In vitro equilibrium partitioning in blood cellsto measure plasma free fraction of PET radioligands

401 Objectives Ultrafiltration is the most common method of determining plasma free fraction (fP) for brain PET radioligands (ratio of free concentration to free plus protein-bound concentrations). This fast centrifugally-driven method separates free radioligand in plasma from that equilibrated with the protein-bound radioligand. However, ultrafiltration is vulnerable to errors when a radioligand that should have passed into the filtrate is adsorbed to some portion of the ultrafiltration apparatus. Here, we propose the use of a distinctly different ‘cell method’ to validate ultrafiltration fP measurements. This method is based on that earlier developed for tritiated compounds and has been compared with results obtained from classic equilibrium dialysis (Biopharm Drug Dispos; 5:281, 1984), which is often viewed as the ‘gold standard’ method for measuring fP. Here we describe a novel cell method for measuring radioligand fP that uses membranes of intact blood cells as surrogates for a synthetic dialysis membrane to equilibrate and separate free from protein-bound radioligand. Methods Measurements of fP were done simultaneously for 11C-(R)-rolipram, 11C-PBR28, and 11C-FPEB by ultrafiltration and cell method. Concentrations of the ultrafiltrates (cpm/mL) were determined volumetrically and gravimetrically. By monitoring their specific concentrations (cpm/mL), specific sites of adsorption to the ultrafiltration apparatus were localized after incubating (30 min) disassembled components of the ultrafiltration apparatus with radioligands in media that mimicked solutions produced during fP determinations. Cell-fP was determinedin vitro by incubating whole blood and PBS-washed blood cells with radioligand in microcentrifuge tubes at room temperature until equilibration (30 min). Cell-fP was the ratio of radioactivity concentration in buffer supernate (free) normalized by hematocrit-corrected cell partitioning to that of the normalized plasma concentration (free plus protein-bound). Results 11C-(R)-rolipram and 11C-PBR28 demonstrated minimal adsorption to the ultrafiltration apparatus, and values for fP were similar between the two methods (11C-rolipram ultrafiltration fP 4.47±0.25 (n=3), cell-fP 4.55±0.46 (n=3); 11C-PBR28 ultrafiltration fP 2.67±0.18 (n=3), cell-fP 2.56±0.36 (n=3)). However, fP values for 11C-FPEB differed by 40% between the two methods (ultrafiltration fP 5.60±0.04 (n=3), cell-fP 8.55±0.13 (n=3)); notably, with the ultrafiltration method, 16% was due to adsorption only to the collection cup. Other adsorptive surfaces included the polycarbonate surfaces and the filter membrane, which accounted for the remaining difference of 24%. Fewer surfaces were involved in the cell method; only one type of microcentrifuge tube was used and it showed no measurable radioligand adsorption. Conclusions This study implemented a novel cell method - analogous to the ‘gold standard’ of equilibrium membrane dialysis - to quickly measure fP for short-lived PET radioligands. Like equilibrium membrane dialysis, the cell method was less prone to misleading loss of radioligand through surface adsorption. The cell method may become the reference against which other methods are evaluated for potential confounds.