Abstract
The (−)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (−)CLA (1 or 5 μmol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (−)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 μmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (−)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 μmol/L had significantly lower intracellular uptake than (−)CLA with a ratio[(−)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 μmol/L. Besides, in the non-induced KB cells, (+)CLA(5 μmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (−)CLA(5 μmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (−)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs.
Highlights
P-glycoprotein(P-gp) belongs to a superfamily of ATP binding cassette transport proteins and plays the role of an energy-dependent drug efflux pump[1,2]
The results from this study indicated that the (−)-isomer could be a P-gp inhibitor, but the (+)-isomer could be a modulator with concentration-dependent biphasic effects and showed stereoselective regulation of P-gp activity and possible drug—drug interactions when combined with other P-gp substrate drugs
When a higher concentration (5 μmol/L) was added to the cells, the stimulatory effects of (+) CLA were converted into inhibitory ones on P-gp activity, which resulted in significantly increased rhodamine 123 (Rh123) accumulation by 58.2% (P < 0.01 vs. control), near its antipode, by 63.0%
Summary
P-glycoprotein(P-gp) belongs to a superfamily of ATP binding cassette transport proteins and plays the role of an energy-dependent drug efflux pump[1,2]. Distributional kinetics in target tissues (e.g. hippocampus, cortex and cerebellum) showed that (+)CLA had greater values for AUC0–1 than did (−)CLA [18]. These data suggest that the absorptive and distributional processes of CLA enantiomers could be involved in P-gp in animal intestines and brain microvessels
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