TY - JOUR T1 - Using in vitro measurements of plasma and tissue free fractions to interpret radioligand uptake JF - Journal of Nuclear Medicine JO - J Nucl Med SP - 246P LP - 246P VL - 48 IS - supplement 2 AU - R. Gunn AU - S. Summerfield AU - C. Salinas AU - K. Read AU - G. Searle AU - A. Ruffo AU - C. Parker AU - A. Stevens AU - T. Bonasera AU - P. Jeffrey AU - M. Laruelle Y1 - 2007/05/01 UR - http://jnm.snmjournals.org/content/48/supplement_2/246P.1.abstract N2 - 1141 Objectives: Until recently, passive diffusion across the BBB has been a general assumption for transport of PET radioligands into the brain. To account for active transport, we propose that the non-displaceable volume of distribution (VN) of a radiotracer be decomposed into non-specific binding and BBB transport components. VN=(f1/f2)(FT/FP); f1 and f2 are the free fractions in plasma and the non-displaceable tissue compartments, and FT and FP are the free concentrations in tissue and plasma. Thus, VN is defined by the product of non-specific binding ratio (f1/f2) and free partition coefficient at equilibrium across the BBB (FT/FP). For tracers that involve passive diffusion FT/FP=1, and VN simplifies to f1/f2, its usual definition. The FT/FP ratio can be experimentally assessed by comparing VN as measured with PET, with the f1/f2 ratio as measured in vitro by equilibrium dialysis. 7 compounds, with known PGP substrate status, were used to validate the proposed method. Methods: Estimates of VN were obtained from pig PET studies for 7 different radiotracers, using either blocked or true reference regions. Free fractions in plasma and brain homogenate were determined by means of 96-well equilibrium dialysis. Test compounds were incubated in plasma or brain (10 μg/mL, 37C) and dialysed against phosphate buffered saline through a 12-14 kDa cutoff semipermeable membrane for five hours. Aliquots of tissue and dialysate were collected and analysed for test compound using HPLC/MS/MS analysis. Results: For predicted non-PGP substrates (n=4), a good agreement between the PET and equilibrium dialysis data was observed consistent with passive diffusion, FT/FP=0.91±0.26, not significantly different from 1 (p=0.57). For predicted PGP substrates, FT/FP=0.43±0.16, significantly lower than 1 (p<0.05), and consistent with active efflux. Conclusions: This work supports the combination of PET and equilibrium dialysis to determine the true free concentration of a radiotracer in brain. This has implications for the assessment of BBB transport, estimation of in vivo KDs and interpretation of biodistribution studies. Also, the ability to estimate f1 and f2 in vitro may provide a useful screening tool for selection amongst novel candidate radioligands. ER -