Two binding sites for [3H]PBR28 in human brain: implications for TSPO PET imaging of neuroinflammation

Abstract

[(11)C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [(11)C]PK11195, another TSPO radioligand. We measured the specific binding signals with [(3)H]PK11195 and [(3)H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [(3)H]PBR28, although all samples showed [(3)H]PK11195 binding. There was a marked reduction in the affinity of [(3)H]PBR28 for TSPO in samples with no visible [(3)H]PBR28 autoradiographic signal (K(i)=188+/-15.6 nmol/L), relative to those showing normal signal (K(i)=3.4+/-0.5 nmol/L, P<0.001). Of this latter group, [(3)H]PBR28 bound with a two-site fit in 40% of cases, with affinities (K(i)) of 4.0+/-2.4 nmol/L (high-affinity site) and 313+/-77 nmol/L (low-affinity site). There was no difference in K(d) or B(max) for [(3)H]PK11195 in samples showing no [(3)H]PBR28 autoradiographic signal relative to those showing normal [(3)H]PBR28 autoradiographic signal. [(3)H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [(11)C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.

Figure 1

Representative images for total and nonspecific binding in a high affinity binder (Donor 1) and a low-affinity binder (Donor 5) with [³H]PBR28 and [³H]PK11195, and colocalisation of [³H]PK11195 binding with major histocompatibility complex class II (MHC II) staining in a low-affinity binder (Donor 5). (A, B) Donor 1 [³H]PK11195 total binding and nonspecific binding, (C, D) donor 1 [³H]PBR28 total binding and nonspecific binding; (E, F) donor 5 [³H]PK11195 total binding and nonspecific binding; (G, H) donor 5 [³H]PBR28 total binding and nonspecific binding. (I) Low MHC II expression from normal appearing white matter ( × 400 magnification) colocalising with low [³H]PK11195 binding; (J) high MHC II expression from white matter lesion ( × 400 magnification) corresponding with high [³H]PK11195 binding. Normal white matter is represented by the white arrow; normal grey matter by the black arrow; and white matter lesion by the red arrow.

Figure 2

K_d and B_max for [³H]PK11195 and [³H]PBR28. (A) K_d for [³H]PK11195, (B) K_d for [³H]PBR28, (C) B_max for [³H]PK11195, (D) B_max for [³H]PBR28. Where data fitted a two-site fit, the K_d for the high-affinity site and the total B_max (for both high- and low-affinity sites) were used to calculate means.

Figure 3

(A) Competition assay with [³H]PK11195 in the presence of increasing concentrations of unlabelled PK11195. Curves show individual fits to each subject's data. The nonspecific binding has been subtracted to leave the specific binding, which has been normalised to 100% specific binding. Blue—low-affinity binder (LAB). Black—high-affinity binder (HAB). Red—mixed-affinity binder (MAB). (B) Competition assay with [³H]PK11195 in the presence of increasing concentrations of unlabelled PBR28. Thin lines show individual fits to each subject's data. Thick lines show results from the population fit defined by the equations in the text, which assume that MABs have both HAB and LAB binding sites (percentage of each unspecified). The nonspecific binding has been subtracted to leave the specific binding, which has been normalised to 100% specific binding. Blue—low-affinity binder (LAB). Black—high-affinity binder (HAB). Red—mixed-affinity binder (MAB).