Estrogen-Like Compounds for Ischemic Neuroprotection
James W. Simpkins, PhD; Shao-Hua Yang, MD, PhD; Ran Liu, MD; Evelyn Perez, PhD;
Zu Yun Cai, PhD; Douglas F. Covey, PhD; Pattie S. Green, PhD
Abstract—We have synthesized a library of estrogen analogues, including enantiomers of estradiol and A-ring substituted
estrogens. These compounds have reduced or no binding to either estrogen receptor-␣ or estrogen receptor-␤, exhibit
enhanced neuroprotective activity in in vitro models, and are potent in protecting brain tissue from cerebral
ischemia/reperfusion injury. These potent, nonfeminizing estrogen analogues are prime candidates for use in stroke
neuroprotection. (Stroke. 2004;35[suppl I]:2648-2651.)
Key Words: estrogens 䡲 estrogen receptors 䡲 neuroprotection 䡲 stroke
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here are ⬎750 000 new strokes per year in the United
States. Unfortunately, there are few available options for
the treatment of stroke-related brain damage. Efforts to
develop effective therapies for acute ischemic stroke
achieved several important successes during the past decade,
the greatest of which is thrombolysis. However, with the
restrictive 3-hour therapeutic window for recombinant tissue
plasminogen activator in stroke therapy,1 only a small number
of acute stroke patients are estimated to receive this intervention.2 Even with the patient educational efforts, institutional
initiatives and the advanced techniques of diffusion and
perfusion magnetic resonance imaging, only 10% of the acute
stroke patients are candidates for the intervention.3,4
We first demonstrated that estrogens are potent neuroprotectants5 and are very effective against ischemia-induced
brain damage.6 Also, sex differences in the incidence and
outcome of stroke suggest that hormonal factors may influence the development and outcome of stroke.7,8 Recently,
estrogens have been found to be associated with a decreased
risk and delayed onset and progression of stroke and enhanced recovery from numerous traumatic and chronic neurological and mental diseases.6,9 –11 Various lines of clinical
and experimental evidence have shown that both endogenous
and exogenous estrogens exert neuroprotective effects.9,12
Protective effects of estrogens have been widely reported in
different types of neuronal cells against different toxicities,
including serum deprivation, oxidative stress, amyloid ␤
peptide (A␤)-induced toxicity, and excitotoxicity.12
The neuroprotective effects of estrogens have been demonstrated in a variety of models of acute cerebral ischemia,
including transient and permanent middle cerebral artery
(MCA) occlusion models,6,13,14 global forebrain ischemia
models,15 photothrombotic focal ischemia models,16 and
T
glutamate-induced focal cerebral ischemia models.17 The
protective effects of estrogens have been described in rats,
mice, and gerbils.6,18,19 Estrogen-induced neuroprotection has
been demonstrated in adult and middle-aged female rats, as
well as in reproductively senescent female rats.20 These
effects of estrogens have been shown despite the presence of
diabetes and hypertension.21,22 The neuroprotective effects of
estrogens have also been demonstrated against subarachnoid
hemorrhage, a highly prevalent form of stroke in females.23
Finally, the neuroprotective action of estrogen is not limited
to the female; estrogen protection is also seen in males.24,25
Collectively, these results indicate that estrogens could be
valuable candidates for brain protection during acute stroke in
males and females.
Concentrations of estrogens ranging from low-physiological to high-pharmacological have been shown to produce
protective effects in stroke models. When estradiol is administration at low physiological levels soon before the onset of
an ischemic event, no protection is seen.14 In contrast,
neuroprotective effects of estradiol were clearly demonstrated
with the acute treatment at the time of or just before an
ischemic event, as well as after its onset.6,25,26 The therapeutic
window of estrogens at the dose of 100 ␮g/kg lasts up to 3
hours after insult,27 and this therapeutic window can be
extended to up to 6 hours after ischemic insult with doses of
500 to 1000 ␮g/kg.28 This long postevent efficacy of estrogens is promising, because the therapeutic window for
estrogen neuroprotection could be insult severity-dependent
and could be different between different species. It has been
shown that the infarct penumbra, which is the infarct area that
can be protected, develops over a longer period in human
subjects than in rodent,29 suggesting that estrogens may have
an even longer therapeutic window in human subjects.
Received June 2, 2004; accepted June 2, 2004.
From the Department of Pharmacology & Neuroscience (J.W.S., S.-H.Y., R.L., E.P.), University of North Texas Health Science Center, Fort Worth,
Texas; the Department of Molecular Biology and Pharmacology (Z.Y.C., D.F.C.), Washington University School of Medicine, St. Louis, Mo; and the
Department of Gerontology and Geriatric Medicine (P.S.G.), School of Medicine, University of Washington, Seattle, Wash.
Correspondence to Dr James W. Simpkins, Department of Pharmacology & Neuroscience, 3500 Camp Bowie Blvd, University of North Texas Health
Science Center, Fort Worth, TX 76107. E-mail [email protected]
© 2004 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000143734.59507.88
2648
Simpkins et al
Estrogens and Ischemic Neuroprotection
2649
but may relate to the clinical study designs. Both clinical
studies used continuous oral estrogen exposure, whereas the
animal studies used a single dose or a short period of dosing
of estrogen that was timed with the experimentally induced
stroke. Continuous oral estrogen therapy in women is well
known to increase clotting factor and to reduce antithrombotic enzymes. Thus, the published continuous estrogen
therapy trials are not a test of the ability of acutely administered estrogens to prevent brain damage from strokes.
Structure–Activity Relationship for Estrogen
Analogues and Neuroprotection
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Using our library of ⬎70 newly synthesized estrogen
analogues, we conducted dose-response assessments to
determine ED50 for neuroprotection against 2 different
toxins. Here, we report on some of the compounds (structures depicted in Figure 1). We observed that estrogens
that are modified to enhance their redox potential also have
2 related and useful effects on the activity of the compounds. First, estrogenicity is reduced or eliminated;
second, the neuroprotective activity of the compounds is
increased, in some cases as much as 50 times that of
17␤-estradiol (17␤-E2) (Figure 2).
Comparison of Estrogens and Nonfeminizing
Estrogen Analogues on Stroke
Neuroprotection Using an In Vivo Model
Figure 1. Structures of selective estrogen analogues.
Clinical studies are at odds with these consistent animal
studies. Both the Women’s Health Initiative (WHI)30 and the
WEST31 trials failed to show a beneficial effect of estrogen
therapy on stroke, and the WEST trials demonstrated an
increase in fatal strokes in subjects using estrogen therapy.31
The differences between animal and clinical studies are many
Effects of Estrone, ZYC-23, and ZYC-26, on Uterine Weights and
Infarct Volume After Transient Middle Cerebral Artery Occlusion
in Ovariectomized Rats
Treatment
Infarct Volume (mm3)
Uterine Weight (mg)
Vehicle
264⫾30
100⫾4
Estrone
142⫾36*
186⫾10*
ZYC-26
112⫾30*
103⫾3
ZYC-23
280⫾25
104⫾4
*P⬍0.05 vs vehicle control group.
All data are expressed as mean⫾SEM for n⫽10 –12 rats per group.
All steroids were administered at 100 ␮g/kg in 2-hydroxypropyl-␤cyclodextrin just before transient (1 hour) middle cerebral artery occlusion.
From these compounds, we selected several for in vivo
assessment against a routinely used model of cerebral ischemia, transient MCA occlusion, in ovariectomized rats. To
date, we have assessed 8 compounds in this model, including
17␤-E2, estrone, 17␣-E2, the enantiomer of 17␤-E2 (ent-E2),
the enantiomer of 17-desoxyestradiol (ZYC-13), 2-(1adamantyl) estrone (ZYC-3), and 2-(1-adamantyl)-4methylestrone (ZYC-26) (Figure 1). Data for 3 of these novel
estrogen analogues are provided.
Ent-E2 was assessed for ER binding and was shown to be
less than one-eighth as active as 17␤-E2 in competitive bind
assays for human recombinant estrogen receptor-␣ (ER␣)
(Figure 2B) and ER␤ (data not shown). Despite this low
affinity for binding to either ER, ent-E2 was as effective as
17␤-E2 in preventing glutamate-induced cell death in HT-22
cells (Figure 2) and in reducing infarct size after MCA
occlusions (Figure 3). We found no evidence of metabolic
conversion of ent-E2 to 17␤-E2 in ovariectomized rats.32
Finally, we observed no effects of ent-E2 on physiological
parameters before, during, or after MCA occlusion.32 These
data suggest that nonfeminizing enantiomers of estrogens can
be potently neuroprotective in vitro and in animal models for
human ischemic damage.
ZYC-3 showed no binding to either ER␣ or ER␤ in
competitive binding assays (Figure 2) but was 6-times more
potent than 17␤-E2 in a test for neuroprotection against
glutamate toxicity (Figure 2). Further, in an assessment of
brain protection from transient MCA occlusion, ZYC-3
performed better than 17␤-E2 (Figure 4) without affecting
physiological parameters.33 The placement of a bulky adamantyl group in the 2 position of the A-ring of the estrone
eliminates estrogenicity, enhances antioxidant potential (un-
2650
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November 2004 (Supplement 1 - New Series)
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Figure 4. Effects of 17␤-estradiol (E2) and ZYC-3 on infarct size
in ovariectomized rats. **P⬍0.05 versus OVX control group. E2
(100 ␮g/kg) was administered in oil 2 hours before and ZYC-3
(100 ␮g/kg) in hydroxypropyl-␤-cyclodextrin immediately before
transient (1 hour) middle cerebral artery occlusion. Modified
from Reference 31, with permission.
Figure 2. Effects of selective estrogen analogues on (A) glutamate (20 mmol/L) toxicity in HT-22 cells and (B) on binding to
human cloned estrogen receptor (ER)-␣. In parentheses are (A)
ED50 values for neuroprotection and (B) IC50 values for inhibition
of binding of estradiol to ER␣; n.p. indicates no protection; n.b.
indicates no binding was detected over the concentration range
studied. When standard error of mean (SEM) bars are not
shown, they are smaller than the symbol used to depict the
mean value.
published observations), and enhances the potent neuroprotective activity of estrogens against ischemic brain damage.
ZYC-26 has an adamantyl moiety on the 2-carbon and a
methyl group on the 4 carbon of estrone. ZYC-23 is similar,
but the phenolic nature of the A-ring is eliminated through
O-methylation of the 3-carbon, a chemical change that we
have previously shown to completely eliminate neuroprotec-
tion of estrogens.34 Neither of these compounds bound to
either estrogen receptor (Figure 2). Consistent with our
previous study,35 estrone significantly decreased ischemic
lesion volume by ⬇47% (Table). Similar to our in vitro study,
ZYC-26 reduced lesion volume by ⬎68% (Table). As expected, no protective effects of ZYC-23 were seen in vitro
(Figure 2) or after transient MCA occlusion (Table).
Estrone treatment significantly induced uterine hypertrophy at 24 hours after treatment (Table). Consistent with our
ER binding assay in vitro, neither estrogen analogue showed
feminizing effects in vivo (Table).
Conclusion and Future Studies
Collectively, these studies demonstrate several principles that
are useful in the further discovery of novel drugs based on the
potent neuroprotective effects of estrogens. First, estrogenicity can be reduced or eliminated from the estrogen molecule
through chiral changes in the steroid, as was performed with
17␣-E2, ent-E2, and ZYC-13, or by adding bulky groups to
the 2 and/or 4 position of the A-ring, as is performed for
ZYC-3 and ZYC-26. Second, in vitro neuroprotection screen-
Figure 3. Effects of 17␤-estradiol (E2) and the
enantiomer of 17␤-estradiol (ent-E2) on infarct size
in ovariectomized rats. Also depicted are representative photographs of TTC-stained brain sections
from an animal in each treatment group. *P⬍0.05
vs oil group. E2 or ent-E2 was administered subcutaneously in oil (100 ␮g/kg) 2 hours before transient (1 hour) middle cerebral artery (MCA) occlusion. Modified from Reference 30, with permission.
Simpkins et al
ing assays are effective in selecting compounds of potential
use in an animal model for cerebral ischemia. Finally,
chemical modifications that eliminate estrogenicity while
enhancing neuroprotection allow for brain protection without
the side effects associated with chronic hormone use.36,37 This
would allow for treatment of women and men with these
estrogen analogues.
Acknowledgements
Supported by National Institutes of Health grants AG10485 and
AG22550, and by a grant from the Texas Higher Education Coordinating Board to J.W.S.
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Estrogen-Like Compounds for Ischemic Neuroprotection
James W. Simpkins, Shao-Hua Yang, Ran Liu, Evelyn Perez, Zu Yun Cai, Douglas F. Covey
and Pattie S. Green
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Stroke. 2004;35:2648-2651; originally published online October 7, 2004;
doi: 10.1161/01.STR.0000143734.59507.88
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