Yoshitane Nozaki

Functional Characterization of Rat Brain-specific Organic Anion Transporter (Oatp14) at the Blood-Brain Barrier HIGH AFFINITY TRANSPORTER FOR THYROXINE

Oatp14/blood-brain barrier-specific anion transporter 1 (Slc21a14) is a novel member of the organic anion transporting polypeptide (Oatp/OATP) family. Northern blot analysis revealed predominant expression of Oatp14 in the brain, and Western blot analysis revealed its expression in the brain capillary and choroid plexus. Immunohistochemical staining indicated that Oatp14 is expressed in the border of the brain capillary endothelial cells. When expressed in human embryonic kidney 293 cells, Oatp14 transports thyroxine (T4; prothyroid hormone) (Km = 0.18 μm), as well as amphipathic organic anions such as 17β estradiol-d-17β-glucuronide (Km = 10 μm), cerivastatin (Km = 1.3 μm), and troglitazone sulfate (Km = 0.76 μm). The uptake of triiodothyronine (T3), an active form produced from T4, was significantly greater in Oatp14-expressed cells than in vector-transfected cells, but the transport activity for T3 was ∼6-fold lower that for T4. The efflux of T4, preloaded into the cells, from Oatp14-expressed cells was more rapid than that from vector-transfected cells (0.032 versus 0.006 min–1). Therefore, Oatp14 can mediate a bidirectional transport of T4. Sulfobromophthalein, taurocholate, and estrone sulfate were potent inhibitors for Oatp14, whereas digoxin, p-aminohippurate, or leukotriene C4, or organic cations such as tetraetheylammonium or cimetidine had no effect. The expression levels of Oatp14 mRNA and protein were up- and down-regulated under hypo- and hyperthyroid conditions, respectively. Therefore, it may be speculated that Oatp14 plays a role in maintaining the concentration of T4 and, ultimately, T3 in the brain by transporting T4 from the circulating blood to the brain.

Species difference in the inhibitory effect of nonsteroidal anti-inflammatory drugs on the uptake of methotrexate by human kidney slices.

Simultaneous use of nonsteroidal anti-inflammatory drugs (NSAIDs), probenecid, and other drugs has been reported to delay the plasma elimination of methotrexate in patients. Previously, we have reported that inhibition of the uptake process cannot explain such drug-drug interactions using rats. The present study quantitatively evaluated the possible role of the transporters in such drug-drug interactions using human kidney slices and membrane vesicles expressing human ATP-binding cassette (ABC) transporters. The uptake of methotrexate by human kidney slices was saturable with a Km of 45 to 49 μM. Saturable uptake of methotrexate by human kidney slices was markedly inhibited by p-aminohippurate and benzylpenicillin, but only weakly by 5-methyltetrahydrofolate. These transport characteristics are similar to those of a basolateral organic anion transporter (OAT) 3/SLC22A8. NSAIDs and probenecid inhibited the uptake of methotrexate by human kidney slices, and, in particular, salicylate, indomethacin, phenylbutazone, and probenecid were predicted to exhibit significant inhibition at clinically observed plasma concentrations. Among ABC transporters, such as BCRP/ABCG2, multidrug resistance-associated protein (MRP) 2/ABCC2, and MRP4/ABCC4, which are candidates for the luminal efflux of methotrexate, ATP-dependent uptake of methotrexate by MRP4-expressing membrane vesicles was most potently inhibited by NSAIDs. Salicylate and indomethacin were predicted to inhibit MRP4 at clinical plasma concentrations. Diclofenac-glucuronide significantly inhibited MRP2-mediated transport of methotrexate in a concentration-dependent manner, whereas naproxen-glucuronide had no effect. Inhibition of renal uptake (via OAT3) and efflux processes (via MRP2 and MRP4) explains the possible sites of drug-drug interaction for methotrexate with probenecid and some NSAIDs, including their glucuronides.

Characterization of the Uptake of Organic Anion Transporter (OAT) 1 and OAT3 Substrates by Human Kidney Slices

The activities of renal multispecific organic anion transporters (OATs) 1 and 3 have not been fully evaluated in human kidneys. In the present study, the uptake of some organic anions was characterized in kidney slices from human intact renal cortical tissues: hOAT1 and hOAT3 substrates [p-aminohippurate (PAH) and 2,4-dichlorophenoxyacetate (2,4-D)] and hOAT3 substrates [benzylpenicillin (PCG), dehydroepiandrosterone sulfate (DHEAS), and estrone sulfate (ES)]. Despite large interbatch differences, hOAT1 and hOAT3 mRNA levels correlated well, and there was a good correlation between the uptake of PAH and PCG by kidney slices. The uptake of organic anions by kidney slices was saturable with Km values of 31 to 48 μM for PAH, 0.73 to 4.9 μM for 2,4-D, 14 to 90 μM for PCG, and 9.2 to 11 μM for ES. These parameters were comparable with those for hOAT1 and/or hOAT3. The uptake of DHEAS consists of two saturable components with Km values of 2.2 to 3.9 and 1300 μM, and the Km value of the high-affinity component was close to that for hOAT3. Furthermore, PAH more potently inhibited the uptake of 2,4-D than that of PCG and DHEAS. PCG had a weaker effect on the uptake of PAH and 2,4-D than expected from its Km value. Taken together, it is likely that the uptake of PAH and 2,4-D is due to OAT1, and the uptake of PCG and ES and part of DHEAS uptake are due to OAT3 in human kidney slices. Human kidney slices are useful tools for characterizing the renal uptake of drugs.

Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions.

The risk assessment of organic anion transporting polypeptide (OATP) 1B1–mediated drug-drug interactions (DDIs) is an indispensable part of drug development. We previously reported that in vitro inhibitory potencies of several inhibitors on OATP1B1 depend on the substrates when prototypical substrates, estradiol-17β-glucuronide (E2G), estrone-3-sulfate, and sulfobromophthalein were used as test substrates. The purpose of this study was to comprehensively investigate this substrate-dependent inhibition of OATP1B1 using clinically relevant OATP1B1 inhibitors and substrate drugs. Effects of cyclosporine A (CsA), rifampin, and gemfibrozil on OATP1B1-mediated uptake of 12 substrate drugs were examined in OATP1B1-expressing human embryonic kidney 293 cells. The Ki values (μM) for CsA varied from 0.0771 to 0.486 (6.3-fold), for rifampin from 0.358 to 1.23 (3.4-fold), and for gemfibrozil from 9.65 to 252 (26-fold). Except for the inhibition of torasemide uptake by CsA and that of nateglinide uptake by gemfibrozil, the Ki values were within 2.8-fold of those obtained using E2G as a substrate. Preincubation potentiated the inhibitory effect of CsA on OATP1B1 with similar magnitude regardless of the substrates. R values calculated based on a static model showed some variation depending on the Ki values determined with various substrates, and such variability could have an impact on the DDI predictions particularly for a weak-to-moderate inhibitor (gemfibrozil). OATP1B1 substrate drugs except for torasemide and nateglinide, or E2G as a surrogate, is recommended as an in vitro probe in the inhibition experiments, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variation.

Substrate-Dependent Inhibition of Organic Anion Transporting Polypeptide 1B1: Comparative Analysis with Prototypical Probe Substrates Estradiol-17β-Glucuronide, Estrone-3-Sulfate, and Sulfobromophthalein

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [3H]estradiol-17β-glucuronide (E2G), [3H]estrone-3-sulfate (E1S), and [3H]sulfobromophthalein (BSP) were studied in OATP1B1-transfected cells. Inhibitory potencies of tested compounds varied depending on the substrates. Ritonavir, gemfibrozil, and erythromycin caused remarkable substrate-dependent inhibition with up to 117-, 14-, and 13-fold difference in their IC50 values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12- and 6-fold variation in their IC50 values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [3H]E2G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E2G and E1S competitively inhibited each other, whereas BSP noncompetitively inhibited E2G uptake. In addition, BSP inhibited E1S in a competitive manner, but E1S caused an atypical kinetics on BSP uptake. This study showed substrate-dependent inhibition of OATP1B1 and demonstrated that E2G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.

Expression and Functional Involvement of Organic Anion Transporting Polypeptide Subtype 3 (Slc21a7) in Rat Choroid Plexus

AbstractPurpose. It has been shown that transport(s) are involved in the uptake of estradiol 17β glucuronide (E217βG) by the choroid plexus (CP). The purpose of this study is to compare the substrate specificity of the transporter in the CP with those of rat organic anion transporting polypeptide 1 (rOatp1) and rOatp3. Methods. The expression of rOatp1 and rOatp3 in rat CP was confirmed by RT-PCR and Western blot analyses. The substrate specificity of rOatp1 and rOatp3 was compared using cDNA-transfected LLC-PK1 cells. The uptake of E217βG by rat isolated CP was determined by centrifugal filtration technique. Results. PCR analyses demonstrated that the mRNA expression of rOatp3 was abundant in the CP, whereas that of rOatp1 was low. Immunohistochemical staining revealed that rOatp3 is expressed on the apical membrane of the CP. Kinetic parameters (Km and Ki values) of rOatp3 were similar to those for rOatp1. The results of mutual inhibition study suggest that E217βG and taurocholate share the same mechanism in the CP. Corticosterone, estrone-3-sulfate and indomethacin are moderate inhibitors, but no effects by digoxin, p-aminohippurate, benzylpenicillin and cimetidine were observed. Conclusions. rOatp3 is most possible candidate transporter involved in the uptake of organic anions on the brush border membrane of the choroid epithelial cells.

Novel BACE1 inhibitor, E2609, lowers Aβ levels in the cerebrospinal fluid and plasma in nonhuman primates

Background: Alzheimer’s disease (AD) is characterized by accumulation of Ab-containing plaques and tau-containing neurofibrillary tangles in the brain. The production of pathological Ab peptides, Ab1-42 in particular, is thought to be an initiating event in AD. BACE1 cleavage of the amyloid precursor protein is the first step in the production of Ab and is therefore a prime target to block the pathogenic amyloid cascade leading to AD and potentially modify the disease progression. We have developed a potent small molecule, the orally bioavailable BACE1 inhibitor, E2609, (cell-based assay IC 50 w7 nmol/L) that has been shown to reduce Ab production in the plasma, brain, and cerebrospinal fluid (CSF) of rodents. Methods: The pharmacokinetic and pharmacodynamic relationship of E2609 was examined in cisterna magna ported cynomolgus monkeys. Plasma and CSF were sampled serially over 72 hours post single oral administration of E2609 so that the temporal dynamics of Ab in plasma and CSF could be evaluated in detail. Plasma pharmacokinetics of E2609 was linked to Ab levels

Investigation of utility of cerebrospinal fluid drug concentration as a surrogate for interstitial fluid concentration using microdialysis coupled with cisternal cerebrospinal fluid sampling in wild-type and Mdr1a(-/-) rats.

In drug discovery, the cerebrospinal fluid (CSF) drug concentration (CCSF) has been used as a surrogate for the interstitial fluid (ISF) concentration (CISF). However, the CCSF-to-CISF gradient suggested for P-glycoprotein (P-gp) substrates in rodents causes uncertainty in CISF estimations and subsequent pharmacokinetic-pharmacodynamic analyses. To evaluate the utility of CCSF as a surrogate for CISF, this study directly compared the CCSF with the CISF of 12 compounds, including P-gp substrates, under steady-state conditions in wild-type and Mdr1a(-/-) rats using microdialysis coupled with cisternal CSF sampling. In wild-type rats, the ISF-to-unbound plasma (Kp,uu,ISF) and CSF-to-unbound plasma (Kp,uu,CSF) concentration ratios of the P-gp substrates, except for metoclopramide, were lower than those of the non-P-gp substrates, and the Kp,uu,CSF values were within or close to 3-fold of the Kp,uu,ISF values for all the compounds examined. The Kp,uu,CSF values of the selected P-gp substrates increased in Mdr1a(-/-) rats with a similar magnitude to the Kp,uu,ISF values, resulting in the Kp,uu,CSF-to-Kp,uu,ISF ratios being unchanged. These results suggested that P-gp-mediated active efflux at the blood-brain barrier is a major determinant not only for CISF, but also for CCSF, and that CCSF can be used as a surrogate for CISF even for P-gp substrates in rats.

Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes

In vitro-in vivo extrapolation based on uptake clearance determined in human hepatocytes has been used to predict in vivo hepatic clearance of organic anion transporting polypeptide (OATP) substrates. This study evaluated the relative activity factor (RAF) approach to extrapolate active uptake clearance in transporter-transfected cell systems (CLuptake) to that in human hepatocyte suspensions (PSinf,act). RAF values for OATP1B1 and OATP1B3 were determined in two batches of cryopreserved human hepatocytes using estrone-3-sulfate and cholecystokinin octapeptide as reference substrates, respectively. Fourteen OATP1B substrate drugs selected (atorvastatin, bosentan, cerivastatin, fexofenadine, fluvastatin, glibenclamide, irbesartan, nateglinide, pitavastatin, pravastatin, rosuvastatin, telmisartan, torasemide, and valsartan) showed temperature-dependent uptake in human hepatocytes. In transporter-transfected cells, OATP1B1- and OATP1B3-mediated uptake was observed in all compounds except for telmisartan. RAF-based net CLuptake was mainly accounted for by OATP1B1 (72.3-99.7%) and fell within the 3-fold of PSinf,act observed in human hepatocytes in 11 out of 13 compounds (excluding telmisartan). This study demonstrated that the RAF approach provides a quantitative index of OATP1B1- and OATP1B3-mediated PSinf,act in human hepatocytes, which will facilitate the optimization of the pharmacokinetic properties of OATP1B substrates at nonclinical stages of drug development.