The mineralocorticoid activity of progesterone derivatives depends on the nature of the C18 substituent.

To investigate the role of the C18 substituents in the agonist/antagonist properties of mineralocorticoids, the activities of certain C18-substituted progesterone (P) derivatives were examined. These compounds were characterized by an unsaturated side-chain in the case of 18-vinylprogesterone (18VP) and 18-ethynylprogesterone (18EP) and by an enone group in the case of 18-oxo-18-vinylprogesterone (18OVP). P and its 18-substituted derivatives bind to the recombinant human MR (hMR) overexpressed in Sf9 cells with the following hierarchy of affinity: P > aldosterone > 18VP > 18EP >> 18OVP. Functional cotransfection assays in CV-1 cells, using mouse mammary tumor virus promoter as a steroid receptor-inducible DNA target sequence, indicated that the mineralocorticoid activity depends on the nature of the C18 substituent. 18VP and 18EP retained the antimineralocorticoid feature of P, with the following order of activity: P = 18VP > 18EP. The antagonist potency of 18VP was higher (IC50, approximately 10(-8) M) than that of spironolactone (IC50, approximately 7 x 10(-8) M), the most widely used aldosterone antagonist. Interestingly, introducing an oxo function at C18 conferred agonist mineralocorticoid properties; 18OVP behaves as a full agonist (ED50, approximately 10(-7) M) with no antagonist activity. In contrast to what was observed when the three 18-substituted P derivatives acted through hMR, they retained the agonist feature of P through the human P receptor, with the following order of potency: P > 18VP = 18OVP > 18EP. The activity of the 18-substituted P derivatives through the human glucocorticoid receptor was only detected at concentrations higher than 10(-6) M; P and 18VP displayed a partial antagonist activity, whereas 18OVP had a full agonist activity (ED50, approximately 2 x 10(-6) M). Thus, the presence of an oxo group at C18(18OVP) does not change the agonist feature of P through human P receptor, but confers to the ligand an agonist activity through hMR, suggesting that the C18 carbonyl group of aldosterone plays a crucial role in its agonist activity.

Short-term pharmacokinetics and brain distribution of mecamylamine as a preliminary to carbon-11 labeling for nicotinic receptor investigation.

As a preliminary to development and evaluation of labeled mecamylamine as a potential in vivo imaging ligand for human central nicotinic receptors (nAchRs), this work was intended to determine whether the pharmacokinetic properties of mecamylamine are suitable for experimental studies using (11)C-radiolabeled mecamylamine preliminary to positron emission tomography (PET) in humans. An original gas chromatographic method for rapid and simple determination of mecamylamine in biological samples was developed and validated (within run precision, 3.8-5.2%; between assay variation, 5.3-6.9%; assay accuracy, 5.6-11.8%). The results of the pharmacokinetic investigation in the rat demonstrated a very fast clearance of mecamylamine from blood [half-life, 1.2 h; clearance (CL), 1.2 L/kg/h) concomitant with an uptake that was higher in kidney, intermediate in lung, and lower in heart, liver, and brain. Brain tissue kinetics of mecamylamine showed a similar pattern for all the regions, with a rapid increase followed by a plateau after 15 min. This plateau differed according to the region of the brain; it was higher in colliculi, hippocampus, and cortex (area of high density of nAchRs) than in cerebellum or white matter (area with a limited population of nAchRs). No other lipophilic metabolites that were able to disturb the specific binding to nAchRs were identified during the investigation. Thus, mecamylamine shows peculiar qualities making it a good candidate for carbon-11 labeling for experimental studies in view of final PET imaging.

PET and SPECT imaging of the NMDA receptor system: an overview of radiotracer development.

Imaging the N-methyl-D-aspartate receptors (NMDARs) in the living human brain by positron emission tomography (PET) or single photon emission computed tomography (SPECT) would provide useful information on the role of these receptors in ischemia and in various neurological disorders such as degenerative diseases, epilepsy or schizophrenia. To assess NMDAR radiotracer development and to propose perspectives, we overviewed the PET and SPECT candidate radioligands developed until now. Labelled molecules of interest were classified in three groups according to their binding site: intrachannel pore site blockers, glycine site inhibitors and NR2B selective subunit antagonists.

Mechanism-based inactivation of bovine cytochrome P-450(11beta) by 18-unsaturated progesterone derivatives.

Two 18-unsaturated progesterone derivatives, 18-vinylprogesterone (18-VP) and 18-ethynylprogesterone (18-EP) have proved to be potent inhibitors of the bovine cytochrome P-450(11beta), the enzyme involved in the last steps of aldosterone biosynthesis [Delorme, C., Piffeteau, A., Viger, A. & Marquet, A. (1995) Eur. J. Biochem. 232, 247-256]. In the present study, we demonstrate that these two compounds exhibit the characteristics of mechanism-based inactivators of this enzyme. Inactivation followed pseudo-first-order and saturation kinetics. The kinetic parameters of inactivation were k(i) = 0.11 min(-1) and Ki = 4 microM for 18-VP, and k(i) = 0.12 min(-1) and 22 microM for 18-EP. Inactivation of P-450(11beta) activity was strictly dependent on the presence of NADPH. Protection by the substrate deoxycorticosterone was observed, demonstrating a selective modification at the substrate-binding site. With radiolabeled 18-VP, inactivation was shown to be irreversible with a stoichiometry of 1.4 mol bound [3H]18-VP/mol inactivated cytochrome P-450(11beta). SDS/PAGE analysis of the [3H]18-VP-inactivated enzyme showed that, under conditions preventing heme dissociation, the P-450(11beta) band was labeled, while no labeling of the apoprotein was observed under denaturating conditions. Furthermore, the loss of catalytic activity could be correlated with the destruction of the P-450 chromophore evaluated by the FeII-CO versus FeII difference spectra. These arguments led us to propose that 18-vinylprogesterone inactivates cytochrome P-450(11beta) by heme destruction rather than by protein modification.

Functional characterization of a new class of odorant-binding proteins in the moth Mamestra brassicae.

A new protocol of binding assay allowed us to functionally characterize two additional odorant-binding proteins in antennae of the moth Mamestra brassicae. These proteins have no N-terminal sequence homology with the moth pheromone-binding proteins and general odorant-binding proteins previously described. One of the two proteins designated MbraAOBP2 is between 60 and 73% similar in N-terminal to several proteins characterized in chemosensory organs of Diptera, Hymenoptera, Lepidoptera, and Phasmids, indicating that these proteins constitute a new group of odorant-binding proteins. A particularly high similarity between MbraAOBP2 and ejaculatory bulb specific protein III of Drosophila suggested that vaccenyl acetate could be a specific ligand for these proteins. As a matter of fact, MbraAOBP2 bound vaccenyl acetate in vitro, but we failed to detect any receptor cell in long and short sensilla trichodea of males. This protocol could be used as a rapid method to identify new odorant-binding proteins in chemosensory organs or tissues.

Radiosynthesis of [18F]Lu29-024: a potential PET ligand for brain imaging of the serotonergic 5-HT2 receptor.

In a previous work, Lu29-024 (2,5-dimethyl-3-(4-fluorophenyl)-1-(1-methyl-4-piperidinyl)-1H-indole), a selective 5-HT2A receptor antagonist with nanomolar affinity and high selectivity, was labeled with carbon-11 to evaluate its behavior as a potential PET ligand for the serotonergic 5-HT2A receptor in the central nervous system. Administration of this tracer to rats was followed by a good brain uptake, no brain labeled metabolites but no specific, regio-selective, binding at 20 and 40 min post injection. Despite this, the data noted at 20 and 40 min suggest that this tracer, if associated with a radioactive emitter with a longer half-life than that of carbon-11, could be useful for the quantification of 5HT2A receptors. For these reasons, we chose to label this compound, bearing a fluorine atom, with [18F]fluoride, in order to perform rat studies over a more prolonged time-scale. The precursor for the radiosynthesis of [18F]Lu29-024 was obtained in an overall yield of 20% by a multi-step synthesis including an acetonylation reaction followed by a Fisher indole reaction. The radiotracer was prepared by an aromatic substitution with activated [18F]fluoride followed by a decarbonylation reaction that employed Wilkinson's catalyst. The radiosynthesis of [18F]Lu29-024 required approximatively 110 min with an overall radiochemical yield of 20-35% and specific activities of 37GBq/micromol. Fluorine-labeled Lu29-024 may thus be envisaged as a potentially useful PET tracer that can be applied to a wide range of neurological and psychiatric diseases.