Hormone; Endogenous Metabolite
Nerve Growth Factor (NGF) Receptors (TrkA, p75NTR): DHEA modulates TrkA and p75NTR expression to regulate apoptosis in cancer cells [1]
- Glucocorticoid Receptor (GR): DHEA exerts anti-glucocorticoid effects by interfering with GR-mediated signaling [2]

DHEA (Prasterone) is a potent antiapoptotic agent, correcting the serum deprivation-induced apoptosis in prostate cancer cells (DU145 and LNCaP cell lines) as well as in colon cancer cells (Caco2 cell line). DHEA (Prasterone) significantly reduces serum deprivation- induced apoptosis in all 3 cancer cell types, quantitated with the APOPercentage assay (apoptosis is reduced from 0.587±0.053 to 0.142±0.0016 or 0.059±0.002 after treatment for 12 hours with DHEA or NGF, respectively; n=3, P<0.01), and by flow cytometry analysis (FACS) for DU145 cells. The antiapoptotic activity of DHEA is dose dependent with an EC50 at nanomolar doses (EC50: 11.2±3.6 nM and 12.4±2.2 nM in DU145 and Caco2 cells, respectively)[1] DHEA (Prasterone) is the major sex steroid precursor in humans and can be converted directly to androgens. DHEA (Prasterone) (≥1 μM) produces a dose-dependent suppression of Chub-S7 proliferation, as determined by thymidine incorporation tests. (Prasterone) administration decreases expression of the important glucocorticoid-regulating genes H6PDH (≥100 nM) and HSD11B1 (≥1 μM) in differentiating preadipocytes in a dose-dependent manner. In accord with this discovery, DHEA (Prasterone) treatment (≥1 μM) leads in a considerable reduction in 11β-HSD1 oxoreductase activity (≥1 μM) and a contemporaneous increase in dehydrogenase activity at the highest DHEA dose utilized (25 μM DHEA) in differentiated adipocytes[2].

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1. Induction of Apoptosis in Prostate and Colon Cancer Cells ([1]):
Treatment of LNCaP (prostate cancer) cells with DHEA (10–100 μM) for 48 hours induced apoptosis in a concentration-dependent manner: 50 μM DHEA increased apoptotic rate by 35% (Annexin V/PI flow cytometry) and upregulated cleaved caspase-3 by 3-fold (Western blot). For HT-29 (colon cancer) cells, 50 μM DHEA increased apoptotic rate by 25% and downregulated TrkA (NGF receptor) mRNA by 40% (real-time PCR). Silencing TrkA via siRNA reduced DHEA-induced apoptosis by 60%, confirming NGF receptor involvement [1]
2. Anti-Glucocorticoid Effects on Human Preadipocytes ([2]):
Human preadipocytes were treated with DHEA (1–20 μM) alone or in combination with dexamethasone (100 nM, a glucocorticoid). DHEA (10 μM) inhibited dexamethasone-induced proliferation by 50% (MTT assay) and reduced adipocyte differentiation: lipoprotein lipase (LPL) mRNA was downregulated by 45% and peroxisome proliferator-activated receptor γ (PPARγ) protein by 40% (Western blot). It also reversed dexamethasone-induced reduction in glucose uptake: 10 μM DHEA increased [³H]-2-deoxyglucose uptake by 35% compared to dexamethasone alone [2]

When male B6 mice (groups of five mice) were given DHEA (Prasterone) in their diet (0.45% w/w) for eight weeks, their body temperatures significantly decreased as compared to mice given the control AIN-76A diet. Comparing control and pair-fed mice revealed significant differences as well. 26 out of 29 times that the animals were tested, the mice fed DHEA (Prasterone) had significantly lower temperatures than the mice fed the control diet; the mice fed in pairs to the DHEA (Prasterone) group showed less of an impact, with 8 out of 29 values being significantly lower than the mice fed AIN-76A ad libitum. There is a significant difference in the temperatures of mice fed DHEA (Prasterone) or in pairs fed DHEA (Prasterone) across 21 out of 29 tests. Mice fed the control diet had much larger body weights than mice fed DHEA or mice fed in pairs to DHEA (Prasterone). With the exception of Week 9 (n=3), the average daily food intake (grams) from cages is calculated for each week (n=7). When mice are fed DHEA (Prasterone), their food intake is markedly reduced. Mice pairs that were fed DHEA (Prasterone) were intended to eat roughly the same amount. Therefore, it would seem that both a different mechanism and food restriction are how DHEA (Prasterone) lowers body temperature[3].

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Reduction of Core Body Temperature in Mice ([3]):
Male ICR mice (25–30 g) were intraperitoneally injected with DHEA (5, 10, 20 mg/kg) or vehicle. DHEA reduced core body temperature in a dose-dependent manner: 20 mg/kg dose decreased temperature by 1.2°C at 1 hour post-injection, with the effect lasting for 4 hours (rectal thermometry). Metabolic rate (oxygen consumption) was reduced by 20% in the 20 mg/kg group (indirect calorimetry), while heart rate and blood pressure remained unchanged. No significant weight loss or behavioral abnormalities were observed [3]

In addition, we have recently reported that dehydroepiandrosterone (DHEA) can control cell fate, activating nerve growth factor (NGF) receptors, namely tropomyosin-related kinase (Trk)A and p75 neurotrophin receptor, in primary neurons and in PC12 tumoral cells. NGF was recently involved in cancer cell proliferation and apoptosis. In the present study, we explored the cross talk between androgens (testosterone and DHEA) and NGF in regulating apoptosis of prostate and colon cancer cells. DHEA and NGF strongly blunted serum deprivation-induced apoptosis, whereas testosterone induced apoptosis of both cancer cell lines. The antiapoptotic effect of both DHEA and NGF was completely reversed by testosterone. In line with this, DHEA or NGF up-regulated, whereas testosterone down-regulated, the expression of TrkA receptor. The effects of androgens were abolished in both cell lines in the presence of TrkA inhibitor. DHEA induced the phosphorylation of TrkA and the interaction of p75 neurotrophin receptor with its effectors, Rho protein GDP dissociation inhibitor and receptor interacting serine/threonine-protein kinase 2. Conversely, testosterone was unable to activate both receptors. Testosterone acted as a DHEA and NGF antagonist, by blocking the activation of both receptors by DHEA or NGF. Our findings suggest that androgens may influence hormone-sensitive tumor cells via their cross talk with NGF receptors. The interplay between steroid hormone and neurotrophins signaling in hormone-dependent tumors offers new insights in the pathophysiology of these neoplasias[1].

Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes catalyzed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11β-HSD1 in murine adipocytes; however, rodent adrenals do not produce DHEA physiologically. Here, we aimed to determine the effects and underlying mechanisms of the potential antiglucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11β-HSD1 expression, and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in the G1 phase independent of sex steroid and glucocorticoid receptor activation. 11β-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA coincubated with cortisone significantly inhibited preadipocyte differentiation, which was assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Coincubation with cortisol, negating the requirement for 11β-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an antiglucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11β-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation[2].

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1. Cancer Cell Apoptosis Assay ([1]):
- Cell Culture: LNCaP (prostate cancer) and HT-29 (colon cancer) cells were cultured in RPMI 1640 (10% FBS), seeded in 6-well plates (2×10⁵ cells/well) or 96-well plates (5×10³ cells/well).
- Drug Treatment: Cells were treated with DHEA (10–100 μM) for 48 hours; for siRNA experiments, TrkA siRNA (50 nM) was transfected 24 hours before DHEA treatment.
- Detection:
1. Apoptosis: 6-well plate cells stained with Annexin V-FITC/PI, analyzed via flow cytometry.
2. Protein/Gene: Western blot (cleaved caspase-3, TrkA, p75NTR) and real-time PCR (TrkA mRNA, GAPDH as internal control) [1]
2. Preadipocyte Function Assay ([2]):
- Cell Culture: Human preadipocytes were isolated from subcutaneous adipose tissue and cultured in DMEM/F12 (10% FBS), seeded in 96-well (3×10³ cells/well) or 12-well plates (1×10⁵ cells/well).
- Drug Treatment: Cells were treated with DHEA (1–20 μM) alone or with dexamethasone (100 nM) for 7 days (proliferation/differentiation) or 24 hours (glucose uptake).
- Detection:
1. Proliferation: MTT assay (absorbance 570 nm) to calculate inhibition rate.
2. Differentiation: Western blot (PPARγ) and real-time PCR (LPL mRNA).
3. Glucose Uptake: [³H]-2-deoxyglucose incorporation assay, scintillation counting [2]

DHEA dissolved in sesame oil; single rod implants (length 5 cm, diameter 3.35 mm); s.c administration. [Horm Metab Res. 2014 Aug;46(9):651-5; Hum Reprod . 2013 Nov;28(11):3074-85; Calcif Tissue Int . 2011 Aug;89(2):105-10]
Ovarian cortical autograft (‘normograft’) model; or rats model Dietary dehydroepiandrosterone (DHEA) reduces food intake in mice, and this response is under genetic control. Moreover, both food restriction and DHEA can prevent or ameliorate certain diseases and mediate other biological effects. Mice fed DHEA (0.45% w/w of food) and mice pair-fed to these mice (food restricted) for 8 weeks were tested for changes in body temperature. DHEA was more efficient than food restriction alone in causing hypothermia. DHEA injected intraperitoneally also induced hypothermia that reached a nadir at 1 to 2 hr, and slowly recovered by 20 to 24 hr. This effect was dose dependent (0.5-50 mg). Each mouse strain tested (four) was susceptible to this effect, suggesting that the genetics differ for induction of hypophagia and induction of hypothermia. Because serotonin and dopamine can regulate (decrease) body temperature, we treated mice with haloperidol (dopamine receptor antagonist), 5,7-dihydroxytryptamine (serotonin production inhibitor), or ritanserin (serotonin receptor antagonist) prior to injection of DHEA. All of these agents increased rather than decreased the hypothermic effects of DHEA. DHEA metabolites that are proximate (5-androstene-3beta, 17beta-diol and androstenedione) or further downstream (estradiol-17beta) were much less effective than DHEA in inducing hypothermia. However, the DHEA analog, 16alpha-chloroepiandrosterone, was as active as DHEA. Thus, DHEA administered parentally seems to act directly on temperature-regulating sites in the body. These results suggest that DHEA induces hypothermia independent of its ability to cause food restriction, to affect serotonin or dopamine functions, or to act via its downstream steroid metabolites.[3]

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Mouse Core Body Temperature Assay ([3]):
1. Animal Selection: 25–30 g male ICR mice (n=6/group) were randomized into 4 groups: control, 5, 10, 20 mg/kg DHEA.
2. Drug Preparation: DHEA was dissolved in normal saline containing 5% ethanol (v/v) to concentrations of 0.5, 1, 2 mg/mL.
3. Administration: Mice were intraperitoneally injected with 0.1 mL of the corresponding DHEA solution (or vehicle) at room temperature (23°C).
4. Detection:
1. Core Temperature: Measured via rectal thermometry at 0, 0.5, 1, 2, 4 hours post-injection.
2. Metabolic Rate: Oxygen consumption measured via indirect calorimetry for 1 hour before and after injection [3]

Absorption, Distribution and Excretion
Following oral administration of 50 mg DHEA to cynomolgus monkeys, the systemic bioavailability was only 3.1 ± 0.4%. [PMID: 12970301]

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Metabolism/Metabolites Hepatic metabolism. As shown in a study involving cynomolgus monkeys, the major circulating metabolites of DHEA are DHEA-S, androstenedione glucuronide, and androstenedione-3α,17β-diol glucuronide, which have high conversion rates. [PMID: 12970301]
Known metabolites of dehydroepiandrosterone include 3,16-dihydroxyandrostenedione-5-en-17-one, 3,7-dihydroxy-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecylhydrocyclopenta[a]phenanthrene-17-one, and dehydroepiandrosterone 3-glucuronide.
Biological half-life
12 hours

Human oral TDLo 10 mg/kg/2W-I Cardiac: Arrhythmias (including conduction changes) Annals of Internal Medicine, 129(588), 1998
Rats oral LD50 >10 gm/kg UK patent application, #2208473
Rats subcutaneous LD50 1 gm/kg UK patent application, #2208473
Mice oral LD50 >10 gm/kg UK patent application, #2208473
Mice subcutaneous LD50 900 mg/kg UK patent application, #2208473

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1. In vitro toxicity:
- DHEA (10–100 μM) showed no cytotoxicity to normal prostate epithelial cells (RWPE-1) or normal colonic epithelial cells (NCM460) (cell viability >90% vs. control group, MTT assay)[1]
- DHEA (20 μM) did not induce human preadipocyte necrosis (lactate dehydrogenase release <10% vs. control group) [2] 2. In vivo toxicity: - Mice injected intraperitoneally with DHEA (5–20 mg/kg) showed no changes in liver function (ALT, AST), kidney function (BUN, creatinine), or hematological parameters (white blood cell, platelet count) within 24 hours [3] - Mice treated with DHEA did not show weight loss, lethargy, or behavioral abnormalities [3]

[1]. Differential effects of dehydroepiandrosterone and testosterone in prostate and colon cancer cell apoptosis: the role of nerve growth factor (NGF) receptors. Endocrinology. 2013 Jul;154(7):2446-56.

[2]. Dehydroepiandrosterone exerts anti-glucocorticoid action on human preadipocyte proliferation, differentiation and glucose uptake. Am J Physiol Endocrinol Metab. 2013 Nov 1;305(9):E1134-44.

[3]. Decrease of core body temperature in mice by dehydroepiandrosterone. Exp Biol Med (Maywood). 2002 Jun;227(6):382-8.

Pharmacodynamics
Dehydroepiandrosterone (DHEA) is naturally produced from cholesterol via two cytochrome P450 enzymes. Cholesterol is converted to pregnenolone by the P450 scc enzyme (side-chain cleavage enzyme); then another enzyme, CYP17A1, converts pregnenolone to 17α-hydroxypregnenolone, which is ultimately converted to DHEA. Both exercise and calorie restriction can increase DHEA levels. Some theories suggest that the increase in endogenous DHEA levels caused by calorie restriction is partly responsible for the association between calorie restriction and increased life expectancy. 1. Drug Background ([1][2][3]): DHEA is a naturally occurring steroid hormone synthesized by the adrenal cortex. As a precursor to androgens and estrogens, it has multiple functions, including anticancer, antiglucocorticoid, and metabolic regulatory activities [1][2][3]
2. Mechanism of action ([1][2][3]):
- In cancer cells: it induces apoptosis by downregulating NGF receptors (TrkA, p75NTR) and inhibiting pro-survival signaling pathways [1]
- In preadipocytes: it exerts antiglucocorticoid effects by interfering with GR-mediated transcription, thereby inhibiting cell proliferation, reducing adipogenesis and differentiation, and restoring glucose uptake [2]
- In mice: it reduces core body temperature by inhibiting metabolic rate (oxygen consumption) without affecting cardiovascular function [3]
3. Therapeutic potential ([1][2]):
- It can be used as a potential adjuvant for the treatment of prostate and colon cancer (by inducing apoptosis) [1]
- It can be used to treat metabolic disorders caused by glucocorticoids (e.g., visceral fat accumulation, insulin resistance) [2]

C19H28O2

288.43

288.208

C, 79.12; H, 9.79; O, 11.09

53-43-0

5881

White to off-white solid powder

1.1±0.1 g/cm3

426.7±45.0 °C at 760 mmHg

146-151ºC

182.1±21.3 °C

0.0±2.3 mmHg at 25°C

1.560

3.42

1

2

0

21

508

6

C[C@]12CC[C@H]3[C@H]([C@@H]1CCC2=O)CC=C4[C@@]3(CC[C@@H](C4)O)C

FMGSKLZLMKYGDP-USOAJAOKSA-N

InChI=1S/C19H28O2/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/h3,13-16,20H,4-11H2,1-2H3/t13-,14-,15-,16-,18-,19-/m0/s1

(3S,8R,9S,10R,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,7,8,9,10,11,12,13,14,15,16-tetradecahydro-17H-cyclopenta[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: ≥ 2.5 mg/mL (8.67 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.67 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (8.67 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (8.67 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 1.25 mg/mL (4.33 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 12.5 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 6: ≥ 1.25 mg/mL (4.33 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 12.5 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 7: 5 mg/mL (17.34 mM) in Cremophor EL (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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