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

Abstract

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 3rd 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.

Re-engineering a neuroprotective, clinical drug as a procognitive agent with high in vivo potency and with GABAA potentiating activity for use in dementia

“…Loss is considered to have occurred if the animal remains on its back for more than 30 s. Righting reflex is considered to be regained when the mice were able to successfully right themselves with all four paws touching the floor, twice within 20 s [ 81 ]. Step through passive avoidance (STPA) STPA has been widely used to test long-term working memory and was performed as we have previously described [ 82 – 85 ]. Each animal was given two ip injections: (1) Amnesic agent (scopolamine 1 mg/kg, MK-801 0.1 mg/kg, diazepam 0.5 mg/kg, L-NAME 50 mg/kg) or saline were given 30 min prior to training plus NMZ or saline in the time and delivery method as indicated in Fig.…”

Discovery of a Bradykinin B2 Partial Agonist Profile of Raloxifene in a Drug Repurposing Campaign

“…Moreover, diverse experiments subsequently confirmed a role of raloxifene inducing endothelium-dependent relaxation as due to the upregulation of nitric-oxide synthase (eNOS) expression [ 38 , 39 , 40 ]. Although these results can be due to the partial agonist profile of raloxifene reported in this work [ 33 ], the effect can also be explained as due to the activation of the GPR30 receptor [ 41 ]. Accordingly, further experimental work is necessary to differentiate both effects.…”

Role of inducible nitric oxide synthase in endothelium‐independent relaxation to raloxifene in rat aorta

“…Furthermore, in another study (Bolego et al, ) in female rat aorta, it was found that the relaxant responses to 17β‐estradiol or the ERα agonist 4,4′,4″‐(4‐propyl‐[1H]pyrazole‐1,3,5‐triyl) tris‐phenol were endothelium‐dependent. Moreover, in male rat aorta, three different SERM agonists, raloxifene, desmethylarzoxifene (DMA) and NO‐DMA, showed similar potency at inducing relaxation, and this effect of DMA was dose‐ and endothelium‐dependent (VandeVrede et al, ). Also, raloxifene at 1 μM has been shown to inhibit iNOS expression and NO levels induced by cytokines in rat aortic smooth cells, effects reversed by specific ERα inhibitors (Pinna et al, ).…”