Hideyasu Murakami

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.

Characteristics of choline transport across the blood-brain barrier in mice: correlation with in vitro data.

AbstractPurpose. We examined the functional properties of choline transport across the blood-brain barrier (BBB) in mice. We compared the kinetic parameters and transport properties with those found in our in vitro uptake experiments using mouse brain capillary endothelial cells (MBEC4). Methods. The permeability coefficient-surface area product (PS) values of [3H]choline at the BBB were estimated by means of anin situ brain perfusion technique in mice.Results. [3H]Choline uptake was well described by a two-component model: a saturable component and a nonsaturable linear component. The [3H]choline uptake was independent of pH and Na+, but was significantly decreased by the replacement of Na+ with K+. Various basic drugs, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [3H]choline uptake. These in situ (in vivo) results corresponded well to the in vitro results and suggest that the choline transporter at the BBB is a member of the organic cation transporter (OCT) family. Conclusion. The choline transport mechanism at the BBB is retained in MBEC4.

Inhibitory effects of angiotensin II receptor antagonists and leukotriene receptor antagonists on the transport of human organic anion transporter 4.

Human organic anion transporter 4 (OAT4) is the only member of the OAT family that is expressed in the placenta and also expressed in kidney. Although OAT4 has been shown to transport certain organic anions as well as other members of the OAT family, fewer numbers of substrates have been identified for OAT4 compared with OAT1 and OAT3, suggesting that the substrate specificity of OAT4 is greater than other OAT members. However, the substrate specificity of OAT4 remains to be investigated in detail. The aim of this study was to examine the effects of various drugs on the OAT4‐mediated transport of estrone‐3‐sulfate, a typical substrate of OAT4, by using human embryonic kidney cells stably transfected with OAT4 (HEK‐OAT4). HEK‐OAT4 cells exhibited concentration‐dependent uptake of estrone‐3‐sulfate, with a Km value of 20.9 ± 3.53 μM. Dehydroepiandrosterone sulfate and probenecid potently inhibited estrone‐3‐sulfate uptake. We also searched for the potential inhibitors of OAT4 and identified candesartan, candesartan cilexetil, losartan, losartan carboxyl (EXP3174) and valsartan as inhibitors of OAT4, with Ki values of 88.9, 135.2, 24.8, 13.8 and 19.6 μM, respectively. The above angiotensin II receptor antagonists and leukotriene receptor antagonists share a common structural feature, that is the tetrazole group. Although pranlukast is devoid of anionic motifs other than the tetrazole group, it potently inhibited the OAT4‐mediated uptake of estrone‐3‐sulfate, indicating that a tetrazole group may be one important structural feature in substrate recognition by OAT4.

Distribution of domperidone into the rat brain is increased by brain ischaemia or treatment with the P-glycoprotein inhibitor verapamil

Domperidone (DOM), a peripheral dopamine D2 receptor antagonist, is a substrate of P‐glycoprotein (P‐gp). Therefore, the transport of DOM into the brain may be restricted by P‐gp function at the blood‐brain barrier, and when the function of P‐gp is impaired by ATP depletion under conditions of brain ischaemia (e.g. cerebral thrombosis), side‐effects may be induced as a result of increased distribution of DOM into the brain. In this study, we investigated the effects of brain ischaemia and verapamil, a P‐gp inhibitor, on the permeability coefficient‐surface area product (PS values) of DOM across the blood‐brain barrier by using an in‐situ rat brain perfusion technique. The PS values of DOM were increased 3.4‐ and 3.6‐fold after brain ischaemia for 10 and 20 min, respectively. Furthermore, co‐administration of verapamil significantly increased the PS values of DOM by 42.6‐ and 43.3‐fold in the normal and ischaemia groups, respectively. Brain vascular volume was not affected by ischaemia or verapamil. These results show that the transport of DOM into the brain is restricted by P‐gp and that the brain distribution of DOM can be increased by cerebral ischaemia or co‐administration of a P‐gp inhibitor.