Yasufumi Sawada

Role of human MDR1 gene polymorphism in bioavailability and interaction of digoxin, a substrate of P-glycoprotein

Our objective was to quantitate the contribution of the genetic polymorphism of the human MDR1 gene to the bioavailability and interaction profiles of digoxin, a substrate of P‐glycoprotein.

Brain pericytes contribute to the induction and up-regulation of blood–brain barrier functions through transforming growth factor-β production

The blood-brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-beta (TGF-beta) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-beta1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-beta1 antibody (10 microg/ml) or a TGF-beta type I receptor antagonist (SB431542) (10 microM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-beta1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-beta production.

Effect of bioflavonoids on vincristine transport across blood-brain barrier.

Several grapefruit juice bioflavonoids, including quercetin, are reported to stimulate P-glycoprotein-mediated drug efflux from cultured tumor cells. To see whether these bioflavonoids alter the permeation of vincristine across the blood-brain barrier, we conducted experiments with cultured mouse brain capillary endothelial cells (MBEC4 cells) in vitro and ddY mice in vivo. The steady-state uptake of [3H]vincristine by MBEC4 cells was decreased by 10 microM quercetin, but increased by 50 microM quercetin. Similarly, the in vivo brain-to-plasma concentration ratio of [3H]vincristine in ddY mice was decreased by coadministration of 0.1 mg/kg quercetin, but increased by 1.0 mg/kg quercetin. Kaempferol had a similar biphasic effect on the in vitro uptake of [3H]vincristine. Other aglycones tested (chrysin, flavon, hesperetin, naringenin) increased [3H]vincristine uptake in the 10-50 microM range, and glycosides (hesperidin, naringin, rutin) were without effect. We then addressed the mechanism of the concentration-dependent biphasic action of quercetin. Verapamil, a P-glycoprotein inhibitor, inhibited the efflux of [3H]vincristine from MBEC4 cells, while 10 microM quercetin significantly stimulated it. The uptake of [3H]vincristine by MBEC4 cells was increased by inhibitors of protein kinase C, but decreased by phorbol 12-myristate-13-acetate (PMA), as well as by 10 microM quercetin. The phosphorylation level of P-glycoprotein was increased in the presence of 5 microM quercetin or 100 nM PMA, but decreased by the protein kinase C inhibitor H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine, 30 microM). We conclude that low concentrations of quercetin indirectly activate the transport of [3H]vincristine by enhancing the phosphorylation (and hence activity) of P-glycoprotein, whereas high concentrations of quercetin inhibit P-glycoprotein. Our results indicate that patients taking drugs which are P-glycoprotein substrates may need to restrict their intake of bioflavonoid-containing foods and beverages, such as grapefruit juice.

Effect of furanocoumarin derivatives in grapefruit juice on the uptake of vinblastine by Caco-2 cells and on the activity of cytochrome P450 3A4.

The presence of inhibitors of drug efflux transporters, such as P‐glycoprotein (P‐gp), in grapefruit juice (GFJ) was confirmed based on the uptake of [3H]‐vinblastine (VBL) by Caco‐2 cells. The uptake of [3H]‐VBL by Caco‐2 cells was significantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [3H]‐VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates. These results show that the major inhibitor of efflux transport of VBL is different from that of CYP3A4. Further separation of the 60% methanol eluate afforded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady‐state [3H]‐VBL uptake by LLC‐GA5‐COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady‐state uptake of [3H]‐VBL by Caco‐2 cells. The order of inhibitory potency of these compounds was FC726>DHBG>bergamottin>bergapten>bergaptol. While, the IC50 values for inhibition of CYP3A4 were 0.075, 0.45, 1.0, 1.0 and >20 μM, respectively. Bergaptol specifically inhibited VBL efflux. DHBG was thus identified as a candidate for inhibitors of VBL transport, together with other furanocoumarins. Moreover, partly involvement of the P‐gp inhibition was suggested. Therefore, the inhibition of efflux transport of drugs as well as of drug metabolism by CYP3A4 could be an important cause of drug‐GFJ interaction.

Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein.

To elucidate the role of P-glycoprotein in human placenta, we examined its expression in placenta, and the transcellular transport and uptake of P-glycoprotein substrates in cultured human placental choriocarcinoma epithelial cells (BeWo cells). The uptake of [(3)H]vinblastine and [(3)H]vincristine into BeWo cells was increased in the presence of a metabolic inhibitor, sodium azide. The basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was greater than the apical-to-basolateral transcellular transport. In the presence of cyclosporin A, the basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was significantly increased, and the apical-to-basolateral transcellular transport was decreased. The uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells was significantly enhanced in the presence of several inhibitors, such as verapamil or mouse monoclonal antibody anti-P-glycoprotein MX-MDR (MRK16) as well as cyclosporin A. Although progesterone significantly enhanced the uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells, the uptake of [(3)H]progesterone was not affected by these inhibitors. Immunoblot analysis revealed that P-glycoprotein with a molecular weight of 172 kDa was expressed in BeWo cells and isolated trophoblast cells. Furthermore, P-glycoprotein was detected in human placental brush-border membrane vesicles, but not in human placental basolateral membrane vesicles. In conclusion, these data suggest that P-glycoprotein is expressed on the brush-border membrane (maternal side) of human placental trophoblast cells. P-Glycoprotein is considered to regulate the transfer of several substances including vinblastine, vincristine and digoxin from mother to fetus, and to protect the fetus from toxic substances.

Effects of grapefruit juice and orange juice components on P‐glycoprotein‐ and MRP2‐mediated drug efflux

We investigated the effects of grapefruit juice (GFJ) and orange juice (OJ) on drug transport by MDR1 P‐glycoprotein (P‐gp) and multidrug resistance protein 2 (MRP2), which are efflux transporters expressed in human small intestine. We examined the transcellular transport and uptake of [3H]vinblastine (VBL) and [14C]saquinavir in a human colon carcinoma cell line (Caco‐2) and in porcine kidney epithelial cell lines transfected with human MDR1 cDNA and human MRP2 cDNA, LLC‐GA5‐COL150, and LLC‐MRP2, respectively. In Caco‐2 cells, the basal‐to‐apical transports of [3H]VBL and [14C]saquinavir were greater than those in the opposite direction. The ratio of basal‐to‐apical transport to apical‐to‐basal transport of [3H]VBL and [14C]saquinavir by Caco‐2 cells was reduced in the presence of MK571 (MRPs inhibitor), verapamil (P‐gp inhibitor), cyclosporin A (inhibitor of both), 50% ethyl acetate extracts of GFJ and OJ, or their components (6′,7′‐dihydroxybergamottin, bergamottin, tangeretin, hepatomethoxyflavone, and nobiletin). Studies of transport and uptake of [3H]VBL and [14C]saquinavir with MDR1 and MRP2 transfectants showed that VBL and saquinavir are transported by both P‐gp and MRP2. GFJ and OJ components inhibited the transport by MRP2 as well as P‐gp. However, their inhibitory potencies for P‐gp or MRP2 were substrate‐dependent. The present study has revealed that GFJ and OJ interact with not only P‐gp but also MRP2, both of which are expressed at apical membranes and limit the apical‐to‐basal transport of VBL and saquinavir in Caco‐2 cells.

Basal Membrane Localization of MRP1 in Human Placental Trophoblast

The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta. ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics. In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp. Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively. Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side. These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast. MRP1 was also detected on the abluminal side of blood vessels in the villi. Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.

Effects of Grapefruit Juice and Orange Juice on the Intestinal Efflux of P-Glycoprotein Substrates

AbstractPurpose. The aim of this study is to investigate the effects of 50% ethyl acetate extracts of grapefruit juice (GFJ) and orange juice (OJ) on the transport activity of P-glycoprotein (P-gp) in the rat small intestine. Methods.The efflux of P-gp substrates from rat everted sac in the absence or presence of verapamil, GFJ, OJ or erythromycin was measured. Rhodamine123, fexofenadine and saquinavir were used as P-gp substrates. P-gp expression levels in the rat jejunum and ileum were determined by Western blot analysis. Results. The efflux of rhodamine123 from the everted sac increased from the apex of the jejunum to the low ileum and the expression of P-gp in the ileum was 2.31-fold higher than that in the jejunum. Verapamil and the 50% GFJ and OJ extracts inhibited the efflux from the intestine of all three drugs tested. Erythromycin decreased the efflux of rhodamine123 and fexofenadine, but did not affect the efflux of saquinavir in the intestine. Conclusions. GFJ and OJ extracts inhibited the efflux of P-gp substrates from the small intestine. Therefore, they may enhance the oral bioavailability of P-gp substrates by increasing absorption in the small intestine.

Transforming growth factor-β1 upregulates the tight junction and P-glycoprotein of brain microvascular endothelial cells

Abstract1. The present study was aimed at elucidating effects of transforming growth factor-β (TGF-β) on blood–brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells.2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-β1 (0.01–10 ng/mL).3. The present study demonstrates that TGF-β lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-β in the brain may keep the BBB functioning.

Transport and uptake of nateglinide in Caco-2 cells and its inhibitory effect on human monocarboxylate transporter MCT1.

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches the maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the gastrointestinal tract. The aim of this work is to clarify the intestinal absorption mechanism of nateglinide by means of in vitro studies. We examined the transcellular transport and the apical uptake of [14C]nateglinide in a human colon carcinoma cell line (Caco‐2). We also examined whether nateglinide is transported via monocarboxylate transport‐1 (MCT1) by means of an uptake study using MCT1‐expressing Xenopus laevis oocytes. In Caco‐2 cells, the transcellular transport of [14C]nateglinide from the apical to basolateral side was greater than that in the opposite direction. The uptake of [14C]nateglinide from the apical side was concentration–dependent, H+‐dependent, and Na+‐independent. Kinetic analysis revealed that the Kt and Jmax values of the initial uptake rate of [14C]nateglinide were 448 μM and 43.2 nmol mg protein−1 5 min−1, respectively. Various monocarboxylates, including salicylic acid and valproic acid, and glibenclamide significantly inhibited the uptake of [14C]nateglinide. The uptake study using MCT1‐expressing oocytes showed that nateglinide inhibits the MCT1‐mediated uptake of [14C]L‐lactic acid, though nateglinide itself is not transported by MCT1. Taken together, these results suggest that the uptake of nateglinide from the apical membranes of Caco‐2 cells is, at least in part, mediated by a proton‐dependent transport system(s) distinct from MCT1.