Kazutoshi Horie

Are MDCK Cells Transfected with the Human MRP2 Gene a Good Model of the Human Intestinal Mucosa

AbstractPurpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (KM,Vmax) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (KI) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [3H]-digoxin than did Caco-2 and MDCK-WT cells. KM values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 μM, respectively, whereas Vmax values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm−2s−1, respectively. Known P-gp substrates/inhibitors showed, in general, lower KI values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.

Isolation and characterization of Caco-2 subclones expressing high levels of multidrug resistance protein efflux transporter.

AbstractPurpose. The purpose of this study was to isolate Caco-2 subclones that express high levels of multidrug resistance protein (MDR1) and to characterize their kinetics and affinity parameters for MDR1 substrate/inhibitors. Methods. The subclones were selected by a dilution cloning technique. The polarized efflux of [3H]-vinblastine across subclone cell monolayers was quantified by measuring the apparent permeability coefficients (Papp) of [3H]-vinblastine in the basolateral (BL)-to-apical (AP) direction and in the AP-to-BL direction (Papp BL-to-AP/Papp AP-to-BL) across the cell monolayers. The expression of MDR1 in the Caco-2 subclones compared with the parental Caco-2 cells was confirmed by Western blotting analysis. The kinetics parameters (Km, Vmax) of [3H]-vinblastine and the inhibitory constants (KI) of several known MDR1 substrates/inhibitors on the transport of [3H]-digoxin determined in the parental Caco-2 cells and Caco-2 subclones were also compared. Results. Three subclones (#1, #20, #21) were selected based on their polarized efflux of [3H]-vinblastine. The Papp BL-to-AP/Papp AP-to-BL ratios for #1, #20, and #21 were 110, 140, and 112, respectively, and were about 6-fold higher than the ratio observed for the parental Caco-2 cells. In the presence of GF-120918 (2 μM), a known MDR1-specific inhibitor, the Papp BL-to-AP/Papp AP-to-BL ratios were significantly decreased, suggesting that these cells were overexpressing MDR1. The Km values observed for vinblastine in the Caco-2 subclones were nearly identical to the value observed in the parental Caco-2 cells. In contrast, the Vmax values observed in the subclones were approximate 26-69% higher. The KI values observed for various known MDR1 substrates/inhibitors on [3H]-digoxin transport were nearly identical to those in the parental Caco-2 cells and Caco-2 subclones. The high functional efflux activities of these subclones were stable up to 6 months. Conclusions. Subclones #1, #20, #21 express high levels of MDR1. These Caco-2 subclones may be useful models for profiling drugs for their MDR1 substrate activity and for establishing structure-transport relationships for this efflux transporter.

Characteristics of Ceftibuten Uptake into Caco-2 Cells

The characteristics of ceftibuten uptake into Caco-2 cells grown in a collagen-coated dish were examined. Ceftibuten showed stereoselective and pH-dependent uptake. The pH-dependency of ceftibuten was more marked than that of cefaclor or cephalexin, but all three antibiotics showed maximal uptake at pH 5.5. Ceftibuten uptake was linear for the initial 1 hr and then reached a plateau. The initial uptake (15 min) was markedly reduced by the addition of 2,4-dinitrophenol or FCCP (a protonophore), or by lowering the incubation temperature. The uptake of ceftibuten into the brush-border membrane vesicles prepared from cultured Caco-2 cells showed an overshoot in the presence of an H+-gradient. These findings indicated that the uptake of ceftibuten was energy-dependent, especially H+-gradient-dependent. Uptake inhibition by various compounds was compared using Caco-2 cells. Amino acids and a tetrapeptide did not inhibit uptake, whereas di- or tri-peptides were effective inhibitors. These observations suggest that ceftibuten is taken up by a carrier-mediated transport system(s) for dipeptides. Various antibiotics differed in their ability to inhibit uptake, with cyclacillin showing maximum inhibition. Differences in the inhibitory effect may be accounted for by the heterogeneity (multiplicity) of the transport systems.

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens.

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen‐delivery systems for modulated and/or effective induction of oral tolerance.

Enhanced Accumulation of Sialyl Lewis X-Carboxymethylpullulan Conjugate in Acute Inflammatory Lesion

AbstractPurpose. E-selectin is a cell adhesion molecule that is specifically expressed in the inflammatory vascular endothelium in response to cytokines such as IL-1β and TNF-α, and interacts with specific ligands containing sialyl Lewis X (Neu5Acα2-3Galβl-4(Fucαl-3)GlcNAc-, SLex). In order to investigate the ability of E-selectin ligands to target the inflammatory site, the tissue distribution of carboxymethylpullulan (CMPul) modified with SLex was studied. Methods. CMPul conjugates with various saccharides containing SLex and monovalent SLex were intravenously administered to mice with ear edema induced by arachidonic acid, and their distributions to the inflamed ear and other tissues were studied. To determine the microdistributions of these compounds, the inflamed ear was subjected to microautoradiography. Results. After intravenous administration AUC0-24h of SLex-CMPul, which binds to E-selectin, in the inflamed ear was about 300-fold and 2.5-fold higher than that of monovalent SLex and CMPul conjugated with other saccharides, which can not serve as ligands for E-selectin. Microautoradiography also revealed SLex-CMPul accumulated at the microvessels in the inflammatory lesions. Conclusions. SLex-CMPul was found to have the potential to target drugs to the inflammatory lesion.

Evaluation of carboxymethylpullulan as a novel carrier for targeting immune tissues.

AbstractPurpose. To demonstrate the potential of carboxymethylpullulan (CMPul) as a carrier for targeting immune tissues, and to find whether immune tissues could be set as the target of an immunosuppressant to treat autoimmune diseases. Methods. The biodistribution of CMPul was investigated to evaluate its potency as a carrier for targeting immune tissues. Furthermore, an immunosuppressant-CMPul conjugate was prepared and its suppressive effect on rat adjuvant arthritis was examined. Results. The disappearance rate of 3H-labeled CMPul from the blood circulation was much slower than that of 3H-labeled pullulan (Pul) after intravenous injection to normal rats. The concentration of 3H-labeled CMPul in the spleen and lymph nodes was much higher than that of 3H-labeled Pul at 24 hours after the injection, whereas the concentration of 3H-labeled CMPul in the liver was significantly lower than that of 3H-labeled Pul. A similar targeting property of 3H-labeled CMPul for these immune tissues was observed in arthritic rats. A conjugate composed of a novel immunosuppressant PA-48153C and CMPul showed a suppressive effect on rat adjuvant arthritis judging from a reduction of the arthritic index and spleen weight and an increase of body weight. Conclusions. CMPul is expected to be a promising carrier for targeting immune tissues with an immunosuppressant to enable treatment of autoimmune diseases.

Oral-antigen delivery via a water-in-oil emulsion system modulates the balance of the Th1/Th2 type response in oral tolerance.

AbstractPurpose. To evaluate the ability of a water-in-oil (W/O) emulsion containing ovalbumin (OVA), a model antigen, to induce oral tolerance and to elucidate the mechanism for the induction of oral tolerance by the emulsion system. Methods. A W/O emulsion containing OVA was prepared and evaluated its ability to induce oral tolerance in mice. Also, the Th1/Th2 balance in the mice tolerized was investigated in terms of the ratios of anti-OVA IgG2a titer to anti-OVA IgG1 titer (IgG2a/IgG1 ratios) and cytokine profiles. Results. Anti-OVA total IgG antibody titer of mice administered OVA in saline was approximately 3.5-fold higher than that of the mice administered OVA in W/O emulsion at a dose of 0.1 mg/mouse/day. Similar total IgG responses were observed between the above two at a dose of 1 mg/mouse/day. The IgG2a/IgG1 ratios decreased as the dose of OVA in W/O emulsion, but not in saline, increased at doses of 0, 0.1, and 1 mg/mouse/day. Interferon-γ secretion of PLN cells from the mice administered OVA in W/O emulsion decreased, whereas their interleukin-4 secretion remained high. Although interferon-γ secretion for the mice administered OVA in saline decreased, interleukin-4 secretion did not change. Conclusions. The present study suggests that oral delivery of OVA via the W/O emulsion system may more efficiently enhance the induction of Th2-dominated imbalance than that of OVA in saline.

Effect of the Sialyl Lewis X (SLe) Moiety on Splenic Accumulation of SLex-Carboxymethylpullulan Conjugate

Sialyl Lewis X (SLex), an E‐selectin ligand, was conjugated with carboxymethylpullulan (CMPul) and the disposition characteristics of this conjugate after intravenous administration were investigated using mice with ear edema. The concentration of 3H‐labeled SLex‐CMPul in the spleen was significantly high. When CMPul was modified with a saccharide unable to bind to E‐selectin, this splenic accumulation was not observed. The uptake of radiolabeled SLex‐CMPul by the spleen was completely inhibited by a 100‐fold molar of cold SLe‐CMPul but not by a sialyl N‐acetyllactosamine‐CMPul conjugate (SLN‐CMPul). Microautoradiography analyses revealed that SLex‐CMPul accumulated in the marginal zone of the spleen.