Ivermectin excretion by isolated functionally intact brain endothelial capillaries.

1. Functionally intact brain endothelial capillaries were isolated from porcine brain. p-Glycoprotein was localized at the lumenal membrane of intact capillaries by immunohistochemistry using a murine monoclonal antibody and a secondary FITC fluorescent labelled anti-mouse IgG. Western blot staining of p-glycoprotein in isolated endothelial cells confirmed the immunohistochemistry. 2. Excretion of the fluorescent labelled anthelmintic drug Ivermectin (BODIPY-Ivermectin) was studied in the isolated brain endothelial capillaries. Drug accumulation in the capillary lumen was visualized by fluorescence confocal laser scanning microscopy and was measured by image analysis. Secretion of BODIPY-Ivermectin into the capillary lumen exhibited characteristics of specific and energy-dependent transport. Steady state lumenal fluorescence intensity averaged 1.6 times cellular fluorescence and was reduced 3--4 times below cellular levels when metabolism was inhibited by NaCN. 3. BODIPY-Ivermectin secretion was inhibited in a concentration-dependent manner by unlabeled Ivermectin. In addition, lumenal but not cellular fluorescence intensity was significantly decreased when capillaries were incubated with PSC-833, Cyclosporin A or Verapamil, all inhibitors of p-glycoprotein. Conversely, unlabelled Ivermectin reduced the p-glycoprotein (Pgp)-mediated secretion of a fluorescent derivative of Verapamil, (BODIPY-Verapamil). 4. BODIPY-Ivermectin secretion was not affected in the presence of Leucotriene C(4) (LTC(4)), a potent inhibitor of multidrug resistance related protein (mrp)-mediated transport processes. In addition, excretion of Fluorescein-Methotrexate, an mrp-substrate, was not inhibited by Ivermectin. 5. Uptake experiments with isolated porcine brain capillary cells showing increased cellular uptake of BODIPY-Ivermectin in the presence of unlabelled drug or PSC-833 supported the findings of a Pgp interaction in intact capillaries. 6. The data are consistent with BODIPY-Ivermectin and Ivermectin being transported across the lumenal membrane of brain capillaries. For the first time Pgp-interaction of Ivermectin at the blood brain barrier is demonstrated on a cellular level in an intact vascular tissue.

Uptake of cationzied albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries.

The suitability of protein-coupled liposomes as drug carriers for brain specific targeting was investigated using albumin (BSA) and cationized albumin (CBSA), respectively, as model proteins. Liposomes coated with polyethylene glycol (sterically stabilized, PEG-liposomes) were prepared from phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid and covalently coupled to thiolated BSA or CBSA. Liposomes were loaded with carboxy-fluorescein and rhodamine-labeled dipalmitoyl-phosphatidylethanolamine as hydrophilic and lipophilic marker compounds, respectively. The interaction of these constructs with monolayers of porcine brain capillary endothelial cells (BCEC) and freshly isolated porcine brain capillaries was studied by means of fluorescence assays and confocal laser scanning fluorescence microscopy (CLSFM). In contrast to BSA, CBSA was rapidly taken up by cultured BCECs. BSA-coupled liposomes did not interact with endothelial cells, whereas CBSA-coupled liposomes bound to cellular surfaces and exhibited time dependently a high intracellular accumulation. CBSA-conjugated liposomes were also taken up by intact brain capillaries. Cellular uptake could be inhibited by free cationized albumin, phenylarsineoxide, nocodazole, and filipin, but not by dansylcadaverine, suggesting a caveolae-mediated incorporation process. Immunostaining demonstrated a high expression of caveolin in the capillary endothelium. In conclusion, liposomes coupled to CBSA are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

Xenobiotic transport across isolated brain microvessels studied by confocal microscopy.

To identify specific transporters that drive xenobiotics from central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from rat and pig brain using confocal microscopy and quantitative image analysis. Luminal accumulation of daunomycin and of fluorescent derivatives of cyclosporine A (CSA) and ivermectin was concentrative, specific, and energy-dependent (inhibition by NaCN). Transport was reduced by PSC 833, ivermectin, verapamil, CSA, and vanadate, but not by leukotriene C(4) (LTC(4)), indicating the involvement of P-glycoprotein. Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein methotrexate was also concentrative, specific, and energy-dependent. LTC(4), chlorodinitrobenzene, and vanadate reduced transport of these compounds, but PSC 833 and verapamil did not, indicating the involvement of a multidrug resistance-associated protein (Mrp). Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the capillary endothelium and quantitative polymerase chain reaction showed Mrp1 and Mrp2 expression. Finally, the HIV protease inhibitors saquinavir and ritonavir were potent inhibitors of transport mediated by both P-glycoprotein and Mrp. These results validate a new method for studying drug transport in isolated brain capillaries and implicate both P-glycoprotein and one or more members of the Mrp family in drug transport from central nervous system to blood.