Kinetic Analysis of 3-Quinuclidinyl 4-[ 125 I]Iodobenzilate Transport and Specific Binding to Muscarinic Acetylcholine Receptor in Rat Brain in vivo: Implications for Human Studies

Radioiodinated R- and S-Quinuclidinyl derivatives of RS-benzilate (R- and S-¹²⁵IQNB) have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor binding in vivo. Two sets of experiments were performed in rats. The first involved determining the metabolite-corrected blood concentration and tissue distribution of tracer R-IQNB (active enantiomer) and S-IQNB (inactive enantiomer) in brain 1 min to 26 h after intravenous injection. The second involved the measurement of brain tissue washout over a 2-min period after loading the brain by an intracarotid artery injection of the ligands. Various pharmacokinetic models were tested, which included transport across the blood–brain barrier (BBB), nonspecific binding, low-affinity binding, and high-affinity binding. Our analysis demonstrated that the assumptions of rapid equilibrium across the BBB and rapid nonspecific binding are incorrect and result in erroneous estimates of the forward rate constant for binding at the high-affinity receptor sites (k₃). The estimated values for influx across the BBB (K₁), the steady-state accumulation rate in cerebrum (K), and the dissociation rate constant at the high-affinity site (k₄) of R-IQNB were independent of the specific compartmental model used to analyze these data (K₁ ≈ 0.23 ml/min/g, K ≈ 0.13 ml/ min/g, and k₄ ≈ 0.0019 min^{− 1} for caudate). In contrast, the estimated values of k₃ and the efflux rate constant (k₂) varied over a 10-fold range between different compartmental models (k₃ ≈ 2.3–22 min^{− 1} and k₂ ≈ 1.6–16 min^{− 1} in caudate), but their ratios were constant (k₃/k₂ ≈ 1.4). Our analysis demonstrates that the estimates of k₃ (and derived values such as the binding potential) are model dependent, that the rate of R-IQNB accumulation in cerebrum depends on transport across the BBB as well as the rate of binding, and that uptake in cerebrum is essentially irreversible during the first 360 min after intravenous administration. Graphical analysis was consistent with compartmental analysis of the data and indicated that steady-state uptake of R-IQNB in cerebrum is established within 1–5 min after intravenous injection. We propose a new approach to the analysis of R-IQNB time-activity data that yields reliable quantitative estimates of k₃, k₄, and the nonspecific binding equilibrium constant (K_eq) by either compartmental or graphical analysis. The approach is based on determining the free unbound fraction of radiolabeled ligand in blood and an estimate of K₁. The analysis can be applied to time-activity data obtained in a clinical setting using either positron emission tomography or single photon emission computed tomography and has general application for other highly lipophilic ligands.