Species Comparison of the Binding Properties for Three D2/3-DAR Radioligands Widely Used in PET Imaging Darren R. Quelch1,2, Sarah L. Withey2, David J. Nutt1, Christine A. Parker2, Robin J. Tyacke1 1. Neuropsychopharmacology Unit, Burlington Danes Building, Imperial College, London, W12 0NN. 2. Department of Biology, GSK, Clinical Imaging Centre, Imperial College, London, W12 0NN Introduction The D2/3-Dopaminergic receptor (D2/3-DAR) system has been extensively investigated with classical and emerging Positron Emission Tomography (PET) ligands such as [11C]Raclopride1 , [11C](+)PhNO2 and [11C]N-methyl-Spiperone3. Pre-clinical PET studies are becoming increasingly popular. As such, potential species differences for the binding parameters of ligands become an important issue when translating data for an application to human clinical studies. Endogenous dopamine release has been measured with ‘Pharmacological Challenge’ imaging protocols with D2/3-DAR ligands4 and there is an increasing desire to measure other neurotransmitters with such paradigms. The interaction between protein target, radioligand and endogenous ligand remains to be fully understood. It is thought for the D2/3-DAR, that rather than DA competing directly with the radioligand, receptor internalisation could play a role in the change of the observed PET signal. Aims Species differences in binding parameters in rat and pig striatum with three widely used D2/3-DAR ligands (PhNO, Raclopride and Spiperone) were determined in vitro. The effect of different ionic conditions involved in the receptor internalisation pathway on the binding parameters of D2/3-DAR ligands were assessed. Methods Buffers representative of different compartments within the receptor internalisation pathway were generated (Table 1). In vitro radioligand binding assays were performed at 37 ⁰C (tissue sources: rat and pig striata) in each buffer to determine Kd and Bmax values: D2/3-DAR Ligands: [3H](+)PhNO (agonist), [3H]Raclopride (antagonist), [3H]Spiperone (antagonist). Specific Binding (SB) determined using Haloperidol (1µM). Results Bmax values for all ligands were significantly higher in rat compared to pig (2-Way ANOVA p<0.001). Kd values tended to be lower in the rat compared to the pig, reaching significance with PhNO EE. Together these data show an increased density of D2/3-DAR in the rat striatum compared to the pig and a decreased affinity in the pig striatum. Bmax values for all ligands in both species were not effected by changes in ionic environment Bmax values for [3H]Spiperone and [3H]Raclopride were approximately double that for [3H](+)PhNO in both rat and pig striatum reflecting PhNO’s agonist binding properties. For [3H](+)PhNO a significant decrease in affinity was seen when exposed to EE compared to the IC or EC in pig. The 2-Way ANOVA results show that BOTH condition AND species had a significant effect on Raclopride Kd (p<0.01, p<0.04 respectively). Changes in ionic condition had no effect on Spiperone’s ability to bind D2/3-DAR. Discussion Differences in Bmax values between rat and pig is driving the differences in BPs generated between these two species (Table 2). PhNO was sensitive to changes in ionic condition in a similar fashion to raclopride. We suggest that in vivo [11C](+)PhNO will be sensitive to endogenous dopamine turnover and show changes in BPs due to direct competition AND a decrease in affinity in the endosomal compartment similar to raclopride. These findings suggest that pre-clinical data produced in pig might be a better indicator of human BPs for D2/3-DAR ligands and highlights the importance of making in vitro species comparisons during tracer development. Table 1. The ionic compositions (mM) of the three cellular buffers used. All pH at 37⁰C. Na+ K+ Mg2+ Ca2+ Cl- Tris-HCl MES pH Extracellular (EC) 140 5 1.5 110 50 ~ 7.4 Intracellular (IC) 10 0.5 7.0 Endosomal (EE) 0.003 20 6.0 Fig. 1A. Bmax (fmol/mg protein) Rat:Pig comparison PhNO Raclopride Spiperone 2-Way ANOVA – Condition: P = N/S. Species: P = <0.001. Species x Condition: P = N/S. 2-Way ANOVA – Condition: P = N/S. Species: P = <0.001. Species x Condition: P = N/S. 2-Way ANOVA - Condition: P = N/S. Species: P = <0.001. Species x Condition: P = N/S. ## ## # # # ## ## ## # Fig. 1B. Kd (nM) Rat:Pig comparison PhNO Raclopride Spiperone 2-Way ANOVA - Condition: P =<0.001.Species: P = N/S. Species x Condition:P = N/S. 2-Way ANOVA – Condition: P =0.01. Species: P = 0.04. Species x Condition: P = N/S. 2-Way ANOVA – Condition: P = N/S. Species: P = N/S. Species x Condition: P = N/S. ** ** * # Figure 1: Bmax (A) and Kd (B) values determined for the three cellular buffers (EC, IC, EE) for the D2-DAR ligands [3H](+)PhNO, [3H] Raclopride and [3H]Spiperone . Vertical errors bars represent SEM from 3-6 independent experiments. (2-Way ANOVA with Tukey post-tests generated using SigmaStat 2.03.* Represent comparisons between conditions, # represent comparisons between tissue types from post-test analysis.* P>0.05, **P>0.01). Table 2. In Vitro generated Binding Potentials (BP = Bmax(pmol/g tissue)/Kd (nM). PhNO Raclopride Spiperone Extracellular (EC) Rodent 14.5 6.93 179 Porcine 2.95 1.32 57.7 Human 2.632 2.901 14.595§ Intracellular (IC) 25.3 3.24 165 3.61 0.993 53.0 Endosomal (EE) 0.627 1.22 68.4 0.158 0.279 35.5 Table 2. § Represents in vivo human Bmax for 11C-N-methylspiperone References: 1. Volkow et al, Synapse, 1994;16(4). 2. Willeit et al, Neuropsychopharmacology, 2008;33(2). 3. Wagner et al. Annals of Neurology, 1984;15(s1). 4. Laruelle, JCBFM, 2000;20(3). 5. Tune et al, Psychiatry Research, 1993;49(3). Acknowledgements: BBSRC CASE award sponsorship with GlaxoSmithKline Contact: darren.quelch09@imperial.ac.uk/ darren-robert.x.quelch@gsk.com