The blood-brain barrier and a bloodcerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood-CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon-emission computed tomography with 99m Tcsestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99m Tc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a͞1b(؊͞؊) and mrp(؊͞؊) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect. P-glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300. Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect. Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2-fold greater than classic MDR modulators and Hill coefficients >> 2. In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells. In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P-glycoprotein and for the quantitative characterization of transporter modulation and regulation. The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation.

The multidrug resistance (MDR1) P-glycoprotein functions as a broad specificity efflux transporter of structurally diverse natural product and xenobiotic compounds. P-glycoprotein also is an important component of the functional blood-brain barrier. To enable further studies of function and modulation of MDR1 P-glycoprotein in vitro and in vivo, two novel phosphine technetium(III) complexes were designed and characterized: trans-[2,2'-(1, 2-ethanediyldiimino)bis(1, 5-methoxy-5-methyl-4-oxo-hexenyl)]bis[methylbis(3-methoxy-1- propyl)ph osphine]Tc(III) (Tc-Q58) and trans-[5,5'-(1,2-ethanediyl diimino)bis(2-ethoxy-2-methyl-3-oxo-4-pentenyl)]bis[dimethyl(3- methox y-1-propyl)phosphine)]Tc(III) (Tc-Q63). In human drug-sensitive KB 3-1 cells and multidrug-resistant KB 8-5 and 8-5-11 derivative cell lines, expressing nonimmunodetectable, low, and high levels of MDR1 P-glycoprotein, respectively, accumulation of Tc-Q58 and Tc-Q63 was inverse to expression of the transporter. Differences between drug-sensitive and multidrug-resistant cells, while detectable at picomolar concentrations of each radiopharmaceutical, were independent of tracer concentration. Ratios of tracer accumulation in KB 3-1 and 8-5 cells were 62.3 and 48.1 for Tc-Q58 and Tc-Q63, respectively. Cell contents of Tc-Q58 and Tc-Q63 were enhanced up to 60-fold in MDR cells by known modulators of MDR1 P-glycoprotein, while drugs not in the multidrug-resistant phenotype had no effect on their accumulation. In KB 8-5 cells, potency of modulators was GF120918 >> cyclosporin A > verapamil. Accumulation of Tc-Q58 and Tc-Q63 in Sf9 insect cells infected with a recombinant baculovirus containing human MDR1 P-glycoprotein was reduced in a GF120918-reversible manner (EC50

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