Abstracts to the 2010 Congress on Steroid Research

Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

C o n te n t s l i s t s ava i l a b l e a t S c i e n c e D i re c t

Journal of Steroid Biochemistry and Molecular Biology j o u r n a l h o m e p a ge : w w w. e l s e v i e r. c o m / l o c a te / j s b m b

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

[P1.71] Biological assessment of novel glycyrrhetinic acid derivatives acting as selective inhibitors for 11ȕ-hydroxysteroid dehydrogenase type 2 D.V. Kratschmar*1, A. Vuorinen2, T. Da Cunha1, G. Wolber3, D. Classen-Houben4, O. Doblhoff4 1 University of Basel, Switzerland, 2Institute of Pharmacy / Pharmaceutical Chemistry, Australia, 3 Free University Berlin, Germany, 4Onepharm Research & Development GmbH, Australia Introduction: Availability of glucocorticoids and activation of glucocorticoid receptors (GR) is tightly regulated by 11ȕ-hydroxysteroid dehydrogenase 1 (11ȕ-HSD1) and 11ȕ-HSD2. 11ȕ-HSD2 catalyzes the conversion of active 11ȕ-hydroxyglucocorticoids (cortisol, corticosterone) into inactive 11-ketoglucocorticoids (cortisone, 11-dehydrocorticosterone). Impaired local glucocorticoid metabolism has been associated with several disease states, and modulation of intracellular glucocorticoid availability is considered to be a promising strategy to treat glucocorticoid-dependent diseases. 18ȕ-glycyrrhetinic acid (GA), the biologically active triterpenoid of licorice (Glycyrrhiza), represents a well-known but non-selective inhibitor of both 11ȕ-HSD enzymes. Selective inhibitors are needed for mechanistic studies and future therapeutic applications. In the present study, we biologically characterized a set of novel and selective 11ȕHSD2 inhibitors. Methods: The activities of selected inhibitors were compared in assays using lysates and intact cells expressing recombinant human enzymes. Species-specific differences were considered by comparing inhibitory activities of the compounds on human and mouse 11ȕ-HSD2. Further, the impact of the GA derivatives on 11ȕ-HSD-dependent modulation of GR transactivation activity was assessed. In an attempt to understand the selectivity of the GA derivatives to inhibit 11ȕHSD1 and 11ȕ-HSD2, respectively, an 11ȕ-HSD2 homology model based on structural information on the related 17ȕ-HSD1 was generated and applied together with our recently constructed pharmacophore of 11ȕ-HSD1. Results: Modifications of functional groups on the triterpenoid backbone led to selective inhibitors. Specific modifications at the 3-hydroxyl and the carboxyl of GA rendered high selectivity for 11ȕ-HSD2. The data generated significantly extend the current knowledge about structure activity relationships of GA derivatives as inhibitors of human 11ȕ-HSDs. Conclusions: The structural analyses provide an explanation for the differences in the selectivity and activity of the GA derivatives investigated. The most potent and selective 11ȕ-HSD2 inhibitors should prove useful as mechanistic tools for further anti-inflammatory and anti-cancer in vitro and in vivo studies. Keywords: 11beta-hydroxysteroid dehydrogenase, pharmacophore, inhibitor, glycyrrhetinic acid

Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

[P2.74] Dehydroepiandrosterone’s antiepileptic action in iron-induced animal model of posttraumatic epilepsy M. Mishra*, R. Singh and D. Sharma Jawaharlal Nehru University, India Introduction: Neuroactive steroids (estrogen, progesterone, and testosterone) are important in the physiology and pharmacology of epileptic disorders. There is an increased incidence of epilepsy in elderly. The level of Dehydroepiandrosterone (DHEA) declines with age. DHEA is an antioxidative agent, involved in neuronal excitability as it affects GABA/glutamate receptors. The purpose of the present study was to determine whether DHEA has an antiepileptic effect. In the present experiments the antiepileptic effect of DHEA was studied in the iron-induced animal model of posttraumatic epilepsy. Method: To determine the antiepileptic action of DHEA, observations were made on epileptic seizure activity in iron-induced chronic epileptogenic foci in rat brain. Antiepileptic influence was ascertained on the basis of epileptiform electrical activity, biochemical parameters, gene expression and cognitive-behavioral parameters. Result: DHEA (30 mg/kg/day) administered for 21 days to epileptic rats prevented the epileptiform electrophysiological activity. DHEA also prevented epileptiform activity-related behavioral alterations studied by Morris water maze and open field tests in epileptic animals. DHEA also augmented the expression of CREB in epileptic rats. Intracerebroventricularly administered DHEA (200 n mole/day for 5 days) also attenuated seizure activity. Alterations in epileptogenesis-related biochemical parameters: lipid peroxidation, protein oxidation, membrane fluidity, acetylcholinesterase and Na+, K+- ATPase activities were also countered by DHEA. Discussion: Our study demonstrated that DHEA suppressed iron-induced experimental seizure activity, and countered epileptogenesis-related cognitive deficits. The results points to that DHEA can be an antiseizure compound clinically. Keywords: Dehydroepiandrosterone, Iron-induced epilepsy, posttraumatic epilepsy, Morris water maze

Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

[P2.76] LC-MSMS method development for steroids analysis at Low Detection Limits H.F. Liu1, J. McFarlane1, B. Fernandez1, R. Huang1, M. Jarvis*2, A. Taylor2 1 AB SCIEX, USA, 2AB SCIEX, Canada Steroid analysis is critical for research into a number of common endocrine disorders and also applied as biomarker for numbers of diseases. Major challenges of steroids analysis include sensitivity, selectivity, and large sample volume. Using liquid chromatography and mass spectrometry to analyze steroids at low level has been becoming a new trend to overcome these challenges. Three major steroid methods will be presented including: testosterone for low level detection; estrogens for pg/mL detection; steroids panel including 11 major steroids for general screening. Testosterone, the major androgenic hormone in humans, is commonly measured for its excess or deficiency. The concentration of testosterone in women, children and men undergoing antiandrogen therapy are typically less than 500 pg/mL. A simple one step liquid-liquid extraction method was developed using 200µL serum with 10 pg/mL detection limit and less than 10% CV. Good correlation was observed between the results obtained from this method and other reference results. Estrogens are a group of steroid compounds functioning as the primary female sex hormone. Accurate low pg/mL level detection is required for estrogen analysis often obtainable through multiple complex sample preparation including derivatization. We developed a two step liquidliquid extraction without derivatization method, coupling with mobile phase modifier to improve the ionization, achieving less than 5pg/mL quantification limit; less than 10%CV. Steroidogenesis is the process wherein desired forms of steroids are generated by transformation of other steroids. An LC-MSMS method for analysis of 11 major steroids products covering the wide concentration range from µg/mL to pg/mL using simple protein precipitation sample preparation was developed and verified. The cross interference from high concentration steroid analytes to low concentration analytes and isomer separation are evaluated and considered in the method. Keywords: LC/MS/MS, Low level testosterone, Steroid panel,

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Abstracts / Journal of Steroid Biochemistry & Molecular Biology 126S (2011) e5–e198

[P2.78] 17Beta-Hydroxysteroid Dehydrogenase Type 1 expression is determinant in the estradiolestrone ratio in breast cancer cells C. Zhang*1,2, J. Chen3, J. Aka1, D. Yin2, S. Lin1 1

Centre Hospitalier de Université Laval Research Center (CHUL, CHUQ) and Laval University, Canada, 2Northwestern Polytechnic University, China, 3Chinese Academy of Sciences, China

Estradiol (E2), the most potent estrogen, stimulates the development of breast cancer cells (BCCs), and the cellular E2/E1 ratio is considered important to the proliferation of these cancer cells. Reductive 17ȕ-hydroxysteroid dehydrogenases (17ȕ-HSDs) are involved in the last step of E2 synthesis. Several breast cancer cell lines were chosen to evaluate E2/E1 ratio, including ER positive cell lines (T47D, MCF-7 and ZR75-1), and ER negative cell line (BT-20), and choriocarcinoma cell line (JEG-3) as the controls. Four different concentrations of E1 and E2 was added in the cell culture to be used as the substrates, the E2/E1 ratio was always 9:1 after incubation for 24 hour in all the chosen cell lines with high expression of 17ȕ-HSD1, while E2/E1 ratio was less than 1:5 in cells with low expression of 17ȕ-HSD1 (e.g., MCF-7). In order to know whether the intracellular environment or 17ȕ-HSD1 expression level was determinate for the high E2/E1 ratio, the conversion of E1 to E2 was tested in MCF-7 over expressing 17ȕ-HSD1 (MCF-71), HEK 293 over expressing 17ȕ-HSD1 (HEK 293-1) were used as control. E2/E1 ratio was modified to 9:1 in both HEK 293-1 and MCF-7-1 cells. E2/E1 ratio was decreased to 5:95 after treatment with specific siRNAs for 17ȕ-HSD1 in ER positive cell line (T47D) and ER negative cell line (BT-20). It demonstrates that 17ȕ-HSD1 expression level determines the high ratio of E2/E1. Further, some kinetic study was carried out in HEK 293-1 and breast cancer cell line (T47D). The Km value were respectively 0.25 uM and 0.12 uM for E1 to E2 conversion in HEK 293-1 and T47D cells, demonstrating a strong apparent affinity of 17ȕ-HSD1 to the estrone substrate. Keywords: 17ȕ-hydroxysteroid dehydrogenases 1, estradiol/estrone ratio, breast cancer cell, intracellular kinetics