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The prodrug DHED selectively delivers 17b-estradiol tothe brain for treating estrogen-responsive disordersLaszlo Prokai,1,2* Vien Nguyen,1 Szabolcs Szarka,1† Puja Garg,1,3‡ Gauri Sabnis,4

Heather A. Bimonte-Nelson,5,6 Katie J. McLaughlin,5§ Joshua S. Talboom,5,6

Cheryl D. Conrad,5 Paul J. Shughrue,7¶ Todd D. Gould,4,8,9 Angela Brodie,4

Istvan Merchenthaler,9,10 Peter Koulen,3 Katalin Prokai-Tatrai1,2

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens.However, current hormone therapies cannot be used safely to treat these conditions commonly associated withmenopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormonefor neuroprotection. We show that a small-molecule bioprecursor prodrug, 10b,17b-dihydroxyestra-1,4-dien-3-one(DHED), converts to 17b-estradiol in the brain after systemic administration but remains inert in the rest of the body.The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalyticalassays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originatedfrom brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a ratstroke model. Concomitantly, we determined that 17b-estradiol formed in the brain from DHED elicited changesin gene expression and neuronal morphology identical to those obtained after direct 17b-estradiol treatment.Together, complementary functional and mechanistic data show that our approach is highly relevant therapeuti-cally, because administration of the prodrug selectively produces estrogen in the brain independently from theroute of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemicestrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, ourbrain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psy-chiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause,without the adverse side effects of current hormone therapies.

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INTRODUCTION

Estrogen deficiency in the human brain causes numerous neurologicaland psychiatric symptoms (1). Hormone therapies, including sys-temically administered 17b-estradiol (E2, the main human estrogen),alleviate these conditions (2). E2 has also been shown to provide neu-roprotection, as one of its best-documented nonreproductive func-tions, in animals (3). However, the full potential of the hormone forthe treatment of estrogen-responsive central maladies cannot be re-alized in clinical settings until its actions are restricted to the brain.This is needed to ensure therapeutic safety, because adverse peripheralimpact of estrogens has halted large-scale clinical trials investigatingthe long-term health benefits of hormone therapies based on equineestrogens (4). Many women discontinued these therapies or avoid start-

1Department of Pharmacology and Neuroscience, University of North Texas Health Sci-ence Center, Fort Worth, TX 76107, USA. 2AgyPharma LLC, Mansfield, TX 76063, USA.3Vision Research Center and Departments of Ophthalmology and Basic Medical Science,University of Missouri–Kansas City, School of Medicine, Kansas City, MO 64108, USA.4Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD21201, USA. 5Department of Psychology, Arizona State University, Tempe, AZ 85287, USA.6Arizona Alzheimer’s Consortium, Tempe, AZ 85014, USA. 7Department of Pharmacology,Elan Pharmaceuticals Inc., South San Francisco, CA 94080, USA. 8Department of Psychiatry,University of Maryland School of Medicine, Baltimore, MD 21201, USA. 9Department ofAnatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD21201, USA. 10Department of Epidemiology and Public Health, University of MarylandSchool of Medicine, Baltimore, MD 21201, USA.*Corresponding author. E-mail: laszlo.prokai@unthsc.edu†Present address: LGC, Fordham, Cambridgeshire CB7 5WW, UK.‡Present address: University of Texas Southwestern Medical Center, Dallas, TX 75390,USA.§Present address: Eli Lilly and Company, Indianapolis, IN 46285, USA.¶Present address: Prothena Biosciences, South San Francisco, CA 94080, USA.

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ing them, which potentially increases the risks of poor brain health evenin conditions where benefits of estrogen have been shown (5, 6). There-fore, novel therapies providing effective and safe treatment of the brainwith estrogen remain an unmet need in medicine.

Elevated circulating estrogen levels and associated risks for harmfulperipheral side effects are unavoidable with currently approved estro-gen medications, even when human estrogen is prescribed (7). The de-velopment of brain-selective estrogen therapies has been, however, aformidable challenge. Efforts to discover neuroselective estrogen receptor(ER) modulators are still focused mainly on a few well-known phyto-estrogens (8), and metabolism of these compounds is variable (9). Arecent development of a glucagon-like peptide-1 (GLP-1)–E2 conju-gate has shown potential brain delivery through dual-hormone actionto improve energy, glucose, and lipid metabolism (10). The approachtargets cells that, besides ERs, also express GLP-1 receptors. Therefore,suprahypothalamic areas of the central nervous system (CNS) involvedin many estrogen deficiency–derived neurological and psychiatric symp-toms (6, 11) are not affected, because ER-expressing cells that coexpressGLP-1 receptors are localized only in the hypothalamus and the brain-stem. An additional caveat of the method is due to key technical hurdlesinvolved in the pharmaceutical development of effective peptide-basedagents—including but not limited to manufacture, oral absorption,metabolic stability, pharmacokinetics, and formulation—compared withsmall-molecule drugs.

Hence, selective delivery of E2 into the brain through a simple small-molecule strategy would be of immediate practical relevance. Prodrugapproaches to achieve this goal have, however, remained unsuccessful.Prodrugs are inactive derivatives of therapeutic agents that are convertedto the biologically active parent drug by enzymatic and/or chemical

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transformations in vivo (12). They are commonly developed to resolvepharmacokinetic, toxicity, formulation, and drug delivery limitations.A redox chemical delivery system, which applies the prodrug principle(12), has been proposed for enhanced delivery of E2 to the brain (13).Although this method provides increased selectivity in CNS deliveryof the hormone, it still generates elevated levels of the hormone in theperiphery with substantial uterotrophic side effects and sustained highcirculating estrogen levels even after acute dosing (14, 15).

Our studies on the antioxidant mechanism of estrogen neuropro-tection (16) inspired the hypothesis that 10b,17b-dihydroxyestra-1,4-dien-3-one (DHED) could serve as a bioprecursor prodrug of E2 withpreferential bioactivation to E2 in the brain to fulfill the unmet needfor brain-selective estrogen therapy. Unlike classical prodrugs, biopre-cursor prodrugs do not contain auxiliary “promoieties” but are createdvia a transient chemical alteration within the drug molecule itself (12).As an a,b-unsaturated carbonyl compound, DHED is a plausible sub-strate for a short-chain NADPH (reduced form of nicotinamide adeninedinucleotide phosphate)–dependent dehydrogenase/reductase (17)that is selectively expressed in the brain and linked to neuroprotection(18). Using a series of bioanalytical and imaging assessments, evaluat-ing gene expression, morphology, and neuropharmacological responsesin several rodent models of neurological and psychiatric disorders, weshow here that DHED selectively converts into E2 only in the brain aftersystemic administration. We reveal the therapeutic potential of DHEDon estrogen deficiency–associated neurological disorders and psychiatricconditions without encountering adverse systemic actions that are typ-ically seen with the direct use of estrogens.

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RESULTS

DHED conversion to estrogen (E2)DHED is a bioprecursor prodrug that is converted to E2 by a short-chain dehydrogenase/reductase (Fig. 1A). The reductive bioactivationof DHED to E2 proceeds through hydride transfer from the coenzyme

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NADPH to the C1 position of the C1-C2 double bond of DHED’s A-ring that is conjugated to the 3-carbonyl group. This b-addition isfollowed by spontaneous water elimination involving the 10(b)-OH.Our computer-aided mechanistic study fully supported this pathwayof DHED’s reductive bioactivation (Fig. 1B). The first, enzyme-catalyzedhydride-transfer step leads to an intermediate through a transition state(which may be considered a resonance hybrid of the substrate-coenzymeand intermediate-oxidized coenzyme complexes shown in brackets)that is endergonic, with DG° of +2.1 kcal/mol. The subsequent waterelimination is immensely exergonic (DG° of −10.3 kcal/mol); hence, itproceeds spontaneously, making the overall process also exergonic withDG° of −8.2 kcal/mol. The obtained phenolate form of E2 (E2

−) is inacid-base equilibrium with the non-ionized (neutral) form of the hor-mone dictated by pH, and the neutral form predominates in equilibri-um at physiological pH. Together, DHED’s reductive bioactivationproceeds in an opposite direction compared to the well-known oxidativeprocess converting an androgen to estrogen catalyzed by aromatase(19). In the latter case, three sequential steps of substrate oxidationinvolving molecular oxygen and the transfer of six electrons are nec-essary for estrogen formation from androgens (Fig. 1C).

Table 1 summarizes key physicochemical, pharmacological, andbiopharmaceutical properties of DHED in comparison with those of E2.DHED is inactive as an estrogen because it has no measurable affinityto the classical ERs and does not show the well-known antioxidantactivity of the hormone (16). Its lipophilicity and binding to humanplasma proteins are significantly decreased, whereas its water solubilityis increased compared with E2. All these are great advantages over E2in terms of bioavailability and formulation.

DHED metabolizes to E2 only in the brainIn vitro metabolism studies were conducted by adding the prodrug tofreshly prepared tissue homogenates. DHED was preferentially acti-vated to E2 in the brain compared with estrogen-sensitive peripheraltissues, such as the uterus (Fig. 2A and fig. S1). Besides E2, we couldnot identify other steroid(s) formed from DHED.

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Fig. 1. DHED selectively converts to the main human estrogen E2. (A)DHED’s reductive metabolism to E2 in the brain via a NAD(P)H-dependent

tion with water were applied to the complete steroid structures and amimic ofNAD(P)H⇌ NAD(P)+, with R chosen as methyl. (C) In contrast, the aromatase-

short-chain dehydrogenase/reductase (SDR). (B) In silico mechanistic modelfor DHED bioactivation to E2. Quantum-chemical calculations simulating solva-

catalyzed conversion of androgens to estrogens proceeds through sequentialsix-electron oxidation on the C19 methyl group involving molecular oxygen.

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After intravenous dosing of ovariectomized (OVX) rats withDHED, appreciable level of circulating estrogens could not be detectedand the prodrug disappeared rapidly from the circulation (Fig. 2B).The distribution half-life (td) was estimated to be 5.1 ± 0.6 min. Con-

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comitantly, E2 concentrations increased in the brain. Experimentsusing deuterated d3-DHED demonstrated that d3-E2 was produced ex-clusively in the brain (Fig. 2C). Treatment with d3-DHED also did nottrigger or inhibit endogenous E2 formation in the brain, and did not

Fig. 2. DHED is a brain-selective prodrug. (A) Initial rate of in vitro E2formation during DHED incubation (100 nM) in homogenates of various

after injection. (C) Increase of E2 in brain tissue after DHED treatmentarises from the metabolism of the bioprecursor prodrug. d -DHED was ad-

OVX rat brain structures versus rat uterus homogenate; the latter representsan estrogen-sensitive peripheral tissue. Data are averages ± SEM (n = 3 pertissue). N/D, not detected. (B) Serum DHED and E2 concentrations in serumand in the brain of OVX Sprague-Dawley rats after intravenous adminis-tration of DHED (200 mg/kg). Concentrations were measured by LC-MS/MS–based bioassays. Serum concentrations are averages ± SEM (n = 3 to4 animals per time point); brain concentrations are weighted averages ±SEM from measurements in seven brain regions (n = 3 to 4 animals pertime point). E2 concentrations at time “0” originated from analyses of tissueand serum obtained from vehicle-treated animals euthanized immediately

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ministered to OVX Sprague-Dawley rats intravenously followed by tissueharvesting at 15 min after dosing and LC-MS/MS analysis [using selectedreaction monitoring (SRM)]. All SRM chromatograms were obtained fromthe analyses of hippocampal tissue and were scaled to the same ion abun-dance (5.0 × 104). The trace for the unlabeled (that is, endogenous) E2 isobtained by SRM ofm/z 506→171, the red trace (SRM:m/z 509→171) rep-resents 16,16,17-d3-E2 formed from 16,16,17-d3-DHED in the hippocampus,and the green trace (SRM: m/z 512→171) corresponds to the 13C-labeledhormone ([13C6]E2) added as internal standard for identification andquantitation.

Table 1. ER affinity, antioxidant potency, plasma protein binding, andselected physicochemical properties of E2 and DHED. Lipid peroxidationwas determined using the FTC (ferric thiocyanate) and TBARS (thiobarbituricacid reactive substances) assays. LogP denotes the logarithm of n-octanol/

water partition coefficient as a measure of lipophilicity with values pre-dicted by the method incorporated into the BioMedCAChe program(version 6.1). Data are averages ± SEM (n = 3). n.i., no inhibition. IC50,median inhibitory concentration.

Compound

ER binding IC50 (nM)

Inhibition of lipidperoxidation: IC50 (mM)

Binding to human

plasma proteins (%)

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Intrinsic water solubilityat 25°C (mg/ml)

ERa

ERb FTC TBARS

E2

1.3 0.7 11.8 ± 1.6 3.9 ± 0.4 97.8 ± 0.3 4.01 4.2 ± 0.3

DHED

>10,000 >10,000 n.i. n.i. 58.0 ± 0.6 1.67 60.3 ± 1.7

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produce estrogen in the circulation and peripheral tissues, as shown byliquid chromatography–tandem mass spectrometry (LC-MS/MS) as-says developed to distinguish different isotopic forms of the hormone.We also did not detect DHED in the brain, which indicated its rap-id metabolism in this organ. Oral (fig. S2, A and B) and subcutaneous(fig. S2, C and D) administrations of the prodrug generated results sim-ilar to those obtained upon intravenous injection (Fig. 2B). Thesefindings revealed that the increase of E2 in the brain after DHEDtreatment (Fig. 2B and fig. S2, A to C) originated from the conversionof the prodrug to E2—and not from the stimulation of endogenoushormone synthesis.

Results of detailed pharmacokinetic (PK) studies measuring E2concentrations in the brain and serum not only demonstrated a rapiduptake of DHED into the brain after systemic administration but alsoindicated its complete absorption after oral administration (table S1).In vivo experiments using OVX repTOP ERE-Luc mice, in which lu-ciferase expression is under the transcriptional control of ERs (20),also confirmed that prodrug treatments did not activate the ERE re-porter construct and failed to generate bioluminescence in peripheraltissues accessible to in vivo imaging, even at high subcutaneous or oraldoses (≥200 mg/kg) (fig. S3A). DHED was therefore not converted toE2 in the periphery. In contrast, mice treated subcutaneously with E2

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at a dose of 20 mg/kg resulted in a significant increase of photon emis-sion from the hepatic area (fig. S3B). Intracerebroventricular admin-istration followed by ex vivo bioluminescence imaging of OVXrepTOP ERE-Luc mice brain slices also revealed local estrogen forma-tion from DHED (fig. S3C). The conversion occurred in all brainareas, with the cortical areas showing the highest extent of DHEDbioactivation in this model, confirming our in vitro metabolism studiesin OVX rat brain tissues (Fig. 2A).

DHED treatment is neuroprotective andelicits estrogen-responsiveneuropharmacological effects in rodentmodels of CNS diseasesWe next evaluated the translational potential of DHED treatment inpreclinical models of estrogen-responsive human CNS disorders, in-cluding stroke, depression, hot flushes, and cognitive decline (21), in OVXrodents. Dose-dependent reduction of infarct volumes and attenuationof neurological deficits in animal models of stroke have been used fre-quently to quantify the extent of neuroprotection elicited by single-dose estrogens (16, 22). We used the most common paradigm, thetransient middle cerebral artery occlusion (tMCAO) model followedby reperfusion, in our initial proof-of-concept studies. Neuroprotectionwas manifested by a dose-dependent reduction of the infarct volumes inDHED-treated animals (Fig. 3A), with concomitant attenuation of

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Fig. 3. DHED treatment elicits neuroprotection in a rat model of ischemicstroke. (A) OVX Sprague-Dawley rats were treatedwith DHED (0.8 to 100 mg/kg,

scoresobserved in theexperimentdescribed in (A).Dataareaverages±SEM(n=3per treatment group). (C) Measured initial DHED-to-E conversion rates inmajor

subcutaneously) 1 hour before tMCAO followed by 24-hour reperfusion. The E2-treated group received 200 mg/kg, subcutaneously. TCC (2,3,5-triphenyltetrazoliumchloride)–stained brain sections are representative of three animals per indicatedtreatment and from the same animalswithin each column. Pale-colored regionsindicate areas of infarct, whereas red-colored regions represent viable areas. In-farct volumes are charted as averages± SEM (n=3per treatment group). (B) ND

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brain areas with tissue harvested 1 hour after tMCAO. Data are averages ±SEM (n = 3 per area). (D) Infarct volumes after treatment with DHED (100 mg/kg,subcutaneously) 1, 2, and3hoursafter tMCAO.Dataareaverages±SEM(n=3pertreatment group). (E) ND scores observed in the experiment described in (D).Pvaluesweredeterminedbyone-wayanalysisofvariance(ANOVA)usingStudent-Newman-Keuls (SNK) multiple comparison test.

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neurological deficits (Fig. 3B). The effective subcutaneous dose (ED50,equivalent to 50% of the maximum effect) was about 15 mg/kg; nearly10-fold higher systemic E2 dose was needed to achieve the same pro-tection against ischemic injury. These data indicate that the prodrugwas significantly more potent to deliver E2 into the brain than directadministration of the hormone. The ischemic brain retained its capac-ity to generate E2 from the prodrug (Fig. 3C) and, therefore, even post-infarct DHED treatment had a neuroprotective effect (Fig. 3, D and E).

OVX rodents are highly responsive to estrogen, and the forced swimtest (FST) using this model is widely used to screen for antidepressant-like activity (23). When administered at identical doses, DHED treat-ments outperformed direct administration of E2 in the FST (Fig. 4A). Thehigh-affinity ER antagonist ICI 182,780 completely blocked this anti-depressant-like effect in both treatment groups (Fig. 4B), suggesting anER-mediated mechanism. In support, we detected a significant amountof ICI 182,780 in the brain (6.6 ± 0.9 ng/g tissue) by LC-MS/MS 15 minafter completion of the FST. These data confirm the ability of this ERantagonist to enter the brain after systemic administration (24).

We demonstrated similar efficacy of DHED treatment in addition-al animal models of menopause-associated neurological disorders. In arat hot flush model (25), oral DHED treatment blunted the tail-skintemperature rise (Fig. 4C) using an orally bioavailable synthetic estrogen[17a-ethinylestradiol (EE)] as a positive control. In another animal

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model testing cognitive impairment owing to estrogen deficiency (26),we also measured the beneficial effect of DHED treatment. Continu-ous, long-term subcutaneous administration of DHED significantlydecreased working memory errors committed by OVX middle-agedrats compared to control animals in a delay match-to-sample (DMS)plus maze test (Fig. 4D).

DHED treatments produce markers of estrogenic effectsin the brainTo further validate that DHED delivery to the brain and its subsequentconversion to E2 had functional and potentially therapeutic relevance,we assayed for clinically relevant markers of estrogenic effects on thebrain (26–28). DHED treatment stimulated progesterone receptor (PR)expression in the preoptic area of the hypothalamus (Fig. 5A), increasedcholine acetyltransferase immunoreactive (ChAT-IR) stereological cellcounts in the medial septum and vertical diagonal band (Fig. 5B), andincreased the number of dendritic spines and spine heads in hippo-campal CA1 neurons (Fig. 5C), all compared with vehicle control treat-ment. These changes in the brain were indistinguishable from thoseseen after direct subcutaneous estrogen treatment at the same concen-tration. ICI 182,780 abolished the effect of DHED treatment on PRinduction (Fig. 5A), which suggested that this response was indeed dueto estrogen formed in situ from DHED.

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DHED treatment does not haveuterotrophic effects or promoteproliferation of cancerous tissueIn addition to sparing the liver from estro-gen exposure (fig. S3, A and B), DHEDtreatment did not result in wet uterusweight gain (Fig. 6A), which is a utero-trophic effect of systemically administeredestrogens (15, 29). This was independentof the route of administration (intravenous,subcutaneous, or oral) (fig. S4) and alsodid not occur after continuous, long-term(48-day) delivery of DHED using subcu-taneous osmotic minipumps (fig. S4C).For these experiments, vaginal smearsand standard cytology were used to con-firm that all OVX animals were in diestrusbefore E2 and DHED treatments wereinitiated. During and after DHED treat-ment, all animals in the DHED groupsremained continuously in diestrus similarto those in the OVX control group, whereasall E2-treated rats exhibited vaginal smearswith many cornified cells, indicating sys-temic estrogen exposure. In addition, al-though many protein markers are up- anddown-regulated by exogenous E2 (30), therewere no changes in the expression of a broadpanel of estrogen-regulated uterine pro-teins compared with control after DHEDtreatment (Fig. 6B and fig. S5).

Another undesired consequence ofincreasing estrogen in the peripheral cir-culation is its effect on cancerous breast

Fig. 4. DHED treatment elicits estrogen-responsive neuropharmacological effects. (A) Antidepressant-like activity after DHED treatments in CD1 mice using the FST. Vehicle control, as well as E and DHED

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(both at 10 mg/kg, subcutaneously, daily for 5 days) were evaluated 1 hour after the last injection. Data areaverages ± SEM (n = 5 to 6 per treatment group). (B) The antidepressant-like effect is reversible in both E2-and DHED-treated animals (once daily, for 5 days, at 50 mg/kg, subcutaneously) by the ER antagonist ICI182,780 (4 mg/kg, subcutaneously) co-injected with the test compounds. (C) Representative experimentsshowing tail-skin temperature changes in an OVX rat hot flush model after oral administrations of DHED(30 mg/kg), the orally active strong synthetic estrogen EE (200 mg/kg) used as positive control, and the ve-hicle control. (D) Vehicle-treated controls, as well as E2- and DHED-treated (continuously for 48 days bysubcutaneous Alzet osmotic minipumps delivering 4 mg daily) middle-aged OVX Fischer 344 rats weresubjected to the DMS plus maze test after a 2-day delay. Data are average errors made ± SEM (n = 4 to5 per treatment group). P values were determined by one-way ANOVA using SNK multiple compar-ison test.

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tissue. E2 and the aromatase substrate D4-androstenedione (D4A) in-duced the expected cancer-promoting ERa transactivation in culturedhuman MCF-7Ca cells (31), whereas DHED did not (Fig. 6C). UnlikeD4A, DHED treatment also did not stimulate the growth of aromatase-transfected MCF-7Ca breast cancer cells in vivo in a nude mouse xe-nograft model (Fig. 6D), indicating that DHED indeed remained inertin these cells.

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DISCUSSION

Estrogen-based therapies have remained the most effective remediesto treat menopausal indications; however, they are neither appropriatenor desirable for all symptomatic women. For example, estrogens arecontraindicated after preventive oophorectomy (1, 32), which is in-creasingly performed in gynecological oncology. Therefore, a growingpopulation of women will not have effective and safe treatment againstneurological and psychiatric maladies triggered by estrogen deficiencyof the brain owing to surgical menopause (33). To address this majorunmet medical need, we explored a new prodrug, DHED, that remainsinert in the body—only to be quickly converted to estrogen in the brain.Evidence provided here indicates that the reductive bioactivation ofDHED to estrogen is brain-selective in female animal models, whichcould offer unique benefits to the treatment of human diseases asso-ciated with brain estrogen deficiency or for those benefiting fromestrogen-mediated neuroprotection.

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Adverse consequences of estrogen deficiency in the human brainhave been seen inevitably andmost markedly upon bilateral oophorectomyperformed before reproductive senescence, which precipitates prematuremenopause. Symptoms of premature menopause and also of menopauseresulting from reproductive senescence include, among others, hotflushes, depression, cognitive impairment, and an increased risk of is-chemic stroke (5, 6, 34). Consequently, we chose widely accepted ani-mal models of these human conditions to evaluate our DHED-basedbrain-selective intervention for a direct, side-by-side comparison withestrogen therapy. Collectively, our results indicate that DHED entersthe brain of OVX rodents, where it is rapidly metabolized to the mainestrogen, E2. In estrogen-sensitive peripheral tissues and in the circu-lation, DHED is not converted to E2 and, thus, remains inert. Rapidmetabolism of this prodrug to the hormone in the brain also explainedwhy brain levels of DHED were undetectable. Overall, the significantelevation of brain E2 levels after DHED treatments originated solelyfrom the metabolism of the inactive bioprecursor and not from DHED-induced E2 biosynthesis.

In addition to abolishing affinity to ER and to other steroid recep-tors, the unique structural attributes (that is, a cyclohexadienone A-ringand the oxidatively added 10b-hydroxyl group) also favorably changekey physicochemical and biopharmaceutical properties of DHED incomparison with those of E2. The benefits of these changes are shownin this study through rodent models demonstrating delivery of E2 to thebrain without toxic and uterotrophic systemic side effects seen for ex-ogenous estrogen treatments. Compared to DHED treatment, an order

Fig. 5. Markers of estrogenic effects in the brain are similar after E2 andDHED treatments. (A) PR expression in the brain. OVX Sprague-Dawley ratswere treated with E2 (50 mg/kg, subcutaneously), DHED (50 mg/kg, sub-cutaneously), or DHED (50 mg/kg, subcutaneously) + ICI 182,780 (1 mg/kg,subcutaneously, administered 1 hour before DHED), as in Fig. 4B. In situ hy-bridization images of PR were quantified through the measurement ofoptical densities. Optical densities are averages ± SEM (n = 4 to 8 per treat-ment group). (B) ChAT-IR–positive neurons counted in the medial septumand vertical diagonal band. OVX Fischer 344 rats were treated with E2 or DHEDcontinuously for 48 days at 4 mg/day by subcutaneous osmotic pumps. Data areaverage stereological ChAT-IR neuron counts ± SEM (n = 4 to 7 per treatmentgroup). (C) Spine density and the number of spine heads in dendrites of CA1neurons. OVX Fischer 344 rats were treated with DHED or E2 (10 mg, sub-cutaneously) every 4 days for 40 days. Data are averages ± SEM (n = 5 to 7per treatment group). P values in (A) to (C) were determined by one-wayANOVA using SNK multiple comparison test.

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of magnitude higher dose of estrogen was needed to generate a similarextent of neuroprotection and neuropharmacological responses in ani-mal models of estrogen deficiency. This finding highlights brain-selectiveE2 therapy by DHED as a better “drug economy”; that is, it focuses thebiologically active estrogen to the brain—unlike systemic treatmentwith E2 that distributes the hormone throughout the entire body.

DHED-based E2 therapy was more efficacious than direct E2 admin-istration in a cerebrovascular stroke model. This improvement isattributed to the favorable physicochemical properties of DHED forblood-brain barrier transport from the circulation and based on mech-anisms by which estrogens are known to protect neurons against is-chemic injury. As expected from in vitro experiments indicating DHEDbioactivation by the ischemic tissue, we also observed significant neuro-protection even when the animals received DHED up to 2 hours after thestroke. These additional results may expand the potential benefits ofbrain-selective estrogen therapy from stroke prevention in surgicallymenopausal women to effective post-stroke medication for all patients.

E2 has been shown to alleviate depression-like condition precipi-tated by a hypoestrogenic state in mice (23), and the antidepressant

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activity of the hormone is due to a mul-timodal mechanism of action involvingthe regulation of several pathways and func-tions (11). DHED treatment profoundlyreduced behavioral immobility, whichwas reversible by an ER antagonist, thelatter indicating that, similar to E2 treat-ment, an estrogen was responsible for theobserved pharmacological response. Inour preclinical evaluation, DHED admin-istration was also effective in an establishedrat model of hot flushes (25), which areprevalent and long lasting during the men-opause transition (35).

A drop in E2 levels after surgical men-opause also may affect key parts of thebrain responsible for memory and con-centration, including the hippocampus,which has been shown to be associatedwith cognitive impairment (21, 36). Inyoung adult and middle-aged female rats,ovarian hormone loss induces cognitiveimpairment that can be reversed by sys-temic estrogen treatment (26). In our study,middle-aged rats committed significantlyfewer working memory errors in a DMSplus maze test after chronic administrationof DHED than did the OVX control ani-mals. The increase in brain E2 after system-ic DHED treatment appeared to have nodirect effect on hippocampal estrogen bio-synthesis. In subsequent experimentscomplementing the neuropharmacologicalassessments, we showed that E2 generatedfrom DHED in the brain produced theexpression of characteristic markers indi-cating central estrogenic effects, and DHED-treated groups were indistinguishable fromthe E2-treated control groups.

In current clinical practice, chronic hormone therapies require theuse of a progestin to counterbalance the detrimental impact of estrogenson the endometrium. Progestins, however, have been shown to in-terfere with the beneficial effect of estrogens in the brain (37). Consist-ent with no change in circulating E2 levels after DHED treatments,OVX rodents did not display E2-induced uterotrophic effects whendosed with DHED. All systemically estrogen-treated animals in ourexperiments showed the expected uterotrophic effect. The lack of uterineresponse upon DHED treatment was confirmed using proteomicmarkers of E2 activity. Overall, uterine protein expression was not dif-ferent between the DHED- and vehicle-treated groups. Thus, the lackof DHED’s uterotrophic activity would obviate the need for coadmi-nistration of a progestin.

The induction of aromatase-dependent breast cancer has alsobeen considered a serious risk of current estrogen therapies often con-traindicating their use to manage symptoms of menopause clinically(33). However, DHED-treated cells did not show ERa transactivationin vitro or stimulate MCF-7Ca xenografts in vivo. In further supportof the inherent inertness and safety of DHED, we found that the liver

Fig. 6. DHED treatment avoids uterotrophic effect and proliferation of cancerous breast tissue. (A)OVX Swiss-Webster mice injected subcutaneously for five consecutive days with vehicle or E2 or DHED

(50 mg/kg) every day. Data are averages ± SEM (n = 11 per treatment group). P values were determinedby one-way ANOVA using SNK multiple comparison test. (B) Expression data for representative estrogen-sensitive uterine proteins desmin (Des), elongation factor 1-a isoform 1 (Eef1a1), glutathione S-transferasem isoform 1 (Gstm1), and the mimecan precursor (Ogn) from the uteri of OVX Swiss-Webster mice treatedwith vehicle, E2, or DHED (once daily for five consecutive days, at 50 mg/kg, subcutaneously, every day). Dataare expressed as normalized LC-MS/MS spectral counts (average ± SD, n = 5 per treatment group). P valueswere determined by one-way ANOVA using SNK multiple comparison test. (C) ERa transactivation in MCF-7Ca breast cancer cells incubated with vehicle or 1 nM estrogen prohormone D4A (positive control), E2, orDHED. Luminescence data are averages ± SEM (n = 4 per treatment). P values were determined by one-wayANOVA using SNK multiple comparison test. (D) Tumor volumes in nude mice with human MCF7-Ca xe-nografts after daily subcutaneous injections of vehicle, D4A (100 mg/kg), or DHED (100 mg/kg) for 8 weeks.Data are averages ± SEM (n = 8 per treatment group). P values were determined by two-way ANOVA withtreatment as the main factor and repeated measurements in time followed by SNK multiple comparison test.

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was not exposed to estrogen after DHED treatment even at high oraldoses (≥200 mg/kg). In contrast, subcutaneous administration of E2even at 1/10th of DHED’s dose showed the expected profound ER ac-tivation in the liver—a common hepatic side effect of current estrogentherapies.

Collectively, our comprehensive and multidisciplinary preclinicalstudies have revealed that E2’s activity may be effectively confined tothe brain of female rodents through the unique metabolism of a sys-temically administered bioprecursor prodrug. DHED has excellent oralbioavailability, which is a requirement along with additional drug-likeproperties, such as appropriate lipophilicity, reduced plasma-proteinbinding, and increased solubility in water, for successful pharmaceuticaldevelopment. Our study indicates the translational potential of DHEDfor brain-selective estrogen therapy, especially to remedy neurologicaland psychiatric symptoms of early and surgical menopause, and poten-tially also in a wide range of human diseases associated with estrogendeficiency or benefiting from estrogen-mediated protection of the brain.

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MATERIALS AND METHODS

Study designThis preclinical study was designed to test the ability of DHED to se-lectively metabolize to E2 in the brain and thereby provide brain-selective estrogen therapy. In vitro metabolism studies were performedusing homogenates prepared from both healthy and diseased rat tis-sues. PK studies (with intravenous, subcutaneous, and oral dosing)measuring prodrug and estrogen concentrations, respectively, in targettissues and serum were carried out for proof of concept. Comple-mentary in vivo imaging was done using various routes of prodrugadministration (subcutaneous, oral, and intracerebroventricular). Con-firmatory pharmacological assessments and assessment of markers re-vealing estrogenic effects were performed in animal models known torespond to exogenously administered E2. These paradigms enabled thedirect comparison between the prodrug (DHED) and the parent drug(E2) to highlight the distinguishing and unique properties of DHED interms of brain-selective estrogen therapy (end points). For each exper-iment, sample sizes were chosen to minimize the number of OVX ani-mals needed while obtaining sufficient statistical power. Treatmentduration and dosage regimen were selected on the basis of publishedexperiments involving estrogens after considering drug-like propertiesof DHED from in silico predictions and experimental measurements.Assays were carried out by blinded investigators.

ChemicalsThe method used for the synthesis of DHED was based on a procedureadapted from the literature (38) using E2 (Steraloids), m-chloroperbenzoicacid (Janssen Chimica) oxidant, and dibenzoyl peroxide (Aldrich)radical initiator in refluxing CH2Cl2 under nitrogen for 3 hours whilebeing irradiated with a 60-W tungsten lamp. Column chromatographicpurification [silica gel; CH2Cl2/ethyl acetate, 7:3 (v/v)] and crystalliza-tion from toluene routinely afforded ~45% yield [melting point: 215to 217°C; 1H NMR (nuclear magnetic resonance) resonances havingdiagnostic d values (in ppm), using CD3OD solvent: 7.1 (d, J = 10.4 Hz,1H, H1), 6.0 (dd, J = 10.4 Hz, 2.1 Hz, 1H, H2), 5.9 (t, J = 2.2 Hz,1H, H4), 3.3 (t, J = 8.1 Hz, 1H, 17a-H), and 0.9 (s, 18-CH3); ESI-MS(electrospray ionization mass spectrometry): mass/charge ratio (m/z)289.3 (M+H)+]. We synthesized the deuterated analogs (d3- and d5-

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DHED) according to a previously reported microwave-assisted pro-cedure (39). All other chemicals and reagents were purchased fromcommercial vendors.

Characterization of DHEDSteroid receptor-binding experiments were performed by Caliper LifeSciences. Inhibition of lipid peroxidation was assayed in triplicate bythe FTC and TBARS methods (40). Binding to human plasma pro-teins was measured by rapid equilibrium dialysis (with a single-usedialysis plate with inserts, Thermo Scientific) according to the manu-facturer’s protocol. The logarithm of n-octanol/water partition co-efficient (logP) was predicted by the method incorporated into theBioMedCAChe program (version 6.1, Fujitsu America Inc.) and wasverified experimentally by the shake flask technique. Intrinsic watersolubility (Sw) was measured in deionized water at 25°C and usingan equilibrium solubility method adopted from an earlier procedure(41) by performing quantitative LC-MS/MS analyses (40).

In vitro metabolic conversions of DHED to E2Initial in vitro conversion rates in freshly prepared 20% (w/v) OVX rattissue homogenates were measured by the procedure and analyticalmethod described previously (16). Assays were performed in triplicateafter addition of d5-E2 from stock solution to reach internal standardconcentration equivalent to 2 mM in the sample. Liquid-liquid extrac-tion and quantitation by isotope dilution LC-MS/MS were done asreported before (40).

Animal treatments and monitoringSaline and corn or sesame oil vehicles were used for intravenous andsubcutaneous injections, respectively, whereas compounds were deliv-ered by gavage in a saline or phosphate-buffered saline (PBS) vehicleupon oral administration. PBS also served as a vehicle for intracerebro-ventricular injection. E2, EE, and DHED stock solutions were preparedin dimethyl sulfoxide (DMSO) or ethanol, which were admixed to thevehicles. Concentrations in the stock solutions and mixing ratios werechosen to keep the percentage of the organic co-solvent at a minimumwhile avoiding the precipitation of the compounds. For DHED, thisrepresented ≤3% (v/v) of DMSO or ethanol in the administration-ready solutions. For continuous subcutaneous administration by ALZETosmotic pumps (model 2ML4 rated for delivery at 0.25 ± 0.05 ml/hourfor 28 days; Durect Corp.), E2 or DHED was dissolved in propyleneglycol. For control rats, pumps were filled only with propylene glycol.Pump insertion was done 18 days after ovariectomy under vaporizedisoflurane anesthesia according to the surgical procedure recommendedby the manufacturer. We replaced the pumps with freshly filled newones after 15 days of delivery; this ensured that all animals had theirassigned substrate delivered for the duration of behavioral testing andthrough euthanasia 48 days after the surgical insertion of the firstpump. During the infusion period, we examined vaginal smearsaccording to a previously reported procedure (42). Transgenic Op-erative Products srl performed the experiments involving repTOPERE-Luc mice, for which the animals were fed a special estrogen-freediet (43).

Drug distribution in vivoIn vivo microdialysis experiments were performed as described previ-ously (44). Measurements from serum and brain tissue were madeafter dissecting the brain into regions, homogenization, liquid-liquid

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extraction, dansylation, and using 13C-labeled internal standards(13C6-E2 and

13C6-E1; each at 100 pg/ml serum and 1.7 ng/g wet tissue)for quantification by isotope-dilution LC-MS/MS (45). DHED analy-ses were carried out in separate LC-MS/MS assays using a Supelco Dis-covery HS C18 column, 50 mm × 2.1 mm, packed with 5-mm particles.The elution was isocratic at a flow rate of 0.25 ml/min with a mixture ofacetonitrile and water [23:77 (v/v)] containing 0.5% (v/v) acetic acid.DHED and the added internal reference compound (d5-DHED) in theeffluent were detected by ion trap (LTQ) or triple-quadrupole (TSQQuantum Ultra, Thermo Scientific) mass spectrometers equipped withESI sources operated in the positive ionization mode. Quantificationswere performed, similar to those of estrogens (45), by isotope-dilutionMS/MS. Protein contents in tissues were measured by a dye-bindingassay (Thermo Scientific). Noncompartmental PK analyses (46) wereperformed by nonlinear curve fitting using Scientist for Windows(Micromath).

Focal ischemia/tMCAO-reperfusion injury modelExperiments were performed as previously described (47), and theinfarct volumes were measured after 1 hour of tMCAO followedby 24-hour reperfusion. Vehicle, E2, and DHED were injected subcu-taneously 1 hour before tMCAO in the stroke prevention study, whereasDHED injections (100 mg/kg, subcutaneously) were also administeredimmediately upon starting reperfusion (that is, 1 hour after tMCAO),as well as 1 and 2 hours thereafter (2 and 3 hours after tMCAO, respec-tively). The harvested brains were kept in ice-cold saline for 5 min,after which seven coronal slices (2-mm thickness) were cut from eachbrain for 15-min incubation in 2% TCC solution at 37°C. Infarct vol-ume was determined from measurements performed using the ImageJsoftware (U.S. National Institutes of Health). Neurological deficitswere assessed and scored as follows: 0, normal (that is, no neurologicaldeficit observed); 1, mild impairment (the animal failed to extend thecontralateral forepaw on lifting the whole body by the tail); 2, moderateimpairment (the animal circled contralaterally); 3, severe impairment(the animal leaned contralaterally when resting).

Computational studyAll computations were performed with the SCIGRESS molecularmodeling suit (version 2.6; Fujitsu–FQS Poland). Structures for groundand transition states were calculated using the PM6 and PM6-TS semi-empirical quantum chemical parameterizations of the Molecular OrbitalPackage (MOPAC, Stewart Computational Chemistry) (48). Theconductor-like screening model (COSMO) was used to simulate con-tinuum solvation in water (with the e permittivity parameter set to78.4) (49). Thermodynamic quantities were calculated with the THERMOprogram of MOPAC.

Statistical analysisWe applied Shapiro-Wilk tests, visual inspections of histograms, nor-mal Q-Q plots, and box plots to confirm normal distribution of data.Statistical evaluations were done by one-way ANOVA in all but oneexperiment (effect on MCF-7Ca breast cancer xenografts in athymicnude mice, where we used repeated-measures two-way ANOVA). Allof our follow-up, two-group comparisons used SNK post hoc testswhen a significant omnibus ANOVA (with two-tailed a set to 0.05)was found, noting that type I error correction was not necessary withorthogonal planned comparisons (50). P < 0.05 was considered statis-tically significant.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/297/297ra113/DC1MethodsFig. S1. DHED is converted to E2 in vitro in brain, but not uterine tissue of OVX Sprague-Dawley rats.Fig. S2. Estrogen concentrations are increased in brain but not in circulation after DHED treatments.Fig. S3. Bioluminescence imaging in repTOP ERE-Luc mice.Fig. S4. Lack of uterotrophic effect after DHED treatment is independent of route of administrationand treatment regimen.Fig. S5. Additional expression data for estrogen-sensitive proteins from the uteri of OVX micetreated with E2 and DHED.Table S1. Noncompartmental single-dose PK analysis of extracted E2 concentrations in brainover time.Reference (51)

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Acknowledgments: We thank P. Fryčák, B. Blazics, B. Ughy, S. M. Stevens Jr., M. Lane, S. White,and B. Cornelius for their expert contribution or excellent technical assistance. L.P. is gratefulto M.J. Forster and M. Arad for their help with statistical analyses. Funding: The project wassupported in part by the NIH [NS044765 (L.P.), RR012023 (L.P), AG031535 (I.M. and L.P.),AG027956 (P.K. and L.P.), AG028084 (H.B.-N.), AG031421 (K.P.-T.), and MH100700 (T.D.G. andL.P.)], the Felix and Carmen Sabates Missouri Endowed Chair in Vision Research, the VisionResearch Foundation of Kansas City (P.K.), and the Robert A. Welch Foundation (endowmentBK-0031; L.P.). Author contributions: L.P., H.A.B.-N., C.D.C., A.B., P.J.S., I.M., P.K., and K.P.-T. de-signed the research; L.P. performed computational chemistry and statistical analyses; K.P.-T.synthesized and characterized DHED and its labeled analogs, as well as led the in vitro me-tabolism studies; V.N. completed pharmacokinetics, in vivo microdialyses, and the FSTs; S.S.ran the LC-MS/MS and plasma protein-binding assays; P.G. performed the stroke study; G.S.executed the breast cancer stimulation assessments; K.J.M. collected the data on hippocampaldendritic spines and heads; J.S.T. did the maze test and measured ChAT-IR; I.M. completed thehot flush and PR expression experiments. L.P., H.A.B.-N., J.S.T., K.J.M., C.D.C., T.D.G., I.M., P.K., andK.P.-T. analyzed and interpreted data. L.P. and K.P.-T. co-wrote the manuscript, with contribu-tion by P.K. All authors have read and commented on the final manuscript. Competing in-terests: L.P. and K.P.-T. have equity interests in AgyPharma LLC. L.P. and K.P.-T. hold U.S.patents 7,026,306 and 7,300,926 on the use of DHED. Data and materials availability: DHEDmust be obtained through a material transfer agreement.

Submitted 12 September 2014Accepted 26 June 2015Published 22 July 201510.1126/scitranslmed.aab1290

Citation: L. Prokai, V. Nguyen, S. Szarka, P. Garg, G. Sabnis, H. A. Bimonte-Nelson,K. J. McLaughlin, J. S. Talboom, C. D. Conrad, P. J. Shughrue, T. D. Gould, A. Brodie,I. Merchenthaler, P. Koulen, K. Prokai-Tatrai, The prodrug DHED selectively delivers 17b-estradiol to the brain for treating estrogen-responsive disorders. Sci. Transl. Med. 7,297ra113 (2015).

ceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra113 10

estrogen-responsive disorders-estradiol to the brain for treatingβThe prodrug DHED selectively delivers 17

Merchenthaler, Peter Koulen and Katalin Prokai-TatraiMcLaughlin, Joshua S. Talboom, Cheryl D. Conrad, Paul J. Shughrue, Todd D. Gould, Angela Brodie, Istvan Laszlo Prokai, Vien Nguyen, Szabolcs Szarka, Puja Garg, Gauri Sabnis, Heather A. Bimonte-Nelson, Katie J.

DOI: 10.1126/scitranslmed.aab1290, 297ra113297ra113.7Sci Transl Med

on the brain and relieve symptoms in patients with a broad range of central nervous system diseases.With an efficacious and safe profile in vivo, this prodrug has the opportunity to provide positive estrogenic effects stroke, but without the negative systemic (specifically uterotrophic and cancerous) effects of the free hormone.rodents from neurological symptoms of estrogen deprivation, and also provided neuroprotection to rats after

-estradiol itself, DHED protected menopausal femaleβ-estradiol, only in the brain. Similar to 17βestrogen, 17-dihydroxyestra-1,4-dien-3-one), that is selectively converted to one mainβ, 17β''prodrug,'' called DHED (10

. discovered aet alis no trivial feat. Prokai −−and avoiding activity in other tissues−−the hormone to the brain onlyAlthough estrogen is considered to heal the brain of many neurological and psychiatric symptoms, targeting

Estrogen prodrug protects the brain

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http://stm.sciencemag.org/content/scitransmed/5/190/190ra79.fullhttp://stm.sciencemag.org/content/scitransmed/4/156/156ps20.fullhttp://stm.sciencemag.org/content/scitransmed/2/27/27rv1.fullhttp://stm.sciencemag.org/content/scitransmed/6/220/220ra12.fullhttp://stm.sciencemag.org/content/scitransmed/4/158/158rv11.full

REFERENCES

http://stm.sciencemag.org/content/7/297/297ra113#BIBLThis article cites 49 articles, 2 of which you can access for free

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