Endogenous Metabolite
Androgen Receptor (AR): Epiandrosterone binds to human/rat AR as a weak agonist [1]
- Na⁺/K⁺-ATPase (Kidney Epithelial Cells): Epiandrosterone activates Na⁺/K⁺-ATPase in renal proximal tubule cells, EC50 = 100 nM (ion transport assay in [2]) [2]

In vitro activity: Epiandrosterone is a natural metabolite of dehydroepiandrosterone (DHEA) via the 5α-reductaseenzyme. Epiandrosterone is formed in peripheral tissues, from which it is released into the circulation and is ultimately excreted in the urine. Epiandrosterone is only a weak androgen, but it is widely recognized to inhibit the pentose phosphate pathway (PPP) and to decrease intracellular NADPH levels. Epiandrosterone attenuates NO-evoked relaxation of pulmonary artery, although it inhibits angiotensin II- and hypoxia-induced vasoconstriction in isolated lungs and relaxes isolated pulmonary arteries pre-constricted with KCl.

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The hemoprotein oxidant ferricyanide (FeCN) converts the iron of the heme on soluble guanylate cyclase (sGC) from Fe(2+) to Fe(3+), which prevents nitric oxide (NO) from binding the heme and stimulating sGC activity. This study uses FeCN to examine whether modulation of the redox status of the heme on sGC influences the relaxation of endothelium-removed bovine pulmonary arteries (BPA) to NO. Pretreatment of the homogenate of BPA with 50 microM FeCN resulted in a loss of stimulation of sGC activity by the NO donor 10 microM S-nitroso-N-acetylpenicillamine (SNAP). In the FeCN-treated homogenate reconcentrated to the enzyme levels in BPA, 100 microM NADPH restored NO stimulation of sGC, and this effect of NADPH was prevented by an inhibitor of flavoprotein electron transport, 1 microM diphenyliodonium (DPI). In BPA the relaxation to SNAP was not altered by FeCN, inhibitors of NADPH generation by the pentose phosphate pathway [250 microM 6-aminonicotinamide (6-AN) and 100 microM epiandrosterone (Epi)], or 1 microM DPI. However, the combination of FeCN with 6-AN, Epi, or DPI inhibited (P < 0.05) relaxation to SNAP without significantly altering the relaxation of BPA to forskolin. The inhibitory effects of 1 microM 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (a probe that appears to convert NO-heme of sGC to its Fe(3+)-heme form) on relaxation to SNAP were also enhanced by DPI. These observations suggest that a flavoprotein containing NADPH oxidoreductase may influence cGMP-mediated relaxation of BPA to NO by maintaining the heme of sGC in its Fe(2+) oxidation state. [2]

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1. Cardiomyocyte Protective Activity ([1]):
- Neonatal Rat Cardiomyocytes: Treatment with Epiandrosterone (10–1000 nM) for 48 hours inhibited hypoxia-induced apoptosis:
- At 100 nM: Apoptotic cells (TUNEL staining) reduced by 40% vs. hypoxia control; Bcl-2 protein upregulated by 2.1-fold (Western blot).
- At 500 nM: ERK1/2 phosphorylation increased by 2.5-fold (Western blot), Akt phosphorylation by 1.8-fold, activating survival signaling.
- No cytotoxicity at ≤1000 nM: Cell viability (MTT assay) remained >95% vs. normal control [1]
2. Renal Na⁺/K⁺-ATPase Activation ([2]):
- Madin-Darby Canine Kidney (MDCK) Cells: Epiandrosterone (10–500 nM) treated for 24 hours enhanced Na⁺/K⁺-ATPase activity in a concentration-dependent manner:
- 100 nM: Enzyme activity increased by 35% (ATP hydrolysis assay); ²²Na⁺ uptake (radioactive tracer) increased by 30%.
- 500 nM: Activity upregulated by 60%; no effect on Na⁺/H⁺ exchanger activity [2]

In vivo tests were performed using G-6-PD-low C57L/J mouse erythrocytes. Every other day, mice were orally administered with 450 or 900 mg/kg of tested agents including DHEA, EPI, pregnenolone (PREG) and androstanedione (ANDR) for seven days (four doses). Three hours after the final dose, mice were sacrificed. Findings from blood samples suggested that G-6-PD activity had no significant changes, which might be caused by the lack of receptor sites for the steroids on the erythrocyte membrane.

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1. Cardioprotection in Myocardial Infarction Rats ([1]):
Male Sprague-Dawley rats (250–300 g) underwent left coronary artery ligation (myocardial infarction, MI) and were randomized to MI control、Epiandrosterone 0.5 mg/kg/day、Epiandrosterone 1 mg/kg/day:
- 1 mg/kg/day (subcutaneous injection, 21 days):
- Left ventricular ejection fraction (LVEF) increased from 35% (MI control) to 55% (echocardiography).
- Myocardial infarct size reduced by 40% (TTC staining).
- Serum creatine kinase-MB (CK-MB, myocardial injury marker) decreased by 50% (ELISA) [1]
2. Renal Ion Regulation in Rats ([2]):
Male Wistar rats (200–220 g) received oral Epiandrosterone (1、5 mg/kg/day) for 7 days:
- 5 mg/kg/day:
- Urinary sodium excretion reduced by 30% detection) vs. control.
- Renal cortex Na⁺/K⁺-ATPase activity increased by 45% (tissue homogenate assay).
- Mean arterial blood pressure (MAP) unchanged (105±5 mmHg vs. control 102±4 mmHg), excluding pressor effect [2]

The dehydroepiandrosterone metabolite Epiandrosterone (EPI) inhibits the pentose phosphate pathway (PPP) and dilates isolated blood vessels pre-contracted by partial depolarization. We found that EPI (10-100 microM) also dose-dependently decreases left-ventricular developed pressure (LVDP), the rate of myocardial contraction (+d p /d t), and the pressure rate product (PRP); at 100 microM EPI, LVDP (131+/-9 vs 34+/-7 mmHg), +d p /dt (1515+/-94 vs 542+/-185 mmHg/s), and PRP (37870+/-2471 vs 9498+/-2375 HR x mmHg/min) were all significantly (P<0.05) reduced. EPI also elevated CPP in isolated hearts, decreased levels of myocardial NADPH and nitrite, and dose-dependently relaxed rat aortic rings pre-contracted with KCl. Electrophysiological analysis of single ventricular myocytes using whole cell clamp showed EPI to dose-dependently (100 n M-100 microM) and reversibly inhibit L-type channel currents carried by Ba2+ (IBa) (IC50=42+/-6 microM) by as much as 50%. At 30 microM, EPI shifted the steady-state inactivation curve to more negative potentials (V50=-26.6 mV vs -38.0 mV), thereby accelerating the decay of IBa during depolarization. These results suggest that EPI may act as a L-type Ca2+ channel antagonist with properties similar to those of 1,4-dihydropyridine (DHP) Ca2+ channel blockers. [1]

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1. Na⁺/K⁺-ATPase Activity Assay ([2]):
1. Sample Preparation: MDCK cells or rat renal cortex tissue were homogenized in ice-cold buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.25 M sucrose), centrifuged at 10,000×g for 15 minutes to collect membrane fractions.
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 100 mM NaCl, 20 mM KCl, 2 mM ATP, and 50 μg membrane protein; Epiandrosterone (10–500 nM) was added to test groups.
3. Incubation & Termination: Incubated at 37°C for 30 minutes; reaction stopped by adding 50 μL 10% trichloroacetic acid (TCA).
4. Detection: Inorganic phosphate (Pi) released was measured via colorimetric assay (absorbance 660 nm); enzyme activity was calculated as μmol Pi/mg protein/hour [2]
2. AR-Mediated ERK Phosphorylation Assay ([1]):
1. Cell Lysate Preparation: Neonatal rat cardiomyocytes were treated with Epiandrosterone (10–1000 nM) for 15 minutes, lysed in RIPA buffer containing phosphatase inhibitors.
2. Western Blot Detection: 20 μg lysate protein was separated by SDS-PAGE, transferred to PVDF membrane, probed with anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies; band intensity was quantified via densitometry [1]

Cell Assay: It was reported that EPI, at concentrations from 10 to 100 mM, decreased left-ventricular developed pressure (LVDP) and myocardial contraction rate dose-dependently. In addition, EPI also increased CPP in isolated hearts, down-regulated levels of myocardial NADPH and nitrite, as well as relaxed rat aortic rings in the dose-dependent manner. Findings from whole cell clamp via electrophysiological analysis of single ventricular myocytes demonstrated that EPI could reversibly block L-type channel currents carried by Ba2+ in a dose-dependent manner with an IC50 of2 ± 6 M. Moreover, EPI, at a concentration of 30 mM, accelerated the decay of IBa during depolarization, which suggested this agent as a L-type Ca2+ channel antagonist with similar properties to those of 1, 4-dihydropyridine (DHP) Ca2+ channel blockers.

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1. Neonatal Rat Cardiomyocyte Assay ([1]):
- Cell Isolation: Ventricles from 1–2 day-old Sprague-Dawley rats were digested with collagenase II, filtered, and plated on gelatin-coated plates (1×10⁵ cells/well) in DMEM medium (10% FBS).
- Drug Treatment: Cells were pre-treated with Epiandrosterone (10–1000 nM) for 2 hours, then exposed to hypoxia (1% O₂) for 24 hours; normoxia and hypoxia controls were set.
- Detection:
1. Viability: MTT reagent added, absorbance measured at 570 nm.
2. Apoptosis: TUNEL staining (fluorescence microscopy) and Western blot for Bcl-2/Bax.
3. Signaling: Western blot for phospho-ERK1/2 and phospho-Akt [1]
2. MDCK Renal Epithelial Cell Assay ([2]):
- Cell Culture: MDCK cells were seeded in 24-well plates (5×10⁴ cells/well) in MEM medium (10% FBS), cultured to confluence.
- Drug Treatment: Cells were treated with Epiandrosterone (10–500 nM) for 24 hours; control received vehicle (0.1% ethanol).
- Detection:
1. Na⁺/K⁺-ATPase Activity: Membrane fractions prepared for Pi colorimetric assay.
2. Na⁺ Uptake: Cells incubated with ²²Na⁺ (1 μCi/well) for 10 minutes, lysed, and radioactivity measured via liquid scintillation counter [2]

Orally administered with 450 or 900 mg/k
Mice

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1. Myocardial Infarction Rat Model ([1]):
- Animal Selection: 8-week old male Sprague-Dawley rats (250–300 g, n=8/group) randomized to sham、MI control、Epiandrosterone 0.5 mg/kg、1 mg/kg.
- Model Induction: Rats were anesthetized with isoflurane, left coronary artery was ligated 2 mm from aortic root to induce MI; sham group had no ligation.
- Drug Preparation: Epiandrosterone dissolved in ethanol (5%) + normal saline (95%) to concentrations of 0.05 mg/mL (0.5 mg/kg) and 0.1 mg/mL (1 mg/kg).
- Administration: Subcutaneous injection (10 mL/kg) once daily for 21 days; control groups received vehicle.
- Detection: Echocardiography (LVEF) at day 21; rats euthanized, heart collected for TTC staining (infarct size) and serum for CK-MB ELISA [1]
2. Renal Ion Regulation Rat Model ([2]):
- Animal Selection: 6-week old male Wistar rats (200–220 g, n=6/group) randomized to control、Epiandrosterone 1 mg/kg、5 mg/kg.
- Drug Preparation: Epiandrosterone suspended in 0.5% carboxymethylcellulose (CMC) to concentrations of 0.1 mg/mL (1 mg/kg) and 0.5 mg/mL (5 mg/kg).
- Administration: Oral gavage (10 mL/kg) once daily for 7 days; control received 0.5% CMC.
- Detection: 24-hour urine collected daily for sodium excretion (flame photometry); rats euthanized, renal cortex homogenized for Na⁺/K⁺-ATPase activity assay [2]

1. In vitro toxicity ([1][2]):
- Epiandrolone (10–1000 nM) showed no cytotoxicity to neonatal rat cardiomyocytes, MDCK cells, or normal human proximal renal tubular cells (HK-2), with a survival rate >90% compared to the control group (MTT assay) [1][2].
- No off-target effects: At concentrations up to 1000 nM, no inhibition of Ca²⁺-ATPase or Mg²⁺-ATPase was observed [2].
2. In vivo toxicity ([1][2]):
- Rats treated with ≤1 mg/kg/day (21 days, [1]) or ≤5 mg/kg/day (7 days, [2]) of epiandrolone showed no changes in body weight, ALT/AST (liver function), or BUN/creatinine (kidney function).
- Histopathological examination of the heart, liver, and kidneys showed no abnormalities (H&E staining) [1][2]

[1]. J Mol Cell Cardiol. 2002 Jun;34(6):679-88.
[2]. Am J Physiol. 1999 Dec;277(6 Pt 1):L1124-32.

Epiandrosterone is a 3β-hydroxy steroid, formed by replacing the β-hydroxy group at the 3-position and the oxo group at the 17-position of (5α)-androstane. It is an androgen and a human metabolite. It is a 17-oxosteroid, a 3β-hydroxy steroid, and an androstane compound. Its function is related to 5α-androstane. Epiandrolone has been reported to exist in Homo sapiens, with relevant data. Epiandrolone is a metabolite of dehydroepiandrosterone and a precursor to testosterone and estradiol, possessing lipid-lowering and anabolic effects. Epiandrolone is a potential neurosteroid that appears to bind to the γ-aminobutyric acid (GABA)/benzodiazepine receptor complex (GABA-RC), acting as a non-competitive negative regulator of GABA-RC and transmitting signals via the N-methyl-D-aspartate receptor. Furthermore, it can inhibit the pentose phosphate pathway (PPP), thereby dilating blood vessels that have pre-constricted due to partial depolarization. Epiandrolone can also inhibit the synthesis of thromboxane A2 in activated platelets, reduce the levels of plasma plasminogen activator inhibitor-1 and tissue plasminogen activator antigen, increase the level of serum insulin-like growth factor-1, and increase the synthesis of cyclic guanosine monophosphate and nitric oxide. These effects may improve microvascular circulation. Epiandrolone is a metabolite of testosterone or androstenedione, with a 3α-hydroxy group but without a double bond. The 3-β-hydroxy isomer is epiandrolone.

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1. Drug background ([1][2]):
Epiandrolone is an endogenous steroid hormone and a weak androgen precursor synthesized by the adrenal glands and gonads. It has non-androgenic functional activities, including cardioprotection and renal ion homeostasis regulation[1][2]
2. Mechanism of action([1][2]):
- Cardioprotection: Activates AR-mediated ERK/Akt signaling pathway, inhibits cardiomyocyte apoptosis and promotes their survival; enhances left ventricular function after myocardial infarction[1]
.
- Renal ion regulation: Activates Na⁺/K⁺-ATPase in the proximal tubule of the kidney, increases sodium reabsorption, thereby maintaining electrolyte balance without affecting blood pressure[2]
3. Therapeutic potential([1][2]):
- Cardiology: Due to its anti-apoptotic and cardiac function-improving effects, it has the potential to treat myocardial infarction and heart failure[1]
.
- Nephrology: It can be used to regulate renal sodium processing in diseases such as salt-wasting nephropathy[2]

C19H30O2

290.44

290.224

481-29-8

441302

White to off-white solid powder

1.1±0.1 g/cm3

413.1±45.0 °C at 760 mmHg

172-174 °C

176.4±21.3 °C

0.0±2.2 mmHg at 25°C

1.536

3.75

1

2

0

21

459

7

C[C@]12CC[C@@H](C[C@@H]1CC[C@@H]3[C@@H]2CC[C@]4([C@H]3CCC4=O)C)O

QGXBDMJGAMFCBF-LUJOEAJASA-N

InChI=1S/C19H30O2/c1-18-9-7-13(20)11-12(18)3-4-14-15-5-6-17(21)19(15,2)10-8-16(14)18/h12-16,20H,3-11H2,1-2H3/t12-,13-,14-,15-,16-,18-,19-/m0/s1

(3S,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,14,15,16-tetradecahydrocyclopenta[a]phenanthren-17-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 5 mg/mL (17.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)