Predicting response to sepantronium bromide (YM155), a survivin suppressant, by PET imaging with [11C]YM155.

INTRODUCTION: Sepantronium bromide (YM155) is a survivin suppressant that induces apoptosis in tumor cells. Although YM155 induces tumor regression in various tumor types in vivo, phase I and II studies demonstrated responding and non-responding patient populations. We investigated 11C-labeled YM155 ([11C]YM155) used as a positron emission tomography (PET) tracer to assess whether tumor uptake of [11C]YM155 correlated with its anti-tumor effect, thereby allowing identification of patients who would respond to YM155 treatment. METHODS: (1) Uptake of YM155 was measured in 39 human cancer cell lines in vitro using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). (2) In vivo tumor uptake was assessed in xenografted mice and total body distribution was evaluated in a cynomolgus monkey using [11C]YM155 with PET/computed tomography (CT) (mice) and PET (monkey) imaging. RESULTS: Intracellular uptake of YM155 in human cancer cell lines correlated well with its in vitro efficacy measured by GI50 (Pearson's r = -0.5709). Similarly, in vivo studies using tumor xenografted mice showed that tumors sensitive to YM155 demonstrated robust uptake of [11C]YM155, whereas insensitive tumors demonstrated low uptake. In the monkey, the biodistribution of [11C]YM155 indicated low accumulation in lung, breast, head, and neck and was only significant in organs involved with drug clearance: i.e. liver, kidneys, and bladder. CONCLUSIONS: Robust uptake of [11C]YM155 by a tumor appears to be a positive predictive marker for a good response to YM155. The findings suggest the potential utility of PET/CT imaging with [11C]YM155 for selection of patients whose tumors are likely to respond to YM155. ADVANCES IN KNOWLEDGE: YM155 efficacy correlated closely with its in vitro intracellular uptake and uptake on [11C]YM155 PET imaging. [11C]YM155 PET may predict tumor sensitivity to YM155. IMPLICATIONS FOR PATIENT CARE: The concept that tumor response can be accurately predicted prior to chemotherapy should be exploited to improve cancer treatment outcomes through judicious patient selection. The small molecule sepantronium bromide (YM155), a survivin suppressant, has been developed for the treatment of several cancers, including non-Hodgkin lymphoma, lung cancer, and breast cancer. The preferentially high in vitro uptake of YM155 by YM155-sensitive cancer cells and the high in vivo uptake of [11C]YM155 in YM155-sensitive tumors demonstrated by PET imaging suggest the potential utility of performing [11C]YM155 PET to allow the identification of patients with YM155-sensitive tumors.

Functional imaging of pharmacological action of SGLT2 inhibitor ipragliflozin via PET imaging using 11C-MDG.

Sodium-dependent glucose cotransporter 2 (SGLT2) is a pharmacological target of type 2 diabetes mellitus. The aim of this study was to noninvasively visualize the pharmacological action of a selective SGLT2 inhibitor ipragliflozin in the kidney using positron emission tomography (PET) imaging with 11C-methyl-d-glucoside (11C-MDG), an SGLT-specific radio-labeled substrate. PET imaging with 11C-MDG in vehicle-treated rats demonstrated that intravenously injected 11C-MDG substantially accumulated in the renal cortex, reflecting that the compound was reabsorbed by SGLTs. In contrast, ipragliflozin-treated rats showed significantly lower uptake of 11C-MDG in renal cortex in a dose-related manner, suggesting that ipragliflozin inhibited the renal reabsorption of 11C-MDG. This method of visualizing the mode of action of an SGLT2 inhibitor in vivo has demonstrated the drug's mechanism in reducing renal glucose reabsorption in kidney in living animals.

Combination of YM155, a survivin suppressant, with bendamustine and rituximab: a new combination therapy to treat relapsed/refractory diffuse large B-cell lymphoma.

PURPOSE: There remains an unmet therapeutic need for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to evaluate the therapeutic potential of sepantronium bromide (YM155), a survivin suppressant, in combination with either bendamustine or both bendamustine and rituximab using DLBCL models. EXPERIMENTAL DESIGN: Human DLBCL cell lines, DB, SU-DHL-8, and WSU-DLCL2, were treated with YM155 in combination with bendamustine. Cell viability, apoptosis induction, protein expression, and cell-cycle distribution were evaluated. Furthermore, antitumor activities of YM155, in combination with bendamustine or both bendamustine and rituximab, were evaluated in mice bearing human DLBCL xenografts. RESULTS: The combination of YM155 with bendamustine showed greater cell growth inhibition and sub-G1 population than either agent alone. YM155 inhibited bendamustine-induced activation of the ATM pathway and accumulation of survivin at G2-M phase, with greater DNA damage and apoptosis than either single agent alone. In a DLBCL DB murine xenograft model, YM155 enhanced the antitumor activity of bendamustine, resulting in complete tumor regression without affecting body weight. Furthermore, YM155 combined with bendamustine and rituximab, decreased FLT-PET signals in lymph nodes and prolonged overall survival of mice bearing disseminated SU-DHL-8, an activated B-cell-like (ABC)-DLBCL xenografts when compared with the combination of either rituximab and bendamustine or YM155 with rituximab. CONCLUSIONS: These results support a clinical trial of the combination of YM155 with bendamustine and rituximab in relapsed/refractory DLBCL.

SGLT2 selective inhibitor ipragliflozin reduces body fat mass by increasing fatty acid oxidation in high-fat diet-induced obese rats.

Ipragliflozin is a novel and selective sodium-glucose cotransporter 2 (SGLT2) inhibitor that induces sustained increases in urinary glucose excretion by inhibiting renal glucose reabsorption and thereby exerting a subsequent antihyperglycemic effect. Here, we examined the effect of ipragliflozin on body weight in high-fat diet-induced (HFD) obese rats. Treatment of ipragliflozin (10mg/kg once daily) reduced body weight despite a slight increase in food intake. Dual-energy X-ray absorptiometry and computed tomography demonstrated that the reduction in body weight was accompanied by reduced visceral and subcutaneous fat masses but not lean mass or bone mineral content. Analysis of plasma and urinary parameters suggested the possibility that ipragliflozin enhanced lipolysis and fatty acid oxidation, and indirect calorimetry showed that ipragliflozin decreased the heat production rate from glucose but increased the rate from fat and lowered the respiratory exchange ratio. In conclusion, these data demonstrate that ipragliflozin-induced urinary glucose excretion specifically reduces fat mass with steady calorie loss by promoting the use of fatty acids instead of glucose as an energy source in HFD rats. By improving hyperglycemia and promoting weight reduction, ipragliflozin may prove useful in treating type 2 diabetes in obese individuals.

Pre-clinical validation of orthotopically-implanted pulmonary tumor by imaging with 18F-fluorothymidine-positron emission tomography/computed tomography.

The development of positron-emission tomography (PET) and X-ray computed tomography (CT) imaging has improved the detection of tumor burden and, in turn, pre-clinical drug development and clinical treatment. In pre-clinical drug development, clinically-relevant murine cancer models, such as orthotopic models of lung cancer, have provided an accurate representation of tumor burden in humans. However, evidence demonstrating the capability of imaging-guided evaluation of these clinically-relevant models is limited. Here, we combined (18)F-fluorothymidine (FLT)-PET/CT imaging and a murine model of human non-small cell lung cancer (NSCLC) to improve the accuracy of anticancer drug evaluation in pre-clinical studies. We found that FLT-PET/CT imaging enabled the progression of pulmonary tumors to be longitudinally monitored rather than FDG-PET/CT. Furthermore, in an efficacy study of a standard treatment of docetaxel in a murine lung cancer model, FLT-PET imaging detected the anticancer response earlier than volumetric analysis by CT imaging. We, thus, observed a relationship between the alteration of FLT signals and Ki-67 index in the pulmonary tumor during the period of chemotherapy. These results indicate that the combination of FLT-PET/CT imaging and an orthotopic NSCLC model is an effective strategy for evaluating clinical efficacy and potential of an anticancer agent during pre-clinical development.

[18F]FDG-PET as an imaging biomarker to NMDA receptor antagonist-induced neurotoxicity.

Positron emission tomography (PET) is an effective tool for noninvasive examination of the body and provides a range of functional information. PET imaging with [(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) has been used to image alterations in glucose metabolism in brain or cancer tissue in the field of clinical diagnosis but not in the field of toxicology. A single dose of N-methyl-d-aspartate (NMDA) receptor antagonist induces neuronal cell degeneration/death in the rat retrosplenial/posterior cingulate (RS/PC) cortex region. These antagonists also increase local cerebral glucose utilization. Here, we examined the potential of [(18)F]FDG-PET as an imaging biomarker of neurotoxicity induced by an NMDA receptor antagonist, MK-801. Using [(18)F]FDG-PET, we determined that increased glucose utilization involved the neurotoxicity induced by MK-801. The accumulation of [(18)F]FDG was increased in the rat RS/PC cortex region showing neuronal cell degeneration/death and detected before the onset of neuronal cell death. This effect increased at a dose level at which neuronal cell degeneration recovered 24h after MK-801 administration. Scopolamine prevented the neurotoxicity and [(18)F]FDG accumulation induced by MK-801. Furthermore, in cynomolgus monkeys that showed no neuronal cell degeneration/death when treated with MK-801, we noted no differences in [(18)F]FDG accumulation between test and control subjects in any region of the brain. These findings suggest that [(18)F]FDG-PET, which is available for clinical trials, may be useful in generating a predictive imaging biomarker for detecting neurotoxicity against NMDA receptor antagonists with the same pharmacological activity as MK-801.

Radiosynthesis, biodistribution and imaging of [11C]YM155, a novel survivin suppressant, in a human prostate tumor-xenograft mouse model.

INTRODUCTION: Sepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [(11)C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts. METHODS: Methods utilizing [(11)C]acetyl chloride and [(11)C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [(11)C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [(11)C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS). RESULTS: Sufficient quantities of radiopharmaceutical grade [(11)C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16-22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29-60 GBq/µmol (EOS). High uptake levels of radioactivity (%ID/g, mean±SE) were observed in tumor (0.0613±0.0056), kidneys (0.0513±0.0092), liver (0.0368±0.0043) and cecum (0.0623±0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [(11)C]YM155, at 40 min after injection, were 26.5 (±2.9) and 25.6 (±3.6), respectively. CONCLUSION: A rapid method for producing a radiopharmaceutical grade [(11)C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [(11)C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.

Sepantronium bromide (YM155) enhances response of human B-cell non-Hodgkin lymphoma to rituximab.

In the treatment of B-cell non-Hodgkin lymphoma (B-NHL) rituximab improves long-term survival in combination with conventional chemotherapy. However, because the majority of patients with B-NHL eventually relapse, the development of more effective therapies is needed. Here, we evaluated the antitumor effects of a combination treatment involving sepantronium bromide (YM155), a first-in-class survivin suppressant, and rituximab in B-NHL xenograft mice models. To determine the efficacy of the combination treatment, YM155- and rituximab-treated B-NHL cell xenografted mice were monitored for tumor size and survival and subjected to 2'-deoxy-2'-(18)F-fluoro-D-glucose ((18)F-FDG) and 3'-(18)F-fluoro-3'-deoxythymidine ((18)F-FLT) positron emission tomography (PET) imaging. In addition, the cell proliferation status of excised tumors was examined by Ki-67 immunohistochemistry. In DB, WSU-DLCL-2, and Mino xenograft-bearing mice, the combination treatment of YM155 and rituximab induced significant tumor growth inhibition and tumor regression compared with either single agent. On day 3 after the initiation of treatment a significant decrease in both (18)F-FDG and (18)F-FLT tumor uptake from pretreatment levels was observed in combination treatment groups. The Ki-67 proliferation index was significantly decreased on day 3 in the xenograft models treated with combination treatment, suggesting that the combination of YM155 and rituximab reduced cell proliferation and glucose metabolism. Furthermore, compared with monotherapy, combination treatment prolonged survival times of severe combined immunodeficient mice with disseminated WSU-FSCCL and Jeko B-NHL tumors. Our findings demonstrate that YM155 and rituximab combination treatment enhances antitumor activity in B-NHL xenografts, and (18)F-FLT and (18)F-FDG PET imaging may allow the early functional evaluation of treatment responses in patients with B-NHL.

Methods to evaluate transporter activity in cancer.

Plasma membrane transporter proteins play an important role in taking up nutrients into and effluxing xenobiotics out of cells to sustain cell survival. In the last decade, a number of studies have shown that these physiologically important transporters affect absorption, distribution, and excretion of major anticancer agents in clinical use. More importantly, many transporters have been reported to be differentially upregulated in cancer cells compared to normal tissues, suggesting that the differential expression of transporters in cancer cells may become good targets for enhancing drug delivery as well as diagnostic markers for cancer therapy. Hence, utilizing the knowledge of transporter functions likely provides us with the possibility of delivering a drug to the target tissues, avoiding distribution to other tissues, and improving oral bioavailability. This chapter focuses on methodology to analyze the activity of transporters that are involved in drug transport.

Inhibition of oligopeptide transporter suppress growth of human pancreatic cancer cells.

Oligopeptide transporters are abundantly expressed in various types of cancer cells. We here synthesized two novel dipeptides, l-phenylalanyl sarcosine (Phe-Sar) and 4-(4-methoxyphenyl)-l-phenylalanyl sarcosine (Bip(OMe)-Sar), and examined their effect on the growth of human pancreatic cancer AsPC-1 cells, which are known to highly express oligopeptide transporter PEPT1/SLC15A1. Growth of AsPC-1 cells was inhibited by these two peptides and a typical PEPT1/SLC15A1 substrate Gly-Sar. Growth inhibition by Gly-Sar, Phe-Sar and Bip(OMe)-Sar was concentration-dependent with half-maximal inhibitory concentration of 50, 0.91 and 0.55mM, respectively. These peptides also inhibited PEPT1-mediated [(3)H]Gly-Sar uptake with half-maximal inhibitory concentration of 2.6, 0.81 and 0.27mM, respectively. Thus, the rank order of the tumor cell growth inhibition by these three peptides was the same as that of PEPT1-inhibitory activity. Growth of AsPC-1 cells was also inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), which is a typical inhibitor of amino acid transporter system L. The growth inhibition by BCH and Gly-Sar was additive, suggesting that these compounds act at distinct loci. Oligopeptide transporters thus appear to be a promising target for inhibition of pancreatic cancer progression. These results also proposed the idea that oligopeptide transporter is required for growth of AsPC-1 cells.

Direct evidence for efficient transport and minimal metabolism of L-cephalexin by oligopeptide transporter 1 in budded baculovirus fraction.

The oligopeptide transporter PEPT1 (SLC15A1) is responsible for absorption of peptidic nutrients in the small intestine. Although the L-diastereomer of the beta-lactam antibiotic cephalexin (L-cephalexin) is likely to be transported by PEPT1, there has been no direct demonstration of PEPT1-mediated L-cephalexin transport. Indeed, after the incubation with L-cephalexin, the intact form of L-cephalexin has not been identified inside vesicles/proteoliposomes prepared from brush border membrane of intestinal epithelial cells or cultured cell lines exogenously transfected with PEPT1 gene. Thus, it appears that L-cephalexin is rapidly metabolized by PEPT1 or PEPT1-associated proteins. Here, we attempted to verify whether L-cephalexin is transported by PEPT1 and whether it is hydrolyzed by PEPT1 itself, by using budded baculovirus expressing PEPT1 protein. Marked uptake of L-cephalexin in PEPT1-expressing budded baculovirus, compared with wild-type virus, indicated that L-cephalexin is a substrate for PEPT1. The uptake was found to be pH sensitive, and was strongly inhibited by the D-diastereomer of cephalexin and glycylsarcosine, but not by glycine. Thus, L-cephalexin is transported by PEPT1 itself. Upon the transport of both L- and D-cephalexin by PEPT1, dose-dependent membrane depolarization was observed; the EC(50) values of 0.18 and 2.9 mM, respectively, indicate that the affinity of L-cephalexin for PEPT1-mediated transport is much higher than that of the D-diastereomer. On the other hand, the L-cephalexin metabolite 7-aminodesacetoxycephalosporanic acid was not detected in PEPT1-expressing or wild-type virus at either pH 6.0 or 7.4. We conclude that L-cephalexin is transported by PEPT1 with high affinity, but is not metabolized by PEPT1 itself.

Organic anion transporter OAT1 is involved in renal handling of citrulline.

Because citrulline plasma concentration is elevated in kidney failure, citrulline could be a biomarker of renal insufficiency, although the mechanism regulating its disposition in the kidney has not been clarified. In rat kidney slices, citrulline uptake was apparently Na(+) dependent, saturable with K(m) 556 microM, and significantly inhibited by anionic (PAH) and cationic (TEA) compounds, but not by probenecid at 1 mM. Preincubation of kidney slices with glutarate increased citrulline uptake, while such an increase was not observed after preincubation of the slices in Na(+)-free buffer. This result suggested that a sodium-dependent dicarboxylate cotransporter is involved in citrulline uptake by rat kidney slices. In studies using transporter-overexpressing cells, human organic anion transporter 1 (OAT1) and rat Oat1 exhibited citrulline transport activity with K(m) values of 238 and 373 microM, respectively, while other OATs and organic cation transporters (OCTs) did not transport citrulline. Based on the relative activity factor method, the contribution of rat Oat1 to the overall uptake of citrulline in rat kidney slices was approximately 70%. Moreover, the interaction among citrulline, PAH, and probenecid uptakes via rat Oat1 suggested that there are multiple functional sites on Oat1 and that the citrulline site may be distinct from the PAH and probenecid site. Thus OAT1/Oat1 appears to be one of the major contributors to renal basolateral uptake of citrulline, and impaired activities of these transporters may contribute substantially to the increase in plasma citrulline in renal failure. Accordingly, citrulline may be useful for diagnosis of kidney function as is creatinine.

Transport characteristics of L-citrulline in renal apical membrane of proximal tubular cells.

L-Citrulline has diagnostic potential for renal function, because its plasma concentration increases with the progression of renal failure. Although L-citrulline extracted by glomerular filtration in kidney is mostly reabsorbed, the mechanism involved is not clearly understood. The present study was designed to characterize L-citrulline transport across the apical membranes of renal epithelial tubular cells, using primary-cultured rat renal proximal tubular cells, as well as the human kidney proximal tubular cell line HK-2. L-Citrulline was transported in a Na(+)-dependent manner from the apical side of both cell types cultured on permeable supports with a microporous membrane. Kinetic analysis indicated that the transport involves two distinct Na(+)-dependent saturable systems and one Na(+)-independent saturable system in HK-2 cells. The uptake was competitively inhibited by neutral and cationic, but not anionic amino acids. Relatively large cationic and anionic compounds inhibited the uptake, but smaller ones did not. In HK-2 cells, mRNA expression of SLC6A19 and SLC7A9, which encode B(0)AT1 and b(0,+)AT, respectively, was detected by RT-PCR. In addition, L-citrulline transport was significantly decreased in HK-2 cells in which either SLC6A19 or SLC7A9 was silenced. Hence, these results suggest that amino acid transporters B(0)AT1 and b(0,+)AT are involved in the reabsorption of L-citrulline in the kidney, at least in part, by mediating the apical membrane transport of L-citrulline in renal tubule cells.

Cancer detection using a PET tracer, 11C-glycylsarcosine, targeted to H+/peptide transporter.

UNLABELLED: H+/peptide transporter, PEPT1, is functionally expressed in some human cancer cell lines and might be a candidate molecular target for detection of cancers in vivo using PET. The aim of the present study was to establish a novel tumor-imaging technology using a PET tracer targeted to H+/peptide transporter(s). We also compared the tracer with 18F-FDG, focusing on the specificity of their accumulation between tumor and inflammatory tissues. METHODS: A dipeptide PET tracer, 11C-glycylsarcosine (11C-Gly-Sar), was injected intravenously into athymic mice transplanted with human pancreatic, prostate, and gastric cancer cells. The distribution patterns of 11C-Gly-Sar and 18F-FDG in the tumor-bearing mice, and in mice with inflammatory tissue, were assessed by imaging with a positron planar imaging system (PPIS). Tissue distributions of tracer radioactivity were also measured. The expression levels of PEPT1 and PEPT2 (PEPTs) proteins in tumor xenografts and inflammatory tissue were examined by immunohistochemical analysis. The messenger RNA expression levels of PEPTs in 58 available cancer cell lines were quantified by means of real-time polymerase chain reaction. RESULTS: All 3 tumor xenografts were well visualized with the PPIS after injection of 11C-Gly-Sar. Expression of PEPTs in those xenografts was confirmed by immunohistochemical analysis. Tumor-to-blood concentration ratios of 11C-Gly-Sar increased in a time-dependent manner and were much higher than unity. Most of the radioactivity found in the tumor tissue was recovered as the intact tracer. These results indicated that 11C-Gly-Sar was taken up by the PEPTs in tumor xenografts. It is noteworthy that 11C-Gly-Sar was minimally present in inflammatory tissues that expressed no PEPT1 or PEPT2 protein, whereas 18F-FDG was highly accumulated, with the values of the selectivity index being >25.1 and 0.72 for 11C-Gly-Sar and 18F-FDG, respectively. The mRNAs of PEPT1 and PEPT2 were expressed in 27.6% and 93.1%, respectively, of the cancer cell lines examined in the present study. CONCLUSION: The present study indicates that 11C-Gly-Sar is a promising tumor-imaging agent and is superior to 18F-FDG for distinguishing between tumors and inflammatory tissue. Because PEPTs were ubiquitously expressed in various types of tumor cells examined, 11C-Gly-Sar could be useful for the detection of many types of cancers.

Functional expression of stereoselective metabolism of cephalexin by exogenous transfection of oligopeptide transporter PEPT1.

Gastrointestinal absorption of the beta-lactam antibiotic cephalexin (CEX) is highly stereoselective: l- and d-CEX are both taken up by intestinal epithelial cells through the brush-border membrane, most likely via oligopeptide transporter PEPT1, but l-CEX is not found in serum or urine after administration p.o. because of its rapid intestinal metabolism, whereas d-CEX is well absorbed in the unchanged form. We examined the contribution of PEPT1 to the stereoselective uptake and metabolism of CEX. We observed stereoselective metabolism of CEX after exogenous transfection of PEPT1 alone into mammalian cell lines: l-CEX, but not d-CEX, was metabolized to 7-aminodesacetoxycephalosporanic acid (7-ADCA) in HeLa and human embryonic kidney 293 cells stably and transiently expressing human PEPT1, respectively, whereas such metabolism was minor in cells expressing the vector alone. The formation rate of 7-ADCA depended on the amount of PEPT1 cDNA transfected. l-CEX metabolism was rapid because only 7-ADCA was found inside and outside the cells during incubation with l-CEX. The characteristics of PEPT1-mediated metabolism of l-CEX were similar, but not identical, to those of PEPT1-mediated transport. PEPT1-mediated metabolism was also observed in permeabilized cells expressing PEPT1, in which PEPT1-mediated intracellular substrate accumulation was negligible, suggesting that the increase in l-CEX metabolism by PEPT1 transfection cannot be fully explained by an increase in uptake and subsequent exposure to intracellular hydrolases. The present findings show that stereoselectivity in CEX absorption can be fully explained in terms of PEPT1, implying that the l-CEX hydrolase is PEPT1 itself or is induced by PEPT1.

Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells.

Human organic anion transporting polypeptide OATP-B (OATP2B1) is a pH-sensitive transporter expressed in the apical membranes of small intestinal epithelial cells. In this study, we have examined the contribution of OATP-B to the uptake of [3H]estrone-3-sulfate in Caco-2 cells in comparison with those of its homologs OATP-D (OATP3A1) and OATP-E (OATP4A1). Immunocytochemical study revealed that OATP-B is expressed in the apical membranes of Caco-2 cells. The uptake of [3H]estrone-3-sulfate by Caco-2 cells was Na+-independent and inhibited by several organic anions. It showed biphasic saturation kinetics with Km values of 1.81 microM and 1.40 mM. The uptake of [3H]estrone-3-sulfate by human embryonic kidney (HEK) 293 cells stably expressing OATP-B (HEK293/OATP-B) was also Na+-independent and inhibited by several organic anions. The Km value for estrone-3-sulfate uptake by OATP-B (1.56 microM) was close to that for the high-affinity component observed in Caco-2 cells. The mRNA expression level of OATP-B was higher than that of OATP-D or OATP-E in Caco-2 cells and in human jejunum biopsies from healthy volunteers. The values of [3H]estrone-3-sulfate uptake normalized to OATP-B mRNA expression were similar in Caco-2 cells and HEK293/OATP-B cells. The specific activity of OATP-B per mRNA expression was much higher than that of OATP-D and OATP-E. [3H]Estrone-3-sulfate uptake by membrane vesicles prepared from HEK293/OATP-B cells exhibited an overshoot phenomenon in the presence of an inwardly directed H+ gradient, suggesting that an H+ gradient is the driving force of estrone-3-sulfate transport by OATP-B. These results suggest that OATP-B is predominantly responsible for the apical uptake of estrone-3-sulfate in Caco-2 cells.