Comparative quantification of pharmacodynamic parameters of chiral compounds (RRR- vs. all-rac-α tocopherol) by global gene expression profiling

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Summary

Pharmacologically active compounds (e.g. from the groups of pharmaceutical drugs, cofactors or vitamins) often consist of two or more stereoisomers (enantiomers or diastereoisomers) which may differ in their pharmacodynamic/kinetic, toxicological and biological properties. A well-known example is vitamin E which is predominantly administered as two different forms, one derived from natural sources (mainly soybeans), and one from production by chemical total-synthesis. While vitamin E from natural sources occurs as a single stereoisomer (RRR-α-tocopherol), synthetic vitamin E (all-rac-α-tocopherol) is an equimolar mixture of eight stereoisomers. Based on a number of animal studies it has been suggested that the biological potency of natural-source vitamin E is 1.36 greater compared to its counterpart produced by chemical synthesis. In this study, we have used the Affymetrix GeneChip® technology to evaluate the feasibility of a new bio-assay where the gene regulatory activities of RRR-α-tocopherol and all-rac-α-tocopherol were quantified and compared on the genome-wide level. For this purpose, HepG2 cells were supplemented with increasing amounts of RRR- or all-rac-α-tocopherol for 7 days. Genes showing a dose-related induction/repression were identified by global gene expression profiling. Our findings show that RRR- and all-rac-α-tocopherol share an identical transcriptional activity, i.e. induce/repress the expression of the same set of genes. Based on the transcriptional dose–response data, EC50 and IC50 values were determined for each of these genes. The feasibility of calculating a “transcriptional potency factor” of RRR- vs. all-rac-α-tocopherol was evaluated by dividing the EC50/IC50 of RRR-α-tocopherol by the corresponding EC50/IC50 of all-rac-α-tocopherol for every of the vitamin E responsive genes. Using this approach we have calculated 215 single biopotency ratios. Subsequently, the mean of all potency ratios was found to be 1.05.

In the present work we propose a new assay for the analysis and comparison of the biological activity and potency of chiral compounds in vivo.

Introduction

Several commonly prescribed drugs, as well as other pharmacologically active compounds such as vitamins are administered as mixtures of stereoisomers. Characterized by their individual three-dimensional configurations, stereoisomers may possess their own unique chemistry, biological activity and pharmacokinetic profile. Such an example is represented by α-tocopherol (vitamin E) which possesses three chiral centres at positions 2, 4′, and 8′, giving rise to four diastereoisomeric pairs of enantiomers, i.e. eight individual stereoisomers (RRR, RSR, RRS, RSS, SRR, SSR, SRS, and SSS) (Kamal-Eldin and Appelqvist, 1996).

While α-tocopherol contained in vegetable oils (nuts, seeds, grains) or industrially produced from natural sources (mainly soybeans), occurs as a single stereoisomer (RRR-α-tocopherol, RRR-α-T), α-tocopherol obtained by chemical total-synthesis (all-rac-α-tocopherol, all-rac-α-T) is an equimolar mixture of all eight stereoisomers (Netscher, 1996, Netscher, 1999). Over the past four decades a number of animal-based assays has been developed in order to characterize and compare the biological activities and biological potencies of the different stereoisomers. The biological activity of a compound describes its specific ability or capacity to achieve an intended biological effect such as, in the case of vitamin E, prevention of fetal resorption (Leth and Sondergaard, 1977; Weiser et al., 1985), prevention of reed blood cell haemolysis (Weiser et al., 1963), curative myopathy (Machlin et al., 1982; Weiser et al., 1985) and more. The biological potency of a substance is defined as the quantitative measure of its biological activity (Hoppe and Krennrich, 2000) and is usually expressed in terms of EC50 and IC50 (concentration or dose of a compound that produces 50% of the maximal possible effect).

Based on these animal studies, it has been suggested that vitamin E stereoisomers possess equal biological activity (Weiser and Vecchi, 1982) but different biological potencies (Weiser et al., 1963, Weiser et al., 1985; Marusich et al., 1968; Hoppe and Krennrich, 2000). In this regard, the biological potency of RRR-α-T was calculated to be 1.36 times of the value of its total-synthetic analogue all-rac-α-T. This factor is believed to reflect the differences in distribution and clearance of the two forms of α-tocopherol in plasma and tissues (Behrens and Madere, 1991; Weiser et al., 1996; Burton et al., 1998).

All these methods share the limitation to measure the biological activity and potency at the level of a single, specific assay. However, it is to assume that any active ingredient, including chiral compounds, might exert more than one biological activity. As a consequence, a given assay can only measure the potency in regard to this one activity, and different values may be obtained if different endpoints are considered.

In 1991, hitherto unknown functions of vitamin E were described for the first time (Boscoboinik et al., 1991). Meanwhile, vitamin E has been shown to regulate enzyme activity (Ricciarelli et al., 1998), cell proliferation (Tasinato et al., 1995) as well as the transcription of numerous genes (Barella et al., 2004), opening new opportunities for the design of alternative assays to measure the biological activity and potency of vitamin E stereoisomers. With regard to the monitoring of gene transcriptional activity, remarkable advances in molecular techniques have made it possible to quantify changes in gene expression at a global, i.e. genome-wide, scale (Schena et al., 1995; Elliott and Ong, 2002). For the first time there is the possibility to identify, quantify and compare all transactivation activities of a given compound, in our case RRR- and all-rac-α-T, on a global level overcoming the limit of a “single assay”-based characterization.

In the current study, we evaluated the feasibility of a novel assay to measure the biological activity and potency of RRR-α-T and all-rac-α-T based on their specific ability to regulate gene transcription at the genome-wide level in human cells.

Section snippets

Cells and cell culture media, vitamin E compounds

HepG2 cells (ATCC HB-8065) were cultured in 6 cm dishes in DMEM medium (GIBCO-Invitrogen, Switzerland) with 10% NU serum™ (Becton Dickinson, Switzerland) containing 1% Penicillin/Streptomycin and undetectable amounts of vitamin E (detection limit 20 nM). Vitamin E compounds were applied as the acetate derivatives: RRR-α-tocopheryl acetate (Sigma and DSM, Switzerland; 99–99.5 weight%, determined by gas chromatography) and all-rac-α-tocopheryl acetate (DSM Nutritional Products Ltd., Kaiseraugst,

Cell growth

Cells were seeded at ∼20% confluence and reached ∼80% confluence after 7 days of culture. No differences in cell growth rate and cell vitality were observed at any time during the experimental procedure between all treatment groups.

Cellular α-tocopherol concentrations

During the supplementation period, cells in all treatment groups were collected every ∼48 h to measure cellular vitamin E content. The mean intracellular concentration (shown are the 10 and 300 μM treatment groups) increased significantly by day 2 reaching a plateau

Discussion

In this study, we aimed at developing a sensitive and universally applicable assay for the analysis of the biological activities and biological potencies of chiral compounds in vivo. Here, we used RRR-α-T and all-rac-α-T as test compounds. In a first step, we investigated the biological activities of α-tocopherol by identifying and comparing the genes that were specifically regulated by these isomers. Thereafter, we determined the biological potency by calculating the EC50 or IC50 of RRR or

Acknowledgments

We would like to thank Dr. D. D’Orazio and Dr. B. Flühmann for the critical review of the experimental data. The technical assistance of D. Crameri and N. Meier is gratefully acknowledged.

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