Tailoring the sensitivity of PET tracers to detect P-glycoprotein-mediated drug-drug interactions at the blood-brain barrier: a comparative in vitro study

Complete text of publication follows: Aim/Introduction: PET imaging using radiolabeled substrates of P-glycoprotein (P-gp, ABCB1) have highlighted the importance of this efflux transporter in restricting the brain distribution of drugs across the human blood-brain barrier (BBB). So far, P-gp substrates with high transport capacity (avid substrates) have been employed to generate maximal contrast between baseline conditions and conditions with impaired P-gp function. However, the sensitivity of different radiolabeled P-gp substrates to detect small changes in P-gp function and/or predict the risk for inhibitor/substrate drug-drug interactions at the BBB remains to be compared. Materials and Methods: Standardized in vitro uptake assays were performed in MDCKIIMDR1 cells over expressing human P-gp to compare the transport properties of the radiolabeled substrates ¹¹C-verapamil, ¹¹C-N-desmethyl-loperamide, ¹¹C-metoclopramide and the newly developed ¹¹C-domperidone. Cell-monolayers were incubated with buffer containing the tested compound (1 MBq/mL) and the P-gp substrate calcein-AM (1 μM, fluorescent internal control for P-gp function). Six concentrations of the clinically validated inhibitors cyclosporine A (CsA, 0-25 μM) and tariquidar (TQD, 0-200 nM) were tested (n=4 per condition). After 30 min incubation, monolayers were washed and cell-lysate was counted for radioactivity and fluorescence. The transport ratio (TR) was estimated as the ratio of the maximal uptake (full inhibition) to the baseline uptake (no inhibition). Sensitivity to inhibition was described by the IC50 of CsA and TQD for each substrate, estimated from the sigmoidal Hill equation. Outcome values were divided by those of calcein-AM to provide relative (/R) values (IC50/R and TR/R) in order to correct for inter-day variability and allow for comparison. Results: ¹¹C-metoclopramide (TR/ R=0.22 and 0.23 for CsA and TQD, respectively) showed a lower transport capacity than ¹¹C-verapamil (TR/R=0.76 and 0.64), ¹¹C-N-desmethyl-loperamide (TR/R=0.54 and 0.39) and ¹¹C-domperidone (TR/R=1.22 and 0.86). Using CsA, the IC50 ranged from 2.4±0.5 μM (¹¹C-metoclopramide) to 6.4±0.6 μM (¹¹C-domperidone). IC50/R was lower for ¹¹C-metoclopramide (IC50/R=0.86)< ¹¹C-N-desmethyl-loperamide (IC50/R=0.96)< ¹¹C verapamil (IC50/R=1.15)<¹¹C-domperidone (IC50/R=1.89). Using TQD, IC50 ranged from 5.2±2.6 nM (¹¹C-metoclopramide) to 82 ±20 nM (¹¹C-domperidone). IC50/R was lower for ¹¹C-metoclopramide. (IC50/R=0.23)<¹¹C-domperidone (IC50/R=0.82 )<¹¹C-N-desmethyl-loperamide (IC50/R=1.0)<¹¹C-verapamil (IC50/R=1.15). Conclusion: Compared with the weak P-gp substrate ¹¹C-metoclopramide, avid P-gp substrates may lack sensitivity to detect small changes in P-gp function and fail to detect partial P-gp inhibition. Interestingly, the sensitivity to inhibition depends on the selected inhibitor. Standardized and normalized in vitro assays are therefore essential to select the radiolabeled substrate with the best ability to detect drug-drug interactions with P-gp inhibitors at the BBB in vivo