Analytical method for simultaneously measuring ex vivo drug receptor occupancy and dissociation rate: Application to (R)-dimethindene occupancy of central histamine H₁ receptors

Abstract

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k₄). [Here, we reference k₄ as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan ()]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [³H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k₄ for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k₄ rate determined by using our ex vivo method was equivalent to k₄determined by in vitro competition kinetics (dissociation half-life t_1/2 ~ 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

Keywords::

• Competition kinetics
• Histimine receptor
• radioligand binding
• receptor occupancy
• rat cortex

Acknowledgments

Declaration of interest: The authors acknowledge Bin-Feng Li and Raymond Gross for synthesis of (R)-dimethindene. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.