GnRH receptor (human GnRH-R) – binding affinity (K_D) = 0.202 ± 0.03 nM (determined by displacement binding assay using [¹²⁵I]Cetrorelix as tracer) [1]
Antagonistic potency IC₅₀ = 1.21 ± 0.33 nM (functional inhibition of [D-Trp⁶]GnRH-induced luciferase activity in reporter cell line) [1]

At 1000 ng/ml, ceterix diacetate inhibits the development of ES-2 cell lines. The antiproliferative effects of ceterprelix diacetate are similar to those of GnRH-I agonists, indicating that the GnRH-I system in cancer cells may not be subject to the GnRH-I agonists and antagonists dichotomy [2].

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In a cellular luciferase reporter gene assay using a recombinant cell line expressing the human GnRH receptor (clone L23.1/5), Cetrorelix diacetate (SB 75) concentration-dependently inhibited the stimulation of luciferase activity induced by 1 nM [D-Trp⁶]GnRH, with an IC₅₀ of 1.21 ± 0.33 nM. The inhibition was specific to GnRH receptor-mediated signaling, as Cetrorelix diacetate (SB 75) did not significantly affect fos-Luc gene transcription induced by 10 nM TPA (phorbol ester) or 0.1 nM bFGF (basic fibroblast growth factor), even at high peptide concentrations. This demonstrates that the compound competes with agonist for binding to the GnRH receptor without interfering with GnRH receptor-independent signaling to the c-fos promoter. [1]
In human ovarian cancer cell lines (except EFO-27), Cetrorelix diacetate (SB 75) exhibits antiproliferative effects comparable to those of GnRH-I agonists, indicating that the classic dichotomy of GnRH-I agonists and antagonists does not apply to tumor cells. In the ES-2 ovarian cancer cell line, Cetrorelix diacetate (SB 75) inhibited cell growth only at a concentration of 1000 ng/ml. After GnRH-I receptor knock-down in EFO-21 and OVCAR-3 human ovarian cancer cell lines, the antiproliferative effects of Cetrorelix diacetate (SB 75) remained present, suggesting that its effects are not mediated through the GnRH-I receptor. In the ovarian cancer cell line EFO-27, which expresses GnRH-I receptor but not putative GnRH-II receptor, the antiproliferative effects of Cetrorelix diacetate (SB 75) were also observed, further supporting a GnRH-I receptor-independent mechanism. [2]

Receptor binding affinity was determined using a radioligand binding assay with intact cells under physiological conditions. For displacement binding assays, 0.25 × 10⁶ cells per 100 μl were incubated with approximately 225 pM [¹²⁵I]Cetrorelix (specific activity 5-10 × 10⁵ dpm/pmol) and various concentrations of unlabeled Cetrorelix diacetate (SB 75) as competitor. Incubation was carried out, and binding data were analyzed using EBDA/ligand analysis software (Biosoft V3.0) to calculate the dissociation constant (K_D). The K_D of Cetrorelix diacetate (SB 75) was determined as 0.202 ± 0.03 nM from ten independent experiments. [1]

A luciferase reporter gene assay was performed using a stable transfected murine L-cell line expressing the human GnRH receptor and the c-fos promoter-driven firefly luciferase gene (clone L23.1/5). Cells were seeded at 5000 cells/well in microtiter plates and cultured for 24 h in DMEM with 10% fetal calf serum. After serum starvation for 24 h in serum-free DMEM, growth-arrested cells were pretreated with increasing concentrations of Cetrorelix diacetate (SB 75) (antagonist) for a period (not specified) prior to stimulation with 1 nM [D-Trp⁶]GnRH (agonist). Following 6 h of stimulation, cells were lysed with 100 μl lysis buffer (25 mM Tris-phosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100) for 15 min at room temperature. Then 10 μl of cell lysate was transferred to a white microplate, and 50 μl of assay buffer (20 mM Tricine pH 7.8, 1.07 mM (MgCO₃)₄Mg(OH)₂, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 μM coenzyme A, 470 μM firefly luciferin, 530 μM rATPNa₂) was added. Luminescence was quantified after 1 min using a luminometer (EG&G Berthold MicroLumat LB96P). The IC₅₀ of Cetrorelix diacetate (SB 75) for inhibiting agonist-induced luciferase activity was calculated as 1.21 ± 0.33 nM from six experiments using nonlinear regression analysis (Hill model). [1]
To test for nonspecific effects on c-fos promoter activity, cells were stimulated in a GnRH receptor-independent manner using 10 nM TPA or 0.1 nM bFGF, in the presence of increasing concentrations of Cetrorelix diacetate (SB 75). No significant effect of the compound on TPA- or bFGF-induced luciferase activity was observed at any concentration tested, confirming that the inhibition is specific to GnRH receptor-mediated signaling. [1]
The effect of DMSO, a common solvent for compound libraries, was assessed by incubating cells with or without 1 nM [D-Trp⁶]GnRH in the presence of increasing DMSO concentrations (0–10 vol%) for 7 h. Only DMSO concentrations >1 vol% (stimulated cells) or >5 vol% (non-stimulated cells) showed significant inhibitory effects on luciferase activity, indicating that DMSO at ≤1 vol% does not interfere with the assay. [1]

Absorption, Distribution and Excretion
Absorption is rapid following subcutaneous injection. The mean absolute bioavailability after subcutaneous administration in healthy female subjects was 85%. Unmetabolized cetrarolix was detected in urine after subcutaneous injection of 10 mg cetrarolix in both men and women. Dosage: 1.16 L/kg Dosage: 1.28 ml/min·kg [Single subcutaneous injection of 3 mg in healthy adult women] Unmetabolized cetrarolix was detected in urine after subcutaneous injection of 10 mg cetrarolix in both men and women. Cetrolix and trace amounts of (1-9), (1-7), (1-6), and (1-4) peptides were detected in bile samples 24 hours later. 2-4% of the dose was excreted unchanged in the urine as cetrarolix, and 5-10% of the dose was excreted in the bile as cetrarolix and its four metabolites. Therefore, only 7-14% of the total dose is recovered in unchanged cetrorelix and its metabolites from urine and bile within 24 hours. Due to the short collection time of bile and urine, the remaining dose may not be recovered. Following a single intravenous injection of 3 mg cetrorelix, its volume of distribution is approximately 1 L/kg. In vitro human plasma protein binding is 86%. In patients undergoing controlled ovarian stimulation, cetrorelix concentrations in follicular fluid and plasma are similar on the day of oocyte retrieval. Following subcutaneous injections of 0.25 mg and 3 mg cetrorelix, plasma cetrorelix concentrations are below or within the lower limit of quantitation on the days of oocyte retrieval and embryo transfer. Cetrorelix is rapidly absorbed after subcutaneous injection, reaching maximum plasma concentrations approximately 1-2 hours after administration. The mean absolute bioavailability of cetrorelix after subcutaneous injection in healthy female subjects is 85%. Pharmacokinetic studies were primarily conducted in rats and dogs. Absorption at the subcutaneous injection site is rapid and complete, regardless of sex or species. Dose-plasma AUC is linear. Cetrorelix distributes rapidly. Its primary target organs are the kidneys, liver, small intestine, and organs containing luteinizing hormone-releasing hormone (LHRH) receptors (pituitary gland, ovaries). Plasma protein binding is 86%. The drug is rapidly cleared from most tissues, primarily within 48 hours. …Cetrolix crosses the placenta in small amounts. The distribution of cetrorelix or its metabolites in breast milk has not been studied. Cetrorelix is excreted unchanged in the urine and metabolized by peptidases in the bile. …Studies in healthy volunteers indicate that cetrorelix is excreted similarly in humans, rats, and dogs. Following subcutaneous injection, the absolute bioavailability of cetrorelix is approximately 85% in both men and women. The apparent volume of distribution is 1.16 ± 0.29 L/kg in women and 1.02 ± 0.33 L/kg in men. The terminal half-life is approximately 10 hours after intravenous injection and approximately 30 hours after subcutaneous injection, with a decreasing trend in women. Human plasma protein binding is approximately 85%. Linear pharmacokinetics were observed after single (0.25, 0.5, and 1.00 mg) and multiple (0.25 to 1.00 mg) doses. Pharmacokinetics were linear within the 3 mg dose range.
Metabolism/Metabolites
In vitro studies showed that cetrorex was stable for both phase I and phase II metabolism. Cetrorex can be converted by peptidases, with peptides (1–4) being the major metabolites.
In rat bile, the major metabolite of cetrorex was identified as heptapeptide (1–7). This metabolite had no pharmacological activity in rats, i.e., it did not inhibit testosterone secretion.
After subcutaneous injection of 10 mg cetrorex into male and female rats, cetrorex and trace amounts of peptides (1–9), (1–7), (1–6), and (1–4) were detected in bile samples within 24 hours. In vitro studies showed that cetrorex was stable for both phase I and phase II metabolism. Cetrotide is converted by peptidase, with (1-4) peptides being the main metabolites.

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Biological half-life
~62.8 hours
In humans, the terminal half-lives after intravenous and subcutaneous injection are 8-9 hours and 24-40 hours, respectively.
In rats, the terminal half-lives after intravenous and subcutaneous injection are 1-2 hours and 7-14 hours, respectively… Elimination half-life: Single 3 mg dose: 62.8 hours (38.2–108 hours); Single 0.25 mg dose: 5.0 hours (2.4–48.8 hours); 0.25 mg daily for 14 days: 20.6 hours (4.1–179.3 hours) /Excerpt from table/
Half-lives greater than or equal to 100 hours were observed in excretory organs (liver, kidney), spleen, and organs containing LHRH binding sites.

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Protein binding 86% hr Non-human toxicity value 68.1 mg/kg was determined as the minimum lethal dose.

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[1]. Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay. Anal Biochem. 1997 Aug 15;251(1):17-23.

[2]. Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer. Reprod Biol Endocrinol. 2003 Oct 7;1:65.

Therapeutic Uses
Cetrorelix is indicated for the suppression of premature luteinizing hormone (LH) surges in women undergoing controlled ovarian stimulation. This randomized, placebo-controlled, single-blind study was conducted in 45 adult female Wistar rats… After peritoneal implantation of endometrial tissue, rats were randomly assigned to three equal intervention groups: (i) a control group, (ii) a leuprorelin group, and (iii) a cetrorelix group. Six weeks later, the implant volume was measured (volume-1) via a second laparotomy. Subsequently, the control group received weekly subcutaneous injections of saline (0.1 mL/rat), the leuprorelin group received twice-daily subcutaneous injections of leuprorelin (0.075 mg/kg), and the cetrorelix group received subcutaneous injections of cetrorelix (0.001 mg/rat/day) for 8 weeks. At the end of treatment, the implant volume was measured again (volume-2) via a third laparotomy, and the implant was completely removed for histopathological examination. Compare the volume-1 and volume-2 values within each group, and the intergroup scores for stromal and glandular tissue. In both the leuprorelin and cetrorelix groups, volume-2 was significantly reduced compared to volume-1 (P < 0.01 and P < 0.01, respectively), while there was no significant change in volume in the control group (P > 0.05). Compared to the control group, both glandular and stromal tissue were significantly reduced in the control group (P < 0.01 and P < 0.01, respectively). Leuprorelin and cetrorelix showed similar efficacy in reducing the size and histological structure of experimental endometriosis lesions. Drug Warnings: Cetrorelix should be prescribed by a healthcare professional experienced in fertility treatment. Pregnancy must be ruled out before initiating cetrorelix acetate treatment.
In patients receiving controlled ovarian stimulation, 1-2% reported elevated liver function test results, including ALT (SGPT), AST (SGOT), gamma-glutamyl transferase (GGT, GGTP), and alkaline phosphatase, up to 3 times the upper limit of normal.
Caution should be exercised in patients with GnRH allergy. These patients should be closely monitored after the first injection. In a study of an indication unrelated to infertility, one patient experienced a severe allergic reaction with cough, rash, and hypotension after 7 months of treatment with cetrorelix 10 mg/day.
Local reactions (e.g., redness, erythema, ecchymosis, pruritus, swelling, and itching) have been reported. These adverse reactions are usually transient, mild, and short-lived.
For more complete data on drug warnings for cetrorelix (8 warnings total), please visit the HSDB record page.

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Pharmacodynamics
Citrolix is a synthetic decapeptide with gonadotropin-releasing hormone (GnRH) antagonistic activity. GnRH induces anterior pituitary gonadotropic cells to produce and release luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH release is enhanced due to positive feedback from estradiol (E2) in the mid-menstrual cycle, leading to an LH surge. This LH surge induces ovulation of the dominant follicle, resumption of meiosis in the oocyte, and subsequent luteinization, manifested as elevated progesterone levels. Cetrorelix competes with natural GnRH for binding to pituitary cell membrane receptors, thereby controlling LH and FSH release in a dose-dependent manner.

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Cetrorelix diacetate (SB 75) is a synthetic decapeptide with the sequence: Ac-D-Nap-Ala-D-ClPhAla-D-Pyr-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH₂. It is a GnRH receptor antagonist that inhibits the action of GnRH and its superagonistic analogs. In the reporter gene assay, Cetrorelix diacetate (SB 75) serves as a tool to validate the specificity of antagonistic effects, as it competes with agonists for receptor binding without interfering with GnRH receptor-independent signaling pathways (e.g., TPA or bFGF stimulation). For storage and handling, Cetrorelix diacetate (SB 75) was dissolved in 0.01 N CH₃COOH at 1 mM final concentration and stored in siliconized polypropylene tubes at -20°C. [1]
In human ovarian cancer cells, Cetrorelix diacetate (SB 75) exhibits direct antiproliferative effects that appear to be independent of the GnRH-I receptor, as shown by receptor knock-down experiments. The compound may act through a putative GnRH-II receptor or other mechanisms. The antiproliferative activity of Cetrorelix diacetate (SB 75) is comparable to that of GnRH-I agonists in most ovarian cancer cell lines (except EFO-27), challenging the classical agonist/antagonist dichotomy in tumor cells. [2]

C70H92CLN17O14.2(C2H4O2)

1429.669

130143-01-0

Cetrorelix Acetate;145672-81-7;Cetrorelix;120287-85-6

25074887

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1768.4ºC at 760 mmHg

1023.3ºC

0mmHg at 25°C

1.668

2.69

16

16

38

102

2840

10

NC(NCCCCC(N1[C@H](C(N([C@H](C)C(N(C([C@H](N)CC2=CC=C(Cl)C=C2)=O)C([C@H](CC2=CC=C(OC(C)=O)C=C2)NC([C@H](COC(C)=O)NC([C@H](N)CC2=CN=CC=C2)=O)=O)=O)=O)C([C@H](NC(C)=O)CC2=CC3=CC=CC=C3C=C2)=O)=O)CCC1)=O)=N

SBNPWPIBESPSIF-MHWMIDJBSA-N

InChI=1S/C70H92ClN17O14/c1-39(2)31-52(61(94)82-51(15-9-28-77-69(73)74)68(101)88-30-10-16-58(88)67(100)79-40(3)59(72)92)83-60(93)50(14-8-29-78-70(75)102)81-63(96)54(34-43-20-25-49(91)26-21-43)86-66(99)57(38-89)87-65(98)56(36-45-11-7-27-76-37-45)85-64(97)55(33-42-18-23-48(71)24-19-42)84-62(95)53(80-41(4)90)35-44-17-22-46-12-5-6-13-47(46)32-44/h5-7,11-13,17-27,32,37,39-40,50-58,89,91H,8-10,14-16,28-31,33-36,38H2,1-4H3,(H2,72,92)(H,79,100)(H,80,90)(H,81,96)(H,82,94)(H,83,93)(H,84,95)(H,85,97)(H,86,99)(H,87,98)(H4,73,74,77)(H3,75,78,102)/t40-,50-,51+,52+,53-,54+,55-,56-,57+,58+/m1/s1

(2S)-1-[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-acetamido-3-naphthalen-2-ylpropanoyl]amino]-3-(4-chlorophenyl)propanoyl]amino]-3-pyridin-3-ylpropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-5-(carbamoylamino)pentanoyl]amino]-4-methylpentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]-N-[(2R)-1-amino-1-oxopropan-2-yl]pyrrolidine-2-carboxamide

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)