Synthesis and evaluation of dopamine D-3 receptor antagonist C-11-GR218231 as PET tracer for P-glycoprotein

While searching for a PET method to determine the density and occupancy of the dopamine D-3 receptor, we found evidence that suggested that the dopamine D-3 antagonist GR218231 could be a substrate of the P-glycoprotein efflux pump. P-glycoprotein protects the brain against toxic substances and xenobiotics, but it also hampers the delivery of various drugs into the brain. In this study, we aimed to explore whether radiolabeled GR218231 could be applied as a PET tracer for monitoring P-glycoprotein activity in the blood-brain barrier. Such an imaging technique could be useful for the development of new drugs and novel strategies to deliver drugs to the brain and for identification of undesirable drug-drug interactions. Methods: As a potential PET tracer, GR218231 was labeled with C-11 by reaction of the newly synthesized desmethyl precursor with C-11-methyl triflate. The biodistribution of C-11-GR218231 was determined in rats. To assess specific binding to the dopamine D3 receptor, blocking experiments with unlabeled GR218231 (0.2 and 2.5 mg/kg) were performed. To demonstrate the influence of P-glycoprotein on cerebral uptake of C-11-GR218231, the efflux pump was modulated with 50 mg/kg cyclosporine A. The sensitivity of C-11-GR218231 for P-glycoprotein modulation was assessed in dose-response studies, using escalating cyclosporine A dosages. Results: C-11-GR218231 was prepared in 53% +/- 8% decay-corrected radiochemical yield and had a specific activity of 15 +/- 10 GBq/mu mol (mean +/- SD). Biodistribution studies in rats revealed a low and homogeneous uptake in the brain. Pretreatment of the animals with unlabeled GR218231 did not demonstrate any specific binding. Modulation of P-glycoprotein with cyclosporine A caused a 12-fold higher C-11-GR218231 uptake in the brain, indicating that the low cerebral tracer uptake was caused by the P-glycoprotein efflux pump in the blood-brain barrier. Cyclosporine A close-escalation studies showed a dose-dependent sigmoidal increase in C-11-GR218231 uptake in brain and spleen (median effective dose [ED50], 23.3 +/- 0.6 and 38.4 +/- 2.4 mg/kg, respectively), whereas a dose-dependent decrease was observed in the pancreas (ED50, 36.0 +/- 4.4 mg/kg). Conclusion: Although C-11-GR218231 is unsuited for dopamine D3 receptor imaging with PET, it appears to be an attractive PET tracer for visualization and quantification of P-glycoprotein activity in the blood-brain barrier

The human norepinephrine transporter in combination with C-11-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy

Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility of the human norepinephrine transporter (hNET) as a reporter gene in combination with the reporter probe C-11-m-hydroxyephedrine (mHED) for PET. Methods: An adenoviral vector (AdTrack-hNET) containing the hNET gene as reporter gene and the enhanced green fluorescent protein (EGFP) as a substitute for a therapeutic gene was constructed. After COS-7, A2780, and U373 cells were transiently transduced with AdTrack-hNET, hNET protein expression, EGFP fluorescence, and cellular uptake of C-11-mHED were determined. In rats, U373 tumor xenografts were grown and transiently transduced with either AdTrack-hNET or an AdTrack-Luc control adenovirus. Intratumoral accumulation of C-11-mHED was determined by PET and ex vivo biodistribution. The tumors were subsequently examined for EGFP fluorescence. Results: 11C-mHED uptake was positively correlated with AdTrack-hNET viral titer and hNET protein expression. However, large differences in transfection efficiency between cell lines were observed. The highest 11C-mHED uptake was found in hNET transfected U373 cells, in which tracer uptake was > 70-fold higher than that in control cells. 11C-mHED accumulation could be inhibited by desipramine, a potent inhibitor of hNET. In all cell lines, C-11-mHED uptake was positively correlated with EGFP fluorescence, implying that imaging of hNET with 11C-mHED would enable monitoring of a coexpressed therapeutic gene. In the animal model, gene transfection efficiencies were very low, as determined by EGFP fluorescence. Still a significantly higher C-11-mHED uptake in hNIET transduced tumors than that in control tumors was demonstrated by ex vivo biodistribution studies. PET with a clinical camera could visualize 1 of 3 hNET transduced tumors, indicating that the transfection efficiency was near the detection limit. Conclusion: These results indicate that monitoring of gene therapy using the hNET/C-11-mHED reporter gene/probe is feasible, but further investigation with regard to the sensitivity of the technique is required