Evaluation of the Near Infrared Compound Indocyanine Green as a Probe Substrate of P‑Glycoprotein

The efflux transporter P-glycoprotein (P-gp) affects the pharmacokinetics of many drugs. Currently used methods for characterization of P-gp’s functional activity <i>in vivo</i> involve the use of radiolabeled substrates, are costly, and are technically demanding. Our objective was to evaluate whether the FDA-approved near-infrared compound indocyanine green (ICG) can be used as a probe substrate of P-gp. We also characterized the interaction of ICG with another efflux transporter, the breast cancer resistance protein (BCRP). We evaluated ICG accumulation and transport in MDCK cells overexpressing P-gp or BCRP (MDCK-MDR1 and MDCK-BCRP, respectively) compared to control MDCK cells, in the presence or the absence of transporter inhibitors. <i>In vivo</i> imaging of ICG biodistribution in mice was conducted over 3.5 h using valspodar as the P-gp inhibitor. The EC<sub>50</sub> values for ICG accumulation in control MDCK and MDCK-MDR1 cells were 9.0 × 10<sup>–6</sup> ± 5.7 × 10<sup>–7</sup> M and 1.5 × 10<sup>–5</sup> ± 1.1 × 10<sup>–6</sup> M, respectively. The efflux ratio for ICG in MDCK-MDR1 cells was 6.8-fold greater than in control cells. P-gp inhibition attenuated ICG efflux from MDR1-MDCK cells, and their effects in those cells were greater than in control MDCK cells. In contrast, BCRP level of expression or pharmacological inhibition did not significantly affect ICG cellular accumulation. <i>In vivo</i> imaging indicated enhanced cerebral ICG distribution with valspodar (brain – foot area under the concentration–time curves of 3.0 × 10<sup>10</sup>, 5.6 × 10<sup>10</sup> and 3.7 × 10<sup>10</sup> h·[p/s/sr]/μW in valspodar-treated mice vs 9.0 × 10<sup>9</sup> and 5.3 × 10<sup>9</sup> h·[p/s/sr]/μW in controls). The findings from this pilot study suggest that near-infrared imaging using ICG as the probe substrate should be further characterized as a methodology for <i>in vivo</i> evaluation of P-gp activity.