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Clenbuterol is a long-acting β2-adrenoceptor agonist and bronchodilator that is used for the treatment of asthma, but the desired activities reside almost exclusively in the (-)-R-enantiomer. This study examined enantioselectivity in the disposition of clenbuterol following administration of clenbuterol racemate to rats. Concentrations of clenbuterol enantiomers in plasma, urine and bile were determined by LC-MS/MS assay with a Chirobiotic T column. This method was confirmed to show high sensitivity, specificity and precision, and clenbuterol enantiomers in 0.1 ml volumes of plasma were precisely quantified at concentrations as low as 0.25 ng/ml. The pharmacokinetic profiles of clenbuterol enantiomers following intravenous and intraduodenal administration of clenbuterol racemate (2 mg/kg) in rats were significantly different. The distribution volume of (-)-R-clenbuterol (9.17 l/kg) was significantly higher than that of (+)-S-clenbuterol (4.14 l/kg). The total body clearance of (-)-R-clenbuterol (13.5 ml/min/kg) was significantly higher than that of the (+)-S-enantiomer (11.5 ml/min/kg). An in situ absorption study in jejunal loops showed no difference in the residual amount between the (-)-R- and (+)-S-enantiomers. Urinary clearance was the same for the two enantiomers, but biliary excretion of (-)-R-clenbuterol was higher than that of the (+)-S-enantiomer. The fractions of free (non-protein-bound) (-)-R- and (+)-S-clenbuterol in rat plasma were 48.8% and 33.1%, respectively. These results indicated that there are differences in the distribution and excretion of the clenbuterol enantiomers, and these may be predominantly due to enantioselective protein binding.

Substrate‐induced upregulation of ATP‐binding cassette subfamily G member 2 (ABCG2) has been well studied in cancer cells, but it is also important to understand whether ABCG2 is upregulated by its substrates in tissues in which it is constitutively expressed. In the present study, we aimed to clarify the regulatory mechanism of Abcg2 expression by its substrate, mitoxantrone, in placental cells. Abcg2 mRNA expression in rat placental TR‐TBT 18d‐1 cells treated with 10 μM mitoxantrone for 24 h was increased, compared with that in nontreated cells, whereas 10 μM pheophorbide‐a had no effect. Methylated CpG level in the promoter region of the Abcg2 gene was low and was not altered by mitoxantrone. On the contrary, mitoxantrone markedly increased the expression of estrogen receptor (ER) α and progesterone receptor (PR) B. Fulvestrant, an ER antagonist, attenuated the mitoxantrone‐induced increase of Abcg2 mRNA expression, whereas mifepristone, a PR antagonist, had little effect. 17β‐estradiol, an ER ligand, positively regulated the mitoxantrone‐induced increase of Abcg2 expression. DNA demethylation by 5‐aza‐2‐deoxycytidine treatment increased ERα expression, but mitoxantrone failed to facilitate the demethylation of ERα promoter in TR‐TBT 18d‐1 cells. In conclusion, Abcg2 expression is induced by mitoxantrone via the induction of ERα in TR‐TBT 18d‐1 cells.

Hepatic metastasis is a common cause of treatment failure following resection of pancreatic cancer. In this study, we report our results of hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) plus 5-fluorouracil (5-FU) or oral S-1 treatment for postoperative liver metastases from pancreatic cancer. Seven patients with postoperative liver metastases from pancreatic cancer received HAI with GEM plus 5-FU or oral S-1 between October, 2008 and September, 2010 at Kanazawa University Hospital (Kanazawa, Japan). Three out of the 7 cases exhibited a partial response (PR) according to Response Evaluation Criteria in Solid Tumors (RECIST) and stable disease (SD) was achieved in 3 out of the 7 cases (response rate, 85.7%). A decrease in serum tumor marker CA 19-9 levels was observed after 10 HAI treatment cycles in 5 out of the 7 cases. The median time to treatment failure was 8 months (range, 0–17 months). Adverse events included grade 3 leukocytopenia in 1 case and anemia in all 7 cases, although 5 out of the 7 patients were anemic prior to HAI therapy. Grade 2 thrombocytopenia was also observed in 2 cases. Non-hematological events, such as nausea, diarrhea, liver injury or neuropathy and life-threatening toxicities were not reported; however, 6 patients (85.7%) developed catheter-related complications and the HAI catheter and subcutaneous implantable port system had to be removed. These findings demonstrated that HAI may deliver high doses of chemotherapeutic agents directly into the tumor vessels, producing increased regional levels with greater efficacy and a lower incidence/severity of systemic side effects. In conclusion, HAI chemotherapy is a safe and effective treatment for liver metastases from pancreatic cancer.

This study was designed to clarify the influence of long-term enteral nutrition (EN) on the pharmacokinetics of digoxin. Rats were fed EN diets (semi-digested, digested, and elemental) for 4 weeks, then digoxin (0.05 mg/kg) was administered orally. The AUC(0-∞) and k(a) of digoxin were significantly reduced in the semi-digested diet group versus the control, while the AUC(0-∞) was significantly increased in the digested and elemental diet groups. The mRNA level of Slco1a4 was significantly reduced at the upper small intestine in all EN groups. Further, the expression levels of P-glycoprotein (P-gp) protein and Abcb1a mRNA were increased at the same site in all EN groups, and the increases were significant in the elemental diet group. Cyp3a2 protein and mRNA expressions were significantly reduced in the liver in the digested and elemental diet groups. Abcb1a mRNA was also significantly reduced in the kidney in these groups. These results indicate that the absorption kinetics at the small intestine is influenced by semi-digested diet, and the elimination kinetics in the liver and kidney are influenced by digested and elemental diet. Semi-digested diet also altered digoxin pharmacokinetics in humans. Thus, the effect of long-term EN on digoxin pharmacokinetics depended on the dietary components.

Methotrexate (MTX) and salazosulfapyridine (SASP) are disease-modifying drugs that are commonly used in the treatment of rheumatoid arthritis (RA), and combination therapy with MTX and SASP is recommended for RA patients who show an inadequate response to monotherapy with either drug. This study was designed to examine the interaction between the two drugs from the viewpoint of serum MTX concentration in Japanese RA patients, who were receiving combination therapy with relatively low doses of MTX and SASP. This is a 24-week open-label intervention study of stable RA patients (n = 10) with low disease activity. In these patients, who had received SASP/MTX combination therapy for at least 12 weeks, SASP was discontinued, and the patients received MTX monotherapy for the next 24 weeks. The primary outcome was change of serum MTX concentration at 12 weeks after discontinuation of SASP. Two disease activity markers, simplified disease activity index (SDAI) and disease activity score-C reactive protein (DAS28-CRP), were assessed as secondary outcomes at 24 weeks after discontinuation of SASP. We also monitored levels of matrix metalloproteinase-3 (MMP-3) and inflammatory cytokines. Patients were asked to complete a questionnaire after the study. Serum MTX concentration in RA patients who discontinued SASP increased more than 2-fold within 4 weeks, and the higher level was maintained thereafter. No significant differences were detected in SDAI, DAS28-CRP, MMP-3 or inflammatory cytokines. Most participants reported no change in physical condition after withdrawal of SASP, and most preferred MTX monotherapy for future treatment. Withdrawal of SASP from patients receiving SASP/MTX caused a rapid, marked increase of serum MTX concentration, without any apparent change in disease parameters or side effects. Our results suggest that SASP can be discontinued without adverse effects in stable RA patients receiving combination therapy, at least among Japanese patients receiving relatively low doses of the two drugs. UMIN000024507 . October 21, 2016 retrospectively registered.

Background Bladder cancer patients receiving methotrexate, vinblastine, adriamycin and cisplatin (MVAC) chemotherapy are co-administered with dexamethasone as an anti-emetic. We examined whether or not dexamethasone affects the severity and onset day of MVAC-induced severe neutropenia. Methods This was a retrospective study of bladder cancer patients treated with MVAC chemotherapy with or without dexamethasone as an antiemetic at Kanazawa University Hospital during January 2005 - December 2009. Patients were categorized into three groups; no dexamethasone use (Dex (−)), dexamethasone on day 2 (Dex 1 day), and dexamethasone on days 2, 3 and 4 (Dex multiday). We evaluated the incidence of grade 3/4 neutropenia and the day of onset of first severe neutropenic episode during the first course of MVAC chemotherapy. Logistic regression was used to investigate whether co-administration of dexamethasone was a risk factor for severe neutropenia. Results Episodes of grade 3/4 neutropenia occurred in 3 out of 6 (50.0%), 11 out of 12 (91.7%) and 6 out of 6 (100%) patients in the Dex (−), Dex 1 day, and Dex multiday groups, respectively. The appearance day of first severe neutropenia in the Dex multiday group (13.2 ± 1.0) was significantly accelerated compared to the Dex (−) group (17.7 ± 2.1). Univariate logistic regression analysis revealed that dexamethasone is a risk factor for severe neutropenia (OR 17.0; 95%CI: 1.3–223.1). Conclusions Co-administration of dexamethasone for anti-emesis brings forward the first appearance of neutropenia, and increases the severity of neutropenia, in bladder cancer patients receiving MVAC chemotherapy.

C-C motif chemokine receptor (CCR)2 and its ligand, monocyte chemoattractant protein (MCP)-1, are pivotal for adipose tissue macrophage (ATM) recruitment and the development of insulin resistance. However, other chemokine systems also may play a role in these processes. In this study, we investigated the role of CCR5 in obesity-induced adipose tissue inflammation and insulin resistance. We analyzed expression levels of CCR5 and its ligands in white adipose tissue (WAT) of genetically (ob/ob) and high-fat (HF) diet–induced obese (DIO) mice. Furthermore, we examined the metabolic phenotype of Ccr5−/− mice. CCR5 and its ligands were markedly upregulated in WAT of DIO and ob/ob mice. Fluorescence-activated cell sorter analysis also revealed that DIO mice had a robust increase in CCR5+ cells within ATMs compared with chow-fed mice. Furthermore, Ccr5−/− mice were protected from insulin resistance, glucose intolerance, and hepatic steatosis induced by HF feeding. The effects of loss of CCR5 were related to both reduction of total ATM content and an M2-dominant shift in ATM polarization. It is noteworthy that transplantation of Ccr5−/− bone marrow was sufficient to protect against impaired glucose tolerance. CCR5 plays a critical role in ATM recruitment and polarization and subsequent development of insulin resistance.

Pharmaceutical agents directed against methicillin-resistant Staphylococcus aureus can be eliminated during haemodiafiltration, not only by diffusion and ultrafiltration, but also by adsorption onto haemofilters. The latter may be affected by the binding of agents to serum albumin. The present study therefore investigated the affinity of anti-methicillin-resistant Staphylococcus aureus agents (teicoplanin, linezolid, vancomycin) for haemofilters and the pharmacokinetic properties of teicoplanin during haemodiafiltration. Linezolid, teicoplanin and vancomycin were first screened for their in vitro affinity for three different kinds of filter membranes: polysulfone, polyacrylonitrile and polymethylmethacrylate. Only teicoplanin showed significant filter-binding activity. An in vitro haemodiafiltration circulation model was then developed that incorporated a one-litre beaker containing Krebs-Ringer's bicarbonate solution with/without human albumin (0 or 3 g/dl) as an artificial plasma. Teicoplanin (initial concentration 50 μg/ml, representing the maximum plasma concentration (Cmax) resulting from a typical clinical dosage) was circulated throughout the beaker. Teicoplanin concentrations in the ‘plasma’ and ultrafiltrate were determined by high performance liquid chromatography. In the screening experiment, teicoplanin was predominantly adsorbed onto polysulfone and polymethylmethacrylate membranes. Furthermore, teicoplanin was primarily eliminated by adsorption onto these filters during in vitro haemodiafiltration. Albumin significantly reduced both haemodiafiltration clearance and the adsorption-dependent elimination, although there were complex but significant interactions between albumin and the filter membrane. Elimination of teicoplanin in an in vitro haemodiafiltration model was largely due to adsorption onto polysulfone and polymethylmethacrylate haemofilters. Future clinical studies should likely be designed to evaluate present recommendations of teicoplanin dosages in patients on haemodiafiltration.

Treatment of cancers in the abdominal cavity, such as peritoneal dissemination, is difficult, but in principle intraperitoneal administration of anticancer drugs is expected to be preferable to systemic administration. Taxane anticancer drugs are used to treat gastric cancer patients with peritoneal dissemination. They are administered as micellar preparations, Taxol and Taxotere, which consist of paclitaxel in Cremophor EL (crEL) and docetaxel in Polysorbate-80 (PS-80), respectively. In this paper we review the disposition kinetics of taxane anticancer drugs after intraperitoneal administration in peritoneal dissemination patients and animal models and also discuss the effect of the surfactant vehicle on the behavior of taxanes. Copyright © 2012 Ken'ichi Miyamoto et al.

Although podocyte damage is known to be responsible for the development of minimal-change disease (MCD), the underlying mechanism remains to be elucidated. Previously, using a rat MCD model, we showed that endoplasmic reticulum (ER) stress in the podocytes was associated with the heavy proteinuric state and another group reported that a mammalian target of rapamycin complex 1 (mTORC1) inhibitor protected against proteinuria. In this study, which utilized a rat MCD model, a combination of immunohistochemistry, dual immunofluorescence and confocal microscopy, western blot analysis, and quantitative real-time RT-PCR revealed co-activation of the unfolded protein response (UPR), which was induced by ER stress, and mTORC1 in glomerular podocytes before the onset of proteinuria and downregulation of nephrin at the post-translational level at the onset of proteinuria. Podocyte culture experiments revealed that mTORC1 activation preceded the UPR that was associated with a marked decrease in the energy charge. The mTORC1 inhibitor everolimus completely inhibited proteinuria through a reduction in both mTORC1 and UPR activity and preserved nephrin expression in the glomerular podocytes. In conclusion, mTORC1 activation may perturb the regulatory system of energy metabolism primarily by promoting energy consumption and inducing the UPR, which underlie proteinuria in MCD.

金沢大学附属病院薬剤部, In the initial treatment of acute myocardial infarction, it is important to administer oral low-dose acetylsalicylic acid (ASA) within 10 min of arrival at the hospital. However, ASA is supplied as an enteric-coated tablet or buffered tablet to prevent gastric irritation. Although current guidelines recommended that patients should chew their initial dose of ASA, there is little evidence as to whether this is efficacious. Therefore, we aimed to make a direct comparison of the pharmacokinetics and pharmacodynamics of ASA after ingestion of intact and chewed nonenteric-coated buffered ASA tablet (NBA) and enteric-coated ASA tablet (ECA) in a quadruple crossover study in healthy volunteers. Chewing ECA accelerated tmax of ASA absorption, which became equivalent to that after ingestion of intact or chewed NBA. A significant decrease in serum thromboxane B2 was observed 20 min after ingestion of chewed ECA, or intact or chewed NBA, and inhibition of platelet aggregation was also observed within 20 min. Thus, chewing of the ECA appears to result in a similar timing of ASA action to that in the case of chewed or unchewed NBA. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci Copyright © 2011 Wiley-Liss, Inc., 発行後1年より全文公開.

Ethoparmacological relevance Salacia reticulata , a herbal medicine which has been used for the treatment of early diabetes in Ayurvedic medicine, is reported to have an anti-obesity effect and to be useful in the treatment of diabetes mellitus, insulin resistance and other metabolic diseases. Aim of the study The present study was performed to elucidate the mechanism of action of Salacia reticulata with special attention to the adipocytes as the tissue primarily involved in the pathology of metabolic diseases. Materials and methods Mouse-derived adipocyte precursor 3T3-L1 cells were treated with differentiation inducers in the presence or absence of Salacia reticulata (SRCD). We determined triacylglycerol accumulations, differentiation makers, released glycerol and adiponectin. Mangiferin, the primary component of SRCD, was also treated to 3T3-L1 cells. Result Concurrent administration of the extract of SRCD and differentiation inducers resulted in a significant inhibition of differentiation into mature adipocytes. SRCD also exhibited significant inhibitory action on the expression of genes and proteins of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT-enhancer binding protein (C/EBP)α, as well as on the activity of glycerol-3-phosphate dehydrogenase (GPDH), a differentiation marker, and caused a reduction in the concentration of released adiponectin. However, SRCD had no influence on lipolysis as indicated by the release of glycerol into the culture medium. The primary component of SRCD, mangiferin, was investigated for its effect on adipocytes; mangiferin caused no suppression of fat accumulation, suggesting that a component of SRCD other than mangiferin may be involved in the inhibition of adipocyte differentiation. Conclusions The above results suggest that the inhibitory action of SRCD on adipocyte differentiation, and not the promotion of lipolysis, is involved in the suppression of fat accumulation.

Accumulation of visceral fat induces various symptoms of metabolic syndrome such as insulin resistance and abnormal glucose/lipid metabolism and eventually leads to the onset of ischemic cerebrovascular diseases. Geniposide, which is iridoid glycoside from the fruit of Gardenia jasminoides ELLIS, is recognized as being useful against hyperlipidemia and fatty liver. In order to clarify the effect of geniposide on metabolic disease-based visceral fat accumulation and the relevant molecular mechanism, experiments were performed in spontaneously obese Type 2 diabetic TSOD mice and the free fatty acid-treated HepG2 cells. In the TSOD mice, geniposide showed suppression of body weight and visceral fat accumulation, alleviation of abnormal lipid metabolism and suppression of intrahepatic lipid accumulation. In addition, geniposide alleviated abnormal glucose tolerance and hyperinsulinemia, suggesting that geniposide has an insulin resistance-alleviating effect. Next, in order to investigate the direct effect of geniposide on the liver, the effect on the free fatty acid-treated HepG2 fatty liver model was investigated using genipin, which is the aglycone portion of geniposide. Genipin suppressed the intracellular lipid accumulation caused by the free fatty acid treatment and also significantly increased the intracellular expression of a fatty acid oxidation-related gene (peroxisomal proliferator-activated receptor: PPARα). From these results, it was confirmed that geniposide has an anti-obesity effect, an insulin resistance-alleviating effect and an abnormal lipid metabolism-alleviating effect, and the metabolite genipin shows a direct effect on the liver, inducing expression of a lipid metabolism-related gene as one of its molecular mechanisms.

The aim of the present study was to characterize the mechanism of erythromycin transport at the blood-placenta barrier, using TR-TBT 18d-1 cells as a model of rat syncytiotrophoblasts. [(14)C]Erythromycin was taken up by TR-TBT 18d-1 cells with a Michaelis constant of 466 microM. Although the uptake was not dependent on extracellular Na(+) or Cl(-), it was increased at weakly alkaline pH. Significant overshoot of [(14)C]erythromycin uptake by placental brush-border membrane vesicles was observed in the presence of an outwardly directed proton gradient. These results indicate that erythromycin is transferred by the H(+)-coupled transport system in syncytiotrophoblasts. To address the physiological transport of erythromycin in rat placenta, fetal-to-maternal transport clearance was estimated by means of the single placental perfusion technique. Clearance of [(14)C]erythromycin was higher than that of [(14)C]inulin, a paracellular pathway marker, and was decreased by the addition of 5 mM erythromycin, indicating that saturable efflux system from fetus to mother is involved. The effect of various transporter inhibitors on [(14)C]erythromycin efflux from TR-TBT 18d-1 cells was evaluated. cyclosporin A, fumitremorgin C, and probenecid had no effect, whereas ethylisopropylamiloride, a specific inhibitor of Na(+)/H(+) exchangers (NHEs), was significantly inhibitory. These results suggest that erythromycin efflux transport at the rat blood-placenta barrier is mediated by an erythromycin/H(+) antiport system, driven by H(+) supplied by NHEs.

The purpose of this study was to investigate whether the trough plasma concentration of voriconazole (VRCZ) in the steady-state (Cmin) influences efficacy and safety in adult and pediatric patients.Treatment was successful in 75% of pediatric patients and 56% of adult patients,and there was no association between Cmin and efficacy.However,liver dysfunction occurred more frequently in the case that Cmin was over 4μg/mL in both adult and pediatric patients.Also,pediatric patients,especially those younger than 2 years,required a higher dose of VRCZ than adults to achieve a similar Cmin.We concluded that the use of VRCZ at a constant dose over a long period may lead to an increase in Cmin.Therefore,careful,continuous monitoring of the Cmin of VRCZ is necessary for pediatric patients,particularly recipients of hematopoietic stem cell transplantation.

The syncytiotrophoblast, which regulates maternal-fetal transfer of drugs, consists of a single layer in humans, but two layers, i.e., SynI and SynII, in rodents. Polar distribution of transporters in the apical and basal plasma membranes of syncytiotrophoblast is important for placental function in terms of vectorial transport of substrates, but the mechanisms that control protein distribution in the syncytiotrophoblast remain unclear. We have previously established rat syncytiotrophoblast cell lines, TR-TBT 18d-1 and TR-TBT 18d-2, which retain characteristics of SynI and SynII, respectively. In this study, we aimed to characterize the gene expression profiles in the two layers by using these cell lines. DNA microarray analysis indicated that more than 25 mRNAs, including cytoskeleton binding proteins, ezrin and CLP36, are differentially expressed between TR-TBT 18d-1 and TR-TBT 18d-2. Quantitative real time-polymerase chain reaction (PCR) analysis indicated that mRNA expression of ezrin, CLP36, CCN1, and CCN2 is higher in TR-TBT 18d-1 and mRNA expression of elf-1a, hsc70 and flot2 is higher in TR-TBT 18d-2, compared with their counterparts. Immunohistochemical analysis indicated that ezrin is expressed in rat placental villi in vivo, and is located on the apical membranes of TR-TBT 18d-1, while CLP36 is located in the apical and basal sides of TR-TBT 18d-1. The expression of ezrin was highest at gestational days 14 and 18 and was highest among the ezrin/radixin/moesin (ERM) family members. These results may help to clarify the mechanisms controlling polarization of the syncytiotrophoblast and the significance of the double epithelial layers in rat and mouse.

金沢大学附属病院薬剤部, The present study examined the influence of cimetidine on the nephrotoxicity and antitumor effects of cisplatin in vitro and in vivo. When the serum concentration of cimetidine was maintained over 20 μg/ml for 4 h by bolus and continuous intravenous infusion, cimetidine prevented nephrotoxicity of cisplatin without influencing antitumor activity. Cimetidine and the antioxidant N-acetylcysteine (NAC) significantly inhibited the in vitro growth inhibition of cisplatin in cells originating from the kidney, but not in SOSN2 osteosarcoma cells. Cimetidine (1mM) also did not influence platinum concentration in the cells, regardless of whether the organic cation transporter 2 (OCT2) was expressed. Cisplatin did induce reactive oxygen species (ROS) in the KN41 kidney cell line and cimetidine and NAC significantly reduced ROS production. However, cisplatin did not produce ROS in osteosarcoma cells. From these results, cimetidine clearly inhibits nephrotoxicity induced by cisplatin without any influence on the antitumor activity of cisplatin on osteosarcoma in vitro and in vivo. © 2010 Pharmaceutical Society of Japan.

The blood–placenta barrier (BPB) serves to protect the fetus from exposure to toxins, and to transport various nutrients, including nucleosides, and hormones from mother to fetus. It is known that nucleoside transporters contribute to the transfer of nucleosides and nucleoside analogues. 2′,3′-Dideoxyinosine (ddI) has a nucleoside structure, and crosses the BPB. Although ddI is a substrate of several transporters, including equilibrative nucleoside transporters (ENT1 and ENT2), the transport mechanism of ddI in the placenta has not yet been characterized. Therefore, the purpose of this study was to clarify the influx mechanisms of ddI from the maternal to the fetal side, and to examine the interaction between ddI and uridine transport at the BPB. We studied ddI and uridine uptakes using a conditionally immortalized rat syncytiotrophoblast cell line, TR-TBT 18d-1, as a BPB model. The ddI uptake was temperature-dependent, Na+-independent and saturable. Kinetic analysis yielded Km values for ddI and uridine of 6.51 mM and 23.4 μM, respectively. Uridine uptake was inhibited by ENT1 and ENT2 substrates, and ddI uptake was also inhibited by substrates or inhibitors at concentrations that inhibit ENT2. Uridine uptake in Xenopus laevis oocytes expressing rat ENT2 was inhibited by 5 mM ddI, in agreement with the results for TR-TBT 18d-1. Our results indicate that ddI and uridine are both taken up in part via ENT2 in TR-TBT 18d-1 cells, and therefore that ENT2 may contribute to their uptake at the BPB.

Objective: Bone marrow (BM)-derived endothelial progenitor cells (EPC) play an important role in neovascularization and tumor growth. It has been reported that docetaxel and paclitaxel inhibit angiogenesis, but the effect of docetaxel and paclitaxel on EPC-induced neovascularization has not been examined. We aimed to clarify the cytotoxic and inhibitory effects of these drugs on EPC. Methods: The effects of drugs on growth, tube formation, and migration of EPC were analyzed in vitro using a rat BM-derived EPC cell line (TR-BME). Fluorescence-labeled TR-BME cells were injected into tumor-bearing rats and accumulation at the tumor site was analyzed by fluorescence-activated cell sorting (FACS). Results: In in vitro cytotoxicity assays of these drugs in TR-BME, rat endothelial cell line TR-BBB and rat tumor cell line Walker 256, the IC50 values for TR-BME were higher than those for TR-BBB or Walker 256. Both drugs inhibited tube formation and migration of TR-BME at lower concentrations than the cytotoxic IC50. In vivo studies showed that a low dose of both drugs inhibited EPC accumulation at the tumor site in tumor-bearing rats, as determined by FACS, and caused a decrease in microvessel density. Conclusion: Docetaxel and paclitaxel directly inhibited EPC-initiated vasculogenesis at low (non-cytotoxic) concentrations, causing suppression of tumor growth.

The ezrin, radixin, and moesin (ERM) proteins represent a family of adaptor proteins linking transmembrane proteins to the cytoskeleton. The seminiferous epithelium undergoes extensive changes in cellular composition, location, and shape, implicating roles of the membrane–cytoskeleton interaction. It remains unknown, however, whether the ERM proteins are expressed and play significant roles in the testis. In the present study, we examined the spatiotemporal expression of ERM proteins in the mouse testis by Western blotting and immunohistochemistry. Ezrin immunoreactivity was demonstrated in the cytoplasm of steps 15 and 16 spermatids from 5 weeks postpartum through adulthood, whereas radixin immunoreactivity was in the apical cytoplasm of Sertoli cells from 1 week through 2 weeks postpartum. No immunoreactivity for moesin was detected at any age. Immunoprecipitation demonstrated that ezrin was bound to the cytoskeletal component actin, whereas radixin was bound to both actin and tubulin. Of the transmembrane proteins known to interact with ERM proteins, only cystic fibrosis transmembrane conductance regulator, a chloride transporter, was bound to ezrin in elongated spermatids. These results suggest that ezrin is involved in spermiogenesis whereas radixin is involved in the maturation of Sertoli cells, through interaction with different sets of membrane proteins and cytoskeletal components. (J Histochem Cytochem 57:351–362, 2009)

AZT (3'-azido-3'-deoxythymidine; zidovudine), which is used for the prevention of mother-to-child transmission of HIV-1, is transplacentally transferred to the fetus across the blood-placenta barrier, which is composed of syncytiotrophoblasts. We recently showed that apical uptake of AZT by syncytiotrophoblasts is mediated by saturable transport system(s) in the TR-TBT 18d-1 cell line, and the cellular accumulation of AZT was increased in the presence of dehydroepiandrosterone sulfate (DHEAS). Here, we aimed to clarify the mechanism of this effect of DHEAS. Inhibitors of efflux transporters, including breast cancer resistance protein, P-glycoprotein, and multidrug resistance proteins, had little effect on the cellular accumulation of AZT in TR-TBT 18d-1. Kinetic study revealed that the rate constant for AZT uptake was greatly increased in the presence of 1 mM DHEAS. These results suggested that the effect of DHEAS was because of enhancement of the uptake process(es), rather than inhibition of efflux. When AZT uptake was analyzed according to the Michaelis-Menten equation, the estimated Michaelis constant, Km, for AZT uptake in the presence of 1 mM DHEAS was lower than that in its absence, whereas maximum uptake velocity, Vmax, and nonsaturable uptake clearance, kns, were similar in the presence and absence of DHEAS, indicating that DHEAS may change the recognition characteristics of the transporter for AZT in TR-TBT 18d-1. Thus, the increase of AZT uptake in TR-TBT 18d-1 cells in the presence of DHEAS was concluded to be because of a DHEAS-induced change in the affinity of AZT uptake system, although the transporter responsible for AZT uptake has not been identified.

Transport of l-[3H]carnitine and acetyl-l-[3H]carnitine at the blood–brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-l-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-l-carnitine and in the absence of sodium ions. Similar transport properties for l-[3H]carnitine and/or acetyl-l-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of l-[3H]carnitine and acetyl-l-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with Km values of 33.1 ± 11.4 µm and 31.3 ± 11.6 µm, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-l-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of l-[3H]carnitine and acetyl-l-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of l-carnitine and acetyl-l-carnitine from the circulating blood to the brain across the BBB.

The placenta requires nucleosides as nutrients for fetal growth, so it is important to examine potential interactions between placental transports of nucleosides and drugs to ensure the safety of pharmacotherapy during pregnancy. The purposes of this study are to clarify the uptake mechanisms of nucleosides from the maternal side of the syncytiotrophoblast and to investigate the inhibitory effect of various drugs on nucleoside uptake, using the rat syncytiotrophoblast cell line TR-TBT 18d-1, which shows syncytial-like morphology and functional expression of several transporters. Initial uptake of [(3)H]uridine or [(3)H]adenosine from the apical side of TR-TBT 18d-1 was markedly reduced by an excess of the respective unlabelled compound, and was slightly reduced by replacement of Na(+) with N-methyl-d-glucamine, indicating that both uptakes were Na(+)-independent. [(3)H]Uridine and [(3)H]adenosine uptakes in the absence of Na(+) were significantly and concentration-dependently inhibited by both 0.1 microM and 100 microM nitrobenzylthioinosine, suggesting the involvement of equilibrative nucleoside transporters (ENTs, SLC29). Kinetic analysis of adenosine uptake yielded a K(m) value of approximately 17 microM. These results are consistent with the reported uptake characteristics of uridine and adenosine by ENT1 and ENT2. The uptakes were significantly reduced by high concentrations of several nucleoside drugs, including cytarabine, vidarabine, zidovudine, mizoribine, caffeine and amitriptyline, but the effects were small within the therapeutic concentration ranges. In summary, our results suggest that ENTs are involved in apical uptake of uridine and adenosine in the syncytiotrophoblast. However, therapeutic concentrations of the drugs tested in this study might have little influence on maternal-to-fetal nucleoside transfer.

This study was designed to clarify the pharmacological role of carnitine/organic cation transporter (Octn) family members in mouse heart. Immunohistochemical analysis revealed that Octn1 was exclusively expressed on endothelial cells in blood vessels. Octn2 was detected on the plasma membrane of cardiac muscle cells by immunoelectron microscopy. Octn3 was not detected in the heart. Integration plot analysis showed that coadministration of unlabeled L-carnitine reduced distribution of L-[3H]carnitine to the heart. L-[3H]Carnitine uptake in heart slices was reduced by carnitine analogs and various Octn2 substrates. L-[3H]Carnitine uptake by heart slices from juvenile visceral steatosis (jvs) mice, which have a hereditary octn2 gene deficiency, was negligible. Distribution of [3H]quinidine, another Octn2 substrate, to the heart was not reduced by L-carnitine, and [3H]quinidine uptake in heart slices was Na(-)-independent and inhibited by cationic drugs, but not carnitine analogs. [3H]Quinidine uptake by heart slices from jvs mice was similar to that of wild-type mice. These results demonstrate that OCTN2 is functionally expressed on the plasma membrane of muscle cells and is involved in distribution of carnitine to the heart. Some mechanism(s) other than OCTN2 is involved in the distribution of quinidine to the heart.

Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na+-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, withKmvalues of 23.6 and 6.57 μM for carnitine and acetylcarnitine respectively in the presence of Na+. Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na+-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c-Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31-positive endothelial cells. c-Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT-PCR, we demonstrated expression of HGF and c-Met in a rat pericyte cell-line, TR-PCT1, and in primary pericytes. HGF treatment of TR-PCT1 cells induced their migration, but not their proliferation, in a dose-dependent manner (10-100 ng/ml, p

Background Continuous hemodiafiltration (CHDF) is used as renal replacement therapy for critically ill patients with renal failure, and to treat hypercytokinemia. Since CHDF also clears therapeutic agents, drug pharmacokinetics (PK) should be dependent upon CHDF conditions. Although the antibiotic biapenem (BIPM) is used in patients undergoing CHDF, the optimal therapeutic regimen in such patients has not been fully clarified. In this study, we investigated the PK of BIPM in patients with various levels of renal function undergoing CHDF with polysulfone (PS) membrane, and used PK models to identify the optimal administration regimen. Methods BIPM (300 mg) was administered by infusion in patients undergoing CHDF (n = 7). Blood and filtrate-dialysate were collected for compartment and non-compartment analysis. Results The sieving coefficient of PS membrane was 1.00 ± 0.06 (mean ± S.D., n = 7), and CHDF clearance of BIPM was found to be the sum of the dialysate flow rate (QD) and filtrate flow rate (QF). Non-CHDF clearance showed inter-individual variability (4.82 ± 2.48 L/h), depending on residual renal function and non-renal clearance. Based on the average PK parameters obtained with a compartmental model, maximal kill end point (over 40 % T > MIC4 μg/mL) was achieved with regimens of 300 mg every 6 h, 300 mg every 8 h, and 600 mg every 12 h. Monte Carlo simulation indicated that 300 mg infusion for 1 h every 6 h was optimal, and the probability of target attainment at MIC2 μg/mL was 90.2 %. Conclusions Our results establish the optimal regimen of BIPM in patients with various levels of renal function undergoing CHDF with a PS membrane.

The purpose of this manuscript is to report the pharmacokinetics of docetaxel during hyperthermic intraperitoneal chemotherapy (HIPEC) after peritonectomy.Eleven patients with peritoneal metastasis (PM) underwent peritonectomies combined with 40 min of HIPEC with 40 mg/body of docetaxel. The pharmacokinetics of docetaxel were studied by using high-performance liquid chromatography.The docetaxel concentration at the start of HIPEC (0 min) was 9.084 ± 0.972 mg/L. The concentration gradually decreased to 5.599 ± 0.458 mg/L 40 min after HIPEC. In contrast, serum docetaxel levels increased during HIPEC, reaching a maximum level of 0.1334 ± 0.0726 mg/L at 40 min. The clearance (CLp) was 3.164 ± 1.383 L/hr, and the area under the curve (AUC) ratio was 95.12 ± 87.32. The AUC ratio of less-extensive peritonectomies was significantly higher than that of extended peritonectomies. The docetaxel concentration in the tumor tissue increased at 40 min (4.45 mg/gr). The apparent permeability (Papp, 40 min) was 1.47 ± 0.67 mm/40 min. No severe adverse effects were observed after HIPEC.From these results, 40 mg is a safe dose for docetaxel combined with HIPEC, and the locoregional intensity of docetaxel is enough to control PM less than 1.47 mm in diameter.

Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3162–3169, 2015

M17055 is under development as a novel loop diuretic for oral administration. To investigate the molecular mechanism of its gastrointestinal absorption, we initially aimed to clarify the mechanism of uptake of M17055 by Caco-2 cells, focusing on possible involvement of OATP-B (SLCO2B1), which is localized in the apical membranes of human intestinal epithelial cells. The uptake of [14C]M17055 by Caco-2 cells cultured on multi-well dishes was measured after cultivation for 14 days. Uptake of [14C]M17055 by HEK293 cells stably expressing OATP-B (HEK293/OATP-B cells) was also examined. M17055 uptake by Caco-2 cells was saturable, and was inhibited by various organic anions, including other loop diuretics, and several bile acids. Uptake of M17055 by HEK293/OATP-B cells was much higher than that by mock cells. The inhibitory profiles of various organic anions and the estimated K m values for M17055 uptake were similar in Caco-2 and HEK293/OATP-B cells. Moreover, the values of inhibition constants of several inhibitors for M17055 uptake were comparable in the two cell lines. Our data suggest that OATP-B plays a major role in the uptake of the novel loop diuretic M17055 from apical membranes in Caco-2 cells.

薬物トランスポーターの詳細で包括的な情報がデータベース化されている. また遺伝子バンクの整備が進み, トランスポーター遺伝子発現系を利用することで, 開発中の化合物のトランスポーターに対する認識性を解析することが可能である. 近年, トランスポーターの発現分布や基質認識特性を考慮したドラッグデザインやプロドラッグ化が注目されている.本稿では, 薬物の体内動態制御に関わるトランスポーターについて例をあげて紹介し, トランスポーターを利用した臓器選択的DDSにおいて考慮すべき諸問題について考察する.

Blood vessels are composed of endothelial cells (EC) and mural cells, and the interaction between EC and mural cells is essential for the development and maintenance of the vasculature. EC differentiate from bone marrow-derived endothelial progenitor cells (EPC). Recently, we established a conditionally immortalized bone marrow EPC-derived cell line, TR-BME2, and a brain capillary EC (BCEC) line, TR-BBB, from temperature-sensitive-SV40 T-antigen gene transgenic rats. To understand the function of EPC, it is important to analyze the difference between EPC and mature EC such as BCEC. In this study, we identified EPC-specific genes by means of subtractive hybridization between TR-BME2 and TR-BBB. There was no significant difference between TR-BME2 and TR-BBB in the mRNA level of annexin II, which is expressed in EC. In contrast, the mRNA level of smooth muscle cell (SMC) markers such as smooth muscle protein 22 (SM22), calvasculin, and platelet-derived growth factor (PDGF) receptor-beta, was higher in TR-BME2 than in TR-BBB. Moreover, the mRNA level of contractile SMC markers, such as smooth muscle alpha-actin and SM22, was increased in the absence of EC growth factors, such as vascular endothelial growth factor. The mRNA level of synthetic SMC markers, such as matrix Gla protein, was increased by the addition of PDGF-BB. The SMC derived from TR-BME2 showed an altered phenotype, from contractile-type to synthetic-type, when they were cultured in the absence of PDGF-BB. These results show that TR-BME2 cells have higher levels of SMC markers compared with mature EC, and can differentiate into contractile- or synthetic-type SMC.

Summary: We previously established a method to predict the drug metabolism capacity of injured liver based on pharmacokinetic estimation of the amount of cytochrome P450 (CYP) in vivo (PKCYP test), by introducing the apparent liver-to-blood free concentration gradient in vivo (qg) as a parameter. Here we show that the amount of CYP3A2 in CCl 4 -treated rats can be estimated appropriately by applying the PKCYP test using midazolam (MDZ) as a probe, assuming that the qg value in control rats does not change. We applied the results to predict the clearance of theophylline as a model drug with a physiologically based pharmacokinetic model. Male Sprague–Dawley rats were pretreated with CCl 4 , and the amount of CYP (A-CYP vivo ) was quantified by Western blotting. The qg value of MDZ was determined in control rats and used to estimate the amounts of CYP3A2 in CCl 4 -treated rats; the result agreed well with the observed values. The qg value of CYP3A2 estimated with MDZ as a probe was used together with our previously reported value for CYP1A2 (theophylline metabolism in the liver is known to be almost entirely mediated by CYP3A2 and CYP1A2) to predict the total body clearance ( CL tot ) of theophylline in CCl 4 -treated rats. The predicted CL tot was about one-third of the observed value, which was considered acceptable. The time-course of theophylline concentration in serum simulated with a physiologically-based pharmacokinetic model agreed well with the observed values. Thus, the PKCYP test using MDZ as a probe can be used to predict the amount of CYP3A2 and the CL tot of theophylline in CCl 4 -treated rats.

Xenobiotic transporters have been proposed to be involved in membrane penetration of various therapeutic agents. As little information is available on molecular mechanism of their functional regulation, we have attempted to clarify the protein-protein interactions of such transporters as a first step to identify their regulators.Yeast two-hybrid screening was performed to examine the interaction between carboxylic terminus of various xenobiotic transporters and PDZ (PSD95, D1g and ZO1) domain-containing proteins. The interaction and functional regulation were also evaluated in pull-down, immunoprecipitation and transport studies.Specific interaction with PDZ proteins was identified for several xenobiotic transporters including PEPT1, PEPT2, OCT3, OCTN1, OCTN2, OAT4, OATP-A, OATP-D, and OATP-F. The potent interaction was observed between PEPT2 and PDZK1, and deletion of the last four amino acids of the PEPT2 C-terminus almost completely abrogated such interaction. Recombinant PEPT2 C-terminus fusion protein can bind to purified His6-tagged PDZK1, confirming the involvement of two of four PDZ domains within PDZK1 in the interaction. Alanine-scanning mutation in PEPT2 revealed the presence of a consensus sequence (-T-X-L) that is responsible for the PDZK1 interaction. Transfection of PDZK1 increased the uptake of glycylsarcosine by PEPT2, whereas such stimulation was not observed for PEPT2 with the last four amino acids deleted.These results first identified the interaction between PDZ proteins and the cytosolic tail of various xenobiotic transporters. PDZK1 directly interacts with PEPT2, exerting functional regulation of its transporting activity. The current findings imply the localization of PEPT2 within a protein network constructed from PDZK1 and other transporter proteins.

M17055 was developed as a novel diuretic that inhibits both Na(+), K(+), and 2Cl(-) cotransport at the thick ascending Henle's loop and Na(+) reuptake at the distal tubule. It is secreted at the renal proximal tubules. The purpose of the present study was to characterize the renal excretion mechanism of M17055. We used the renal cortical slices and brush border membrane vesicles (BBMVs) to investigate the transport mechanisms across the basolateral and brush border membranes, respectively. M17055 uptake by rat renal slices increased with time and was saturable. Several organic anions including probenecid, para-aminohippurate (PAH), and estrone-3-sulfate, decreased M17055 uptake. The uptake of M17055 was also observed into HEK293 cells expressing rat OAT1, and was inhibited by PAH. M17055 uptake by BBMVs was time-dependent, saturable, osmolarity-sensitive, and inhibited by several organic anions, but not by PAH. These results suggest that plural organic anion transport systems are involved in M17055 transport via both basolateral and brush border membranes of proximal tubule epithelial cells, a part of the renal uptake being mediated by OAT1.

Evidence is accumulating to strongly suggest that drug transporters are one of the determining factors governing the pharmacokinetic profile of drugs. To date, a variety of drug transporters have been cloned and classified as solute carriers and ATP-binding cassette transporters. Such drug transporters are expressed in various tissues such as the intestine, brain, liver, and kidney, and play critical roles in the absorption, distribution and excretion of drugs. However, at the present time, information is limited regarding the genetic polymorphism of drug transporters and its impact on their function. In this context, we have undertaken the functional analyses of the polymorphisms identified in drug transporter genes. This article aims to provide an overview on the functional aspects of the non-synonymous polymorphisms of drug transporters and to present standard methods for the evaluation of the effect of polymorphisms on their function.

We previously demonstrated that P-glycoprotein and MRP2 contribute to the secretory transport of grepafloxacin in the small intestine. Although inhibitors of these secretory transporters increased absorptive transport of grepafloxacin, secretory transport was not altered in Caco-2 cells, as determined by a conventional Transwell method. Because the value of the permeability coefficient of grepafloxacin is high, permeation through the unstirred water layer (UL) might be the rate-limiting step. To examine the possibility that the UL effect may mask the involvement of membrane transporters in the transport of drug with high permeability in Caco-2 cells, transport experiments were performed by agitating the experimental solution to decrease the thickness of the UL, and by lowering the temperature to decrease permeation via active transporters. Under these conditions, the UL effect was not rate limiting, and the inhibitory effects of transporter modulators were reflected in the apparent permeability as a decrease in secretory transport as well as an increase in absorptive transport. In conclusion, it was demonstrated that the UL can be the rate-limiting factor for transport of drugs with high membrane permeability in Caco-2 cells. When the UL affects the apparent permeability in an experimental apparatus in vitro, careful analysis is required to evaluate the contributions of transporters from the apparent permeability of drugs.

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [d-Ala2, d-Leu5]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [3H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [3H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [3H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [3H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [3H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.

Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80–130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 sperma...

The feasibility of using adenovirus-mediated human oligopeptide transporter (hPEPT1) gene transfer to achieve peptide drug delivery to the brain across the blood-brain barrier was tested by examining the accumulation of model peptides in a rat brain endothelial cell line (RBEC1) and rat brain after transduction with a recombinant adenovirus encoding hPEPT1-enhanced yellow fluorescent protein fusion gene (AdhPEPT1-EYFP). In vitro uptake of [(3)H]GlySar was determined in RBEC1 transduced with AdhPEPT1-EYFP. In vivo, the accumulation of cefadroxil in rat brain was evaluated after transduction of AdhPEPT1-EYFP. At pH 6.0, the uptake of [(3)H]GlySar by RBEC1 transduced with AdhPEPT1-EYFP was increased 4-fold compared with that of nontransduced cells. At pH 7.4, uptake of [(3)H]GlySar in AdhPEPT1-EYFP transduced RBEC1 was 1.5 times higher than that of nontransduced cells. Unlabeled glycylsarcosine (10 mM) reduced the uptake of [(3)H]GlySar to a level comparable with that of nontransduced cells. At 30 min after intravenous administration of cefadroxil to rats transduced with AdhPEPT1-EYFP at 3.2 x 10(9) plaque-forming units/rat by an in situ brain perfusion method, the brain-to-plasma concentration ratio (Kp) of cefadroxil was increased about 2 times compared with that of nontransduced or AdGFP (control vector)-transduced rats, although this was not statistically significant. In contrast, Kp of [(14)C]inulin, a marker for extracellular fluid space, remained unchanged after adenoviral transduction. In conclusion, our results suggest that adenovirus-mediated heterologous expression of hPEPT1 in vivo could be a useful approach to deliver oligopeptides to the brain.

The characteristics of beta-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the beta-[3H]alanine transport indicated that the transporter for beta-alanine functions with Kt of 25.3 +/- 2.5 microM and Jmax of 6.90 +/- 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. Beta-[3H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN3) and low temperature reduced the uptake significantly. Furthermore, the uptake of beta-[3H]alanine required Na+ and Cl- in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one beta-alanine molecule. The Na+ and Cl--dependent uptake of beta-[3H]alanine was stimulated by a valinomycin-induced inside-negative K+-diffusion potential. beta-Amino acids (beta-alanine, taurine, and hypotaurine) inhibited strongly the uptake of beta-[3H]-alanine, whereas alpha- and gamma-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of beta-[3H]alanine was stimulated by preloading of beta-alanine or taurine but not L-leucine. These results show that beta-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to beta-amino acids.

To explore the feasibility of drug delivery to the liver by the use of adenovirus-mediated human oligopeptide transporter (hPEPT1) gene transfer, we examined the accumulation of L-[(3)H]carnosine in the hepatoma cell line (HepG2 and WIFB9) and mouse liver. We constructed a recombinant adenovirus encoding hPEPT1-enhanced yellow fluorescent protein (EYFP) fusion gene (AdhPEPT1-EYFP). In vitro uptake of L-[(3)H]carnosine was determined in HepG2 and WIFB9 cells transduced with AdhPEPT1-EYFP. In vivo, the accumulation of L-[(3)H]carnosine in mouse liver was evaluated after transduction of AdhPEPT1-EYFP. At pH 6.0, the uptake of L-[(3)H]carnosine by HepG2 and WIFB9 cells transduced with AdhPEPT1-EYFP was increased 15- and 2-fold, respectively, compared with the cells without transduction. At pH 7.4, uptake of L-[(3)H]carnosine in AdhPEPT1-EYFP transduced HepG2 cells was 3 times greater than that of nontransduced cells. In the presence of carnosine or glycylsarcosine as an inhibitor at 20 mM, the uptake of L-[(3)H]carnosine was reduced to a level comparable to that of nontransduced cells. At 30 min after intravenous administration of L-[(3)H]carnosine to mice transduced with AdhPEPT1-EYFP at 1 x 10(10) plaque-forming units/mouse, the tissue-to-plasma concentration ratio (K(p)) of L-[(3)H]carnosine in the liver was significantly increased to 7 times that of nontransduced mice. In contrast, the K(p) value of [(14)C]inulin, a marker for extracellular fluid space, remained unchanged after adenoviral transduction suggesting minimal pathological damage of tissues. hPEPT1-EYFP was localized at both the basolateral and apical membranes in HepG2 cells, WIFB9 cells, and mouse liver. In conclusion, our results suggest that delivery of oligopeptide to the liver by adenovirus-mediated heterologous expression of hPEPT1 in vivo is feasible.

The effect of polyrotaxane-dipeptide (Val-Lys) conjugates on the uptake of a model dipeptide (Gly-Sar) was examined via human peptide transporter (hPEPT1) on HeLa cells. Here, Val-Lys groups are introduced to alpha-CDs, which are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups (polyrotaxane). The Gly-Sar uptake via hPEPT1 was significantly inhibited in the polyrotaxane conjugates, and this inhibitory effect was not explained by the sum of interaction between hPEPT1 and alpha-CD-Val-Lys conjugates. Further, the inhibition was significantly greater than those observed in dextran-Val-Lys conjugates. Therefore, our data clearly suggests that supramolecular structure in the polyrotaxane conjugates contributes considerably to the inhibitory effect via multivalent binding of Val-Lys groups with hPEPT1.

We investigated the contribution of multidrug resistance-associated protein 2 (MRP2) to the secretory transport of grepafloxacin and compared its functional role with that of P-glycoprotein (P-gp) by using Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs), in which MRP2 is hereditarily defective. In intestinal tissue from SDRs mounted in Ussing chambers, the level of transport in the direction from the serosal layer to the mucosal layer was twofold greater than that in the direction from the mucosal layer to the serosal layer. This secretory transport of grepafloxacin was diminished by both probenecid, an MRP2 inhibitor, and cyclosporine, a P-gp inhibitor. In intestinal tissue from EHBRs, the secretory transport of grepafloxacin was lower than that in intestinal tissue from SDRs and was inhibited by cyclosporine but not by probenecid. The absorption of grepafloxacin from intestinal loops in SDRs was in the order of duodenum > jejunum > ileum and was increased by cyclosporine but not by probenecid. The absorption in EHBRs was not higher than that in SDRs. The intestinal secretory clearance in SDRs after intravenous administration of grepafloxacin was shown to be greater for the ileum than for the duodenum, which is in good agreement with the previously reported regional expression profile of MRP2 mRNA. The intestinal secretory clearance was lower in EHBRs than in SDRs. Accordingly, in addition to P-gp, MRP2 might play a role in the secretory transport of grepafloxacin. The function of MRP2 in facilitating grepafloxacin transport in the secretory direction is more pronounced both in vitro and in vivo, while the restriction of entry from the lumen into the cell by MRP2 seems to be negligible, compared with that by P-gp, in the case of grepafloxacin.

Purpose. To establish how closely intestinal transport activity for beta-lactam antibiotics is correlated with PepT1 expression, absolute expression level of PepT1 mRNA and transport activity were determined longitudinally in the small intestine of fed and starved rats.