In vitro transport studies with kaempferol and its anxiolytic metabolite 4-HPAA in human cell models

Previous pharmacological investigations have shown that the flavonoid kaempferol induces anxiolytic activities in mice after oral administration, but not after intraperitoneal (i.p.) application. However, i.p. administration of 4-hydroxyphenylacetic acid (4-HPAA), a major metabolite of kaempferol formed by the intestinal microflora, induces behavioral changes. Kaempferol was thus considered to be a prodrug. Orally administered CNS active compounds have to be absorbed in the intestine and reach the CNS as target tissue. With the aid of in vitro cell models, we investigated the ability of kaempferol and its main metabolite 4-HPAA to cross the intestinal and the blood brain barrier (BBB). Established BBB (human brain capillary endothelial cell line; hBMEC) and human intestinal transport (Caco-2 cells) models were selected for compound permeability prediction. As a first step, reliable and robust quantitative UPLC-MS/MS assays were developed and validated for determination of kaempferol and 4-HPAA in the transport media. Methods were validated according to FDA guidance over the range of 20 ng/ml (LLOQ) to 2000 ng/ml (ULOQ). Quantitation of kaempferol was carried out in positive electrospray ionization (ESI) mode with ¹³C-labeled kaempferol as internal standard (IS). The method for quantification of 4-HPAA was developed in negative ESI mode and vanillic acid served as IS. The concentration/response data of both analytes fitted with a quadratic curve with 1/X² weighing. The average coefficients of determinations (R ²) were greater than 0.995. The assays were used for transport studies with cell layers grown in transwell plates. The results from this study, combined with bioanalytical findings from in vivo studies with oral application of kaempferol and phenylacetic acids, will help to clarify the role of flavonoids and their metabolites in the behavioral effects observed in vivo.