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

Abstract

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 </= 70 nM) compared with cells infected with a wild-type baculovirus. By contrast, cell contents of Tc-Q58 or Tc-Q63 in Sf9 cells expressing the homologous MDR3 P-glycoprotein did not differ from wild-type virus. Demonstrating molecular targeting of these complexes in vivo, distribution and retention of Tc-Q58 in brain tissue of FVB mice treated with a saturating dose of GF120918 and mice deficient in the mdr1a gene [mdr1a (-/-)] were enhanced 180% and 520% over control, respectively. Exploiting the gamma-emission spectrum of 99mTc, increased uptake of Tc-Q58 in brain tissue of mdr1a (-/-) mice was readily detected noninvasively by scintigraphic imaging. Thus, both Tc-Q58 and Tc-Q63 are demonstrated to be substrates for 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 activity and modulation of MDR1 P-glycoprotein in vitro and to directly monitor the functional status of P-glycoprotein at the blood-brain barrier in vivo.

MDR1 P-glycoprotein Reduces Influx of Substrates without Affecting Membrane Potential

“…LY335979 did not enhance accumulation of Tc-Sestamibi in MCF-7 cells, providing further evidence that this cell line does not express MDR1 Pgp (6,24). Previously, our laboratory has shown that KB 8-5 cells accumulate ϳ50-fold less TcSestamibi than Pgp-negative parental KB 3-1 cells (17). Thus, these functional data are consistent with differences in relative expression of Pgp in MCF-7/MDR1 and KB 8-5 cells.…”

“…Clones of transfected cells were isolated and maintained in medium containing 1 mg/ml G418. Drug-sensitive (Pgp-negative) KB 3-1 and MDR (Pgp-positive) KB 8-5 human epidermoid carcinoma cells were grown and maintained as described (17).…”

“…Western Blots-Pgp was detected in enriched membrane fractions of cells with monoclonal antibody C219 (Signet Laboratories, Inc., Dedham, MA) as described previously by our laboratory (17).…”

P-glycoprotein deficiency at the blood-brain barrier increases amyloid- deposition in an Alzheimer disease mouse model

“…Sep-Pak purification (>95%) and quality control of the tracer were performed as previously described (25). Following intravenous injection, distribution of [ 99m Tc]Sestamibi in the brain and blood of Pgp WT and Pgp-null mice with or without coexpression of APPsw +/-was determined 5 minutes after injection as previously described (26,27 and are reported as mean ± SEM (n = 3-4) or ± range (n = 2).…”

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“…Following administration of a gamma-emitting compound, gamma scintigraphic images can be acquired using a gamma camera, and distribution of the compound throughout the body can be examined. Of major significance to BBB transport, it has been shown that 99 m Tc-sestamibi and other related compounds, such as 99 m Tc-tetrofosmin and a novel technetium (III) complex ( 99 m Tc-Q58), have higher brain uptake in P-gp knockout mice compared with wild-type mice (Luker et al 1997;Chen et al 2000;Dyszlewski et al 2002), indicating that such technetium-labelled compounds may be used to assess P-gp transport activity in-vivo. Although this technique may be useful in characterizing efflux transporters and BBB permeability in disease states, it will have a limited role in screening of compounds for potential brain uptake.…”