Biotransformation of letrozole in rat liver microsomes: effects of gender and tamoxifen.

The in vitro metabolic kinetics of letrozole were investigated by incubating letrozole (10-500 microM) in female or male rat liver microsomes to assess the effect of gender and to predict the in vivo biotransformation characteristics of letrozole in rats. The effects of tamoxifen (TAM) on the metabolic kinetics of letrozole were also examined by incubating letrozole in female rat liver microsomes in the presence or absence of TAM. The effects of chronic pretreatment of female rats with TAM (0.5, 1.0, 5.0 mg/kg/day, i.p. for 7 consecutive days) on liver microsomal protein content and metabolic activity were also examined. The formation rate of the carbinol metabolite of letrozole, CGP44 645, was significantly higher (p<0.05) in male rat liver microsomes in comparison to female. The V(max)/K(m) ratio for letrozole metabolism in female rat liver microsomes did not change significantly (p>0.05) in the presence of TAM. After chronic pretreatment of female rats with TAM (up to a dose of 1.0mg/kg/day), the hepatic microsomal protein content was significantly increased but the formation rate of CGP44 645, when normalized for protein content, did not change significantly. These results suggest that there is a marked gender difference in letrozole metabolism in rats. It also appears that acute treatment of female rat liver microsomes with TAM produces negligible inhibitory effect on the CYP mediated metabolic clearance of letrozole. However, chronic pretreatment of female rats with TAM appear to induce CYPs, but does not significantly impact the metabolic activities of the enzymes associated with the formation of the carbinol metabolite of letrozole.

Characterization of ethyl (3-quinuclidinyl) acetate (EQA) as a ligand for acetylcholine receptors.

Recent studies suggest that neuronal nicotinic acetylcholine receptors (nAChRs) may play a role in several CNS disorders and that subtype selective nicotinic ligands may be useful in the treatment of these disorders. Ethyl (3-quinuclidinyl)acetate (EQA) is a bulky, reverse-ester analog of ACh, that produces signs of cholinergic stimulation that may be nicotinic in origin The objective of the present study was to further evaluate EQA as a potential cholinergic ligand. Behavioral studies, smooth muscle assays, and radioligand binding assays were performed on this novel ligand. The effects of EQA on blood pressure and acetylcholinesterase activity were also evaluated. The results of the study suggest that EQA is an agonist at peripheral nicotinic acetylcholine receptors and may have antagonist properties at central nicotinic receptors.

Structure-activity studies of fluoroalkyl-substituted gamma-butyrolactone and gamma-thiobutyrolactone modulators of GABA(A) receptor function.

Dihydro-2(3H)-furanones (gamma-butyrolactones) and dihydro-2(3H)-thiophenones (gamma-thiobutyrolactones) containing fluoroalkyl groups at positions C-3, C-4, and C-5 of the heterocyclic rings were prepared. The anticonvulsant/convulsant activities of the compounds were evaluated in mice. Brain concentrations of the compounds were determined and the effects of the compounds on [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding to the picrotoxin site on GABAA receptors were investigated. The effects of the compounds on GABAA receptor function were studied using electrophysiological methods and cultured rat hippocampal neurons. Fluorination at C-3 results in either subtle or pronounced effects on the pharmacological activity of the compounds. When hydrogens are replaced with fluorines at the methylene carbon of an ethyl group, as in 3-(1,1-difluoroethyl)dihydro-3-methyl-2(3H)-furanone (1), the anticonvulsant actions of the compound are not much changed from those found for the corresponding alkyl-substituted analogue. In marked contrast, fluorination at the methyl carbon of the ethyl group, as in dihydro-3-methyl-3-(2,2,2-trifluoroethyl)-2(3H)-furanone (3), produces a compound having convulsant activity. This convulsant activity seems to be due to an increased affinity of the compound for the picrotoxin site on GABAA receptors caused by an interaction that involves the trifluoromethyl group. Results obtained with gamma-butyrolactones containing either a 3-(1-trifluoromethyl)ethyl or a 3-(1-methyl-1-trifluoromethyl)ethyl substituent indicate that the interactions of the trifluoromethyl group with the picrotoxin binding site are subject to both stereochemical and steric constraints. Sulfur for oxygen heteroatom substitution, as in the corresponding gamma-thiobutyrolactones, affects the type (competitive, non-competitive, etc.) of binding interactions that these compounds have with the picrotoxin site in a complex manner. Fluorination of alkyl groups at the C-4 and C-5 positions of gamma-butyrolactones having convulsant activity increases convulsant potency.

Amino- and amido-tetrabenazine derivatives: synthesis and evaluation as potential ligands for the vesicular monoamine transporter.

Tetrabenazine (TBZ) and dihydrotetrabenazine are well known inhibitors of the CNS vesicular monoamine transporter (VMAT), which is responsible for the packaging of monoamine neurotransmitters in presynaptic vesicles. Amino and amido derivatives of tetrabenazine were prepared as potential ligands for the vesicular monoamine transporter. Ultimately, organotin derivatives of promising ligands were prepared for radiolabeling with 125I. The compounds were evaluated for their ability to inhibit the specific binding of a selective radioligand to the transporter in rat striatal homogenates. Of the compounds evaluated, three amine derivatives of TBZ (primary, secondary and tertiary) were found to have modest to high affinity for the transporter, while two amides exhibited low to undectable affinity. The secondary propargyl amine was found to possess the highest affinity (Ki = 7.6 nM) and was chosen for further evaluation. The organotin derivative of this compound was synthesized in order to prepare the corresponding radioiodinated ligand. However, our inability to synthesize and characterize the iodinated amine precluded its evaluation as a potential radioiodinated ligand for the transporter. Alternative approaches for decreasing the lipophilicity of TBZ analogs while maintaining high binding affinity are currently being explored.

Binding of 125I-iodovinyltetrabenazine to CNS vesicular monoamine transport sites.

Binding characteristics of a novel radioiodinated tetrabenazine (TBZ) analog (iodovinyltetrabenazine; 125I-TBZ-Fraction I) were evaluated. In rat striatal homogenates, 125I-TBZ-I displayed a pharmacological profile consistent with specific binding to vesicular monoamine transport (VMAT) sites. In vitro autoradiographic studies using rat brain sections further demonstrated that 125I-TBZ-I labeled the regions rich in VMAT sites, and it may be a useful marker for these sites. This novel radioiodinated ligand, with high specific activity and high binding affinity, may provide a powerful tool for the in vitro assessment of neuronal loss in various neurodegenerative diseases.

Dicarboxylate diamide dimercaptide (N2S2) technetium-99m complexes: synthesis and biological evaluation as potential renal radiopharmaceuticals.

Novel diamide dimercaptide (N2S2) ligands 4, 5, and 8 have been synthesized and evaluated as potential renal radiopharmaceuticals. The target compounds were prepared in modest overall yields of 22%, 19%, and 20%, respectively, using readily available starting materials. Following in situ deprotection, 99mTc complexes of high radiochemical purity were obtained in excellent yield and were found to be stable for up to 6 h. The 99Tc complex of ligand 8 was isolated as the AsPh4 salt. The X-ray crystallographic data for [99TcO(8)]AsPh4 (space group P2(1)/n: Z = 4, a = 9.342(3) A; b = 18.594(5) A; c = 18.417(7) A; beta, deg = 90.61(3); V, A3 = 3199.1(20)) show that the Tc is bound to both thiolate sulfur atoms and to two deprotonated amide nitrogen atoms. The coordination geometry about the Tc is square-pyramidal with an -yl oxygen atom in the apical position. The Tc-N bond distances (2.002(12) and 1.984(12) A), the Tc-S bond distances (2.300(5) and 2.286(5) A), and the Tc-O bond distance (1.667(11) A) are in good agreement with bond lengths reported for similar complexes. The carboxylate groups are not bonded to the Tc atom in the solid state, nor in CDCl3 solution, as evidenced by X-ray crystal data and solution NMR data, respectively. In the solid state, [99TcO(8)]AsPh4 is monoanionic, therefore, at physiological pH, [99mTcO(8)] is presumably trianionic. Biodistribution studies performed in rats with the 99mTc complexes revealed slow blood clearance and high muscle uptake for these agents. Modest hepatobiliary excretion was observed, and low quantities of the complexes were found in the heart, lungs, and spleen after 1 h. The urinary excretion of the 99mTc complexes of ligands 4, 5, and 8 was found to be slow when compared to the excretion of [131I]OIH in rats (22%, 22%, and 32% vs 85-86%, respectively). Protein binding of 99mTc complexes of ligands 4, 5, and 8 in both rat and monkey plasma was found to be similar to MAG3. While the synthetic schemes reported here supply facile routes to novel N2S2 ligands, biodistribution studies of the 99mTc complexes performed on rats revealed slow renal excretion rates, accompanied by slow blood clearance and high uptake in muscle tissue. Preliminary planar imaging studies in monkeys also revealed slow renal excretion for these agents. The 99mTc complexes evaluated here are poor candidates as renal radiopharmaceuticals.

Direct observation of a fluorinated anticonvulsant in brain tissue using 19F-NMR techniques.

A fluorinated derivative of an anticonvulsant gamma-butyrolactone [alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone; alpha-DFGBL] was synthesized as a probe for NMR spectroscopic observation of the drug in brain tissue. The fluorinated compound is an efficacious anticonvulsant in mice, and inhibits the specific binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to mouse brain membranes with a concentration dependence similar to that of the non-fluorinated compound alpha-ethyl-alpha-methyl-gamma-butyrolactone. Quantitative 19F-NMR spectroscopic studies, coupled with chromatographic measurements of drug tissue concentration, showed that virtually all of the alpha-DFGBL in brain was NMR-observable and that, following intraperitoneal injection, alpha-DFGBL rapidly achieved millimolar concentrations in brain. The 19F-NMR spectra of a alpha-DFGBL in brain and liver tissue were broad (1-2 ppm) and complex, exhibiting multiple chemical shift features. The major chemical shift features in these spectra were assigned on the basis of differential extraction and comparison of 19F spin-spin relaxation times (T2s) and 19F chemical shifts of alpha-DFGBL in tissue to those in pure solvents. The major feature at 10.4 ppm in the tissue spectra was assigned to a weakly polar, membrane-associated environment for the fluorinated compound, while the feature at 11.2 ppm was assigned to an aqueous environment for alpha-DFGBL. The drug was in slow exchange between these two environments in brain. In addition, the feature at lowest field (9.7-9.8 ppm) was identified as a water-soluble hydroxy-acid metabolite of alpha-DFGBL produced by the liver. These data indicate that gamma-butyrolactone anticonvulsants achieve high concentrations in brain, where they exist in several, largely membrane-associated, environments. These findings are consistent with the purported action of the gamma-butyrolactones as low-affinity modulators of gamma-aminobutyric acid-A channels.

Relative anticonvulsant effects of GABAmimetic and GABA modulatory agents.

Anticonvulsant properties of compounds that enhance GABA-mediated inhibition through modulatory sites on the GABAA receptor [phenobarbital (PB), clonazepam (CZP), alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL)] were compared with anticonvulsant effects of compounds believed to be antagonists at these modulatory sites (Ro15-1788 and alpha-isopropyl-alpha-methyl-gamma-butyrolactone gamma-IMGBL)] and to 4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol (THIP, GABAA receptor agonist), (+/-) baclofen (GABAB receptor agonist), and gamma-vinyl GABA, a compound that increases endogenous GABA. The compounds were tested for their ability to block experimental seizures caused by maximal electroshock, pentylenetetrazol, picrotoxin, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), bicuculline (BIC), aminophylline, strychnine, and t-butyl-bicyclophosphorothionate (TBPS) in mice. CZP blocked all but strychnine seizures. PB was also highly effective, blocking all but TBPS seizures. alpha-EMTBL, representing a new class of experimental anticonvulsant drugs, prevented all seizures except strychnine (STR)- and aminophylline-induced seizures. The antagonists are effective only against one convulsant stimulus. Ro15-1788 and alpha-IMGBL prevented only DMCM- and pentylenetetrazol (PTZ)-induced seizures, respectively. THIP and gamma-vinyl GABA both blocked only BIC and picrotoxin seizures. Baclofen had no anticonvulsant activity. These data demonstrate that compounds that increase neuronal inhibition by potentiating the action of GABA have a broader spectrum of anticonvulsant action than either antagonists or GABAmimetic agents or compounds that increase endogenous GABA.

Synthesis and structure-activity studies of alkyl-substituted gamma-butyrolactones and gamma-thiobutyrolactones: ligands for the picrotoxin receptor.

A series of gamma-butyrolactones and gamma-thiobutyrolactones possessing a variety of alkyl groups and alkyl-substitution patterns was prepared and evaluated for anticonvulsant and convulsant activity. Behavioral studies performed on these compounds suggest that maximal anticonvulsant activity (against maximal electroshock and pentylenetetrazol) results when three or four carbon atoms are present at the alpha-position. For convulsant potency, a similar dependence on the size of the alkyl chain at the beta-position was observed. Additional gamma-dimethyl groups were found to increase the convulsant potency of a beta-substituted compound and to cause an alpha-substituted anticonvulsant to become a convulsant. In general, sulfur for oxygen heteroatom substitution in the alpha-substituted lactones resulted in improved anticonvulsant potency and spectrum of activity. Binding of these compounds to the picrotoxin site of the GABA receptor complex was demonstrated with a [35S]-tert-butylbicyclophosphorothionate radioligand binding assay. Measurements of brain concentrations for selected compounds supports a hypothesis that correlates binding to the picrotoxin site with the pharmacological effects of these compounds.

Modulation of the picrotoxin receptor by fluorinated ethyl, methyl-butyrolactones.

A number of investigators have shown that gamma-butyrolactones bind to the picrotoxin site on the gamma-aminobutyric acid (GABA) receptor. We examined the effects of three fluorinated gamma-butyrolactone derivatives on GABA currents and synaptic currents in cultured hippocampal neurons. alpha-(2,2,2-trifluoroethyl)-alpha-methyl-gamma-butyrolactone was a partial picrotoxin agonist, meaning that like picrotoxin, it blocked GABA currents, but not completely. This action is in marked contrast to that of its parent compound, alpha-ethyl-alpha-methyl-gamma-butyrolactone, which is a mixed inverse agonist/antagonist, enhancing GABA currents at low GABA concentration (less than or equal to 1 microM) and antagonizing picrotoxin block of GABA currents at higher GABA concentrations (approximately 30 microM). The properties of two difluorinated derivatives were found to be very different from the trifluorinated compound. Both alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone and alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-thiobutyrolactone+ ++ dramatically increased the current produced by low concentrations of GABA. The former had less marked inverse agonist effects at higher GABA concentrations but behaved instead like a picrotoxin antagonist. In addition, alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone reduced both excitatory and inhibitory synaptic currents suggesting a separate effect on presynaptic transmitter release through an unknown mechanism. These experiments support the hypothesis that gamma-butyrolactones can have a variety of effects at the picrotoxin receptor and indicate that specific properties of these compounds can be altered drastically by fluorination.

Physiological modulation of the GABA receptor by convulsant and anticonvulsant barbiturates in cultured rat hippocampal neurons.

The actions of convulsant and sedative barbiturates on responses to gamma-aminobutyric acid (GABA) application and on inhibitory postsynaptic currents were compared using voltage-clamp techniques in cultured rat hippocampal neurons. The convulsant barbiturates, 5-ethyl-5-(3-methylbut-2-enyl) barbituric acid (3M2B), and (+)-5-ethyl-5-(1,3-dimethylbutyl) barbituric acid [+)-DMBB), and the sedative barbiturate, 5-ethyl-5-(3-methylbutyl) barbituric acid (3MB), all potentiated GABA-mediated chloride currents. In addition, these compounds prolonged the duration of GABAergic inhibitory postsynaptic currents. The similarity between the action of convulsant and sedative barbiturates suggests that the convulsant activity of 3M2B and (+)-DMBB are not mediated by their actions at GABAergic synapses.