Quantification of the purinergic P2X7 receptor with [11C]SMW139 improves through correction for brain-penetrating radiometabolites

The membrane-based purinergic 7 receptor (P2X7R) is expressed on activated microglia and the target of the radioligand [11C]SMW139 for in vivo assessment of neuroinflammation. This study investigated the contribution of radiolabelled metabolites which potentially affect its quantification. Ex vivo high-performance liquid chromatography with a radio detector (radioHPLC) was used to evaluate the parent and radiometabolite fractions of [11C]SMW139 in the brain and plasma of eleven mice. Twelve healthy humans underwent 90-min [11C]SMW139 brain PET with arterial blood sampling and radiometabolite analysis. The volume of distribution was estimated by using one- and two- tissue compartment (TCM) modeling with single (VT) and dual (VTp) input functions. RadioHPLC showed three major groups of radiometabolite peaks with increasing concentrations in the plasma of all mice and humans. Two radiometabolite peaks were also visible in mice brain homogenates and therefore considered for dual input modeling in humans. 2TCM with single input function provided VT estimates with a wide range (0.10–10.74) and high coefficient of variation (COV: 159.9%), whereas dual input function model showed a narrow range of VTp estimates (0.04–0.24; COV: 33.3%). In conclusion, compartment modeling with correction for brain-penetrant radiometabolites improves the in vivo quantification of [11C]SMW139 binding to P2X7R in the human brain.

was released from the trap by heating with pressurized air and subsequently [ 11 C]CH 4 was mixed with iodine crystal vapor at 60 °C followed by a radical reaction at 720 °C. The formed [ 11 C]CH 3 I was collected in a Porapak Q trap at room temperature and the unreacted [ 11 C]CH 4 was recirculated for 3 min. [ 11 C]CH 3 I was released from the Porapak Q trap by heating the trap using a custom-made oven at 180 °C. [ 11 C]CH 3 OTff was produced by online transfer of [ 11 C]CH 3 I through a glass column packed with silver triflate at 165 °C. Carbon-11 labelled [ 11 C]SMW139 was obtained by trapping [ 11 C]CH 3 OTff at room temperature in a reaction vessel containing the desmethyl precursor SMW167 (2-chloro-5-hydroxy-N-(((3s,5s,7s)-3,5,7trifluoroadamantan-1-yl)methyl) benzamide), (0.5 -1 mg, 1.3 µmol-2.6 µmol) and NaOH (0.5M, 3 μL) in acetone (400 μL). The reaction mixture was diluted with sterile water (500 μL) before injecting to the built-in high-performance liquid chromatography (HPLC) system for the purification of the desired radiolabelled product. The HPLC system consisted of a semipreparative reverse phase XBridge column (C18, 10 × 250 mm, 5 μm particle size) and a Merck Hitachi UV detector (λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection.
Acetonitrile / 0.1% Trifluoroacetic acid (TFA), 50:50 (v/v) was used as HPLC mobile phase with a flow rate of 6 mL/min. The radioactive fraction corresponding to pure [ 11 C]SMW139 was collected from HPLC and evaporated to dryness. The final purified [ 11 C]SMW139 was formulated in 6-mL phosphate buffered saline (pH7.4) and the formulated product was then sterile filtered through a Millipore Millex® GV filter unit (0.22 μm) for further use in vivo.
Turku PET Centre. Radiochemistry was performed as previously described 2 .

Quality control and molar activity (MA) determination.
Karolinska Institutet. The radiochemical purity, identity, and stability of [ 11 C]SMW139 was determined by an analytical HPLC system which included an XBridge RP column (C18, 4.6µm Turku PET Centre. The radiochemical purity, identity, stability and the MA of [ 11 C]SMW139 was determined as previously described 2 .

Small animal study
Radio detector high-performance liquid chromatography analysis. The supernatant was

Arterial plasma input for [ 11 C]SMW139 and radioactive [ 11 C]SMW139 metabolites. At
Karolinska Institutet, the individual blood curve over the first 5 min from the ABSS was merged with the curve from manual blood samples. Radioactivity concentrations in plasma and blood were divided yielding a plasma/blood ratio curve. Using linear interpolation, the plasma/blood ratio curve was extrapolated from 0 s to the end of sampled ABSS data. Plasma time-activity curves covering the whole scan were generated by multiplying the extrapolated plasma/blood ratio curve with the ABSS blood curve and by fusing the result with the plasma curve from manual samples. At Turku PET Centre, input processing assumed that parent radioligand concentration in blood cells is zero during the time of ABSS data collection. Plasma timeactivity curves covering the whole scan were generated by dividing the ABSS blood curve by (1 -hematocrit) and by merging the result with the plasma curve from manual samples.
Subsequent processing was performed concordantly for both study sites with PMOD (version 3.7, PMOD Technologies LLC). Data for parent fraction of [ 11 C]SMW139 were fitted using a mono-exponential fit and were multiplied with the uncorrected plasma time-activity-curve to obtain the parent input curve (i.e. the plasma radioactivity of unchanged [ 11 C]SMW139).
Furthermore, we estimated an input curve representing the activity of presumably brainpenetrant [ 11 C]SMW139 radiometabolites in the plasma. Therefore, the parent fraction and the fraction of the radiometabolite, which was assumed not to be present within the brain, were combined, fitted with a mono-exponential function and multiplied with the plasma time-activitycurve. The obtained curve was subsequently subtracted from the plasma input curve, which resulted in the radiometabolite input curve. Finally, blood and plasma input curves were delay corrected.