In vitro metabolism study of the promising anticancer agent the lignan (−)-grandisin

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Abstract

The lignan (−)-grandisin has shown important pharmacological activities, such as citotoxicity and antiangiogenic, antibacterial and trypanocidal activities. So, it has been considered as a potential drug candidate. In the early drug development process, drug metabolism is one of the main parameters that should be evaluated; therefore, the biotransformation of this lignan by rat liver microsomes was investigated for the first time. In order to perform the biotransformation study and to determine the kinetic parameters, a simple, sensitive and selective HPLC method was developed and fully validated. After method validation, the biotransformation study was accomplished and the kinetic parameters were determined. The biotransformation study obeyed the Michaelis–Menten kinetics. The Vmax and Km were 1.46 ± 0.034 μmol/mg protein/h and 8.99 ± 0.488 μM, respectively. In addition, the formation of dihydro-grandisin, characterized by GC–MS, by mammalian systems indicated the involvement of a CYP450 enzyme type.

Introduction

(−)-Grandisin (Fig. 1) is a known tetrahydrofuran lignan isolated from Piperaceae, Lauraceae, Myristicaceae and Schisandraceae species [1], [2], [3]. This compound has displayed different biological properties, such as larvicidal activity against the mosquito Aedes aegypti [4], trypanocidal activity against the trypamastigote form of Trypanossoma cruzi [5], [6], [7], and antinociceptive and anti-inflammatory activities [8]. In the last years, many studies on cytotoxicity and antitumoral activity showed that (−)-grandisin is a potential candidate as an anticancer agent [9], [10], [11].

The oxidative metabolism has been extensively investigated in the last two decades by means of biological simulating models, using perfused organs, isolated cells or cell fragments (microsomes) [12]. Its importance lies on the fact that the drug candidates, before the final developmental steps as drugs, must be evaluated as safe and effective, including the evaluation of the pharmacokinetic process involved. In addition, the knowledge on interactions of new drug candidates towards oxidative enzymes, their affinity and speed of metabolism is of fundamental importance, particularly when investigations of drug interactions is concerned.

The in vivo study of hepatic metabolism can be estimated from the “turnover” or intrinsic clearance, derived from in vitro tissue preparations. Species-specific metabolism allows for the extrapolation from animals to humans with an additional level of assurance. Since human studies are generally impractical, unethical, or too costly, in vitro systems are the most relevant way to compare metabolic rates between species. To best utilize this approach, the mechanism needs to be conserved across species. For metabolism studies, the enzymatic processes need to be qualitatively the same [13]. Many laboratories have used liver microsomes to perform such metabolism studies. Hepatic microsomes are a flexible system, are a straightforward preparation and they can be stored, with long-term availability [12]. They probably represent one of the most widely used in vitro system for investigating the metabolic profile of a new drug candidate [14], [15]. The recent appreciation of the metabolism study became apparent that understanding the fundamental properties of the enzyme kinetics of cytochrome P450, and thus the rate of biotransformation of a new drug candidate, becomes a parameter of crucial importance in shaping the therapeutic profile [16]. The advantages arising from these studies are mainly to provide information on the biochemical characteristics of a new drug candidate substance increasing the relevance for future clinical studies [17].

Due to the potential of (−)-grandisin as a new lead compound mainly against cancer, the aim of this paper was to characterize its metabolism employing rat liver microsomal fraction (a mammalian species) by evaluating the Michaelis–Menten kinetics parameters (Vmax, Km), the intrinsic clearance (CLint) and the formation of metabolites.

Section snippets

Chemical and reagents

The tetrahydrofuran lignan (−)-grandisin used in the present work was extracted and purified from Piper solmsianum (Piperaceae) according to previous procedures [18]. Standard stock solution of the lignan (−)-grandisin was prepared at the concentration of 1.15 mmol L−1 in methanol. Calibration curve solutions of the lignan (−)-grandisin in the range of 0.00115–1.15 mmol L−1 were obtained by dilutions in the same solvent mixture. These solutions were stored at −20 °C and protected from light. HPLC

Chromatographic determination and method validation

The method used to determine the (−)-grandisin metabolism was validated according to FDA guidelines for the analysis of drugs in biological fluids [20]. Since there is not any official guide for analytical method validation in metabolism studies, it was decided to follow the FDA guideline (at least partially) due to the characteristic of the matrix employed in this study (microsomes) and due to the high degree of acceptability of this guideline. Method validation is an important step in the

Conclusion

This paper describes, for the first time, the CYP450-mediated biotransformation of a promising natural product hit, the lignan (−)-grandisin, using rat microsomal preparation. The kinetic parameters indicated that the biotransformation of this natural product obeys the Michaelis–Menten kinetics and the metabolism indicated the formation of 2,5-dihydro-3,4-dimethyl-2,5-bis(3,4,5-trimethoxyphenyl)-furan as metabolite. It is the first report about the metabolism of this lignan using mammalian

Acknowledgments

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for financial support and for granting research fellowships. The authors also would like to thanks Prof. P.S. Bonato for her technical support.

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