Rate and Equilibrium Constants for Bupivacaine’s Binding to Isolated Alpha-1-Acid Glycoprotein: An In vitro study.

Binding of local anesthetics to plasma proteins has been presented as an important determinant of their bioavailability. Local anesthetics with a high potential for systemic toxicity, e.g. bupivacaine (BUP), are bound strongly by alpha₁-acid glycoprotein (AAG), more weakly by serum albumin, but drug dissociation may be rapid, thus limiting the importance of protein binding. The purpose of this study was to determine the binding kinetics of BUP to AAG. Bupivacaine binding to AAG was monitored by its displacement of the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS). The increased fluorescence of ANS (λ _excit/em = 380/480 nm) upon binding to AAG was used to determine the equilibrium and kinetic characteristics of this reaction. By studying how BUP altered the binding kinetics of ANS to AAG it was possible to calculate the BUPs equilibrium and kinetic rate constants for AAG binding. ANS fluorescence increased ca. 50-fold when bound to AAG.  Increasing [BUP] with a constant [AAG] + [ANS] returned ANS fluorescence to its unbound status, due to complete displacement of ANS from AAG; bupivacaine’s competitive equilibrium constant, K_i , equals 1-2 μM (pH 7.4, 23^oC). Pre-equilibrating AAG with BUP before the rapid (0.008s) addition of excess ANS slowed the binding of ANS to a rate limited by BUP’s dissociation: k_off = 12.0 ± 0.5 s^-1, corresponding to a half-time ~0.06 seconds. Therefore, although much of the total serum BUP at toxic levels (2-4 µg/mL) will be bound by plasma proteins, dissociation from the tightest binding protein shows that drug is rapidly freed during organ perfusion, allowing newly unbound drug to permeate into the perfused tissues. The very rapid dissociation of BUP from AAG means that equilibrium binding is a very poor index of bio-availability and systemic toxicity of that local anesthetic.