An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

<div><p>Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3^rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.</p></div

Relaxation of isolated aortic rings by SERMs and NO-SERM.

<p>(<b>A</b>) The EC₅₀ values for relaxation were not significantly different for raloxifene, arzoxifene, DMA, and FDMA (p>0.05, one-way ANOVA and Newman-Keul's post-hoc test), whereas for NO-DMA potency was significantly different from all other SERMs (F_(4,43) = 4.085, p<0.01). The maximal relaxation responses for arzoxifene and FDMA were significantly less than those for DMA and raloxifene (F_{(3, 37)} = 11.77 p<0.05, one-way ANOVA and Newman-Keul's post-hoc test). Each value represents the mean ± S.E.M. (n = 7–13). (<b>B</b>) Removal of the endothelium or inhibition of NOS with L-NAME reduced the maximal relaxation response to DMA (F_{(2, 18)} = 28.22, p<0.001, one-way ANOVA and Newman-Keuls post-hoc test). Each value represents the mean ± S.E.M. (n = 7). (<b>C</b>) The EC₅₀ values for relaxation were significantly increased in the presence of L-NAME or after endothelium removal (F_(2,18) = 7.753, p<0.05, one-way ANOVA and Newman-Keuls post-hoc test). Each value represents the mean ± S.E.M. (n = 7).</p

SERM reversal of memory deficits in WT mice is retained by NO-DMA in eNOS (−/−) mice.

<p>Amnestic memory deficit was induced by i.p. injection of either scopolamine (1 mg/kg) or L-NAME (50 mg/kg) 30 min prior to training in C57Bl/6 male mice. SERMs (2 mg/kg) were given 20 min prior to training and latency was assessed 24 h after training with animals being removed from the task if latency >300 s. All SERMs, except F-DMA, restored scopolamine-induced deficits in STPA in C57Bl/6 animals. Against L-NAME-induced deficit, only NO-DMA showed efficacy in reversing memory deficits. In eNOS (−/−) animals subject to scopolamine-induced amnesia, only NO-DMA showed efficacy. Data show mean and S.E.M. (n = 4–10); *** = p<0.001 compared to non-insult wild type vehicle control using one-way ANOVA with Dunnett's post hoc test; F_(14,124) = 29.26, p<0.0001.</p

SERM-elicited neuroprotection in primary cortical neurons exposed to OGD is GPR30 dependent and retained by NO-DMA.

<p>Primary neuronal cultures were subjected to 2 h OGD with compounds added at the start of OGD and inhibitors added 45 min prior to OGD. Cell survival was measured at 24 h. Use of pathway-selective inhibitors indicates that neuroprotection of DMA and NO-DMA is mediated through PI3K-dependent GPR30 signaling in an ER- and NOS-independent manner. Data show mean and S.E.M. normalized to veh. control and estradiol (n = 6); * = p<0.05, ** = p<0.01, *** = p<0.001 compared to untreated vehicle control using one-way ANOVA with Dunnett's post hoc test within each treatment group; no blocker F_(4,67) = 169.5, p<0.0001; ICI 182780 F_(4,61) = 58.65, p<0.0001; pertussis F_(4,61) = 6.78, p = 0.0001; G15 F_(4,61) = 0.63, p = 0.64; LY294002 F_(4,61) = 6.29, p<0.001; L-NAME F_(4,61) = 89.33, p<0.0001.</p

Effects of SERM and NO-SERMon NO levels in plasma and brain of WT and eNOS (−/−) mice.

<p>Levels of NO were assessed by measuring breakdown products 1 h after i.p. injection of SERMs (2 mg/kg) using chemiluminescence detection. Both DMA and NO-DMA increased levels of NO in WT mice. The diminished response in eNOS (−/−) was significantly attenuated in DMA relative to NO-DMA treated animals. Data show mean and S.E.M. (n = 4–12); * = p<0.05, ** = p<0.01, *** = p<0.001 compared to wild type vehicle control using one-way ANOVA with Dunnett's post hoc test within each group: hippocampus F_(4,40) = 7.79, p<0.0001; plasma F_{(4, 23)} = 21.76, p<0.0001.</p

Reversal of LTP deficit in aged 3×Tg mice by SERMs is GPR30 dependent.

<p>LTP was measured after TBS in the CA1 region of hippocampal sections from 16 month male 3×Tg mice or WT controls. Test compounds (SERMs 100 nM; G15 100 nM) were added 30 min prior to TBS. (<b>A, B</b>) DMA and NO-DMA restored deficits in LTP to WT levels and G15 blocked the actions of DMA. (<b>C</b>) Secondary analysis of theta bursts indicate action both during induction and stabilization of LTP, through a GPR30 dependent mechanism. Data show mean and S.E.M. normalized to baseline (n = 4–9); for end fEPSP: * = p<0.05, ** = p<0.01 compared to wild type transgenic background controls using one-way ANOVA with Dunnett's post hoc test; F_(4,32) = 8.21, p = 0.0001.</p