Research ArticleNeurology

The prodrug DHED selectively delivers 17β-estradiol to the brain for treating estrogen-responsive disorders

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Science Translational Medicine  22 Jul 2015:
Vol. 7, Issue 297, pp. 297ra113
DOI: 10.1126/scitranslmed.aab1290
  • 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 short-chain dehydrogenase/reductase (SDR). (B) In silico mechanistic model for DHED bioactivation to E2. Quantum-chemical calculations simulating solvation with water were applied to the complete steroid structures and a mimic of NAD(P)H ⇌ NAD(P)+, with R chosen as methyl. (C) In contrast, the aromatase-catalyzed conversion of androgens to estrogens proceeds through sequential six-electron oxidation on the C19 methyl group involving molecular oxygen.

  • Fig. 2. DHED is a brain-selective prodrug.

    (A) Initial rate of in vitro E2 formation during DHED incubation (100 nM) in homogenates of various OVX rat brain structures versus rat uterus homogenate; the latter represents an estrogen-sensitive peripheral tissue. Data are averages ± SEM (n = 3 per tissue). N/D, not detected. (B) Serum DHED and E2 concentrations in serum and in the brain of OVX Sprague-Dawley rats after intravenous administration of DHED (200 μg/kg). Concentrations were measured by LC-MS/MS–based bioassays. Serum concentrations are averages ± SEM (n = 3 to 4 animals per time point); brain concentrations are weighted averages ± SEM from measurements in seven brain regions (n = 3 to 4 animals per time point). E2 concentrations at time “0” originated from analyses of tissue and serum obtained from vehicle-treated animals euthanized immediately after injection. (C) Increase of E2 in brain tissue after DHED treatment arises from the metabolism of the bioprecursor prodrug. d3-DHED was administered to OVX Sprague-Dawley rats intravenously followed by tissue harvesting at 15 min after dosing and LC-MS/MS analysis [using selected reaction monitoring (SRM)]. All SRM chromatograms were obtained from the analyses of hippocampal tissue and were scaled to the same ion abundance (5.0 × 104). The trace for the unlabeled (that is, endogenous) E2 is obtained by SRM of m/z 506→171, the red trace (SRM: m/z 509→171) represents 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-labeled hormone ([13C6]E2) added as internal standard for identification and quantitation.

  • Fig. 3. DHED treatment elicits neuroprotection in a rat model of ischemic stroke.

    (A) OVX Sprague-Dawley rats were treated with DHED (0.8 to 100 μg/kg, subcutaneously) 1 hour before tMCAO followed by 24-hour reperfusion. The E2-treated group received 200 μg/kg, subcutaneously. TCC (2,3,5-triphenyltetrazolium chloride)–stained brain sections are representative of three animals per indicated treatment and from the same animals within each column. Pale-colored regions indicate areas of infarct, whereas red-colored regions represent viable areas. Infarct volumes are charted as averages ± SEM (n = 3 per treatment group). (B) ND scores observed in the experiment described in (A). Data are averages ± SEM (n = 3 per treatment group). (C) Measured initial DHED-to-E2 conversion rates in major 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 μg/kg, subcutaneously) 1, 2, and 3 hours after tMCAO. Data are averages ± SEM (n = 3 per treatment group). (E) ND scores observed in the experiment described in (D). P values were determined by one-way analysis of variance (ANOVA) using Student-Newman-Keuls (SNK) multiple comparison test.

  • 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 E2 and DHED (both at 10 μg/kg, subcutaneously, daily for 5 days) were evaluated 1 hour after the last injection. Data are averages ± 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 μg/kg, subcutaneously) by the ER antagonist ICI 182,780 (4 mg/kg, subcutaneously) co-injected with the test compounds. (C) Representative experiments showing tail-skin temperature changes in an OVX rat hot flush model after oral administrations of DHED (30 μg/kg), the orally active strong synthetic estrogen EE (200 μg/kg) used as positive control, and the vehicle control. (D) Vehicle-treated controls, as well as E2- and DHED-treated (continuously for 48 days by subcutaneous Alzet osmotic minipumps delivering 4 μg daily) middle-aged OVX Fischer 344 rats were subjected to the DMS plus maze test after a 2-day delay. Data are average errors made ± SEM (n = 4 to 5 per treatment group). P values were determined by one-way ANOVA using SNK multiple comparison test.

  • Fig. 5. Markers of estrogenic effects in the brain are similar after E2 and DHED treatments.

    (A) PR expression in the brain. OVX Sprague-Dawley rats were treated with E2 (50 μg/kg, subcutaneously), DHED (50 μg/kg, subcutaneously), or DHED (50 μg/kg, subcutaneously) + ICI 182,780 (1 mg/kg, subcutaneously, administered 1 hour before DHED), as in Fig. 4B. In situ hybridization images of PR were quantified through the measurement of optical densities. Optical densities are averages ± SEM (n = 4 to 8 per treatment group). (B) ChAT-IR–positive neurons counted in the medial septum and vertical diagonal band. OVX Fischer 344 rats were treated with E2 or DHED continuously for 48 days at 4 μg/day by subcutaneous osmotic pumps. Data are average stereological ChAT-IR neuron counts ± SEM (n = 4 to 7 per treatment group). (C) Spine density and the number of spine heads in dendrites of CA1 neurons. OVX Fischer 344 rats were treated with DHED or E2 (10 μg, subcutaneously) every 4 days for 40 days. Data are averages ± SEM (n = 5 to 7 per treatment group). P values in (A) to (C) were determined by one-way ANOVA using SNK multiple comparison test.

  • 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 μg/kg) every day. Data are averages ± SEM (n = 11 per treatment group). P values were determined by one-way ANOVA using SNK multiple comparison test. (B) Expression data for representative estrogen-sensitive uterine proteins desmin (Des), elongation factor 1-α isoform 1 (Eef1a1), glutathione S-transferase μ isoform 1 (Gstm1), and the mimecan precursor (Ogn) from the uteri of OVX Swiss-Webster mice treated with vehicle, E2, or DHED (once daily for five consecutive days, at 50 μg/kg, subcutaneously, every day). Data are expressed as normalized LC-MS/MS spectral counts (average ± SD, n = 5 per treatment group). P values were determined by one-way ANOVA using SNK multiple comparison test. (C) ERα transactivation in MCF-7Ca breast cancer cells incubated with vehicle or 1 nM estrogen prohormone Δ4A (positive control), E2, or DHED. Luminescence data are averages ± SEM (n = 4 per treatment). P values were determined by one-way ANOVA using SNK multiple comparison test. (D) Tumor volumes in nude mice with human MCF7-Ca xenografts after daily subcutaneous injections of vehicle, Δ4A (100 μg/kg), or DHED (100 μg/kg) for 8 weeks. Data are averages ± SEM (n = 8 per treatment group). P values were determined by two-way ANOVA with treatment as the main factor and repeated measurements in time followed by SNK multiple comparison test.

  • Table 1. ER affinity, antioxidant potency, plasma protein binding, and selected physicochemical properties of E2 and DHED.

    Lipid peroxidation was determined using the FTC (ferric thiocyanate) and TBARS (thiobarbituric acid reactive substances) assays. LogP denotes the logarithm of n-octanol/water partition coefficient as a measure of lipophilicity with values predicted 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.

    CompoundER binding IC50 (nM)Inhibition of lipid peroxidation: IC50 (μM)Binding to human plasma proteins (%)Lipophilicity (logPcalc)Intrinsic water solubility at 25°C (μg/ml)
    ERαERβFTCTBARS
    E21.30.711.8 ± 1.63.9 ± 0.497.8 ± 0.34.014.2 ± 0.3
    DHED>10,000>10,000n.i.n.i.58.0 ± 0.61.6760.3 ± 1.7

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/297/297ra113/DC1

    Methods

    Fig. 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 administration and treatment regimen.

    Fig. S5. Additional expression data for estrogen-sensitive proteins from the uteri of OVX mice treated with E2 and DHED.

    Table S1. Noncompartmental single-dose PK analysis of extracted E2 concentrations in brain over time.

    Reference (51)

  • Supplementary Material for:

    The prodrug DHED selectively delivers 17β-estradiol to the brain for treating estrogen-responsive disorders

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

    *Corresponding author. E-mail: laszlo.prokai{at}unthsc.edu

    Published 22 July 2015, Sci. Transl. Med. 7, 297ra113 (2015)
    DOI: 10.1126/scitranslmed.aab1290

    This PDF file includes:

    • Methods
    • Fig. 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 administration and treatment regimen.
    • Fig. S5. Additional expression data for estrogen-sensitive proteins from the uteri of OVX mice treated with E2 and DHED.
    • Table S1. Noncompartmental single-dose PK analysis of extracted E2 concentrations in brain over time.
    • Reference (51)

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