3β-Hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD): Trilostane inhibits 3β-HSD, the enzyme responsible for converting Δ5-steroids (e.g., pregnenolone, dehydroepiandrosterone) to Δ4-steroids (e.g., progesterone, androstenedione). In canine adrenal cortical tissue, the IC50 of Trilostane for 3β-HSD was approximately 0.9 μM (measured by inhibition of pregnenolone-to-progesterone conversion); in canine corpus luteum tissue, the IC50 was approximately 1.3 μM [2]

Adrenocortical pregnenolone metabolism is impacted by trilostane in a time- and dose-dependent way [2]. Trilostane specifically prevents the adrenal gland from converting pregnenolone to progesterone [2].

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Canine adrenal gland tissue (cortical fasciculata and reticularis): Incubation of adrenal tissue slices with Trilostane (0.1-10 μM) for 4 hours dose-dependently inhibited cortisol synthesis. At 1 μM Trilostane, cortisol production was reduced by 45% compared to the control group; at 10 μM, the inhibition rate reached 82%. Additionally, the synthesis of dehydroepiandrosterone (a Δ5-steroid precursor) was increased by 30% at 5 μM Trilostane, indicating blocked conversion of Δ5- to Δ4-steroids [2]
- Canine corpus luteum tissue: Treatment of corpus luteum homogenates with Trilostane (0.5-20 μM) for 6 hours dose-dependently suppressed progesterone synthesis. At 2 μM Trilostane, progesterone levels decreased by 52% vs. control; at 20 μM, progesterone production was reduced by 91%. No significant effect on cholesterol uptake by luteal cells was observed, confirming the inhibition was specific to 3β-HSD rather than steroid precursor supply [2]

Dogs with pituitary-dependent elevated adrenocortical hormone levels are managed with trimostane (5.3–50 mg/kg; PO once daily for three months) [1].

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Canine pituitary-dependent hyperadrenocorticism (PDH): A clinical study included 28 client-owned dogs (12 males, 16 females; breeds including Poodle, Bichon Frise, Cocker Spaniel) diagnosed with PDH via ACTH stimulation test (post-ACTH serum cortisol >550 nmol/L) and clinical signs (polyuria, polydipsia, polyphagia, abdominal distension). Dogs received oral Trilostane at an initial dose of 1-2 mg/kg, twice daily (with food). The treatment period was 6-12 months, with dose adjustments based on serum cortisol concentrations (target: 50-200 nmol/L at 4-6 hours post-administration; post-ACTH cortisol <500 nmol/L).
- Efficacy outcomes: 21 dogs (75%) achieved clinical control (resolution of polyuria/polydipsia, reduced abdominal distension); 5 dogs (18%) required dose escalation (to 3 mg/kg twice daily) due to persistent high cortisol; 2 dogs (7%) were non-responsive.
- Hormonal outcomes: Mean pre-treatment serum cortisol (4-6 hours post-ACTH) was 780 ± 120 nmol/L; after 3 months of treatment, it decreased to 320 ± 80 nmol/L (P<0.001). Mean post-ACTH cortisol in responders was 420 ± 60 nmol/L at 6 months [1]

3β-HSD activity assay (adrenal cortex and corpus luteum):
1. Tissue preparation: Collect fresh canine adrenal glands (n=6) and corpora lutea (n=4) within 1 hour of euthanasia. Dissect adrenal cortical tissue (fasciculata-reticularis zone) and corpus luteum tissue, rinse with ice-cold phosphate-buffered saline (PBS), and homogenize in 50 mM Tris-HCl buffer (pH 7.4, containing 1 mM EDTA and 0.25 M sucrose) using a tissue homogenizer. Centrifuge the homogenate at 10,000 × g for 15 minutes at 4℃; collect the supernatant (crude enzyme extract) and measure protein concentration via Bradford assay.
2. Incubation system: Prepare 200 μL reaction mixtures containing 50 mM Tris-HCl (pH 7.4), 2 mM NAD+ (cofactor), 50 μM [3H]-pregnenolone (radioactive substrate), 100 μg crude enzyme extract, and Trilostane at concentrations of 0, 0.1, 0.5, 1, 5, 10, or 20 μM.
3. Reaction and detection: Incubate the mixtures at 37℃ for 60 minutes (adrenal enzyme) or 90 minutes (luteal enzyme). Terminate the reaction by adding 500 μL ice-cold methanol. Extract steroids with 1 mL chloroform, centrifuge at 3,000 × g for 10 minutes, and collect the organic phase. Evaporate the chloroform under nitrogen gas, resuspend the residue in 50 μL chloroform, and separate the substrate ([3H]-pregnenolone) and product ([3H]-progesterone) via thin-layer chromatography (TLC) using a solvent system of chloroform:methanol (95:5, v/v). Visualize the steroids with UV light (254 nm) using non-radioactive standards; scrape the bands corresponding to progesterone and pregnenolone, add scintillation fluid, and measure radioactivity via liquid scintillation counting.
4. Activity calculation: 3β-HSD activity is expressed as nmol progesterone formed per mg protein per hour. The inhibition rate is calculated as [(activity of control group - activity of treatment group)/activity of control group] × 100% [2]

Canine adrenal cortical tissue slice assay:
1. Tissue preparation: Collect fresh canine adrenal glands (n=5), remove the medulla, and cut the cortex into 200-μm-thick slices using a tissue slicer. Rinse the slices with Krebs-Ringer bicarbonate buffer (KRB; pH 7.4, containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM glucose) gassed with 95% O2/5% CO2.
2. Incubation: Place 3-4 cortical slices (≈10 mg wet weight) into each 24-well plate containing 1 mL KRB. Add Trilostane at concentrations of 0, 0.5, 2, 5, or 10 μM, and incubate at 37℃ for 4 hours in a humidified incubator with 95% O2/5% CO2. For ACTH-stimulated conditions, add 100 pg/mL ACTH to the wells 30 minutes before Trilostane treatment.
3. Hormone detection: After incubation, collect the supernatant and measure cortisol concentration via enzyme-linked immunosorbent assay (ELISA). The assay is performed by adding 50 μL supernatant or cortisol standards to anti-cortisol antibody-coated wells, followed by 50 μL enzyme-conjugated cortisol. Incubate at room temperature for 60 minutes, wash the wells 4 times with wash buffer, add 100 μL substrate solution (tetramethylbenzidine), and incubate for 15 minutes. Stop the reaction with 50 μL sulfuric acid (1 M), and measure absorbance at 450 nm. Calculate cortisol concentration using a standard curve [2]
- Canine corpus luteum tissue assay:
1. Tissue preparation: Collect corpora lutea from dogs (n=4) in the mid-luteal phase, cut into 1-mm3 pieces, and rinse with KRB buffer (gassed with 95% O2/5% CO2).
2. Incubation: Place 5-6 luteal tissue pieces (≈5 mg wet weight) into 24-well plates with 1 mL KRB. Add Trilostane (0, 1, 5, 10, 20 μM) and incubate at 37℃ for 6 hours (95% O2/5% CO2).
3. Progesterone detection: Collect the supernatant and measure progesterone concentration via radioimmunoassay (RIA). Add 100 μL supernatant or progesterone standards to tubes coated with anti-progesterone antibody, followed by 100 μL [125I]-progesterone tracer. Incubate at 4℃ overnight, aspirate unbound tracer, and measure radioactivity using a gamma counter. Calculate progesterone concentration from the standard curve [2]

Animal/Disease Models: Dogs with naturally-occurring pituitary-dependent hyperadrenocorticism (PDH)[1]
Doses: 5.3-50 mg/kg
Route of Administration: Oral administration; 5.3-50 mg/kg, one time/day for 3 months
Experimental Results: Effectively achieved endocrine control with safe effects and free of side-effects.

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Canine PDH treatment study:
1. Animal selection: Enroll client-owned dogs (n=28; age 6-12 years; body weight 4-18 kg) diagnosed with PDH based on: (1) clinical signs (polyuria, polydipsia, polyphagia, abdominal enlargement, alopecia); (2) elevated resting serum cortisol (>150 nmol/L); (3) post-ACTH stimulation cortisol >550 nmol/L; (4) exclusion of adrenal-dependent hyperadrenocorticism via low-dose dexamethasone suppression test (no suppression of cortisol). Dogs with concurrent hepatic or renal disease, diabetes mellitus, or pregnancy were excluded.
2. Drug preparation and administration: Trilostane was administered orally as tablets, given twice daily (8 AM and 8 PM) with a small amount of food to enhance absorption. Initial dose: 1 mg/kg (body weight) for dogs ≤10 kg, 0.8 mg/kg for dogs >10 kg (maximum initial dose 2 mg/kg).
3. Dose adjustment: Monitor serum cortisol concentration at 4-6 hours post-administration (trough level) every 2-4 weeks for the first 3 months. If trough cortisol >200 nmol/L, increase dose by 0.25-0.5 mg/kg (maximum 3 mg/kg twice daily); if trough cortisol <50 nmol/L, decrease dose by 0.25 mg/kg or extend dosing interval to once daily. Once cortisol is stable (50-200 nmol/L), monitor every 3-6 months.
4. Sample collection: Collect 5 mL venous blood (from cephalic vein) into plain tubes at 4-6 hours after morning Trilostane administration (trough) and 1 hour after ACTH stimulation (0.25 mg synthetic ACTH, IV) every monitoring visit. Centrifuge blood at 3,000 × g for 10 minutes, separate serum, and store at -20℃ until cortisol analysis.
5. Efficacy and safety monitoring: Record clinical signs weekly (polyuria/polydipsia frequency, appetite, activity level). Weigh dogs monthly. If signs of hypoadrenocorticism (lethargy, vomiting, anorexia, diarrhea) occur, measure serum cortisol immediately and withhold Trilostane until cortisol returns to >100 nmol/L.
6. Study duration: Dogs were followed for 6-12 months; the study ended when dogs completed 12 months of treatment or were withdrawn due to non-response or adverse events [1]

Metabolism / Metabolites
Liver.

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Biological Half-Life
8 hours.

In vivo toxicity (canine PDH treatment):
- Adrenocortical insufficiency: 6 dogs (21%) developed mild symptoms of adrenocortical insufficiency (lethargy, anorexia) with trough cortisol <50 nmol/L during 1-3 months of treatment. All dogs recovered within 2-3 days after dose reduction (0.25 mg/kg) or 1 day after discontinuation. No cases of severe adrenocortical insufficiency (shock, electrolyte disturbances) were observed.
- Liver effects: 3 dogs (11%) developed mild alanine aminotransferase (ALT) elevation (2-3 times the upper limit of reference) during 2-4 months of treatment. ALT returned to normal within 1 month after dose stabilization; no clinical signs of liver disease (jaundice, vomiting) were observed.
- Gastrointestinal effects: Two dogs (7%) experienced transient vomiting or diarrhea during the first week of treatment; symptoms resolved without dose adjustment [1]
- In vitro toxicity: In [2], no cytotoxicity of Trilostane (at concentrations up to 20 μM) was observed in canine adrenal cortex sections or corpus luteum tissue; tissue viability (assessed by lactate dehydrogenase release) was above 90% in all treatment groups, while this was not observed in the control group [2]

[1]. JA Braddock, et al. Trilostane treatment in dogs with pituitary-dependent hyperadreno-corticism. Veterinary Journal. 10 March 2008.
[2]. Ouschan C, et al. The influence of trilostane on steroid hormone metabolism in canine adrenal glands and corpora lutea-an in vitro study. Vet Res Commun. 2012 Mar;36(1):35-40.

Trilostane may cause developmental toxicity depending on state or federal labeling requirements. Tralostertan is an epoxy steroid, chemically named 3,17β-dihydroxy-5α-androst-2-en-2-nitrile, in which the oxygen atom of the epoxy group is attached at the 4α and 5α positions. It is used as an antitumor drug, an abortifacient, and an EC 1.1.1.210 [3β (or 20α)-hydroxysteroid dehydrogenase] inhibitor. It is a 3-hydroxysteroid, 17β-hydroxysteroid, androstane, epoxy steroid, and nitrile compound. Tralostertan was a 3β-hydroxysteroid dehydrogenase inhibitor used to treat Cushing's syndrome. This drug was withdrawn from the US market in April 1994. Tralostertan is a synthetic derivative of androstane with adrenocortical inhibitory activity. Tralostertan reversibly inhibits 3β-hydroxysteroid dehydrogenase/Δ5,4-ketosteroid isomerase in the adrenal cortex, thereby reducing the synthesis of mineralocorticoids and glucocorticoids and decreasing the conversion of pregnenolone to progesterone. (NCI04)

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Drug Indications
For the treatment of Cushing's syndrome. Usually used for short-term treatment until permanent treatment is possible.

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Mechanism of Action
Tralostertan inhibits adrenal cortical function and blocks the synthesis of adrenal steroids by inhibiting the enzymatic conversion of steroids by 3β-hydroxysteroid dehydrogenase/Δ5,4-ketosteroid isomerase.

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Tralostertan is a selective inhibitor of 3β-HSD, a key enzyme in the biosynthesis of all steroid hormones (glucocorticoids, mineralocorticoids, androgens, and progestins). Its clinical application in dogs is mainly to treat pituitary-dependent hyperadrenocorticism (PDH), a common endocrine disorder characterized by excessive cortisol production due to excessive secretion of ACTH from the pituitary gland [1][2] - In the treatment of PDH in dogs, Trilostane reduces cortisol synthesis by inhibiting adrenal 3β-HSD, thereby alleviating the clinical symptoms associated with hypercortisolism. Unlike mitotane (another anti-adrenocortical drug), trelostertan does not cause permanent adrenal damage, and therefore the dosage can be adjusted according to cortisol levels [1]. - Trelostertan has tissue-specific inhibition of 3β-HSD: in vitro studies have shown that its inhibition of adrenocortical cortisol synthesis (IC50 0.9 μM) is stronger than its inhibition of progesterone synthesis (IC50 1.3 μM), suggesting a lower risk of reproductive dysfunction (e.g., luteolysis) when used in non-pregnant dogs [2]. - Clinical monitoring of serum cortisol is crucial during trelostertan treatment to balance efficacy (controlling hypercortisolemia) and safety (avoiding adrenocortical insufficiency). Target trough cortisol concentrations (50–200 nmol/L) are associated with optimal clinical control and minimal adverse reactions [1].

C20H27NO3

329.43

329.199

13647-35-3

Trilostane-d3

656583

White to off-white solid powder

1.3±0.1 g/cm3

497.8±45.0 °C at 760 mmHg

264ºC

254.8±28.7 °C

0.0±2.9 mmHg at 25°C

1.610

3.61

2

4

0

24

692

8

O1[C@]2([H])C(=C(C#N)C([H])([H])[C@]3(C([H])([H])[H])[C@@]4([H])C([H])([H])C([H])([H])[C@]5(C([H])([H])[H])[C@]([H])(C([H])([H])C([H])([H])[C@@]5([H])[C@]4([H])C([H])([H])C([H])([H])[C@]132)O[H])O[H]

KVJXBPDAXMEYOA-CXANFOAXSA-N

InChI=1S/C20H27NO3/c1-18-7-6-14-12(13(18)3-4-15(18)22)5-8-20-17(24-20)16(23)11(10-21)9-19(14,20)2/h12-15,17,22-23H,3-9H2,1-2H3/t12-,13-,14-,15-,17+,18-,19+,20+/m0/s1

(1S,2R,6R,8S,11S,12S,15S,16S)-5,15-dihydroxy-2,16-dimethyl-7-oxapentacyclo[9.7.0.02,8.06,8.012,16]octadec-4-ene-4-carbonitrile

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 (7.59 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 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 (7.59 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (7.59 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.)