In vivo evidence for ATP-dependent and P-glycoprotein-mediated transport of cyclosporin A at the blood-brain barrier.

To evaluate the significance of P-glycoprotein (P-gp)-mediated active efflux on the blood-brain barrier (BBB) permeability of cyclosporin A (CsA) in vivo, we investigated the effects of ATP depletion in the brain and of a multidrug-resistant (MDR) reversing agent on the transport of CsA across the BBB. Using transient brain ischemia obtained by 4-vessel occlusion of vertebral and common carotid arteries in rats to deplete ATP content in the brain, the estimated permeability surface area product (PS) value of [3H]CsA was increased 2.7-fold compared with that in normal rats, whereas the PS value of [14C]sucrose was not altered. Additionally, when quinidine hydrochloride (QND) was infused into the brain through a microdialysis probe implanted in the rat hippocampus, the extravascular extraction of CsA was increased to approximately 2.5-fold of the control, whereas no difference in the extravascular extraction between control and normal rats having no implanted dialysis probe was observed. Furthermore, the efflux rate from brain to blood of CsA was decreased remarkably to 5% of control at steady-state by co-administration of CsA with QND directly into the brain through the dialysis probe. The ATP-dependent and QND-sensitive efflux of CsA from the brain strongly indicates that P-gp in the brain capillary endothelial cells functions as an efflux pump under the physiological state, and that P-gp-mediated efflux of CsA is a major mechanism of the restricted transfer from blood into the brain.

Transport mechanism of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors at the blood-brain barrier.

The transport mechanism of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors through the blood-brain barrier was studied in vitro by using primary cultures of bovine brain capillary endothelial cells (BCEC). The uptake of HMG-CoA reductase inhibitors with the lactone form, [14C]lovastatin and [14C]simvastatin, was slightly decreased to 65% of the control uptake (37 degrees C) at low temperature (4 degrees C) and was not affected by pretreatment of the BCEC with metabolic inhibitors (2,4-dinitrophenol and rotenone). [14C]Simvastatin acid (the lactone ring-opened form) was taken up in a markedly temperature- and concentration-dependent fashion, whereas the uptake of [14C] pravastatin was negligible. At pH below 7.4, the uptake rate of [14C]simvastatin acid by the BCEC increased markedly with decreasing medium pH, whereas almost pH-independent uptake was observed in the presence of 1 mM simvastatin acid. Additional studies using an in situ rat brain perfusion method showed that the in vivo cerebrovascular permeation of [14C]simvastatin acid in rats was significantly inhibited in the presence of 1 mM simvastatin acid, demonstrating that the transport system for the acid forms of HMG-CoA reductase inhibitors functions under in vivo conditions. Several monocarboxylic acids significantly inhibited the uptake of [14C]simvastatin acid by the BCEC, whereas dicarboxylic acids did not. The uptake of [14C]simvastatin acid by the BCEC was competitively inhibited by 15 mM acetic acid. Accordingly, we concluded that HMG-CoA reductase inhibitors in lactone form are transported via simple diffusion, whereas those having an acid form are transported across the blood-brain barrier via a carrier-mediated transport mechanism for monocarboxylic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro blood-brain barrier transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors.

Among the HMG-CoA reductase inhibitors, lovastatin and simvastatin have central nervous system (CNS) side effects, such as sleep disturbance, whereas pravastatin does not. This difference in CNS side effects may be due to a difference in blood-brain barrier (BBB) permeability among these inhibitors. To test this hypothesis, we compared the BBB transport ability of HMG-CoA reductase inhibitors by using an in vivo brain perfusion technique in rats and an in vitro culture system of bovine brain capillary endothelial cells. The in vivo BBB permeability coefficients of the lipophilic inhibitors, [14C]lovastatin and [14C]simvastatin, were high. In contrast, that of the hydrophilic inhibitor, [14C]pravastatin, was low and not significantly different from that of [14C]sucrose, an extracellular space marker. Similarly, the in vitro BBB permeability coefficients of [14C]lovastatin and [1C]simvastatin were high, while that of [14C]-pravastatin was low. The in vivo and in vitro transcellular permeabilities obtained for HMG-CoA reductase inhibitors were comparable. This study shows that the BBB permeability correlates with the CNS side effects of the HMG-CoA reductase inhibitors.

Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells.

The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [125I]ebiratide (H-Met(O2)-Glu[125I]His-Phe-D-Lys-Phe-NH(CH2)2NH2), was examined in cultured monolayers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [125I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [125I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [125I]ebiratide. The acid-resistant binding of [125I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM-1 mM). The maximal internalization capacity (Bmax) at 30 min was 7.96 +/- 3.27 pmol/mg of protein with a half-saturation constant (Kd) of 15.9 +/- 6.4 microM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.

Nonlinear intestinal absorption of 5-hydroxytryptamine receptor antagonist caused by absorptive and secretory transporters.

The mechanism of the nonlinear concentration dependence of intestinal absorption of the 5-hydroxytryptamine receptor antagonist azasetron was studied by use of rat in situ intestinal perfusion, as well as an in vitro Ussing-type chamber method mounted with rat intestinal tissue and cultured monolayers of human adenocarcinoma Caco-2 cells. The intestinal absorption rate constant of azasetron evaluated by the Doluisio method increased significantly with increasing concentration of azasetron up to 10 mM in a nonlinear fashion and tended to decrease at higher concentrations. Mucosal-to-serosal directed permeation of [14C]azasetron across rat ileal sheets evaluated by the in vitro Ussing-type chamber method also increased in a nonlinear fashion in a low concentration range, followed by a decrease as the concentration was further increased, whereas serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. Vectorial transport of [14C]azasetron across a Caco-2 cell monolayer was observed, with higher transport in the basolateral-to-apical direction at a trace concentration of azasetron. When the initial uptake rate of azasetron by Caco-2 cells was measured, it was saturable with an apparent half-saturation concentration of 15 mM and was reduced in the presence of several cationic compounds. These observations suggest that azasetron is taken up by a carrier-mediated transport mechanism across the intestinal epithelial cells. When the steady-state uptake of [14C]azasetron was measured, it was increased in the presence of unlabeled azasetron and ondansetron. In addition, the steady-state uptake was enhanced in the presence of a P-glycoprotein inhibitor, cyclosporin A, and by ATP-depletion of the cells, although these treatments had no effect on the initial uptake of [14C]azasetron. Furthermore, the multidrug-resistant cancer cell line K562/ADM that overexpresses P-glycoprotein accumulated azasetron less extensively than did the parental drug-sensitive K562 cells. These results strongly suggest that azasetron is secreted into the intestinal lumen predominantly by P-glycoprotein. We conclude that intestinal transport of azasetron involves specialized transporters in both the absorptive and secretory directions, and the complex nonlinear intestinal absorption characteristics can be ascribed to the participation of multiple transport mechanisms.