Estimation of unbound concentrations of morphine from microdialysate concentrations by use of nonlinear regression analysis in vivo and in vitro during steady state conditions

Abstract

The unbound concentration of morphine in striatum of rats was estimated during a constant rate infusion of morphine 14 mumol/h*kg, by use of the microdialysis technique and nonlinear regression analysis. The concentrations in plasma of morphine and its metabolite, morphine-3-glucuronide, were 4.2 +/- 1.4 microM and 7.7 +/- 4.0 microM, respectively, during the constant rate infusion. The corresponding estimated unbound concentrations of morphine in striatum varied between 0.06 and 0.11 microM. No morphine-3-glucuronide was detected in the brain dialysates. The unbound concentration in striatum was lower than expected based on unbound plasma concentrations and could be an indication of active transport from the brain. Five different equations were tested to find the best empirical description of the relationship between microdialysate concentration and perfusion rate by nonlinear regression analysis. The equations were validated by a serum in vitro study, where three unbound concentrations of morphine estimated from microdialyis were compared to estimates obtained from equilibrium dialysis. The precision of the parameter estimates obtained from the five equations was tested by Monte Carlo simulations. One of the equations (Eq. 4) was selected in preference to the others, because of the good agreement with the estimated unbound concentration obtained by equilibrium dialysis in vitro, and good precision of the parameter estimates. The method described in this paper is valuable when estimating the unbound concentration of drug from microdialysate concentrations during steady state conditions. Furthermore, the method is easily accessible when working in the pharmacokinetic and pharmacodynamic field.