Raloxifene and estradiol benzoate both fully restore hippocampal choline acetyltransferase activity in ovariectomized rats.

Selective estrogen receptor modulators (SERMs) demonstrate tissue-specific estrogen receptor (ER) agonist or antagonist properties. Raloxifene, a prototypical SERM, has ER agonist properties in bone and on cholesterol metabolism but full antagonist properties in the uterus and breast. To characterize the ER agonist/antagonist profile of raloxifene in the brain, we have examined its effect on the activity of a known estrogen-responsive gene product, choline acetyltransferase (ChAT), in the hippocampus and other brain regions of 6-month-old ovariectomized (OVX) Sprague-Dawley rats. Three weeks post-ovariectomy, animals received estradiol benzoate (EB, 0.03 mg or 0.3 mg kg(-1) day(-1) for 3 or 10 days); raloxifene HCl (3.0 mg kg(-1) day(-1) for 3 or 10 days), tamoxifen (3.0 mg kg(-1) day(-1) for 10 days) or vehicle (20% CDX). As previously reported, ChAT activity decreased by approximately 20%-50% in the hippocampus of OVX compared with SHAM-operated control rats with no change in ChAT activity observed in the hypothalamus. Raloxifene or EB reversed the OVX-induced decrease in ChAT activity in the hippocampus but did not change ChAT activity in the hypothalamus. Animals that received combined EB (0.03 mg/kg) plus raloxifene (1 mg/kg) or tamoxifen alone (3.0 or 10 mg/kg) also showed increased hippocampal ChAT activity. Raloxifene failed to increase uterine weight and blocked the estrogen-induced increase in uterine weight, while another SERM, tamoxifen, increased uterine weight. These data demonstrate that raloxifene has estrogen-like properties on hippocampal ChAT activity in vivo, and suggest that benzothiophene SERMs may exert estrogen-like beneficial effects on cholinergic neurotransmission in brain without producing peripheral stimulation of breast or uterine tissue.

Synthesis and pharmacology of conformationally restricted raloxifene analogues: highly potent selective estrogen receptor modulators.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.

Effects of prolonged ethinyl estradiol treatment on calcium channel binding and in vivo calcium-mediated hemodynamic responses in ovariectomized rats.

Ethinyl estradiol (EE2), administered orally to ovariectomized (ovex) rats, has been shown to prevent loss of bone mineral density and to decrease serum cholesterol levels. Radioligand binding studies with the dihydropyridine (DHP) [3H]PN200-110 were undertaken to characterize calcium (Ca++) channels in cardiac and aortic tissues from ovex rats treated for 35 days with EE2 (0.1 mg/kg day p.o.) or vehicle, and from vehicle-treated sham-operated controls (sham). Cardiac tissues from EE2-treated rats displayed significant increases in the density (Bmax) of high-affinity DHP binding sites (505 +/- 46 fmol/mg) compared with vehicle-treated ovex rats (303 +/- 35 fmol/mg); DHP Bmax values from EE2-treated cardiac tissues were not significantly different from vehicle-treated shams (385 +/- 76 fmol/mg). Cardiac Ca++ efflux channels from sarcoplasmic reticulum were assessed with [3H]ryanodine. [3H]Ryanodine Bmax values were not affected by EE2 treatment. However, [3H]ryanodine Kd values in preparations from EE2-treated rats were significantly decreased (10 +/- 2 nM) compared with ovex rats (35 +/- 11 nM) and were similar to values in sham rats (8 +/- 2 nM). Cardiac beta adrenoceptors were not affected by EE2 treatment, which thus confirmed the selective regulation of DHP receptors by EE2. Aortic preparations from EE2-treated rats exhibited significant increases in DHP receptors (125 +/- 37 fmol/mg) compared with both vehicle-treated ovex rats (32 +/- 3 fmol/mg) and vehicle-treated shams (24 +/- 9 fmol/mg). There were no differences in the binding affinity (Kd) of [3H]PN200-110 for cardiac or aortic sites among the three groups. To ascertain if EE2-mediated increases in Ca++ channel density and ryanodine binding affinity affected in vivo responses to agonists that use extracellular and intracellular Ca++ stores, responses to BAY k 8644 and norepinephrine were examined in pithed rats from the same three groups. No significant differences in hemodynamic responses occurred among the three groups to BAY k 8644 or norepinephrine. Thus, in female ovex rats, prolonged treatment with EE2 resulted in increased density of cardiac and aortic calcium channels which did not translate into increased calcium-mediated inotropic, rate or pressor responses. Conversely, EE2 treatment in ovex rats prevented the decrease in cardiac [3H]ryanodine binding affinity evident in vehicle-treated ovex rats. These data suggest that EE2 treatment in normotensive ovex rats resulted in modulation of both the L-type and sarcoplasmic reticulum Ca++ channels, and these alterations maintained cardiovascular homeostasis.

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.

Hypocholesterolemic activity of raloxifene (LY139481): pharmacological characterization as a selective estrogen receptor modulator.

After once-daily oral dosing in ovariectomized rats, raloxifene (LY139481) hydrochloride produced dose- and time-dependent reductions in serum cholesterol and high-density lipoprotein-cholesterol. Paired-feeding studies demonstrated that effects of raloxifene on serum lipids were not secondary to effects on food consumption. Maximal reductions in serum cholesterol occurred within 4 days of raloxifene administration or sooner, depending on the administered dose. The ED50 for 50% reduction in serum cholesterol by raloxifene was 0.13 +/- 0.04 mg/kg/day (mean +/- S.E.M., n = 17); maximal cholesterol reduction by raloxifene (68%) was significantly less than that produced by estrogen (17 alpha-ethinylestradiol; 89%) after 4 to 7 days of daily dosing. Dose-response curves for cholesterol lowering by raloxifene were generated in the presence of varying doses of 17 alpha-ethinylestradiol; two-way analysis of variance revealed significant interactions between estrogen and raloxifene with respect to cholesterol lowering (P < .001). Furthermore, a high dose of raloxifene (10 mg/kg/day) prevented further reduction of serum cholesterol by estrogen (1-100 micrograms/kg/ day) beyond that produced by raloxifene alone. For a series of closely related structural analogs of raloxifene, log(ED50) values for cholesterol lowering were highly correlated with log(relative binding affinity) for the estrogen receptor (r = 0.93; P < .0001). Thus, cholesterol lowering by raloxifene in ovariectomized rats is mediated primarily via partial agonist effects at estrogen receptors. Taken together with previous observations in uterine tissue of estrogen antagonism by raloxifene in the absence of significant agonism, the present findings support the classification of raloxifene as a selective estrogen receptor modulator.

A novel class of 5-HT2A receptor antagonists: aryl aminoguanidines.

Local delivery of serotonin (5-HT) produces a rapid edematous response in soft tissues via increased fluid extravasation which is prevented by 5-HT2 antagonists such as ketanserin or mianserin. Here we report the effects of a new class of aminoguanidine 5-HT2 antagonists, with relative selectivity for 5-HT2A receptors which are potent inhibitors of 5-HT-induced paw edema in the rat. Radioligand binding studies with 125I DOI on human 5-HT2A and 5-HT2C receptors and with 3H-5-HT on human 5-HT2B receptors demonstrated that, LY314228, and LY320954 displayed some selectivity for the 5-HT2A receptor. When compared to binding at other 5-HT2 receptor subtypes, LY314228 had an 18.6-fold greater affinity for the 5-HT2A site over the 5-HT2B site, and 2.6 fold greater at the 5-HT2C site. LY320954 displayed similar preference for 5-HT2A sites. Both compounds also inhibited 5-HT-induced paw swelling in rats, with ED50's of 6.4 and 4.8 mg/kg (for LY314228 and LY320954, respectively). These studies offer evidence for a novel class of pharmacophores for the 5-HT2 receptor family which show greater relative affinities for the 5-HT2A receptor subclass.

Serotonin-induced paw edema in the rat: pharmacological profile.

1. Serotonin (5-HT) induced a linear increase in paw weight in rats within 1 hr of an intraplantar injection (50 microliters vol) over a concentration range of 0.005-0.2 mg/ml. At the 0.2 mg/ml concentration, a 16-fold increase in paw weight was observed as compared to saline-injected controls. 2. Serotonin antagonists, such as LY53857, were the most effective antagonists of 5-HT induced paw swelling, producing near complete antagonism and an approximate ED50 of 0.1 mg/kg. A mixed 5-HT/histamine antagonist, cyproheptadine, also produced a nearly complete inhibition of the 5-HT response with an approximate ED50 of 1.3 mg/kg. 3. Dopamine agonists (pergolide, quinpirole), yohimbine, dexamethasone and nifedipine also produced a significant degree of antagonism of the 5-HT response. 4. Clonidine, prazocin, chlorpheniramine, cimetidine, various dopamine antagonists, imipramine, cyclosporine A, piroxicam and superoxide dismutase were all ineffective at altering the paw swelling response to 5-HT.

Interleukin 1 mediated acceleration of type II collagen-induced arthritis: effects of anti-inflammatory or anti-arthritic drugs.

We previously demonstrated that treatments with rIL-1 beta accelerated the onset and progression of CIA in mice. In the present study, it was observed that IL-1 also enhanced the development of CIA in rats. Like the mouse model, maximal incidence (80-100%) of arthritis occurred within 7 days after the first treatment with IL-1 in rats. Thus, the acceleration of CIA by IL-1 (IL-1 CIA) may be an improved model for the rapid screening of anti-inflammatory and/or anti-arthritic drugs. As a first step to determining the utility of the IL-1 CIA model as a drug screen, we examined the ability of various known anti-inflammatory and anti-arthritic drugs to modify the IL-1 mediated enhancement of CIA in both rats and mice. The results of these studies showed that when analyzed in the IL-1 CIA model, rats and mice exhibited differences in their responses to several of these drugs. For example, dexamethasone, cyclophosphamide, azathioprine, various non-steroidal anti-inflammatory drugs (NSAIDs) as well as methotrexate were found active in the IL-1 CIA of rats. By contrast, the NSAIDs were found to be less effective in suppressing the IL-1 accelerated disease in mice. In both rats and mice, cyclosporine A and several disease modifying anti-arthritic drugs failed to the prevent the development of CIA that was potentiated by IL-1. Thus, in the IL-1 CIA model NSAIDs appeared to be less active in mice than rats. In conclusion, because of the shorter latent period required for the development of arthritis in the IL-1 treated animals, the IL-1 accelerated CIA model in both mice and rats may be useful for screening anti-inflammatory or anti-arthritic compounds.

EEG correlates of emotional tasks related to attentional demands.

This research brings together two separate areas: that of EEG processes associated with positive and negatively valenced emotional material; and that of traditional psychophysiological research related to the "intake" and "rejection" of environmental stimuli. Forty males on each of two days were presented with tasks reflecting both attentional demands and affectual processing. Heart rate and bilateral EEG measures from frontal, parietal and temporal sites were recorded. Using a FFT (fast Fourier transform) electrocortical activity in the 2-7 Hz, 8-15 Hz, and 16-24 Hz was determined and analyzed. The results suggest emotional valence (i.e. positive and negative) and attentional demands (i.e. intake vs rejection) are differentially represented in terms of EEG functioning. An interaction of attentional demand with hemisphere was found for EEG alpha activity in the temporal and parietal areas. For emotional valence there was a significant main effect for EEG beta activity in both the temporal and parietal areas. Differential hemispheric activity was found using a factor analytic technique (PARAFAC) with positively valenced tasks being associated with right temporal beta. Heart rate changes for the attentional dimension were consistent with previous research.

EEG activity during cognitive processing: influence of attentional factors.

The research draws upon two separate areas of inquiry: that of lateralized electrocorical processing of cognitive material; and that of psychophysiological information processing studies related to foci of attentional demands. Eighteen subjects on each of three separate days were presented with tasks considered to be 'right hemispheric' or 'left hemispheric'. These tasks were paired in a 2 X 2 design with an attentional factor requiring attention to the environment or to internal processing. All subjects received all types of tasks. Bilateral EEG measures were taken from the frontal and parietal areas referenced to linked ears. Major frequency bands were determined by a Fast Fourier Transform (FFT) and appropriate ANOVAs performed. The results suggest that task factors (left vs right hemisphere tasks) and attentional demands (internal vs external) are differentially represented in terms of EEG functioning. In general the higher beta frequencies were more sensitive to the hemispheric tasks demands and the middle frequencies (alpha and low beta) more sensitive to the attentional demands especially in the parietal areas.

EEG alpha activity reflects attentional demands, and beta activity reflects emotional and cognitive processes.

Two experiments were designed to examine the effects of attentional demands on the electroencephalogram during cognitive and emotional tasks. We found an interaction of task with hemisphere as well as more overall parietal alpha for tasks not requiring attention to the environment, such as mental arithmetic, than for those requiring such attention. Differential hemispheric activation for beta was found most strongly in the temporal areas for emotionally positive or negative tasks and in the parietal areas for cognitive tasks.