Organic Anion Transporting Polypeptide 1A2 Research Articles

Abstract WP433: Targeting Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) Expression at the Blood-brain Barrier: Implications for Treatment of Ischemic Stroke

Introduction: Stroke is a leading cause of mortality and morbidity. Several drugs with neuroprotective properties have been proposed for stroke treatment but many have failed in clinical trials. These failures may be due to limited drug permeability across the blood-brain barrier (BBB). Targeting endogenous BBB uptake transporters (i.e., organic anion transporting polypeptides (Oatps)) is a novel approach that can improve CNS delivery of drugs relevant to stroke therapy (i.e., statins). Optimal CNS drug delivery via Oatp in the setting of stroke requires characterization of regulatory pathways such as transforming growth factor (TGF)-β signaling. The goal of the present study was to investigate, in vivo , involvement of TGF-β signaling via the activin-like kinase (ALK)-1 receptor on Oatp1a4 expression at the BBB. Methods: Female Sprague-Dawley rats (200-250 g) were administered bone morphogenic protein-9 (BMP-9; 0-5 μg/kg, i.p.), an ALK1 agonist, or vehicle (i.e., 0.9% saline). Inhibition experiments were performed using LDN193189 (0-5 mg/kg, i.p.), an ALK1 antagonist. Western blot analysis and fluorescence microscopy of isolated brain microvessels were used to determine protein expression and localization in rat brain microvessels respectively. Results: Fluorescence staining demonstrated localization of Oatp1a4 and ALK1 in rat brain microvessels. Western blot analysis showed a dose dependent increase in Oatp1a4 protein expression in brain microvessels isolated from BMP-9 treated rats as compared to controls. Treatment with 0.5 μg/kg and 5 μg/kg BMP-9 resulted in a 55% and 116% increase in Oatp1a4 protein expression, implying that activation of ALK1 signaling can up-regulate Oatp1a4 at the brain microvasculature. In contrast, 6 h treatment with LDN193189 did not alter Oatp1a4 expression across a dose range of 0-5 mg/kg, suggesting that ALK1 inhibition does not modulate basal Oatp1a4 expression at the BBB. Conclusions: Taken together, our data implies that TGF-β/ALK1 signaling may play a role in altering Oatp1a4 protein expression at the BBB. Studies are currently being undertaken in our laboratory to fully characterize the role of TGF-β/ALK1 signaling in determining CNS delivery of drugs relevant to stroke treatment (i.e., statins).

An Appraisal of Drug-Drug Interactions with Green Tea (Camellia sinensis)

This review summarizes published in vitro, animal, and clinical studies investigating the effects of green tea (Camellia sinensis) extract and associated catechins on drug-metabolizing enzymes and drug transporters. In vitro studies suggest that green tea extract and its main catechin, (-)-epigallocatechin-3-gallate, to varying degrees, inhibit the activity of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4. UGT1A1 and UGT1A4 isoforms were also inhibited by (-)-epigallocatechin-3-gallate. Animal studies suggest green tea extract and/or (-)-epigallocatechin-3-gallate significantly increase the bioavailability of diltazem, verapamil, tamoxifen simvastatin, 5-fluorouracil, and nicardipine. Conversely, green tea extract and/or (-)-epigallocatechin-3-gallate reduce the bioavailability of quetiapine, sunitinib, clozapine, and nadolol. Of the few clinical studies available for review, it appears neither green tea extract nor (-)-epigallocatechin-3-gallate inhibit any major cytochrome P450 enzyme. Regarding drug transporters, in vitro studies indicate P-glycoprotein, organic anion transporting polypeptide 1A1, organic anion transporting polypeptide 1B1, organic anion transporting polypeptide 1B3, organic anion transporting polypeptide 2B1, organic cation transporter 1, organic cation transporter 2, multidrug and toxin extrusion 1, and multidrug and toxin extrusion 2-K are potentially inhibited by green tea extract. A clinical study indicates the organic anion transporting polypeptide 1A1 transporter is inhibited by (-)-epigallocatechin-3-gallate while P-glycoprotein is unaffected. In conclusion, the ingestion of green tea extract or its associated catechins is not expected to result in clinically significant influences on major cytochrome P450 or uridine 5'-diphospho-glucuronosyltransferase enzyme substrates or drugs serving as substrates of P-glycoprotein. However, some caution is advised in the consumption of significant amounts of green tea beverages or green tea extract in patients prescribed known substrates of organic anion transporting polypeptide, particularly those with a narrow therapeutic index.

Expression of Organic Anion Transporting Polypeptide 1A2 in Red Blood Cells and Its Potential Impact on Antimalarial Therapy.

Important antimalarial drugs, including quinolines, act against blood schizonts by interfering with hemoglobin metabolism. To reach their site of action, these compounds have to cross the plasma membrane of red blood cells (RBCs). Organic cation transporters (OCTs) and organic anion transporting polypeptides (OATPs) are important uptake transporters and interesting candidates for local drug transport. We therefore studied their interaction with antimalarial compounds (quinine, chloroquine, mefloquine, pyrimethamine, artemisinin, and artesunate) and characterized the expression of OATP1A2 and OATP2B1 in RBCs. Competition assays using transporter-overexpressing Madin-Darby canine kidney (MDCKII) cells and the model substrate estrone-3-sulfate identified quinine and chloroquine as potent inhibitors of OATP1A2 function (IC50 quinine: 0.7 ± 1.2 µM; chloroquine: 1.0 ± 1.5 µM), but no or only moderate effects were observed for OATP2B1. Subsequently, quinine was identified as a substrate of OATP1A2 (Km 23.4 µM). The OATP1A2-mediated uptake was sensitive to the OATP1A2-specific inhibitor naringin. Both OATPs were expressed in human RBCs, and ex vivo transport studies demonstrated naringin-sensitive accumulation of quinine in these cells (60 pmol versus 38 pmol/5 × 10(5) RBCs). Additional transport studies using OCT1-3 and organic cation transporter novel type 1 (OCTN1) indicated only significant quinine uptake by OCT1, which was not detected in RBCs. In conclusion, our data demonstrate expression of OATP2B1 and OATP1A2 in RBCs as well as OATP1A2-mediated uptake of quinine. Therefore, modulation of OATP1A2 function may affect quinine uptake into erythrocytes.

Total saponins from dioscorea septemloba thunb reduce serum uric acid levels in rats with hyperuricemia through OATP1A1 up-regulation.

The aim of this study is to evaluate the efficacy of total saponins of Dioscorea (TSD), an extract of the Chinese herbal Bi Xie, on hyperuricemia and to elucidate the underlying mechanisms. The rat hyperuricemia model was established by administration of adenine. Thirty-two rats were randomly allocated into 4 groups: model group, low/high-dose TSD-treated groups, and allopurinol-treated group. Meanwhile, 8 rats were used as normal controls. Serum uric acid (UA), blood urea nitrogen (BUN), serum creatinine (Scr), and organic anion transporting polypeptide 1A1 (OATP1A1) levels were measured. Comparison between the model group and treatment (allopurinol and TSD) groups showed the serum UA levels were significantly decreased in treatment groups. TSD had similar effects to allopurinol. It was found that the OATP1A1 protein expression levels in treatment groups were higher than in model group and normal controls. And different from the allopurinol-treated groups, TSD-treated group had elevated OATP1A1 expression levels in the stomach, liver, small intestine and large intestine tissues. It was suggested that TSD may facilitate the excretion of UA and lower UA levels by up-regulating OATP1A1 expression.

Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2

Chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used to treat malaria and inflammatory diseases, long-term usage of which often causes severe side effects, especially retinopathy. Solute carrier transporters (SLCs) are important proteins responsible for the cellular uptake of endogenous and exogenous substances. Inhibitors competing with transporter substrates for SLCs often results in unfavorable toxicities and unsatisfactory therapeutic outcomes. We investigated the inhibitory effect of CQ and HCQ on substrate uptake mediated through a range of important SLC transporters in overexpressing human embryonic kidney (HEK293) cells. Our data revealed that both CQ and HCQ potently inhibit the uptake activity of organic anion transporting polypeptide 1A2 (OATP1A2). We recently reported OATP1A2 to be expressed in human retinal pigment epithelium (RPE), where it mediates cellular uptake of all-trans-retinol (atROL), a key step in the classical visual cycle. In this study, we demonstrate that CQ and HCQ could markedly impair atROL uptake in OATP1A2-expressing HEK293 cells and more importantly, in primary human RPE cells. Our study shows that CQ and HCQ are novel inhibitors of OATP1A2 and significantly impair OATP1A2-mediated substrate uptake, particularly transport of atROL into the RPE. This effect may compromise the function of the classic visual cycle leading to vision impairment and contribute to the retinopathy observed clinically in patients using CQ or HCQ.

Solute Carrier Family of the Organic Anion-Transporting Polypeptides 1A2- Madin-Darby Canine Kidney II: A Promising In Vitro System to Understand the Role of Organic Anion-Transporting Polypeptide 1A2 in Blood-Brain Barrier Drug Penetration.

Organic anion-transporting polypeptide (OATP) 1A2 has the potential to be a target for central nervous system drug delivery due to its luminal localization at the human blood-brain barrier and broad substrate specificity. We found OATP1A2 mRNA expression in the human brain to be comparable to breast cancer resistance protein and OATP2B1 and much higher than P-glycoprotein (P-gp), and confirmed greater expression in the brain relative to other tissues. The goal of this study was to establish a model system to explore OATP1A2-mediated transcellular transport of substrate drugs and the interplay with P-gp. In vitro (human embryonic kidney 293 cells stably expressing Oatp1a4, the closest murine isoform) and in vivo (naïve and Oatp1a4 knock-out mice) studies with OATP1A2 substrate triptan drugs demonstrated that these drugs were not Oatp1a4 substrates. This species difference demonstrates that the rodent is not a good model to investigate the active brain uptake of potential OATP1A2 substrates. Thus, we constructed a novel OATP1A2 expressing Madin-Darby canine kidney (MDCK) II wild type and an MDCKII-multidrug resistance protein 1 (MDR1) system using BacMam virus transduction. The spatial expression pattern of OATP1A2 after transduction in MDCKII-MDR1 cells was superimposed to P-gp, confirming apical membrane localization. OATP1A2-mediated uptake of zolmitriptan, rosuvastatin, and fexofenadine across monolayers increased with increasing OATP1A2 protein expression. OATP1A2 counteracted P-gp efflux for cosubstrates zolmitriptan and fexofenadine. A three-compartment model incorporating OATP1A2-mediated influx was used to quantitatively describe the time- and concentration-dependent apical-to-basolateral transcellular transport of rosuvastatin across OATP1A2 expressing the MDCKII monolayer. This novel, simple and versatile experimental system is useful for understanding the contribution of OATP1A2-mediated transcellular transport across barriers, such as the blood-brain barrier.

Human organic anion transporting polypeptide 1A2 (OATP1A2) mediates cellular uptake of all-trans-retinol in human retinal pigmented epithelial cells.

Vision depends on retinoid exchange between the retinal pigment epithelium (RPE) and photoreceptors. Defects in any step of the canonical visual cycle can lead to retinal degenerations. All-trans-retinol (atROL) plays an important role in visual signal transduction. However, how atROL enters human RPE from the apical membrane remains unclear. This study investigated the role of human organic anion transporting polypeptide 1A2 (OATP1A2) in atROL uptake in human RPE. Immunoblotting and immunostaining elucidated the expression and localization of OATP1A2 in human RPE. Transporter functional studies were conducted to assess the interaction of OATP1A2 with atROL. Our study revealed OATP1A2 is expressed in human RPE, mainly at the apical membrane. Our data also indicated atROL inhibited the uptake of the typical OATP1A2 substrate, oestrone-3-sulfate (E3S), in over-expressing cells. Studies on the uptake of (3) H-atROL in these over-expressing cells revealed atROL is a substrate of OATP1A2. We confirmed these findings in human primary RPE cells. The transport of E3S and atROL was significantly reduced in human primary RPE cells with OATP1A2 siRNA silencing. Our data provides the first evidence of OATP1A2 expression in human RPE and more importantly, its novel role in the cellular uptake of atROL, which might be essential to the proper functioning of the canonical visual cycle. Our findings contribute to the understanding of the molecular mechanisms involved in retinoid transport between the RPE and photoreceptors and provide novel insights into potential pharmaceutical interventions for visual cycle disruption associated with retinal degenerations.

Features of acute liver congestion on gadoxetate disodium-enhanced MRI in a rat model: Role of organic anion-transporting polypeptide 1A1.

To evaluate the features of hepatic congestion on gadoxetate disodium (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) and the mechanisms responsible for the radiological findings in a rat model of partial liver congestion. A conventional T1 -weighted spin-echo sequence of the liver was performed using a 1.5T magnetic resonance imager with an 80-mm magnetic aperture for animal studies. We induced regional congestion using partial left lateral hepatic vein ligation (n = 5) and evaluated the following in both congestive liver (CL) and noncongestive liver (non-CL): 1) chronological changes in the relative enhancement (RE) up to 60 minutes after Gd-EOB-DTPA administration, and 2) mRNA and protein expression of rat organic anion transporting protein 1a1 (Oatp1a1). The RE in the CL reached a small peak (18%) at 5 minutes, corresponding to approximately half of the value observed in the non-CL, then slowly decreased in a linear manner thereafter. The degree of RE in the CL was significantly lower than that in the non-CL for up to 30 minutes (P < 0.05). An immunohistological examination showed that Oatp1a1 protein expression was downregulated in the CL. The mRNA level of Oatp1a1 in the CL was significantly upregulated, compared with that in control rat liver (P = 0.046), whereas no significant difference was observed between the CL and the non-CL (P = 0.698). The reduced signal intensity in the CL on Gd-EOB-DTPA-enhanced MRI could be explained by the decreased uptake of Gd-EOB-DTPA via Oatp1a1 protein in the congestive area.

Effects of β-blockers and tricyclic antidepressants on the activity of human organic anion transporting polypeptide 1A2 (OATP1A2).

The organic anion transporting polypeptide 1A2 (OATP1A2), a membrane drug transporter expressed on important organs (such as the brain, kidney, and intestine) may be a key element in the disposition of drugs. Previous studies demonstrated that it could transport a broad spectrum of substrates, including endogenous molecules and clinically relevant drugs, such as several β-blockers and 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. The primary objective of this study was to investigate OATP1A2 transport activity using rosuvastatin as a probe substrate and evaluate competitive inhibition of its transport by β-blockers. Rosuvastatin transport was saturable, with a Km of 60.2 µM. With the exception of carvedilol (IC50 of 3.2 µM), all of the other β-blockers that were evaluated had a small or insignificant effect on OATP1A2-mediated uptake of rosuvastatin. Carvedilol differs from the other β-blockers by the tricyclic moiety in its chemical structure. As a secondary objective, the transport of a series of tricyclic compounds by OATP1A2 and their potential for rosuvastatin transport inhibition were evaluated. Tricyclic compounds were not OATP1A2 substrates. On the other hand, tricyclic compounds with a short aliphatic amine chain inhibited OATP1A2-mediated rosuvastatin transport. Our data suggest that these drugs may modulate the transport of OATP1A2 substrates and may affect drug actions.

Does Ethnic Variability Exist in the Systemic Exposures of OATP1A2 Substrates-Fexofenadine in Taiwanese?

The aim of this study was to investigate differences in organic anion transporting polypeptide 1A2 activity among the Taiwanese population via an analysis of 3 pharmacokinetic studies completed in a total of 103 healthy male Taiwanese subjects. The pharmacokinetics of fexofenadine was measured as an indicator of organic anion transporting polypeptide 1A2 activity. Using the Kolmogorov-Smirnov test and quantile plots, the frequency distributions of area under the concentration-time curve and concentration were shown to be tri-modal and to represent 3 pharmacokinetic phenotypes. In a comparison with published data, the mean area under the concentration-time curve of fexofenadine in the Taiwanese subjects was similar to that in American, German, and Indian subjects, but significantly different from that in some Asian populations, including Korean and Japanese ethnic groups. These results suggested that Taiwanese subjects showed genetic variation in fexofenadine pharmacokinetics that was associated with differences in organic anion transporting polypeptide 1A2 activity.

Advanced fibrosis: Correlation between pharmacokinetic parameters at dynamic gadoxetate-enhanced MR imaging and hepatocyte organic anion transporter expression in rat liver.

To compare the value of enhancement and pharmacokinetic parameters measured at dynamic gadoxetate-enhanced magnetic resonance (MR) imaging in determining hepatic organic anion transporter expression in control rats and rats with advanced liver fibrosis. Institutional animal review board approval was received before the study began. Advanced liver fibrosis was created in rats by means of carbon tetrachloride injections over an 8-week period. In 17 rats with liver fibrosis and eight control rats, dynamic gadoxetate-enhanced MR images of the liver were obtained during 1 hour after injection of 0.025 mmol gadoxetate per kilogram of body weight. Enhancement parameters (maximum enhancement [Emax], time to peak [Tmax], and elimination half-life) were measured on enhancement-versus-time curves, and pharmacokinetic parameters (hepatic extraction fraction [HEF] and mean residence time [MRT]) were obtained by means of deconvolution analysis of the concentration-versus-time curves in the liver and the portal vein. The parameters were correlated at simple and multiple regression analysis with the expression of the hepatic anion uptake transporter organic anion-transporting polypeptide 1A1 (Oatp1a1), the hepatobiliary transporter multidrug resistance-associated protein 2 (Mrp2), and the backflux transporter Mrp4, as determined with reverse transcription polymerase chain reaction. In rats with advanced liver fibrosis, the Emax, Tmax, HEF, and MRT decreased significantly relative to those in control rats, whereas the elimination half-life increased significantly. The enhancement and pharmacokinetic parameters correlated significantly with the expression of the transporters at simple regression analysis. At multiple regression analysis, HEF was the only parameter that was significantly associated with the expression of Oatp1a1 and Mrp2 (P < .001, r = 0.74 and P < .001, r = 0.70, respectively). The pharmacokinetic parameter HEF at dynamic gadoxetate-enhanced MR imaging is independently correlated with hepatic organic anion transporter expression.

Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats.

Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats.

PDZK1 and NHERF1 regulate the function of human organic anion transporting polypeptide 1A2 (OATP1A2) by modulating its subcellular trafficking and stability.

The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)−1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.

Genetic Profile and Cancer-Related Pain: A Tale from Two Outlier Cases with Bone Metastatic Disease

Dear Editor, Morphine is the mainstay of pharmacological treatment for moderate-to-severe cancer-related pain. However, different analgesic response is an important problem in palliative care [1]. Genetic variations seems to represent an important cause of this interindividual variability in polymorphisms of opioid receptors, transporters, and metabolizing enzymes, as well as in modulators/suppressors involved in perception and processing of pain information [1]. Therefore, genetic study of outlier cases might be an excellent opportunity to analyze the influence of some single-nucleotide polymorphisms (SNP) in nociception and morphine requirements. Here, we present the study of a genetic profile of two cases: one patient considered a low responder (Patient 1) and one considered sensitive to morphine (Patient 2), requiring about 40-fold less morphine. The difference in morphine requirements prompted us to study SNP that include different phases of analgesic response: μ-opioid receptor (OPRM1; rs1799971), catechol-O-methyltransferase (COMT; rs4680), multidrug resistance protein 1 (ABCB1; rs1128503, rs1045642), organic anion-transporting polypeptides 1A2 (OATP1A2; rs11568563), and UDP-glucuronosyltransferase-2B7 (UGT2B7; hCV32449742: rs7439366, rs7438284). Plasma concentrations of morphine and major metabolites (morphine-3-glucuronide (M3G) and morphine-6-glucuronide [M6G]) were also determined [2] and metabolic ratios were calculated. The first patient, a 23-year-old female presenting an osteosarcoma, bone metastasis, and complains of mixed pain (nociceptive and neuropathic pain), was receiving 800 mg/day of morphine. Coadministered drugs were gabapentin (1700 mg/day) and prednisolone (20 mg/day). Despite medication, the pain relief was not adequate, …

Expression of organic anion-transporting polypeptide 1A2 and organic cation transporter 6 as a predictor of pathologic response to neoadjuvant chemotherapy in triple negative breast cancer

Organic anion-transporting polypeptide 1A2 (OATP1A2) and organic cation transporter 6 (OCT6) are involved in the uptake of taxanes and anthracyclines, respectively. The aim of this study was to evaluate expression levels of OATP1A2 and OCT6 as a predictor of response to neoadjuvant chemotherapy (NAC) in breast cancer. A total of 124 patients who received anthracycline/taxane-based NAC were included. Expression levels of OATP1A2 and OCT6 were immunohistochemically assessed in core needle biopsies obtained prior to NAC. A pathologic good response (pGR) and a pathologic complete response (pCR) were achieved in 24 and 10 % of patients, respectively. In univariate analysis of the entire cohort, negative hormone receptor (HR) status (pGR and pCR, P < 0.001), high Ki-67 level (pGR, P = 0.03; pCR, P = 0.02), triple negative (TN) subtype (pGR, P = 0.001; pCR, P < 0.001), and high OCT6 (pGR, P = 0.003) were associated with the response. In combined analysis, high OATP1A2/high OCT6 level was also a significant factor for pGR (P = 0.001) and pCR (P = 0.001). Two separate multivariate analyses showed that HR status, TN subtype and combined high OATP1A2/high OCT6 level were significant independent predictors. When TN and non-TN tumors were assessed separately in univariate analysis, high Ki-67 level (P = 0.04) were associated with pGR and combined high OATP1A2/high OCT6 level was associated with both pGR (P = 0.005) and pCR (P = 0.03) in the TN group. Multivariate analysis identified the combined high OATP1A2/high OCT6 level as the sole independent predictor of pGR. In the non-TN group, negative HR status (P = 0.03) and positive HER2 status (P = 0.005) were associated with pGR, but HER2 status was the sole independent predictor of pGR. These results suggest that response-associated predictors may differ between the TN and non-TN tumors. Combined high OATP1A2/high OCT6 may be a potential predictor of response to anthracycline/taxane-based chemotherapy in breast cancer, especially in TN tumors.

Personalized therapeutics for levofloxacin: a focus on pharmacokinetic concerns

BackgroundPersonalized medicine should be encouraged because patients are complex, and this complexity results from biological, medical (eg, demographics, genetics, polypharmacy, and multimorbidities), socioeconomic, and cultural factors. Levofloxacin (LVX) is a broad-spectrum fluoroquinolone antibiotic. Awareness of personalized therapeutics for LVX seems to be poor in clinical practice, and is reflected in prescribing patterns. Pharmacokinetic–pharmacodynamic studies have raised concerns about suboptimal patient outcomes with the use of LVX for some Gram-negative infections. Meanwhile, new findings in LVX therapeutics have only been sporadically reported in recent years. Therefore, an updated review on personalized LVX treatment with a focus on pharmacokinetic concerns is necessary.MethodsRelevant literature was identified by performing a PubMed search covering the period from January 1993 to December 2013. We included studies describing dosage adjustment and factors determining LVX pharmacokinetics, or pharmacokinetic–pharmacodynamic studies exploring how best to prevent the emergence of resistance to LVX. The full text of each included article was critically reviewed, and data interpretation was performed.ResultsIn addition to limiting the use of fluoroquinolones, measures such as reducing the breakpoints for antimicrobial susceptibility testing, choice of high-dose short-course of once-daily LVX regimen, and tailoring LVX dose in special patient populations help to achieve the validated pharmacokinetic–pharmacodynamic target and combat the increasing LVX resistance. Obese individuals with normal renal function cleared LVX more efficiently than normal-weight individuals. Compared with the scenario in healthy subjects, standard 2-hour spacing of calcium formulations and oral LVX was insufficient to prevent a chelation interaction in cystic fibrosis patients. Inconsistent conclusions were derived from studies of the influence of sex on the pharmacokinetics of LVX, which might be associated with sample size and administration route. Children younger than 5 years cleared LVX nearly twice as fast as adults. Patients in intensive care receiving LVX therapy showed significant pharmacokinetic differences compared with healthy subjects. Creatinine clearance explained most of the population variance in the plasma clearance of LVX. Switching from intravenous to oral delivery of LVX had economic benefits. Addition of tamsulosin to the LVX regimen was beneficial for patients with bacterial prostatitis because tamsulosin could increase the maximal concentration of LVX in prostatic tissue. Coadministration of multivalent cation-containing drugs and LVX should be avoided. For patients receiving warfarin and LVX concomitantly, caution is needed regarding potential changes in the international normalized ratio; however, it is unnecessary to seek alternatives to LVX for the sake of avoiding drug interaction with warfarin. It is unnecessary to proactively reduce the dose of cyclosporin or tacrolimus when comedicated with LVX. Transporters such as organic anion-transporting polypeptide 1A2, P-glycoprotein, human organic cation transporter 1, and multidrug and toxin extrusion protein 1 are involved in the pharmacokinetics of LVX.ConclusionPersonalized LVX therapeutics are necessary for the sake of better safety, clinical success, and avoidance of resistance. New findings regarding individual dosing of LVX in special patient populations and active transport mechanisms in vivo are opening up new horizons in clinical practice.

Hypoxia/Reoxygenation Stress Signals an Increase in Organic Anion Transporting polypeptide 1a4 (Oatp1a4) at the Blood–Brain Barrier: Relevance to CNS Drug Delivery

Cerebral hypoxia and subsequent reoxygenation stress (H/R) is a component of several diseases. One approach that may enable neural tissue rescue after H/R is central nervous system (CNS) delivery of drugs with brain protective effects such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (i.e., statins). Our present in vivo data show that atorvastatin, a commonly prescribed statin, attenuates poly (ADP-ribose) polymerase (PARP) cleavage in the brain after H/R, suggesting neuroprotective efficacy. However, atorvastatin use as a CNS therapeutic is limited by poor blood-brain barrier (BBB) penetration. Therefore, we examined regulation and functional expression of the known statin transporter organic anion transporting polypeptide 1a4 (Oatp1a4) at the BBB under H/R conditions. In rat brain microvessels, H/R (6% O2, 60 minutes followed by 21% O2, 10 minutes) increased Oatp1a4 expression. Brain uptake of taurocholate (i.e., Oap1a4 probe substrate) and atorvastatin were reduced by Oatp inhibitors (i.e., estrone-3-sulfate and fexofenadine), suggesting involvement of Oatp1a4 in brain drug delivery. Pharmacological inhibition of transforming growth factor-β (TGF-β)/activin receptor-like kinase 5 (ALK5) signaling with the selective inhibitor SB431542 increased Oatp1a4 functional expression, suggesting a role for TGF-β/ALK5 signaling in Oatp1a4 regulation. Taken together, our novel data show that targeting an endogenous BBB drug uptake transporter (i.e., Oatp1a4) may be a viable approach for optimizing CNS drug delivery for treatment of diseases with an H/R component.

Differential Regulation of Hepatic Organic Cation Transporter 1, Organic Anion-Transporting Polypeptide 1a4, Bile-Salt Export Pump, and Multidrug Resistance-Associated Protein 2 Transporter Expression in Lymphocyte-Deficient Mice Associates with Interleukin-6 Production

Cholestasis results from interrupted bile flow and is associated with immune-mediated liver diseases. It is unclear how inflammation contributes to cholestasis. The aim of this study was to determine whether T and B cells contribute to hepatic transporter expression under basal and inflammatory conditions. C57BL/6J wild-type mice or strains lacking T, B, or both T and B cells were exposed to lipopolysaccharide (LPS) or saline, and livers were collected 16 hours later. Branched DNA signal amplification was used to assess mRNA levels of organic anion-transporting polypeptides (Oatp) 1a1, 1a4, and 1b2; organic cation transporter (Oct) 1; canalicular bile-salt export pump (Bsep); multidrug resistance-associated proteins (Mrp) 2 and 3; and sodium-taurocholate cotransporting polypeptide (Ntcp). Real-time polymerase chain reaction analysis was used to correlate changes of transporter expression with interleukin-1b (IL-1b), IL-6, IL-17A, IL-17F, tumor necrosis factor-α (TNF-α), and interferon-γ expression in the liver. LPS treatment inhibited Bsep and Oct1 mRNA expression, and this was abrogated with a loss of T cells, but not B cells. In addition, the absence of T cells increased Mrp2 mRNA expression, whereas B cell deficiency attenuated Oatp1a4 mRNA in LPS-treated mice. Oatp1a1, Oatp1b2, Ntcp, and Mrp3 were largely unaffected by T or B cell deficiency. Lymphocyte deficiency altered basal and inflammatory IL-6, but not TNF-α or IL-1b, mRNA expression. Taken together, these data implicate lymphocytes as regulators of basal and inflammatory hepatic transporter expression and suggest that IL-6 signaling may play a critical role.

Functional Analysis of Novel Polymorphisms in the Human SLCO1A2 Gene that Encodes the Transporter OATP1A2

The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20-50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.

Gabapentin and Diclofenac Reduce Opioid Consumption in Patients Undergoing Tonsillectomy: A Result of Altered CNS Drug Delivery?

Dear Editor, We have read with great interest the article by Mogadam and colleagues on utilization of gabapentin and diclofenac for management of post-operative pain in patients undergoing tonsillectomy (1). Perhaps the most intriguing aspect of this study was the observation that pre-operative administration of gabapentin or diclofenac resulted in reduced post-operative utilization of meperidine, an opioid analgesic. Optimal therapeutic efficacy of opioids requires that they cross the blood-brain barrier (BBB) and attain effective concentrations in the CNS (2). CNS delivery of opioids is determined by putative membrane transporters localized to the BBB endothelium (3, 4). Both pathophysiological factors (i.e., inflammatory signaling in pain) and pharmacological factors (i.e., use of ancillary pain medications) can modulate mechanisms involved in BBB opioid transport, an effect that can cause profound changes in CNS delivery of traditional opioids (i.e., morphine, meperidine). In fact, our research group has demonstrated that painful and/or inflammatory stimuli in the periphery can significantly change transport mechanisms for opioids at the BBB such as the drug efflux transporter P-glycoprotein (P-gp) (5) and the drug influx transporter organic anion transporting polypeptide 1a4 (6). Of particular note, we have also shown that diclofenac itself can attenuate pain-induced changes in BBB transporter activity (6). Our data suggest that a modulation in post-operative meperidine efficacy may, in part, be the result of altered CNS opioid delivery induced by diclofenac. An additional factor to consider is that some ancillary pain medications may act as chemical inhibitors of the same BBB transporters. In the context of the study by Mogadam and colleagues, this effect may involve the critical BBB efflux transporter P-gp. Specifically, gabapentin is a known P-gp inhibitor (7) while in vitro studies have suggested that meperidine is a P-gp transport substrate (8). Although in vivo studies in mdr1a knockout mice showed no difference in meperidine antinociception (9), a direct analysis of P-gp-mediated transport of meperidine in intact laboratory animals has not been undertaken. Furthermore, this study measured analgesic efficacy using only tail-pinch, a technique that may not have been sensitive enough to detect differences in meperidine analgesia between P-gp-deficient and P-gp-competent mice. Nonetheless, it is highly plausible that gabapentin blocked P-gp-mediated efflux transport at the BBB, an effect that effectively increased CNS meperidine delivery with the clinical manifestation of reduced opioid consumption in the post-operative period. Overall, results obtained from basic science studies of our laboratory and others point towards an explanation of modified post-operative opioid efficacy in terms of altered BBB transport and/or CNS delivery of meperidine induced by ancillary pain medications. Additionally, the paper by Mogadam and colleagues emphasizes the absolute importance of novel translational studies aimed at understanding specific mechanisms involved in CNS opioid delivery, opioid efficacy, and/or drug-opioid interactions.