-fold lower liver-plasma ratio in the in Slco1b2(-/-) mice. This is the first report of altered drug disposition profile in the Slco1b2 knockout mice and suggests the utility of this model for understanding the in vivo role of hepatic OATP transporters in drug disposition.

Obstructive cholestasis induces TNF-α- and IL-1β-mediated periportal downregulation of Bsep and zonal regulation of Ntcp, Oatp1a4, and Oatp1b2

Inverse acinar regulation of Mrp2 and 3 represents an adaptive response to hepatocellular cholestatic injury. We studied whether obstructive cholestasis (bile duct ligation) and LPS treatment affect the zonal expression of Bsep (Abcb11), Mrp4 (Abcc4), Ntcp (Slc10a1), and Oatp isoforms (Slco1a1, Slco1a4, and slco1b2) in rat liver, as analyzed by semiquantitative immunofluorescence. Contribution of TNF-alpha and IL-1beta to transporter zonation in obstructive cholestasis was studied by cytokine inactivation. In normal liver Bsep, Mrp4, Ntcp, and Oatp1a1 were homogeneously distributed in the acinus, whereas Oatp1a4 and Oatp1b2 expression increased from zone 1 to 3. Glutamine synthetase-positive pericentral hepatocytes exhibited markedly lower Oatp1a4 expression than the remaining zone 3 hepatocytes. In cholestatic liver Bsep and Ntcp immunofluorescence in periportal hepatocytes significantly decreased to 66 +/- 4% (P < 0.01) and 67 +/- 7% (P < 0.05), whereas it was not altered in pericentral hepatocytes. Oatp1a4 was significantly induced in hepatocytes with a primarily low expression, i.e., in periportal hepatocytes and in glutamine synthetase-positive pericentral hepatocytes. Likewise, Oatp1b2 was upregulated in periportal hepatocytes. Mrp4 zonal induction was homogeneous. Inactivation of TNF-alpha and IL-1beta prevented periportal downregulation of Bsep. Recruitment of neutrophils and polymorphonuclear cells mainly occurred in the periportal zone. Likewise, IL-1beta induction was largely found periportally. No significant transporter zonation was seen following LPS treatment. In conclusion, zonal downregulation of Bsep in obstructive cholestasis is associated with portal inflammation and is mediated by TNF-alpha and IL-1beta. Periportal downregulation of Ntcp and induction of Oatp1a4 and Oatp1b2 may represent adaptive mechanisms to reduce cholestatic injury in hepatocytes with profound downregulation of Bsep and Mrp2.

The expression of the solute carriers NTCP and OCT‐1 is regulated by cholesterol in HepG2 cells

Drug disposition and response are greatly determined by the activities of drug-metabolizing enzymes and transporters. While the knowledge in terms of CYP enzymes and efflux ABC transporters (such as MDR1, P-glycoprotein) is quite extensive, influx transporters are increasingly being unveiled as key contributors to the process of drug disposition. There is little information on the regulation of these proteins in human cells, especially as regards the effect of endogenous compounds. In this study, we analysed the expression of CYP3A4 and three uptake transporters NTCP (SLC10A1), OATP-A/OATP1A2 (SLCO1A2) and OCT-1 (SLC22A1) in HepG2 cells following treatment with cholesterol. While CYP3A4 and OATP1A2 expression was unaffected, cholesterol treatment led to increased levels of NTCP and OCT-1 mRNAs. Alterations in the functional characteristics and/or expression levels of drug transporters in the liver may conceivably contribute to the variability in drug oral bioavailability often observed in the clinical settings.

Intestinal Drug Transporter Expression and the Impact of Grapefruit Juice in Humans

The goals of this study were to assess the extent of human intestinal drug transporter expression, determine the subcellular localization of the drug uptake transporter OATP1A2, and then to assess the effect of grapefruit juice consumption on OATP1A2 expression relative to cytochrome P450 3A4 and MDR1. Expression of drug uptake and efflux transporters was assessed using human duodenal biopsy samples. Fexofenadine uptake by different transporters was measured in a transporter-transfected cell line. We investigated the influence of grapefruit juice on pharmacokinetics of orally administered fexofenadine. The effect of grapefruit juice on the expression of intestinal transporters was determined using real-time polymerase chain reaction and Western blot analysis. In the duodenum of healthy volunteers, an array of CYP enzymes as well as uptake and efflux transporters was expressed. Importantly, uptake transporters thought to be liver-specific, such as OATP1B1 and 1B3, as well as OATP2B1 and 1A2 were expressed in the intestine. However, among OATP transporters, only OATP1A2 was capable of fexofenadine uptake when assessed in vitro. OATP1A2 colocalized with MDR1 to the brush border domain of enterocytes. Consumption of grapefruit juice concomitantly or 2 h before fexofenadine administration was associated with reduced oral fexofenadine plasma exposure, whereas intestinal expression of either OATP1A2 or MDR1 remained unaffected. In conclusion, an array of drug uptake and efflux transporters are expressed in the human intestine. OATP1A2 is likely the key intestinal uptake transporter for fexofenadine absorption whose inhibition results in the grapefruit juice effect. Although short-term grapefruit juice ingestion was associated with reduced fexofenadine availability, OATP1A2 or MDR1 expression was unaffected.

Regulation of mRNA Expression of Xenobiotic Transporters by the Pregnane X Receptor in Mouse Liver, Kidney, and Intestine

Multiple transporter systems are involved in the disposition of xenobiotics and endogenous compounds. The pregnane X receptor (PXR) is a major chemical sensor known to activate the expression of CYP3A/Cyp3a in humans and rodents. The purpose of this study is to systematically determine whether the major xenobiotic transporters in liver, kidney, duodenum, jejunum, and ileum are induced by pregnenolone-16alpha-carbonitrile (PCN), and whether this increase is mediated by the nuclear receptor PXR. In liver, PCN induced the expression of Oatp1a4 and Mrp3 mRNA in wild-type (WT) mouse liver, but not in PXR-null mice. In kidney, PCN did not alter the expression of any drug transporter. In duodenum, PCN increased Abca1 and Mdr1a mRNA expression in WT mice, but not in PXR-null mice. In jejunum and ileum, PCN increased Mdr1a and Mrp2 mRNA, but decreased Cnt2 mRNA in WT mice, but none of these transporters was altered when PCN was administered to PXR-null mice. Therefore, PCN regulates the expression of some transporters, namely, Oatp1a4 and Mrp3 in liver, as well as Abca1, Cnt2, Mdr1a, and Mrp2 in small intestine via a PXR-mediated mechanism.

TISSUE DISTRIBUTION AND ONTOGENY OF MOUSE ORGANIC ANION TRANSPORTING POLYPEPTIDES (OATPS)

Organic anion-transporting polypeptides (Oatps) are Na(+)-independent solute carriers for cellular uptake of organic compounds. The purpose of this study is to determine 1) the constitutive mRNA expression of the 15 mouse Oatp genes in 12 tissues, 2) whether there are gender differences in Oatp expression, and 3) the ontogenic expression of Oatps in liver and kidney. The mRNA expression of the 15 mouse Oatps was quantified using the branched DNA technique. Oatp1a1, 1a4, 1b2, and 2b1 are expressed in liver at relatively high levels, with Oatp1b2 being exclusively expressed in liver. Oatp1a1, 1a6, 3a1, and 4c1 are highly expressed in kidney. Oatp1a4 and 1c1 are highly expressed in brain. Oatp1a5, 6b1, 6c1, and 6d1 are predominant in testes. Oatp2a1, 4a1, and 5a1 are predominantly expressed in placenta. In liver, expression of Oatp1a1 was male-predominant, whereas expression of Oatp1a4 and 1a6 was female-predominant. In kidney, expression of Oatp1a1, 3a1, and 4c1 was higher in males than in females. Hepatic expression of Oatp1a1, 1a4, 1a6, 1b2, and 2b1 gradually increased after birth and reached adult levels by 6 weeks of age. Only Oatp2a1 was expressed at adult levels at birth. In kidney, expression of mouse Oatp1a1, 1a6, and 3a1 was lower at birth than at 6 weeks of age, whereas expression of mouse Oatp1a4, 2a1, and 2b1 was similar at birth and at 6 weeks of age. These data on the tissue distribution and ontogenic expression of mouse Oatps will aid in understanding the pharmacokinetics and toxicokinetics of drugs and other chemicals.

Fexofenadine pharmacokinetics are associated with a polymorphism of the SLCO1B1 gene (encoding OATP1B1)

Fexofenadine pharmacokinetics are associated with a polymorphism of the <i>SLCO1B1</i> gene (encoding OATP1B1)

Molecular cloning and functional characterization of the bovine ( Bos taurus) organic anion transporting polypeptide Oatp1a2 (Slco1a2)

We describe the cloning, functional characterization and tissue localization of a novel membrane transporter of the OATP/Oatp-gene family obtained from liver and kidney of cattle ( Bos taurus). The carrier protein exhibits highest sequence identity to the human OATP1A2 (previously called OATP-A) and is, therefore, named bovine Oatp1a2. Bovine Oatp1a2 received the gene symbol Slco1a2 that is identical to the SLC classification of human OATP1A2 (SLCO1A2, previously called SLC21A3) and is likely an orthologue of the human gene. Two different full-length bOatp1a2 cDNAs of 2316-bp and 3504-bp were obtained and encoded for a 666 amino acid membrane protein, which contains twelve putative transmembrane spanning domains. Bovine Oatp1a2 expression was detected in liver, kidney, brain and adrenal gland. Uptake studies in cRNA-injected oocytes demonstrated that bOatp1a2 transports estrone-3-sulfate and taurocholate, with K m values of 9.6 μM and 51 μM, respectively, and estradiol-17β-glucuronide. However, the structurally-related heart glycosides ouabain (1 μM) and digoxin (1 μM) are neither transported by bovine Oatp1a2 nor by human OATP1A2. We conclude that based on the tested substrates bovine Oatp1a2 shows functional homology to human OATP1A2.