GnRH; Triptorelin acetate binds with high affinity to the gonadotropin-releasing hormone receptor on anterior pituitary cells. Binding affinity studies show that triptorelin exhibits a pKi of 9.6 ± 0.09, corresponding to a Ki value of 0.3 nM, compared to native GnRH with a pKi of 7.9 ± 0.05 (Ki of 13 nM). For kinetic parameters, triptorelin has an association rate constant (k_on) of 0.1 ± 0.01 nM⁻¹·min⁻¹, a dissociation rate constant (k_off) of 0.03 ± 0.008 min⁻¹, and a receptor residence time of 39 ± 12 minutes, whereas native GnRH has a residence time of only 6.3 ± 0.6 minutes. These data demonstrate that triptorelin has significantly enhanced receptor binding affinity and prolonged receptor occupancy.
The primary molecular target of Triptorelin Acetate is the gonadotropin-releasing hormone (GnRH) receptor, also known as the LHRH receptor. As a potent agonist of the GnRH receptor, triptorelin binds to and activates the receptor on pituitary gonadotroph cells. Initial stimulation leads to a transient increase in the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn stimulates the production of sex hormones. However, with continued administration, triptorelin causes downregulation of GnRH receptors and desensitization of the pituitary, leading to a sustained reduction in LH and FSH secretion and consequently a decrease in sex hormone levels. This chemical castration effect is the basis for its use in hormone-sensitive cancers such as prostate cancer.

Triptorelin has protection effects against tripterygium polyglycoside-induced damage to ovarian function on mouse ovarian cells [2]. In cultured human granulosa luteinized cells, triptorelin acetate (1 nM and 3 nM for 48 hours) induces a slight increase in cell viability, but the higher concentration (3 nM) reduces estradiol secretion levels. Radioligand binding assays show that triptorelin has high affinity for the GnRH receptor, with significantly greater binding affinity than native GnRH. In vitro release studies using silica microparticles encapsulating triptorelin acetate were prepared by spray-drying, and their release kinetics were characterized.

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In vitro studies of Triptorelin Acetate have demonstrated its potent agonist activity at the GnRH receptor. The compound binds to GnRH receptors on pituitary cells and stimulates the release of LH and FSH. In vitro assays typically involve treatment of cultured pituitary cells with triptorelin followed by measurement of LH and FSH secretion. The compound has been shown to be a more potent agonist than the natural GnRH due to its enhanced stability and receptor binding affinity. Receptor binding studies have characterized the affinity of triptorelin for the GnRH receptor.

Triptorelin has a protective effect on tripterygium polyglycoside-induced damage to ovarian function in female mice [2].
Central Precocious Puberty Treatment: In five girls with CPP (mean age 7.3 years), intramuscular administration of triptorelin acetate at 60-100 μg/kg every four weeks for 12-20 months reduced growth rate from 8.1 cm/year to 6.2 cm/year, with bone age increment decreasing from 1.7 to 0.94 years per year. Adult height prediction improved from 158.6 cm to 159.0 cm after one year of treatment. Ovulation Induction: In jennies during oestrus with a follicle diameter ≥32±2 mm, subcutaneous administration of 0.1 mg triptorelin acetate achieved an ovulation rate of 83.3% within 24-48 hours, with a mean ovulation time of 37.3 ± 8.3 hours, significantly superior to controls (66.8 ± 25.9 hours, p = .0032). Sustained Release Formulation Study: In rats, subcutaneous administration of a biodegradable silica triptorelin acetate depot formulation gave 5-fold lower Cmax values than the reference product Pamorelin®, with comparable sustained release characteristics. Detectable triptorelin plasma concentrations were maintained throughout the 91-day study period, and testosterone levels remained below castration levels for the same period.

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In vivo studies of Triptorelin Acetate have demonstrated its efficacy in reducing sex hormone levels and treating hormone-sensitive cancers. The compound is administered via subcutaneous or intramuscular injection. Upon subcutaneous administration, triptorelin is rapidly absorbed, with peak plasma concentrations reached within 15-60 minutes and maximal effect achieved within 1 hour. Sustained-release formulations administered intramuscularly can maintain therapeutic effects for more than 28 days. The compound has been shown to effectively reduce testosterone levels in prostate cancer patients, leading to tumor regression and improved clinical outcomes.

Radioligand binding assays are used to evaluate the binding affinity of triptorelin acetate to the GnRH receptor. Membrane preparations expressing the GnRH receptor are incubated with radiolabeled triptorelin and varying concentrations of unlabeled triptorelin. Bound and free radioligands are separated by filtration or centrifugation, and radioactivity is measured to calculate Ki, Kd values, and kinetic parameters (k_on and k_off). Additionally, TR-FRET competition binding assays are performed to determine pKi and pKD values.

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In vitro receptor binding assays for Triptorelin Acetate typically involve studying its interaction with the GnRH receptor. Radioligand binding assays using labeled GnRH or labeled triptorelin can be performed to measure the binding affinity (IC50 or Ki) of the compound for the GnRH receptor. Functional assays, such as LH and FSH secretion assays using pituitary cell cultures, can be used to evaluate the agonist activity of the compound. These assays typically involve treatment of cells with triptorelin followed by measurement of hormone release using immunoassays.

Human granulosa luteinized cells are collected from women undergoing IVF treatment and seeded at a density of 20,000 live cells/well in 96-well plates. Cells are cultured in RPMI-1640 medium containing 6% fetal calf serum at 37°C in 5% CO₂ for 48 hours for recovery. Subsequently, cells are treated with 1 nM or 3 nM triptorelin acetate in serum-free medium for an additional 48 hours. Cell viability is assessed using the MTT assay, and estradiol and progesterone concentrations in culture supernatants are measured by ELISA.

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In vitro cell-based assays for Triptorelin Acetate typically involve treatment of cultured pituitary cells with the compound followed by assessment of GnRH receptor activation. Cells are treated with triptorelin, and the release of LH and FSH is measured using ELISA or other immunoassay techniques. Dose-response experiments are performed to determine the potency (EC50) of the compound in stimulating hormone release. Additionally, receptor binding assays using cells expressing GnRH receptors can be performed to evaluate the affinity of triptorelin for the receptor.

For qualified, healthy SD female mice, the vaginal exfoliated cell method was used to select 30 mice with normal estrous cycle as test animals, which were randomly divided into 3 groups of 10 mice each: • Group A: blank control group, where 0.35 mL of saline was administered to the stomach once daily for 11 weeks; • Group B: tripterygium glycoside group, where 0.35 mL of tripterygium glycoside solution was administered to the stomach from the 8th day; once a day for 10 weeks; • Group C: triptolide + triptorelin group: 0.1 mg/kg daily subcutaneous injection of triptorelin injection; once a day; continuous injection for 11 weeks; from the 8th day, the triptolide solution was administered to the stomach 0.35 mL, once daily for 10 weeks. From the first day of the experiment, the general conditions of the mice were observed and recorded, including energy, activity, hair, food intake, water intake, stomach appetite, second stool, etc. The mice were weighed once a week to observe changes in body weight. The vagina exfoliation cell method, simple to operate, was used to observe the estrous cycle. After 11 weeks of treatment, the drug was stopped for 3 weeks and all mice were sacrificed. The ovaries were then obtained by laparotomy, and the ovarian wet weight was measured using an electronic analytical balance. The ovarian index was calculated by ovarian wet weight (mg) / mouse weight (g) × 100%. After weighing, the ovaries were fixed in a 4% paraformaldehyde solution for 3 days, and were routinely dehydrated, xylene-transparented, wax-impregnated, embedded, sectioned (4 µm), and operated according to the instructions of immunohistochemistry kit. Immunohistochemical average optical density (average optical) analysis method: each slice in each group randomly selected at least three positions with a 200× field of view (FoV) for photographing. When taking pictures, FoV was selected to ensure that the testing tissue fully filled the view. In addition, the background illumination of each photo was kept as consistent as possible. Image-Pro Plus 6.0 software was used to select the same brown-yellow color as the uniform standard for determining the positives of all photos. Each photo was analyzed to obtain the Integrated Optical Density (IOD) and the pixel area (AREA). The average optical density (AO) was obtained by AO = IOD/AREA. (1) The larger the AO value, the higher the positive expression level. [2]

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Rat Sustained Release Formulation Study: Sprague-Dawley male rats (9 weeks old, 250-300 g) are subcutaneously administered 5 mg/kg or 10 mg/kg triptorelin acetate. Blood samples are collected for up to 91 days, and triptorelin and testosterone plasma concentrations are analyzed by UPLC-MS/MS. The reference product Pamorelin® requires reconstitution of the powder in 2 mL of water for injection, using a 20 G needle, while the silica depot formulation can be administered using a 23 G needle. Jenny Ovulation Induction Study: Oestrous jennies are monitored by ultrasound for follicle development. When follicle diameter reaches ≥32±2 mm, 0.1 mg triptorelin acetate is administered subcutaneously (TRIP group). Ultrasound examinations are performed at 14, 24, 38, 42, 48, 62 hours, and then every 24 hours until ovulation to assess ovulation time and rate.

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In vivo animal studies for Triptorelin Acetate typically involve administration to animal models to evaluate its effects on hormone levels and disease progression. In prostate cancer models, triptorelin is administered to tumor-bearing animals to assess tumor growth inhibition and reduction in testosterone levels. Typical endpoints include measurement of serum testosterone and LH levels, tumor volume measurement, and survival analysis. The compound has demonstrated efficacy in reducing tumor growth in hormone-sensitive cancer models.

Absorption and Distribution: Triptorelin has a steady-state volume of distribution of 30-33 L. It exhibits high solubility and low permeability (BDDCS Class 3). Clearance and Half-Life: In healthy volunteers, total clearance is 210 mL/min with an elimination half-life of 2.8 hours. In patients with renal impairment, clearance decreases to 113 mL/min (moderate) and 86.8 mL/min (severe), with half-life prolonged to 7.7 hours; in patients with hepatic impairment, clearance is 57.3 mL/min with a half-life of 7.6 hours. Pharmacokinetics after intravenous bolus administration follow a three-compartment model. Metabolism and Excretion: Triptorelin is primarily metabolized via the hepatic route, with approximately 41.7% of the drug excreted unchanged in urine. Sustained Release Formulation: The silica depot formulation demonstrates sustained release characteristics, maintaining detectable plasma concentrations for 91 days in rats while providing 5-fold lower Cmax values compared to Pamorelin®.

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The pharmacokinetic properties of Triptorelin Acetate include rapid absorption following subcutaneous administration, with peak plasma concentrations reached within 15-60 minutes and maximal effect achieved within 1 hour. Sustained-release formulations administered intramuscularly can maintain therapeutic effects for more than 28 days. The compound has a molecular formula of C64H82N18O13·C2H4O2 and a molecular weight of approximately 1353.49. Triptorelin is metabolized through proteolytic degradation and excreted primarily via the kidneys.

Serious Adverse Reaction Incidence: Among 621 children with CPP treated with GnRH agonists, the serious adverse reaction rate was 0.9% (6 events). These included 4 cases of sterile abscess formation (0.6%), 1 case of anaphylaxis, and 1 case of unilateral slipped capital femoral epiphysis. ADR Types and Time Distribution: In an analysis of 38 suspected ADR cases, the most common ADRs involved cutaneous tissues (29.3%) and systemic and digestive systems (20.7%). 61.1% of ADRs occurred within 2 hours of administration, with 18.4% occurring within 5 minutes post-injection. Severe ADRs included anaphylactic reactions, hip synovitis, and arthralgia. Anaphylactic Reactions: Although rare, anaphylactic reactions can be life-threatening, typically presenting as immediate-type IgE-mediated responses. In a retrospective study, an 8.4-year-old girl experienced dizziness, headache, whole body redness, chest tightness, and loss of consciousness immediately after her sixth triptorelin acetate injection, with spontaneous recovery within minutes. When switching pharmaceutical manufacturers, some patients experienced ADRs, and skin prick tests and intradermal tests are recommended when changing manufacturers. Sterile Abscess Formation: The prevalence of sterile abscess formation in CPP children treated with GnRH agonists is 0.6%. Injection site reactions including erythema, nodules, and sterile abscesses typically occur after multiple injections, potentially leaving residual scarring upon healing.

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The toxicity profile of Triptorelin Acetate has been extensively evaluated in preclinical and clinical studies. Common adverse effects include those related to the initial testosterone surge (tumor flare) and the subsequent reduction in testosterone levels, which may cause hot flashes, decreased libido, and other symptoms of androgen deprivation. Long-term use may be associated with bone density loss and other metabolic effects. The compound is contraindicated in patients with certain medical conditions.

[1]. Efficacy and safety of triptorelin 6-month formulation in patients with central precocious puberty.2016 Nov 1;29(11):1241-1248.
[2]. www.ejgo.net/articles/10.31083/j.ejgo.2021.02.2299

Triptorelin acetate (CAS: 140194-24-7) is the acetate salt form of triptorelin (CAS: 57773-63-4), and the two are essentially therapeutically equivalent in clinical use. The acetate group improves the stability and solubility of the drug. This medication is commercially available under various brand names, including Decapeptyl®, Diphereline®, Pamorelin®, among others. Dosing regimens vary among different formulations: for the treatment of CPP, it is typically administered once every 4 weeks (3.75 mg intramuscular injection), while sustained-release formulations can achieve longer dosing intervals.

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Drug Indications
For synchronizing ovulation in weaned sows for timed artificial insemination.

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Triptorelin acetate (CAS: 140194-24-7) is the acetate salt form of triptorelin (CAS: 57773-63-4), and the two are essentially therapeutically equivalent in clinical use. The acetate group improves the stability and solubility of the drug. This medication is commercially available under various brand names, including Decapeptyl®, Diphereline®, Pamorelin®, among others. Dosing regimens vary among different formulations: for the treatment of CPP, it is typically administered once every 4 weeks (3.75 mg intramuscular injection), while sustained-release formulations can achieve longer dosing intervals.

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Triptorelin Acetate (CAS 140194-24-7) is a potent agonist of LHRH/GnRH used in the treatment of prostate cancer and other hormone-responsive conditions. It is sold under the brand names Decapeptyl and Gonapeptyl. The compound is administered via subcutaneous or intramuscular injection, with sustained-release formulations maintaining therapeutic effects for more than 28 days. It has a molecular formula of C64H82N18O13·C2H4O2 and a molecular weight of approximately 1353.49. Triptorelin is used as part of androgen deprivation therapy for prostate cancer.

C64H82N18O13.C2H4O2

1371.50064

1370.652

C, 57.80; H, 6.32; N, 18.38; O, 17.50

140194-24-7

140194-24-7 (acetate);57773-63-4;124508-66-3 (pamoate);2240176-35-4 (TFA);

25080282

H-Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2.CH3CO2H
L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolyl-glycinamide acetic acid

XHWSYWLRPG

White solid powder

1.52g/cm3

1.723

3.291

18

17

33

99

2750

9

CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N)NC(=O)[C@@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC4=CC=C(C=C4)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC5=CNC6=CC=CC=C65)NC(=O)[C@H](CC7=CN=CN7)NC(=O)[C@@H]8CCC(=O)N8.CC(=O)O

pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 acetate

HPPONSCISKROOD-OYLNGHKZSA-N

AY 25650; CL-118532; Wy42422;BIM 21003; CL118,532; Wy 42462; Arvekap; Triptorelin Acetate; D-Tryptophan-LH-RH; Diferelin; pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2; Trade names Decapeptyl; Trelstar; Diphereline; Gonapeptyl; Variopeptyl.

Triptorelin acetate; AY 25650; BIM 21003; CL118,532; CL-118532; Wy42422; Wy 42462; AY-25650; BIM-21003; CL-118,532; CL-118532; Wy-42422; Arvekap; D-Tryptophan-LH-RH; Diferelin; Triptorelin Acetate; pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2; brand names Decapeptyl; Diphereline; Gonapeptyl; Trelstar; Variopeptyl; 140194-24-7; 43OFW291R9; OvuGel; RefChem:57247;

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)

DMSO: ~250 mg/mL (182.3 mM)
H2O: 0.031 mg/mL
Methanol: Slightly

Formulation 1: ~10 mg/mL (7.3 mM) in 10% DMSO + 90% Saline, Clear solution

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Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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