human GnRH ( IC50 = 0.33 nM ); monkey GnRH ( IC50 = 0.32 nM )
Human gonadotropin-releasing hormone receptor (GnRHR) antagonist (binding IC50 = 0.33 nM in the presence of serum).
Monkey GnRHR antagonist (binding IC50 = 0.32 nM).
Rat GnRHR (low affinity, binding IC50 = 9800 nM).
TAK-385 (relugolix) targets the human gonadotropin-releasing hormone receptor (hGNRHR) . The paper cites prior work (Miwa et al., 2011) reporting that TAK-385 has a binding IC50 of 0.33 nM for the human GnRH receptor in the presence of serum, and an IC50 of 0.32 nM for the monkey GnRH receptor. It has low affinity for the rat GnRH receptor (IC50 = 9800 nM). [2]

Relugolix shows a 30000-fold decrease for the rat receptor (IC50=9800 nM) but a strong binding affinity (IC50=0.32 nM) for the monkey receptor, which is comparable to that of the human receptor (IC50=0.33 nM). In the presence of 40% serum, TAK-385's antagonistic in vitro activity against the human receptor (IC90=18 nM) was 95 times greater than that against the monkey receptor (IC90=1700 nM)[1].

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Relugolix possesses higher affinity and more potent antagonistic activity for human and monkey GnRH receptors compared to its predecessor TAK-013. The binding IC50 for human GnRHR is 0.33 nM (in the presence of serum) and for monkey GnRHR is 0.32 nM.

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In vitro studies demonstrate that Relugolix is a highly potent antagonist of the GnRH receptor. Radioligand binding assays show that Relugolix binds to the human GnRH receptor with equilibrium dissociation constants (Kd) as low as 0.37–0.84 nM. In functional assays, Relugolix potently inhibits GnRH-induced intracellular calcium mobilization (IC50 of approximately 4.05 nM) and arachidonic acid release (IC50 as low as 0.33–0.82 nM), effectively blocking downstream signaling pathways. Notably, its antagonistic activity is maintained in the presence of 40% human serum, indicating its efficacy under physiological conditions.

Relugolix (oral administration; 1-3 mg/kg; single dose for pharmacokinetic study) shows clear suppression of circulating LH levels in monkeys at a dose of 1 mg/kg and a good pharmacokinetic profile. Male cynomolgus monkeys show a pharmacokinetic profile with Cmax, Tmax, and AUCo values of 16.0 ng/mL, 2.7 h, and 90.1 ng, respectively[1].
Relugolix (oral administration; 3, 10, or 30 mg/kg; twice daily; 4 weeks) dramatically lowers the weight of the testicles, lowers the weight of the ventral prostate (3 mg/kg), and lowers the weight of the prostate to castrate levels (10 mg/kg) in male hGNRHR-knock-in mice[2].
Relugolix (oral administration; 30, 100, or 200 mg/kg; twice daily; 4 weeks) causes all mice to enter a constant diestrous phase during the first week at 100 mg/kg, and after 4 weeks in female hGNRHR-knock-in mice, this dose significantly reduces the weights of the uteri and ovaries[2].

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Male Human GnRHR Knock-in Mice: Twice-daily oral administration of relugolix for 4 weeks significantly decreased testis weight at 10 mg/kg. It significantly reduced ventral prostate weight at 3 mg/kg and decreased it to castrate levels at 10 mg/kg.[1]
Female Human GnRHR Knock-in Mice: Twice-daily oral administration of relugolix at 100 mg/kg induced constant diestrous phases within the first week. After 4 weeks, it significantly decreased the weights of ovaries and uteri to levels comparable to ovariectomized mice. The expression of GnRH receptor mRNA in the pituitary was downregulated at doses effective in suppressing gonadal function.[1]
Recovery Studies: The suppressive effect of relugolix was reversible. In female mice treated with 100 or 200 mg/kg twice daily for 28 days, estrous cycles resumed approximately 5 days after drug withdrawal, and the decreased weights of ovaries and uteri completely recovered within 14 days after withdrawal.[1]
In male mice treated with 30 mg/kg twice daily for 28 days, the ventral prostate weight and serum testosterone levels recovered from a completely suppressed state within 14 days after drug withdrawal, although testis weight did not recover within the 28-day observation period.

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TAK-385 suppressed gonadal function in a dose-dependent manner in both male and female hGNRHR knock-in mice. In male knock-in mice, twice-daily oral administration for 4 weeks significantly decreased testis weight at ≥10 mg/kg and reduced ventral prostate weight to castrate levels at ≥10 mg/kg. A significant effect on prostate weight was observed even at the lowest dose tested (3 mg/kg, twice daily). [2]
In female knock-in mice, twice-daily oral administration of TAK-385 at 100 mg/kg induced constant diestrous phases in all animals within the first week, and significantly decreased ovary and uterus weights to ovariectomized levels after 4 weeks. TAK-385 also downregulated human GnRH receptor mRNA expression in the pituitary at doses effective at suppressing gonadal function. [2]
The suppressive effects of TAK-385 were reversible. In female mice treated with TAK-385 (100 or 200 mg/kg, twice daily for 28 days), constant diestrus began to resolve approximately 5 days after drug withdrawal; the animals subsequently showed continuous estrus for several days, and then normal estrous cyclicity resumed. The decreased ovary and uterus weights recovered to near intact levels within 14 days after withdrawal. In male mice treated with TAK-385 (30 mg/kg, twice daily for 28 days), ventral prostate weight and serum testosterone levels recovered to control levels within 14 days after drug withdrawal, confirming functional recovery of the male reproductive axis. [2]

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In human GnRH receptor knock-in mouse models, oral administration of Relugolix potently, continuously, and reversibly suppresses the hypothalamic-pituitary-gonadal axis. In male mice, twice-daily oral administration of 10 mg/kg for four weeks reduced prostate weight to castrate levels. In female mice, the same regimen at 100 mg/kg induced constant diestrous phases within one week and decreased uterine weight to ovariectomized levels. Following drug withdrawal, gonadal function began to recover within five days and was almost completely restored within 14 days in both sexes, demonstrating reversibility. In patients with advanced prostate cancer, Relugolix rapidly reduces testosterone to castration levels without causing testosterone surge.

The binding affinity of Relugolix for the GnRH receptor is typically assessed using radioligand competition binding assays. A standard protocol involves incubating membrane homogenates from cells expressing recombinant human GnRH receptors with a fixed concentration of a radiolabeled tracer ligand (e.g., [¹²⁵I]leuprorelin or ¹²⁵I-labeled [D-Trp6]-LH-RH) and increasing concentrations of Relugolix. Incubation is performed at room temperature or 37°C for approximately 60 minutes. Bound and free ligands are then separated by rapid vacuum filtration or centrifugation, followed by multiple washes with buffer to remove unbound radioactivity. The radioactivity retained on filters or in pellets is quantified using a gamma counter or liquid scintillation counter. Competition curves are generated to calculate the half-maximal inhibitory concentration (IC50), which is then converted to a dissociation constant (Ki or Kd) using the Cheng-Prusoff equation.

The cellular antagonistic activity of Relugolix is commonly assessed using cell lines expressing the human GnRH receptor, such as CHO or HEK293 cells. A representative protocol for calcium flux assays involves seeding cells in multi-well plates and culturing overnight. Cells are then loaded with a calcium-sensitive fluorescent dye (e.g., Fluo-4 Direct) and pre-incubated with varying concentrations of Relugolix (typically for 15–30 minutes). Following stimulation with GnRH agonist, changes in intracellular calcium concentration are monitored in real-time using a fluorescence imaging plate reader (FLIPR). For arachidonic acid release assays, cells are pre-incubated overnight with ³H-labeled arachidonic acid, washed, and then incubated with Relugolix and GnRH for 45 minutes. Supernatants are collected, and released radioactivity is measured by scintillation counting. The IC50 values are calculated from dose-response curves to evaluate the inhibitory effect of Relugolix.

Male hGNRHR-knock-in mice
3, 10 or 30 mg/kg
Oral administration; 3, 10 or 30 mg/kg; twice daily; 4 weeks

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Model: All efficacy studies were conducted in human GnRH receptor (hGNRHR) knock-in mice, where the mouse Gnrhr gene was replaced with human GNRHR cDNA.
Drug Formulation: Relugolix was dissolved in 0.5% methylcellulose solution containing 6 mg/mL citric acid monohydrate.
Male Mouse Studies: Compounds were administered orally (by gavage) twice daily (BID) for 4 weeks at specified doses. Organ weights (testes, ventral prostate) were evaluated at endpoint.
Female Mouse Studies: Compounds were administered orally twice daily for 4 weeks at specified doses. Estrous cycles were monitored via vaginal cytology. Organ weights (ovaries, uterus) and pituitary GnRHR mRNA expression were evaluated at endpoint.
Recovery Studies: After 28 days of twice-daily oral administration, drug treatment was stopped. Estrous cycles (females) and organ weights (both sexes) were monitored at specified time points post-withdrawal (e.g., days 3, 7, 10, 14, 28). Blood was collected from male mice for serum testosterone measurement.

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hGNRHR knock-in mouse generation: A targeting vector was constructed by replacing the first exon (containing the initiation codon) of the mouse Gnrhr locus with human GNRHR cDNA and a neomycin resistance unit. The vector was electroporated into embryonic stem cells, and correctly recombined cells were selected with G418. Recombinant cells were injected into C57BL/6J blastocysts, and chimeric males were backcrossed to C57BL/6J females. [2]
Male mouse efficacy study: 6-11 week-old male hGNRHR knock-in mice were orally administered TAK-385 (3, 10, or 30 mg/kg) suspended in 0.5% methylcellulose containing 6 mg/mL citric acid, twice daily for 4 weeks. At day 28, mice were decapitated, and testes and ventral prostates were weighed. [2]
Female mouse efficacy study: 10-week-old female hGNRHR knock-in mice were orally administered TAK-385 (30, 100, or 200 mg/kg) twice daily for 4 weeks. Estrous cycles were monitored via Giemsa-stained vaginal smears throughout the study. At day 28, mice were decapitated, and pituitaries, ovaries, and uteri were collected. Ovaries and uteri were weighed; pituitary GnRH receptor mRNA expression was measured by real-time PCR. [2]
Recovery study in females: 8-week-old female knock-in mice received TAK-385 (100 or 200 mg/kg, twice daily for 28 days). Subsets were dissected at day 28 (end of treatment), day 42 (14 days post-withdrawal), and day 56 (28 days post-withdrawal) to evaluate recovery of ovary and uterus weights. Estrous cycles were monitored during and after treatment. [2]
Recovery study in males: 14-week-old male knock-in mice received TAK-385 (30 mg/kg, twice daily for 28 days). Subsets were dissected at day 28, 31, 35, 38, 42, and 56 (0, 3, 7, 10, 14, and 28 days post-withdrawal). Testes and ventral prostates were weighed, and serum testosterone levels were measured. [2]

Absorption, Distribution and Excretion
Following a single oral dose of regrugolide, its Cmax and AUC increase proportionally; conversely, with repeated dosing, AUC remains dose-proportional, while the increase in Cmax is greater than dose-proportional. After a once-daily dose of 120 mg, the steady-state AUC and Cmax of regrugolide are 407 (± 168) ng·hr/mL and 70 (± 65) ng/mL, respectively. The absolute oral bioavailability of regrugolide is approximately 12%, and the median time to peak concentration (Tmax) after oral administration is 2.25 hours. Approximately 81% of the oral dose is excreted in feces, of which 4.2% is unchanged; 4.1% is excreted in urine, of which 2.2% is unchanged. The mean renal clearance of regrugolide is 8 L/h, and the total clearance is 26.4 L/h.

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Metabolism/Metabolites
Relugoline is primarily metabolized by the P450 enzyme CYP3A subfamily, with a smaller contribution from CYP2C8.

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Biological Half-Life
The average effective half-life of relugoline is 25 hours, and the average terminal elimination half-life is 60.8 hours.

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Relugolix has good oral bioavailability and is rapidly absorbed. In healthy female subjects following a single 40 mg oral dose, the median time to maximum concentration (t_max) is 1.50–2.26 hours, the geometric mean peak concentration (C_max) ranges from 9.36–16.1 ng/mL, and the elimination half-life (t_1/2) is approximately 15.9–56.6 hours (mean approximately 25–33 hours). In male Beagle dogs, the long-acting microcrystalline formulation administered intramuscularly exhibits sustained-release characteristics with significantly extended AUC and t_max. Relugolix is a substrate of P-glycoprotein (P-gp); co-administration with oral P-gp inhibitors significantly increases relugolix exposure, necessitating dosing interval adjustments. Co-administration with combined strong CYP3A and P-gp inducers decreases relugolix exposure, potentially requiring dose adjustments.

Hepatotoxicity
In patients receiving regorafenib, 1% to 3% experienced serum transaminase elevations exceeding 3 times the upper limit of normal (ULN), a similar proportion observed in patients receiving control drugs such as leuprorelin or degarelix. Serum enzyme elevations are usually mild and self-limiting, resolving spontaneously even without dose adjustment. Less than 1% of patients had ALT values exceeding 5 times the ULN. No symptomatic or jaundice-related ALT elevations were observed in pre-registration clinical trials of regorafenib monotherapy or in combination with estradiol and norethindrone. Since its approval and widespread use, there have been no published reports of clinically significant liver injury caused by regorafenib. Probability Score: E (Unlikely a cause of clinically significant liver injury). Protein Binding Regorafenib has a protein binding rate of 68-71% in plasma, primarily binding to albumin, and secondarily to α1-acid glycoprotein.

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In clinical practice, the common adverse events associated with Relugolix are linked to its pharmacological mechanism (testosterone suppression). The most common adverse reactions (≥10%) include hot flush (54%), increased glucose (44%), increased triglycerides (35%), musculoskeletal pain (30%), decreased hemoglobin (28%), increased alanine aminotransferase (27%), fatigue (26%), increased aspartate aminotransferase (18%), constipation (12%), and diarrhea (12%). Serious adverse events include myocardial infarction (0.8%), acute kidney injury (0.6%), arrhythmia (0.6%), and hemorrhage (0.6%). QT/QTc interval prolongation is a potential risk, particularly for patients with congenital long QT syndrome, congestive heart failure, or electrolyte abnormalities. Post-marketing cases of angioedema, including pharyngeal edema, have been reported. Relugolix is contraindicated in patients with severe hypersensitivity to relugolix or any of its components. In animal studies, based on its mechanism of action, Relugolix can cause fetal harm and pregnancy loss.

[1]. Discovery of 1-{4-[1-(2,6-Difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (TAK-385) as a Potent, Orally Active, Non-Peptide Antagonist of the Human Gonadotropin-Releasing Hormone Receptor. J. Med. Chem. 2011, 54, 14, 4998–5012

[2]. Suppression of the hypothalamic-pituitary-gonadal axis by TAK-385 (relugolix), a novel, investigational, orally active, small molecule gonadotropin-releasing hormone (GnRH) antagonist: studies in human GnRH receptor knock-in mice. Eur J Pharmacol . 2014 Jan 15:723:167-74.

Pharmacodynamics
Approximately 56% of patients achieved castration-level testosterone concentrations (<50 ng/dL) by day 4 of treatment, and 97% maintained this level during the 48-week treatment period. Relugoli needs to be taken orally once daily to maintain the required testosterone concentration. Androgen deprivation therapy may prolong the QTc interval; therefore, caution should be exercised in patients at high risk of baseline QTc interval prolongation, such as those with electrolyte disturbances, congestive heart failure, or those taking other medications known to prolong the QTc interval. Based on its mechanism of action and animal studies, use of relugolix in pregnant women may cause fetal harm—male patients with female partners should be advised to use effective contraception throughout treatment and for 2 weeks after discontinuation of treatment to prevent unintended fetal exposure. Relugolix (TAK-385) is an investigational novel non-peptide, orally active gonadotropin-releasing hormone (GnRH) antagonist. It is a thienopyrimidine derivative. [1]
It is based on previous studies of TAK-013 (shufogoli), but has a higher affinity for human and monkey GnRH receptors. [1]
Due to its low affinity for rat GnRH receptors, in vivo evaluation using human GnRH receptor knock-in mice is required. [1]
This study shows that daily oral administration of relugolix can effectively, persistently and reversibly inhibit the hypothalamic-pituitary-gonadal axis in this humanized model. [1]
relugolix is considered a potential therapeutic intervention for hormone-dependent diseases such as endometriosis, uterine fibroids, prostate cancer, and premenopausal breast cancer. [1]
The downregulation of pituitary GnRH receptor mRNA by relugolix may be another pharmacological mechanism of action besides competitive receptor inhibition. Hostile.

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Compound nomenclature: TAK-385 is also known as relugolix. It is a thienopyrimidine derivative. [2]
Advantage over peptide antagonists: Unlike peptide GnRH antagonists (e.g., cetrorelix, ganirelix, degarelix) which require subcutaneous injection, TAK-385 is orally active. This offers potential advantages in patient convenience and the ability to discontinue treatment rapidly if adverse effects occur. [2]
Sex difference in effective dose: A 10-fold higher dose of TAK-385 (100 mg/kg vs. 10 mg/kg in males) was required to completely suppress gonadal function in female knock-in mice compared with males. The authors suggest this may be due to sex differences in the regulation of the HPG axis rather than differences in pharmacokinetics. [2]
Receptor downregulation: TAK-385 downregulated GnRH receptor mRNA expression in the pituitary at doses effective for suppressing reproductive function, which may contribute to its pharmacological action in addition to competitive antagonism. [2]
Reversibility of testis weight: Although prostate weight and testosterone levels recovered rapidly (within 14 days), testis weight did not recover within the 28-day observation period after drug withdrawal. This is likely due to the time required for regeneration of the spermatogenic epithelium, which comprises the majority of testicular weight. [2]

C29H27F2N7O5S

623.630391359329

623.18

C, 55.85; H, 4.36; F, 6.09; N, 15.72; O, 12.83; S, 5.14

737789-87-6

Relugolix-d6

10348973

White to off-white solid powder

3.966

2

11

9

44

1010

0

O=C(NOC)NC1=CC=C(C(S2)=C(CN(C)C)C(C(N3C4=NN=C(OC)C=C4)=O)=C2N(CC5=C(F)C=CC=C5F)C3=O)C=C1

AOMXMOCNKJTRQP-UHFFFAOYSA-N

InChI=1S/C29H27F2N7O5S/c1-36(2)14-19-24-26(39)38(22-12-13-23(42-3)34-33-22)29(41)37(15-18-20(30)6-5-7-21(18)31)27(24)44-25(19)16-8-10-17(11-9-16)32-28(40)35-43-4/h5-13H,14-15H2,1-4H3,(H2,32,35,40)

1-[4-[1-[(2,6-difluorophenyl)methyl]-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea

TAK 385; TAK385; TAK-385; trade names: Orgovyx; Relumina

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: 61~100 mg/mL (97.8~160.4 mM)

Solubility in Formulation 1: ≥ 0.83 mg/mL (1.33 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (1.33 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 8.3 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: ≥ 0.83 mg/mL (1.33 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 8.3 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.)