Improvement of Oral Bioavailability of Ginsenosides via Mechanism-based Biopharmaceutical Approaches

Objective: The overall goal is to determine the major factors limiting oral bioavailability of ginsenosides and utilizing the knowledge gained to increase the oral bioavailability of ginsenosides via a machenism-based biopharmaceutical approach. The objectives of this project were 1) to determine the major reasons responsible for low oral bioavailability of ginsenosides using various ADME assays including presystemic stability, solubility, permeability and metabolism; 2) to delineate the absorption mechanism of various ginsenosides including the identification of responsible transporters; 3) to enhance oral bioavailability of ginsenosides based on the knowledge obtained in the first two aims.

Method: For objective 1), saturated solubility measurement in different aqueous matrics, permeability in Caco-2 cells were determined along with stability in GI tract and in vitro metabolism in pooled intestinal/liver s9 fractions prepared from A/J mice. For objective 2), in vitro transcellular transport experiments employing Caco-2 and MDCK II cell monolayers were performed, together with in situ intestinal perfusion and in vivo pharmacokinetic experiments utilizing wild-type and MDR1 knockout mice. For objective 3), in vitro transcellular transport model using Caco-2 cell monolayers were used as a screening tool for identifing effective inhibitors or inhibitor combo using fourth generation P-glycoprotein inhibitors that are considered to be safe, and the most effective inhibitor combo was then used for the enhancement of bioavailability of ginsenosides in A/J mice.

Results: 1) Poor solubility and permeability were identified to be the major reasons for low oral bioavailability of ginsenosides. Although CYP metabolism occurred at in vitro system, the phase I metabolite was not found in vivo. 2) Rh2 and C-K were found to be good substrates of P-gp and inhibition/knockout of P-gp can significantly enhance their oral bioavailability. A structure-transport mechanism study utilizing twenty-two ginsenosides demonstrated that P-glycoprotein-mediated efflux mechanism was responsible for the efflux of ginsenosides with one glucose attached to one or both OH groups of the aglycone. 3) A combination of fourth generation P-gp inhibitors (i.e., biochanin A, wogonin and piperine @ 50 µM each) decreased efflux of ginsenosides Rh2s and C-K in Caco-2 cells. The effective and efficient inhibition of P-gp by the same inhibitor combo led to an increased oral bioavailability of Rh2s and C-K in A/J mice, primarily through increased volume of distribution.

Conclusion: The poor solubility and slow permeation were the major reasons causing low oral bioavailability of ginsenosides. Systematic studies showed that P-gp is the exclusive efflux transporter for ginsenosides Rh2 and C-K and that structure-dependent P-gp-mediated ginsenoside efflux is mainly due to difference in the number of sugar moieties. In vitro and in vivo study demonstrated that inhibition of P-gp can significantly increase oral bioavailability of ginsenosides. A triple combination of biochanin A, wogonin and piperine led to an increased oral bioavailability of Rh2s and C-K in vivo.