Characterization of silvestrol pharmacokinetics in mice using liquid chromatography-tandem mass spectrometry

Abstract

A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the plant natural product silvestrol in mice, using ansamitocin P-3 as the internal standard. The method was validated in plasma with a lower limit of quantification of 1 ng/mL, accuracy ranging from 87 to 114%, and precision (coefficient of variation) below 15%. The validated method was used to characterize pharmacokinetics in C57BL/6 mice and metabolism in mouse, human and rat plasma, and liver microsomes. Mice were dosed with silvestrol formulated in hydroxypropyl-β-cyclodextrin via intravenous, intraperitoneal, and oral routes followed by blood sampling up to 24 h. Intraperitoneal systemic availability was 100%, but oral administration resulted in only 1.7% bioavailability. Gradual degradation of silvestrol was observed in mouse and human plasma, with approximately 60% of the parent drug remaining after 6 h. In rat plasma, however, silvestrol was completely converted to silvestric acid (SA) within 10 min. Evaluation in microsomes provided further evidence that the main metabolite formed was SA, which subsequently showed no cytotoxic or cytostatic activity in a silvestrol-sensitive lymphoblastic cell line. The ability of the analytical assay to measure tissue levels of silvestrol was evaluated in liver, brain, kidney, and spleen. Results indicated the method was capable of accurately measuring tissue levels of silvestrol and suggested it has a relatively low distribution to brain. Together, these data suggest an overall favorable pharmacokinetic profile of silvestrol in mice and provide crucial information for its continued development toward potential clinical testing.

Fig. 1

Structures of silvestrol and silvestric acid

Fig. 2

Mass spectra of silvestrol and AP-3. Spectra were obtained from direct infusion of 10 μg/mL of each analyte in positive ion electrospray ionization mode. The chemical structures of silvestrol and AP-3 and their putative fragments are shown in the insets: a mass spectrum and chemical structure of silvestrol with m/z 672 @ 25 V; b chemical structure and MS/MS spectrum of the putative silvestrol fragment at m/z 535; c mass spectrum and chemical structure of AP-3 at m/z 635 @ 25 V; d chemical structure and MS/MS spectrum of the putative fragment ion of AP-3 fragment at m/z 547. Dashed lines in panels a and c indicate putative fragmentation sites

Fig. 3

Total ion chromatograms of silvestrol in mouse plasma. Chromatograms from (a) blank plasma and (b) plasma spiked with 1 ng/mL silvestrol (1.94 min retention time)

Fig. 4

Total ion chromatogram of silvestric acid, silvestrol and AP-3. LC-MS/MS chromatogram showing relative separation of silvestric acid (1.40 min), silvestrol (1.84 min), and AP-3 (2.72 min)

Fig. 5

Silvestrol degradation and silvestric acid formation in rat plasma. Data show relative quantities of compound and metabolite normalized to the average response at 0 (silvestrol, black bars) or 30 (silvestric acid, gray bars) minutes. Error bars are SD from triplicate samples

Fig. 6

Plasma concentration–time profiles of silvestrol. Data points are means + SD of silvestrol in mouse plasma after a IV and IP administration or b oral administration. IV (circles) and IP (triangles) doses were each 5 mg/kg, and oral (squares) doses were 25 mg/kg. N = 6 mice per time point

Fig. 7

Metabolic profile of silvestrol in microsomes. Extracted ion chromatograms showing the abundance of silvestrol and silvestric acid in human, rat, and mouse microsomes. Silvestrol was incubated in: a functional microsomes for 0 min; b inactivated microsomes for 60 min; and c functional microsomes for 60 min