Stroke. 2004;35:2648-2651
Published online before print October 7, 2004,
doi: 10.1161/01.STR.0000143734.59507.88
(Stroke. 2004;35:2648.)
© 2004 American Heart Association, Inc.
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
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 jsimpkin{at}hsc.unt.edu
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Abstract
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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-ß, 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.
Key Words: estrogens • estrogen receptors • neuroprotection • stroke
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Introduction
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There 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 ß peptide (Aß)-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 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 µg/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 µg/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.
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 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.
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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 ED
50 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 17ß-estradiol
(17ß-E2) (
Figure 2).
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Comparison of Estrogens and Nonfeminizing Estrogen Analogues on Stroke Neuroprotection Using an In Vivo Model
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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 17ß-E2, estrone,
17
-E2, the enantiomer of 17ß-E2 (
ent-E2), the enantiomer
of 17-desoxyestradiol (ZYC-13), 2-(1-adamantyl) estrone (ZYC-3),
and 2-(1-adamantyl)-4-methylestrone (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 17ß-E2 in competitive bind assays for human recombinant estrogen receptor- (ER) (Figure 2B) and ERß (data not shown). Despite this low affinity for binding to either ER, ent-E2 was as effective as 17ß-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 17ß-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.
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Figure 3. Effects of 17ß-estradiol (E2) and the enantiomer of 17ß-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 µg/kg) 2 hours before transient (1 hour) middle cerebral artery (MCA) occlusion. Modified from Reference 30, with permission.
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ZYC-3 showed no binding to either ER or ERß in competitive binding assays (Figure 2) but was 6-times more potent than 17ß-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 17ß-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 (unpublished observations), and enhances the potent neuroprotective activity of estrogens against ischemic brain damage.
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Figure 4. Effects of 17ß-estradiol (E2) and ZYC-3 on infarct size in ovariectomized rats. **P<0.05 versus OVX control group. E2 (100 µg/kg) was administered in oil 2 hours before and ZYC-3 (100 µg/kg) in hydroxypropyl-ß-cyclodextrin immediately before transient (1 hour) middle cerebral artery occlusion. Modified from Reference 31, with permission.
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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 neuroprotection 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).
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Effects of Estrone, ZYC-23, and ZYC-26, on Uterine Weights and Infarct Volume After Transient Middle Cerebral Artery Occlusion in Ovariectomized Rats
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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).
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Conclusion and Future Studies
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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 screening 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.
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Acknowledgments
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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.
Received June 2, 2004;
accepted June 2, 2004.
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References
|
|---|
- The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995; 333: 1581–1587.[Abstract/Free Full Text]
- Clark WM, Albers GW, Madden KP, Hamilton S. The rtPA (alteplase) 0- to 6-hour acute stroke trial, part A (A0276g): results of a double-blind, placebo-controlled, multicenter study. Thrombolytic therapy in acute ischemic stroke study investigators. Stroke. 2000; 31: 811–816.[Abstract/Free Full Text]
- Hacke W, Brott T, Caplan L, Meier D, Fieschi C, von Kummer R, Donnan Heiss WD, Wahlgren NG, Spranger M, Boysen G, Marler JR. Thrombolysis in acute ischemic stroke: controlled trials and clinical experience. Neurology. 1999; 53: S3–S14.
- Fisher M, Albers GW. Applications of diffusion-perfusion magnetic resonance imaging in acute ischemic stroke. Neurology. 1999; 52: 1750–1756.[Abstract/Free Full Text]
- Bishop J, Simpkins JW. Estradiol treatment increases viability of glioma and neuroblastoma cells in vitro. Molecular and Cellular Neuroscience. 1994; 5: 303–308.[CrossRef][Medline]
[Order article via Infotrieve]
- Simpkins JW, Rajakumar G, Zhang YQ, Simpkins CE, Greenwald D, Yu CJ, Bodor N, Day AL. Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. J Neurosurg. 1997; 87: 724–730.[Medline]
[Order article via Infotrieve]
- Stegmayr B, Asplund K, Kuulasmaa K, Rajakangas AM, Thorvaldsen P, Tuomilehto J. Stroke incidence and mortality correlated to stroke risk factors in the WHO MONICA Project. An ecological study of 18 populations. Stroke. 1997; 28: 1367–1374.[Abstract/Free Full Text]
- Thorvaldsen P, Asplund K, Kuulasmaa K, Rajakangas AM, Schroll M. Stroke incidence, case fatality, and mortality in the WHO MONICA project. World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease. Stroke. 1995; 26: 361–367.[Abstract/Free Full Text]
- Garcia-Segura LM, Azcoitia I, DonCarlos LL. Neuroprotection by estradiol. Prog Neurobiol. 2001; 63: 29–60.[CrossRef][Medline]
[Order article via Infotrieve]
- Honjo H, Kikuchi N, Hosoda T, Kariya K, Kinoshita Y, Iwasa K, Ohkubo T, Tanaka K, Tamura T, Urabe M, Kawata M. Alzheimer’s disease and estrogen. J Steroid Biochem Mol Biol. 2001; 76: 227–230.[CrossRef][Medline]
[Order article via Infotrieve]
- Panidis DK, Matalliotakis IM, Rousso DH, Kourtis AI, Koumantakis EE. The role of estrogen replacement therapy in Alzheimer’s disease. Eur J Obstet Gynecol Reprod Biol. 2001; 95: 86–91.[CrossRef][Medline]
[Order article via Infotrieve]
- Green PS, Simpkins JW. Neuroprotective effects of estrogens: potential mechanisms of action. Int J Dev Neurosci. 2000; 18: 347–358.[CrossRef][Medline]
[Order article via Infotrieve]
- Alkayed NJ, Harukuni I, Kimes AS, London ED, Traystman RJ, Hurn PD. Gender-linked brain injury in experimental stroke. Stroke. 1998; 29: 159–165.[Abstract/Free Full Text]
- Dubal DB, Kashon ML, Pettigrew LC, Ren JM, Finklestein SP, Ran SW, Wise PM. Estradiol protects against ischemic injury. J Cereb Blood Flow Metab. 1998; 18: 1253–1258.[CrossRef][Medline]
[Order article via Infotrieve]
- Sudo S, Wen TC, Desaki J, Matsuda S, Tanaka J, Arai T, Maeda N, Sakanaka M. ß-estradiol protects hippocampal CA1 neurons against transient forebrain ischemia in gerbil. Neurosci Res. 1997; 29: 345–354.[CrossRef][Medline]
[Order article via Infotrieve]
- Fukuda K, Yao H, Ibayashi S, Nakahara T, Uchimura H, Fujishima M, Hall ED. Ovariectomy exacerbates and estrogen replacement attenuates photothrombotic focal ischemic brain injury in rats. Stroke. 2000; 31: 155–160.[Abstract/Free Full Text]
- Mendelowitsch A, Ritz MF, Ros J, Langemann H, Gratzl O. 17beta-Estradiol reduces cortical lesion size in the glutamate excitotoxicity model by enhancing extracellular lactate: a new neuroprotective pathway. Brain Res. 2001; 901: 230–236.[CrossRef][Medline]
[Order article via Infotrieve]
- Chen J, Xu W, Jiang H. 17 beta-estradiol protects neurons from ischemic damage and attenuates accumulation of extracellular excitatory amino acids. Anesth Analg. 2001; 92: 1520–1523.[Abstract/Free Full Text]
- Culmsee C, Vedder H, Ravati A, Junker V, Otto D, Ahlemeyer B, Krieg JC, Krieglstein J. Neuroprotection by estrogens in a mouse model of focal cerebral ischemia and in cultured neurons: evidence for a receptor-independent antioxidative mechanism. J Cereb Blood Flow Metab. 1999; 19: 1263–1269.[CrossRef][Medline]
[Order article via Infotrieve]
- Wise PM, Dubal DB, Wilson ME, Rau SW, Bottner M, Rosewell KL. Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies. Brain Res Brain Res Rev. 2001; 37: 313–319.[CrossRef][Medline]
[Order article via Infotrieve]
- Toung TK, Hurn PD, Traystman RJ, Sieber FE. Estrogen decreases infarct size after temporary focal ischemia in a genetic model of type 1 diabetes mellitus. Stroke. 2000; 31: 2701–2706.[Abstract/Free Full Text]
- Carswell HV, Dominiczak AF, Macrae IM. Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol. 2000; 278: H290–H294.[Abstract/Free Full Text]
- Yang SH, He Z, Wu SS, He YJ, Cutright J, Millard WJ, Day AL, Simpkins JW. 17-ß estradiol can reduce secondary ischemic damage and mortality of subarachnoid hemorrhage. J Cereb Blood Flow Metab. 2001; 21: 174–181.[CrossRef][Medline]
[Order article via Infotrieve]
- Hawk T, Zhang YQ, Rajakumar G, Day AL, Simpkins JW. Testosterone increases and estradiol decreases middle cerebral artery occlusion lesion size in male rats. Brain Res. 1998; 796: 296–298.[CrossRef][Medline]
[Order article via Infotrieve]
- Toung TJ, Traystman RJ, Hurn PD. Estrogen-mediated neuroprotection after experimental stroke in male rats. Stroke. 1998; 29: 1666–1670.[Abstract/Free Full Text]
- McCullough LD, Alkayed NJ, Traystman RJ, Williams MJ, Hurn PD. Postischemic estrogen reduces hypoperfusion and secondary ischemia after experimental stroke. Stroke. 2001; 32: 796–802.[Abstract/Free Full Text]
- Yang SH, Shi J, Day AL, Simpkins JW. Estradiol exerts neuroprotective effects when administered after ischemic insult. Stroke. 2000; 31: 745–749.[Abstract/Free Full Text]
- Yang SH, Liu R, Wu SS, Simpkins JW. The use of estrogens and related compounds in the treatment of damage from cerebral ischemia. Steroids and the nervous system. Ann N Y Acad Sci. 2004; 1007: 101–108.
- De Keyser J, Sulter G, Luiten PG. Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing? Trends Neurosci. 1999; 22: 535–540.[CrossRef][Medline]
[Order article via Infotrieve]
- Wassertheil-Smoller S, Hendrix SL, Limacher M, Heiss G, Kooperberg C, Baird A, Kotchen T, Curb JD, Black H, Rossouw JE, Aragaki A, Safford M, Stein E, Laowattana S, Mysiw WJ. Effect of estrogen plus progestin on stroke in postmenopausal women: the Women’s Health Initiative: a randomized trial. JAMA. 2003; 289: 2673–2684.[Abstract/Free Full Text]
- Viscoli CM, Brass LM, Kernan WN, Sarrel PM, Suissa S, Horwitz RI. A clinical trial of estrogen-replacement therapy after ischemic stroke. N Engl J Med. 2001; 345: 1243–1249.[Abstract/Free Full Text]
- Green PS, Yang SH, Nilsson KR, Kumar AS, Covey DF, Simpkins JW. The nonfeminizing enantiomer of 17beta-estradiol exerts protective effects in neuronal cultures and a rat model of cerebral ischemia. Endocrinology. 2001; 42: 400–406.
- Liu R, Yang SH, Perez E, Yi KD, Wu SS, Eberst K, Prokai L, Prokai-Tatrai K, Cai ZY, Covey DF, Day AL, Simpkins JW. Neuroprotective effects of a novel non-receptor binding estrogen analogue: in vitro and in vivo analysis. Stroke. 2002; 33: 2485–2491.[Abstract/Free Full Text]
- Green PS, Gordon K, Simpkins JW. Phenolic A ring requirement for the neuroprotective effects of steroids. J Steroid Biochem Mol Biol. 1997; 63: 229–235.[CrossRef][Medline]
[Order article via Infotrieve]
- Prokai L, Prokai-Tatrai K, Perjesi P, Zharikova A, Perez E, Liu R, Simpkins J. Quinol-based cyclic antioxidant mechanism in estrogen neuroprotection. Proc Natl Acad Sci U S A. 2003; 100: 11741–11746.[Abstract/Free Full Text]
- Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, Hendrix SL, Jones BN III, Assaf AR, Jackson RD, Kotchen JM, Wassertheil-Smoller S, Wactawski-Wende J, WHIMS Investigators. Estrogen plus progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women: the Women’s Health Initiative Memory Study: a randomized controlled trial. J Am Med Assoc. 2003; 289: 2651–2662.[Abstract/Free Full Text]
- Rapp SR, Espeland MA, Shumaker SA, Henderson VW, Brunner RL, Manson JE, Gass ML, Stefanick ML, Lane DS, Hays J, Johnson KC, Coker LH, Dailey M, Bowen D, WHIMS Investigators. Effect of estrogen plus progestin on global cognitive function in postmenopausal women: the Women’s Health Initiative Memory Study: a randomized controlled trial. J Am Med Assoc. 2003; 289: 2663–2672.[Abstract/Free Full Text]
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Abstract
Introduction
