Hirotami Matsuo

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 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.

Effect of cyclosporin A or tacrolimus on the function of blood-brain barrier cells.

Recently, it has been reported that continuous treatment with cyclosporin A or tacrolimus induces encephalopathy in transplant patients. The mechanism of immunosuppressant-induced encephalopathy is unclear. We investigated the cytotoxicity to brain capillary endothelial cells and the effect of these two drugs on P-glycoprotein function using mouse brain capillary endothelial (MBEC4) cells. The transcellular transport of [3H]sucrose was significantly increased and the cellular viability, based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion test, was decreased by cyclosporin A (approximately 50% at 5 microM; P<0.005), while tacrolimus showed a much smaller effect. These findings indicate that the toxicity of cyclosporin A was greater than that of tacrolimus. The uptake of [3H]vincristine, a substrate of P-glycoprotein, was increased by these two drugs. The expression of P-glycoprotein in MBEC4 cells was reduced, but there was no effect on mdr1b mRNA levels. The decrease in the expression of P-glycoprotein may be due to the inhibition of the turnover of P-glycoprotein, which involves translation. In conclusion, the direct cytotoxic effect on the brain capillary endothelial cells and the inhibition of P-glycoprotein may be partly involved in the occurrence of immunosuppressant-induced encephalopathy.

Induction of apoptosis in mouse brain capillary endothelial cells by cyclosporin a and tacrolimus

Although cyclosporin A and tacrolimus are used clinically as potent immunosuppressants, there have been reports of neurotoxicity and encephalopathy. A possible mechanism is that these drugs damage the blood-brain barrier (BBB), inducing dysfunction and increased permeability, and are then able to enter the brain. We studied the cytotoxicity of cyclosporin A and tacrolimus, focused on apoptosis induction, using an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells (MBEC4). We found that these two drugs induced cell shrinkage, chromatin condensation and DNA fragmentation, which are characteristics of apoptosis. Our data suggest that the induction of apoptosis on the brain capillary endothelial cells may be at least partly involved in the occurrence of immunosuppressant-induced encephalopathy.

Possibility of the reversal of multidrug resistance and the avoidance of side effects by liposomes modified with MRK-16, a monoclonal antibody to P-glycoprotein

For cancer chemotherapy, avoiding the side effects of chemotherapeutic agents is difficult. Multidrug resistance is one of the major obstacles to successful cancer chemotherapy. P-Glycoprotein (P-gp) serves as an efflux pump and plays a key role in the multidrug resistance. We examined the effect of MRK-16, a monoclonal antibody against P-gp, modified liposomes (MRK-Lip) on the human myelogenous leukemia K-562 cells and its adriamycin resistance cell line K-562/ADM cells to avoid the side effects and to reverse the multidrug resistance. The uptake of vincristine (VCR) by K-562/ADM cells was lower than that by K-562 cells. This low uptake was increased in the presence of verapamil and MRK-16, however, it was not increased in the presence of control antibody, IgG2A. The binding of MRK-Lip to K-562/ADM cells was higher than that of IgG2A-modified liposome (IgG-Lip) and liposome without modification (Cont-Lip). Moreover, the cytotoxicity of VCR-encapsulated MRK-Lip to K-562/ADM cells was higher than that of VCR-encapsulated IgG-Lip and Cont-Lip. These results suggest that the interaction between liposomes and multidrug resistance cells was increased by the modification of liposomes with MRK-16. Consequently, the usefulness of MRK-Lip in cancer chemotherapy as a potent carrier was suggested.

Choline uptake by mouse brain capillary endothelial cells in culture.

Choline, a precursor of the neurotransmitter acetylcholine, is synthesized in only small amounts in the brain, so the choline concentration in the brain may vary depending on the plasma concentration and the transport rate across the blood‐brain barrier. To elucidate the transport mechanism of choline, we carried out uptake experiments with mouse brain capillary endothelial cells in culture (MBEC4).

Efflux Transport of Tolbutamide Across the Blood‐brain Barrier

In an attempt to determine the reason for the low brain distribution of tolbutamide, we have demonstrated the transport of tolbutamide from the brain to the blood via a non‐P‐glycoprotein efflux transport system which is inhibited by sulphonamides. We evaluated the directional transport of tolbutamide across the blood‐brain barrier by means of an in‐vivo brain‐tissue distribution study and experiments on in‐vitro transcellular transport and uptake in cultured mouse‐brain capillary endothelial cells (MBEC4).

Contribution of P-glycoprotein to efflux of ramosetron, a 5-HT3 receptor antagonist, across the blood-brain barrier

In‐situ rat and mouse brain perfusion data indicated that the brain distribution of ramosetron (R‐ramosetron), a 5‐hydroxytryptamine3 (5‐HT3) receptor antagonist, was extremely low compared with that expected from its lipophilicity. We hypothesized the involvement of an efflux system(s) and investigated the contribution of P‐glycoprotein to efflux transport of ramosetron across the blood‐brain barrier by means of an in‐vitro uptake study in cell lines that over‐express P‐glycoprotein. We examined the contributions of mdr1a, mdr1b and MDR1 P‐glycoprotein by using LV500 cells, MBEC4 cells and LLC‐GA5‐COL300 cells, which over‐express mdr1a P‐glycoprotein, mdr1b P‐glycoprotein and MDR1 P‐glycoprotein, respectively. The uptake of [14C]ramosetron by LV500 cells and LLC‐GA5‐COL300 cells was significantly lower than that by the respective parental cells. Next, we studied the effects of P‐glycoprotein inhibitors, verapamil and ciclosporin, on uptake of [14C] ramosetron by these cell lines. The uptake of [14C]ramosetron by LV500 cells and LLC‐GA5‐COL300 cells was significantly increased in the presence of verapamil or ciclosporin, while verapamil did not affect the uptake of [14C]ramosetron by MBEC4 cells. These results indicate that the efflux of [14C]ramosetron is partly mediated by mdr1a P‐glycoprotein, but not by mdr1b P‐glycoprotein, and that there is a difference in substrate specificity between mdr1a P‐glycoprotein and mdr1b P‐glycoprotein. Further, [14C]ramosetron was confirmed to be effluxed by human MDR1 P‐glycoprotein. We conclude that the limited distribution of ramosetron to the brain is due, at least in part, to efflux mediated by the P‐glycoprotein at the blood‐brain barrier.