CHARACTERIZATION OF PHOSPHINE COMPLEXES OF TECHNETIUM(III) AS TRANSPORT SUBSTRATES OF THE MULTIDRUG-RESISTANCE P-GLYCOPROTEIN AND FUNCTIONAL MARKERS OF P-GLYCOPROTEIN AT THE BLOOD-BRAIN-BARRIER

The multidrug resistance (MDR1) P-glycoprotein functions as a broad sp ecificity efflux transporter of structurally diverse natural product a nd xenobiotic compounds. P-glycoprotein also is an important component of the functional blood-brain barrier. To enable further studies of f unction and modulation of MDR1 P-glycoprotein in vitro and in vivo, tw o novel phosphine technetium(III) complexes were designed and characte rized: bis[methylbis(3-methoxy-1-propyl)phosphine]Tc(III) (Tc-Q58) and trans-[5,5'-(1,2-ethanediyl bis[dimethyl(3-methoxy-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 nonimmu nodetectable, low, and high levels of MDR1 P-glycoprotein, respectivel y, accumulation of Tc-Q58 and Tc-Q63 was inverse to expression of the transporter. Differences between drug-sensitive and multidrug-resistan t cells, while detectable at picomolar concentrations of each radiopha rmaceutical, were independent of tracer concentration, Ratios of trace r accumulation in KB 3-1 and 8-5 cells were 62.3 and 48.1 for Tc-Q58 a nd Tc-Q63, respectively. Cell contents of Tc-Q58 and Tc-Q63 were enhan ced up to 60-fold in MDR cells by known modulators of MDR1 P-glycoprot ein, while drugs not in the multidrug-resistant phenotype had no effec t on their accumulation. In KB 8-5 cells, potency of modulators was GF 120918 much greater than cyclosporin A > verapamil. Accumulation of Tc -Q58 and Tc-Q63 in Sf9 insect cells infected with a recombinant baculo virus containing human MDR1 P-glycoprotein was reduced in a GF120918-r eversible manner (EC50 less than or equal to 70 nM) compared with cell s infected with a wild-type baculovirus. By contrast, cell contents of Tc-Q58 or Tc-Q63 in Sf9 cells expressing the homologous MDR3 P-glycop rotein did not differ from wild-type virus, Demonstrating molecular ta rgeting of these complexes in vivo, distribution and retention of Tc-Q 58 in brain tissue of FVB mice treated with a saturating dose of GF120 918 and mice deficient in the mdr1a gene [mdr1a (-/-)] were enhanced 1 80% and 520% over control, respectively. Exploiting the gamma-emission spectrum of Tc-99m, increased uptake of Tc-Q58 in brain tissue of mdr 1a (-/-) mice was readily detected noninvasively by scintigraphic imag ing. Thus, both Tc-Q58 and Tc-Q63 are demonstrated to be substrates fo r transport by MDR1 P-glycoprotein, broadening the specificity of this transporter to include phosphine-containing metal complexes. As shown with Tc-Q58, these Q complexes can be used to detect transport activi ty and modulation of MDR1 P-glycoprotein in vitro and to directly moni tor the functional status of P-glycoprotein at the blood-brain barrier in vivo.