Jacky Van Dun

Comparative effects of the aromatase inhibitor R76713 and of its enantiomers R83839 and R83842 on steroid biosynthesis in vitro and in vivo.

R76713 (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole) is a selective, non-steroidal aromatase inhibitor containing an asymmetric carbon atom. In this paper, we compare the effects of R76713 (racemate) with its enantiomers R83839 (the levo-isomer) and R83842 (the dextro-isomer) on steroid biosynthesis in rat cells in vitro and in the rat in vivo. In rat granulosa cells, aromatase activity was inhibited by 50% at concentrations of 0.93 nM of R76713, 240 nM of R83839 and 0.44 nM of R83842, revealing a 545-fold difference in activity between both enantiomers. Up to 1 microM, none of the compounds had any effect on steroid production in primary cultures of rat testicular cells. Above this concentration all three compounds showed a similar slight inhibition of androgen synthesis with a concomitant increase in the precursor progestins, indicative for some effect on the 17-hydroxylase/17,20-lyase enzyme. In rat adrenal cells none of the compounds showed any effect on corticosterone synthesis. At concentrations above 1 microM there was an increase in the levels of 11-deoxycorticosterone pointing towards an inhibition of the 11-hydroxylase enzyme. This increase was more pronounced for R83839 than for R76713 and R83842. In vivo, in PMSG-primed rats, R83842 reduced plasma estradiol by 50%. 2 h after oral administration of 0.0034 mg/kg, whereas 0.011 mg/kg of R76713 and 0.25 mg/kg of R83839 were needed to obtain the same result. Oral administration of up to 20 mg/kg of the compounds did not significantly affect plasma levels of adrenal steroids in LHRH/ACTH-injected rats. Plasma testosterone was lowered at 10 and 20 mg/kg of R83842 and at the highest dose (20 mg/kg) of R76713 and R83839. In conclusion, the present study shows that the aromatase inhibitory activity of R76713 resides almost exclusively in its dextro-isomer R83842. R83842 exhibits a specificity for aromatase as compared to other enzymes involved in steroid biosynthesis of at least a 1000-fold in vitro as well as in vivo. This confirms the extreme selectivity previously found for the racemate.

Neurotrophic and Antileukemic Daphnane Diterpenoids from Synaptolepis kirkii

Biological assay guided fractionation of a dichloromethane extract of Synaptolepis kirkii led to the isolation of four new and five known daphnane-type diterpene orthoesters, whose structure was established by spectroscopic data. Full spectroscopic data of the new and known natural products are reported here for the first time. Pronounced neurotrophic and substantial antileukaemia activities of these compounds were found in in vitro assays.

Solubilization of rat brain serotonin-S2 receptors using chaps/salt

Serotonin-S2 receptors from rat frontal cortex were solubilized with a mixture of 6.8 mM CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate) and 1.4 M sodium chloride. This new solubilization procedure solubilized about 40% of the membrane-bound serotonin-S2 receptors in an active form. The solubilized receptors were not sedimented after 1 h of centrifugation at 100 000 X g and they passed freely through 0.20 micron filters. The solubilized preparation showed high affinity binding of [3H]ketanserin and revealed a typical serotonin-S2 receptor profile: binding could be displaced by nanomolar concentrations of different serotonin antagonists and by micromolar concentrations of serotonin agonists. Compounds belonging to other pharmacological classes were poorly, or not active. Upon density gradient sedimentation, Svedberg coefficients of approximately 5 S were found on sucrose gradients made with H2O or D2O as the solvent. This was much lower than the value of 11.5 S previously reported from lysophosphatidylcholine-solubilized serotonin-S2 receptors.

7-Amino-8-[125I]-ketanserin ([125I]AMIK), a highly sensitive, serotonin-S2 receptor ligand

Abstract Several [ 3 H] radioligands have been proposed for In vitro biochemical characterization of serotonin-S 2 receptors. Among the most widely used are [ 3 H]spiperone and [ 3 H]ketanserin. Recently, a derivative of ketanserin, [ 3 H]7-aminoketanserin has been Introduced as a high affinity selective serotonin-S 2 receptor ligand with a very low non-specific binding [1]. A major drawback of all these ligands, however. Is the low specific radioactivity especially if one wants to study tissues with low receptor contents. [ 125 I]-Labelled compounds have much greater sensitivity than tritiated probes. The available iodinated serotonin-S 2 receptor ligands [ 125 I]LSD [2] and [ 125 I]MIL (a methylated derivative of [ 125 I]LSD) [3] show, however, the major disadvantage of interacting with other serotonin binding sites at nanomolar ligand concentrations [4]. It therefore seemed desirable to develop a [ 125 I]-labelled compound, based on a selective ligand for the serotonin-S 2 receptor subtype, such as 7-aminoketanserin.

Characterization of cholate-solubilized dopamine receptors from human, dog and rat brain

[3H]Spiperone binding sites were solubilized in high yield from human, dog and rat brain with a mixture of sodium cholate (0.3% w/v) and sodium chloride (1.4 M). The binding sites were not sedimented after one hour at 100,000 g, they passed freely through 0.20 micron filters, migrated as a single peak in gradient sedimentation and were retarded upon gel filtration, proving that they were truly solubilized. The solubilized binding sites were definitely of dopaminergic nature. They showed saturable, reversible, high affinity binding of [3H]spiperone; displacement of [3H]spiperone binding by nanomolar concentrations of dopamine antagonists and micromolar concentrations of serotonin antagonists; stereo-specificity and a good correlation with drug affinities for membrane preparations. The non-displaceable, non-specific [3H]spiperone binding was very low. Gradient sedimentation analysis revealed a sedimentation coefficient of 12 S for dog solubilized preparations, 9 S for rat solubilized preparations and only 2.5 S for human solubilized preparations (values, uncorrected for detergent binding). Gel filtration experiments seem to confirm these molecular characteristics. Therefore the present results show that the dopamine receptor reveals the same pharmacological properties when solubilized with cholate-salt from rat, dog or human brain, while physico-chemical properties seem to indicate some differences.

Real-time gene expression analysis in human xenografts for evaluation of histone deacetylase inhibitors

Real-time analysis of gene expression in experimental tumor models represents a major tool to document disease biology and evaluate disease treatment. However, monitoring gene regulation in vivo still is an emerging field, and thus far it has not been linked to long-term tumor growth and disease outcome. In this report, we describe the development and validation of a fluorescence-based gene expression model driven by the promoter of the cyclin-dependent kinase inhibitor p21waf1,cip1. The latter is a key regulator of tumor cell proliferation and a major determinant in the response to many anticancer agents such as histone deacetylase inhibitors. In response to histone deacetylase inhibitors, induction of fluorescence in A2780 ovarian tumors could be monitored in living mice in a noninvasive real-time manner using whole-body imaging. Single p.o. administration of the histone deacetylase inhibitor MS-275 significantly induces tumor fluorescence in a time- and dose-dependent manner, which accurately predicted long-term antitumoral efficacy in individual mice following extended treatment. These findings illustrate that this technology allows monitoring of the biological response induced by treatment with histone deacetylase inhibitors. In addition to providing experimental pharmacokinetic/pharmacodynamic markers for investigational drugs, this model provides insight into the kinetics of in vivo regulation of transcription, which plays a key role in causing and maintaining the uncontrolled proliferation of tumor tissue. [Mol Cancer Ther 2006;5(9):2317–24]

Identification of the serotonin‐S2 receptor ligand binding site by photoaffinity labelling with 7‐azido‐8‐[125I]ketanserin ([125I]AZIK)

7‐Azido‐8‐[125I]ketanserin ([125I]AZIK) was characterized as a potent photoaffinity probe for serotonin‐S2 receptors. In reversible binding experiments, [125I]AZIK bound with high affinity (Kd = 0.69 nM) to rat frontal cortex membranes. When incubation with [125I]AZIK was followed by UV irradiation, the binding was found to be irreversible. Protection experiments with various drugs demonstrated the serotonin‐S2 nature of the photoaffinity labelling. SDS‐polyacrylamide gel electrophoresis of the photolabelled membranes allowed one to identify the serotonin‐S2 receptor ligand binding site as a single polypeptide with a molecular mass of approx. 67500 Da. [125I]AZIK will be a valuable tool for the elucidation of the serotonin‐S2 receptor structure.

Differential photoaffinity labelling of serotonin‐S2 receptors and histamine‐H1 receptors using 7‐azidoketanserin

7‐Azidoketanserin, a potent photoaffinity probe for serotonin‐S2 receptors was shown to irreversibly photoinactivate histamine‐H1receptors as well. The photolabelling of H1‐receptors could be prevented by several selective histamine‐H1 antagonists. In guinea pig cerebellum, a brain area that is highly enriched in H1‐receptors, photolabelling could be directed exclusively to these receptors by adding a high concentration of pipamperone, which selectively blocks serotonin‐S2 receptors. In rat pre‐frontal corex, a region that is enriched in serotonin‐S2 receptors, pyrilamine was used to block H1‐receptors, thereby directing the photolabelling exclusively to S2‐receptors.

Identification of a series of substituted 2-piperazinyl-5-pyrimidylhydroxamic acids as potent histone deacetylase inhibitors

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Photoaffinity labeling of human brain dopamine receptors

Abstract During recent years, a considerable amount of research has been directed towards the characterization and purification of hormone and drug receptors. Neuroleptlc drugs are thought to act by blocking brain dopamine receptors; in vitro binding assays using either [3H] haloperidol [1,2] or [3H] spiperone [3] have provided more direct evidence for this interaction. Although [3H] spiperone was also found to label serotonin-S2 receptors [4], it remains the more appropriate ligand when used with selective displacers since it is capable of labeling dopamine receptors both in vitro and in vivo [5] and even when the receptor has been reduced to a macromolecular form [6]. Dopamine receptors have also been identified in human brain by in vitro binding assays [7]. Photoaffinity labeling has been a valuable tool for the elucidation of the molecular structure of several receptor sites [8,9]. Recently, we developed a photoaffinity compound azapride (4-azido-5-chloro-2-methoxy-N-[l-(phenylmethyl)-4-piperidinyl]benzamide) which is an azide analogue of clebopride, a selective dopamine antagonist [10,11]. The synthesis and the binding characteristics in dog striatum will be described elsewhere [12]. In the present paper, we report on the use of this photoaffinity probe to label dopamine receptors from human brain.