In vitro and in vivo studies on the binding and permeation of ketotifen and norketotifen atropisomers in the central nervous system

Abstract

Ketotifen (K) is a first-generation antihistamine with antiinflammatory potency. It penetrates the blood-brain barrier (BBB) and causes a sedative side effect. Norketotifen (N) is an active metabolite of K. The S-atropisomer of N (SN), however, has antihistaminic and antiinflammatory properties but less sedating side effect than RN and K. This may be due to: (1) higher concentrations of K and RN than SN in the central nervous system (CNS) and/or (2) higher affinity of K and RN than SN for rat brain H1 receptors. The aim of this thesis was to investigate the mechanism of why SN lacks a sedative side effect. To determine concentrations of racemic K and N and atropisomers of K and N in buffer solutions and bio-matrices, nonchiral and chiral high-performance liquid chromatography (HPLC) assays were developed and validated. Log P and log D of K and N (both octanol/buffer and liposomes/buffer) were determined to aid in interpretation of in vitro cell studies. Rat brain endothelial (RBE4) and colorectal adenocarcinoma (Caco-2) cell monolayers were used as in vitro models of the BBB to study the stereoselective uptake and permeability of K and N atropisomers. To investigate the distribution of K and N atropisomers between brain tissue and plasma, the total and free brain-to-plasma (B/P) ratios of K and N atropisomers were measured 5 min post-administration of racemic K and N through the rat tail vein. The affinity of K and N atropisomers to brain H1 receptors was investigated by determining the extent of inhibition of [3H] mepyramine binding to H1 receptors in rat brain cell membranes. The in vitro studies indicated active mechanisms for transporting K and N in both RBE4 and Caco-2 cell lines; however, none of these mechanisms were stereoselective. Interestingly, for both cell lines, more N was found binding non- specifically to cell membranes than that of K, though in a non-stereoselective manner. Liposomes/buffer distribution studies aided in interpretation of these results. Similarly, the total and free B/P ratios of K and N atropisomers suggested a predominant influx mechanism involved in transporting of K and N through the rat BBB. However, this mechanism was not stereoselective for either K or N atropisomers. In addition, K and N non-specifically bound to rat plasma protein and brain tissues in different degree but the non-specific binding was not stereoselective either. In contrast, H1 receptor affinity results suggested a stereoselective binding of SN for the H1 receptors in rat brain, in that SN had a lowest affinity compared with RN, SK and RK. Significant differences in the affinity for the H1 receptors were found between SN and SK, SN and RK, moreover, between SN and RN. Although the difference was significant but not substantial compared to some published stereoselective affinity for the H1 receptors, a similar degree of difference was observed and published by other research groups. Thus the lowest affinity of SN for the H1 receptors could participate in the observed less sedative effect caused by SN. In conclusion, there was no stereoselective transport of SN through the BBB either in vitro or in vivo, and there was no stereoselective non-specific binding of SN to rat plasma proteins or brain tissues. The lower sedative effect of SN is due to a lower uptake of N than K into the brain and reduced binding of SN to CNS H1 receptors.