Analytical method development for synthesized conjugated metabolites of trans-resveratrol, and application to pharmacokinetic studies
Highlights
► Synthesis and purification of R3S, R4′S, R3G, and R4′G. ► Development and validation of highly sensitive LC–MSn assay for all analytes. ► Application to pharmacokinetic studies in a mouse model.
Introduction
Trans-3,5,4′-trihydroxystilbene (trans-resveratrol, RES) is a dietary phytochemical thought to have beneficial health effects via pleiotropic mechanisms [1], [2]. One major hurdle to its development is its extremely low oral bioavailability due to metabolism [3], [4]. RES is known to be efficiently and almost completely converted in humans to its glucuronidated and sulfated metabolites. Human studies indicate that systemic exposure of RES is predominantly in the form of its conjugated metabolites [5], [6]. This has led to the hypothesis that the conjugates of RES might themselves be active, and therefore need to be evaluated.
While a few studies have attempted evaluation of RES conjugates in vitro [7], it is critical to evaluate the in vivo disposition of these metabolites. The pharmacokinetics (PK) of metabolites are expected to be different from those of the parent. Thus, knowledge of systemic levels of metabolites is necessary in order to correlate metabolite exposure to any observed pharmacologic activity.
As stated above, one hypothesis is that conjugated metabolites of RES are themselves active. However, the possibility of inactive metabolites also exists. Conjugation of RES is a complex process. Sulfation and glucuronidation are reversible processes and reversible metabolism can result in these metabolites acting as ‘depots’ for the active parent. Further, these conjugates are good candidates for enterohepatic recirculation. Recirculation of RES metabolites has indeed been suggested in preclinical studies [8]. The pharmacokinetics and pharmacodynamics of RES and its metabolites is therefore complicated by extensive first-pass metabolism, reversible conjugation, and enterohepatic recirculation.
The comprehensive study of RES and its metabolites in vivo requires two critical tools: (i) synthesis of adequate amounts of pure RES metabolites for in vivo dosing experiments, and (ii) a validated bioanalytical assay with low detection capability in order to quantitate low circulating levels of RES and its metabolites. The present work aimed at developing these two tools. Synthetic methods were developed for four monoconjugates of RES: the 3- and 4′-monosulfates (R3S and R4′S respectively), and the 3- and 4′-monoglucuronides (R3G and R4′G respectively). Our synthetic methods allowed us to produce adequate levels of pure metabolites that had application in subsequent bioanalytical assay development as standards, as well as in vivo PK studies. It should be noted that while these monoconjugates have recently become commercially available, their cost is prohibitive to conducting in vivo studies in replicates necessary for statistical power. We developed and validated an LC–MSn assay for the quantitation of RES and each of its monoconjugates at low concentrations. While methods have been reported for the quantitation of RES and qualitative identification of its metabolites [9], to our knowledge direct quantitation of RES metabolites has not been conducted to date. RES metabolites have previously been evaluated by hydrolysis and against a RES standard curve. Finally, we applied our synthetic metabolite standards and validated bioanalytical assay to a pharmacokinetic analysis of RES and its metabolites in a mouse model.
Section snippets
Materials
Resveratrol (trans-resveratrol, purity >99%; RES), chlorosulfonic acid, anhydrous pyridine, sulfur trioxide pyridine complex, sodium carbonate, acetobromo-alpha-d glucuronic acid methyl ester, sodium methoxide, acetic acid, tetrahydrofuran (THF) and methanol were purchased from Sigma-Aldrich (St. Louis, MO). R3S, R3G and R4′G for initial calibration were purchased from Toronto Research Chemicals (North York, Canada). Ammonium acetate was purchased from ThermoFisher Scientific (Pittsburgh, PA).
Synthesis of RES glucuronides and sulfates
The synthesis of the glucuronides was accomplished using a modification of the procedure reported by Vitaglione and co-workers [15]. The route used to prepare R3G and R4′G is shown in Fig. 1. RES mono-sulfates were prepared using modifications to two precedented procedures [11], [12], [13], [19]. Fig. 2A depicts the use of SO3·Py complex while Fig. 2B involves the use of chlorosulfonic acid. Yields and spectroscopic data for RES mono-sulfates and glucuronides are listed in Table 1.
LC–MS/MS assay for quantitation of RES and its metabolites
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Synthesis of RES metabolites
For RES glucuronidation, facile synthesis of the 3 and 4′ glucuronide products was achieved with the use of 1eq of acetobromo glucuronic acid methyl ester. This method is a modification of the procedure reported by Vitaglione and co-workers [15]. This one pot reaction involves the use of NaOMe in methanol which serves as the protonating agent for RES and the deprotecting agent for the acetyl groups.
The majority of reports on the chemical synthesis of RES sulfates [17], [23], [24], [25] use a
Conclusion
All four monoconjugates of RES – R3S, R4′S, R3G, and R4′G – have been successfully synthesized, purified, and characterized. These metabolites were utilized as synthetic standards to develop and validate a highly sensitive LC–MSn assay for concomitant quantitation of RES and all its monoconjugates. Together, synthetic metabolites and validation of a bioanalytical method were applied to characterize the plasma PK of RES in mice. Future studies will include characterization of biological activity
Acknowledgments
The work was partially supported by Award number R03CA133943 from the National Cancer Institute to SN and DJC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NCI. The authors are grateful also for support supplied by Temple University Graduate School (OFI) and to the School of Pharmacy (SS).
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These authors contributed equally to this work.