Characterization of a ⁶⁷Ga/⁶⁸Ga radiopharmaceutical for SPECT and PET of MDR1 P-glycoprotein transport activity in vivo: Validation in multidrug-resistant tumors and at the blood-brain barrier

Overexpression of multidrug resistance (MDR1) P-glycoprotein (Pgp) remains an important barrier to successful chemotherapy in cancer patients and impacts the pharmacokinetics of many important drugs, thus evoking a need to noninvasively interrogate Pgp transport activity in vivo. Methods: Cell tracer transport experiments as well as mouse biodistribution and microPET imaging studies were performed to characterize a nonmetabolized gallium(III) complex, gallium(III)-(bis(3-ethoxy-2-hydroxybenzylidene)-N,N′-bis(2, 2-dimethyl-3-amino-propyl)ethylenediamine) (Ga-[3-ethoxy-ENBDMPI])⁺, as a candidate SPECT (⁶⁷Ga) and generator-produced PET ( ⁶⁸Ga) radiopharmaceutical recognized by MDR1 Pgp. Results: The ⁶⁷Ga-complex showed high membrane potential-dependent accumulation in drug-sensitive KB3-1 cells and modulator-reversible low accumulation in MDR KB8-5 cells. In KB8-5 cells, the median effective concentrations (EC ₅₀) of MDR modulators LY335979, PSC 833, and cyclosporin A were 69 nmol/L, 1 μmol/L, and 3 μmol/L, respectively. Using a variety of cells stably expressing MDR1 Pgp, multidrug resistance-associated proteins (MRP1-MRP6), or the breast cancer resistance protein (BCRP/MXR), the ⁶⁷Ga-complex was shown to be readily transported by MDR1 Pgp and, to a much lesser extent, by MRP1, but not MRP2-MRP6 or BCRP/MXR. In a nude mouse xenograft tumor model, the ⁶⁷Ga-complex produced a readily detected 3-fold difference between Pgp-expressing tumors and drug-sensitive tumors in the opposite flank. In mdr1a/1b(-/-) gene-deleted mice, the ⁶⁷Ga-complex showed 17-fold greater brain uptake and retention compared with wild-type mice with no net difference in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. This could be readily observed with microPET using the ⁶⁸Ga-complex. Incidentally, wild-type mice showed heart-to-blood ratios of >100 by 1 h after injection and heart-to-liver ratios of 2.2 by 120 min. Conclusion: Molecular imaging of the functional transport activity of MDR1 Pgp with (^67/68Ga-[3- ethoxy-ENBDMPI])⁺ may enable noninvasive SPECT/PET monitoring of the blood-brain barrier, chemotherapeutic regimens, and MDR1 gene therapy protocols in vivo. These Pgp-directed properties of the radiopharmaceutical may also translate favorably to myocardial perfusion imaging.