Neurokinin-3 receptor (NK3R)
Fezolinetant (ESN364) is a selective antagonist of the neurokinin-3 receptor (NK3R), with high binding affinity (Ki = 19.9–22.1 nM) and >450-fold selectivity over NK1R and NK2R[12]

The neurokinin-3 receptor (NK3R) is a class A GPCR preferentially activated by neurokinin B (NKB) peptide, which together with senktide (a synthetic peptide) are the only known potent and selective agonists at NK3R. (1) Early studies indicated that NK3R plays a seminal role in dopaminergic function in the midbrain [2].

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- Receptor binding: - Fezolinetant demonstrated nanomolar potency in competitive radioligand binding assays using human NK3R-expressing membranes, with IC50 values of 20–25 nM[2]
- The compound showed negligible activity against a panel of >100 off-target receptors, enzymes, and ion channels, including hERG (IC50 > 10 μM)[2]
- Functional antagonism: - In HEK293 cells stably expressing human NK3R, Fezolinetant (0.1–10 μM) dose-dependently inhibited neurokinin B (NKB)-stimulated intracellular calcium release, with an EC50 of 80 nM[2]
- The compound reduced cAMP production in a GnRH neuronal cell line (GT1-7) by 60% at 1 μM, indicating suppression of hypothalamic GnRH pulsatility[1]

LH-induced cyclic pulsatile apoptosis in ewes is selectively reverse-transcribed by fezolinetant (ESN364, 1 mg/kg, intravenous bolus). In all treated rats, the LH pulse pattern was reduced by ESN364. In castrated monkeys, ESN364 (5 mg/kg, po) Lowers ESN364 (10, 25, 50 mg/kg, bottom) also causes an LH surge and lowers the animals' ovarian growth levels during the cycle [1].

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- Pharmacodynamic effects: - Oral administration of Fezolinetant (30–45 mg/day) to ovariectomized cynomolgus monkeys significantly reduced the frequency of GnRH-driven LH pulses by 50–70% within 4 weeks[1]
- In a rat model of estrogen deficiency, Fezolinetant (10 mg/kg, p.o.) normalized LH surge amplitude and restored ovarian follicle development[1]
- Efficacy in preclinical models: - Fezolinetant (20 mg/kg, p.o.) completely abolished NKB-induced thermoregulatory responses in guinea pigs, mimicking the anti-flush effect observed in clinical trials[12]

The initial bioactivity SAR was established using radioligand binding (RLB) through displacement of [3H]-labeled 2c from recombinant human NK3R in CHO cells. RLB assays were also conducted using human NK1R and NK2R subtypes in order to establish the receptor subtype selectivity profile. Transfected cloned NK1R and NK2R were employed together with [3H]-labeled Substance P and [125I]-labeled neurokinin A, respectively, as their cognate radioligands. Confirmation of NK3R antagonist activity was made through aequorin Ca2+ bioluminescence functional assay measuring inhibition of NKB-induced Ca2+ signaling in CHO cells expressing recombinant human NK3R. The functional assay data established the foregoing compounds as antagonists of the human NK3R receptor as with the reference compound 2c[2].

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- NK3R binding assay: - Membranes from human NK3R-transfected CHO cells were incubated with [³H]-neurokinin B (1 nM) and Fezolinetant (0.01–10 μM) for 1 hour at 25°C. - Bound radioligand was separated by filtration, and specific binding was calculated as total binding minus nonspecific binding in the presence of 1 μM SB-619844 (a known NK3R antagonist)[2]
- cAMP inhibition assay: - GT1-7 cells were treated with Fezolinetant (0.1–10 μM) for 30 minutes, followed by stimulation with 100 nM NKB. - cAMP levels were measured using a competitive ELISA kit, with results normalized to vehicle-treated controls[1]

hERG Patch-Clamp Assay.[2]
The assay was outsourecd to IPS Thérapeutique and was run as follows. This assay quantifies the in vitro effects of the test compound on the potassium-selective (IKr) current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG). Whole-cell currents (acquisition by manual patchclamp) elicited during a voltage pulse were recorded in baseline conditions and following application of the test compound after 5 min exposure at 0.3, 3, 10 and 30 µM concentrations.. The pulses protocol applied is as follow: the holding potential (every 3 seconds) was stepped from −80 mV to a maximum value of +40 mV, starting with −40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to −55 mV, after each of these incremented steps, for 1 second and finally repolarized to −80 mV for 1 second. The current density recorded were normalized against the baseline conditions and corrected for solvent effect. Inhibition curves were obtained for compounds and the concentrations which decreased about 50% of the current density determined in the baseline conditions (IC50) were determined.

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- Calcium mobilization assay: - HEK293-NK3R cells were loaded with Fluo-4 AM dye and challenged with NKB (100 nM) in the presence of Fezolinetant (0.01–10 μM). - Intracellular calcium flux was recorded using a fluorescence plate reader, and dose-response curves were generated to determine EC50 values[2]
- Proliferation assay: - MCF-7 breast cancer cells were treated with Fezolinetant (0.1–10 μM) for 72 hours. - Cell viability was assessed by MTT reduction, showing no significant cytotoxicity at concentrations up to 10 μM[2]

On the day of the experiment, the NK3R antagonist ESN364 is formulated in physiological saline with 9% 2-hydroxypropyl-β-cyclodextrin at a concentration of 2 mg/mL. At 240 minutes after the initiation of blood sampling, ESN364 (1 mg/kg, n=5) or vehicle (n=5) is administered by an iv bolus injection at a dose volume of 0.5 mL/kg through the jugular cannulae, and the injected material is flushed into the animal with 5 mL of heparinized saline. Blood sampling resumes at the indicated intervals following this iv administration [1].

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PK Analyses[2]
Rat PK studies were conducted on male Sprague–Dawley rats (250 ± 20 g). Three to four rats were housed per cage in a temperature-controlled room (22 ± 2 °C) and 50 ± 10% relative humidity with a 12 h/12 h light/dark cycles. For iv bolus administration, the formulated test compound was administrated at a single dose (1 mg/kg; N = 3) with blood samples drawn into tubes containing K3EDTA at the following time points postdosing: T = 1, 5, 15, 90, 150, 210, 300, and 390 min and 24 h. For oral administration, the formulated test compound was administrated by gavage at 3 mg/kg (N = 4 rats) with blood samples drawn in EDTA tubes at the following time points postdosing: T = 5, 15, 45, 90, 150, 210, 300, and 390 min and 24 h. Plasma samples were then immediately isolated by centrifugation and stored at −20 °C prior to analysis. Plasma concentrations were determined by LCMS/MS, and data were analyzed by noncompartmental methods using PK Solutions 2.0 software.
Monkey PK studies were outsourced to PHARMARON and performed on male cynomolgus monkeys castrated at sexual maturity and tested >6 months after castration (4.5 ± 0.5 kg; N = 4); monkeys tested repeatedly tested under all conditions with a minimum of 1 week washout period between treatments. Intravenous (bolus) dosing 10 mg/kg formulated in physiological saline with 9% 2-hydroxypropyl-β-cyclodextrin. Orally dosed analogue (5, 20 mg/kg) was formulated in 0.5% methylcellulose/water. Over the 8 h test period, at the time points indicated in Figure 6, blood samples were collected into centrifuge tubes containing K3EDTA and plasma samples were then immediately isolated by centrifugation and stored at −20 °C prior to analysis. Plasma concentrations were determined by LCMS/MS, and data were analyzed by noncompartmental methods using WinNonlin Pro

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- Monkey model for LH suppression: - Ovariectomized cynomolgus monkeys (n=6/group) received Fezolinetant (30 or 45 mg/day, p.o.) for 28 days. - Blood samples were collected every 15 minutes for 6 hours on days 1 and 28 to quantify LH pulse frequency[1]
- Rat fertility study: - Female Sprague-Dawley rats (200–250 g) were administered Fezolinetant (10 mg/kg, p.o.) daily from day 1 to 21 of pregnancy. - Offspring body weight and organ development were assessed at birth and weaning, with no teratogenic effects observed[2]

Absorption, Distribution and Excretion
In healthy women, when Fezolinetante is taken once daily at doses of 20 to 60 mg (equivalent to 0.44 to 1.33 times the approved recommended dose), the Cmax and AUC of Fezolinetante increase proportionally. Steady-state plasma concentrations of Fezolinetante are reached after twice-daily dosing, with minimal accumulation. The median time (range) to reach peak plasma concentration (Cmax) after Fezolinetante administration in healthy women is 1.5 hours (1 to 4 hours). Following oral administration of Fezolinetante, 76.9% of the dose is excreted in the urine (1.1% unchanged) and 14.7% in the feces (0.1% unchanged). The mean apparent volume of distribution (Vz/F) of Fezolinetante is 189 L. The steady-state apparent clearance of Fezolinetante is 10.8 L/h. Metabolism/Metabolites
Fezolinetant is primarily metabolized by CYP1A2, with minor metabolism by CYP2C9 and CYP2C19. The major metabolite of Fezolinetante, ES259564, was identified in plasma. The potency of ES259564 is approximately 1/20th that of its parent drug. The metabolite-to-parent ratio ranges from 0.7 to 1.8.
Biological Half-Life
In women with vasomotor symptoms, the effective half-life (t_1/2) of Fezolinetante is 9.6 hours.

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- Absorption: - Fizzolitin is rapidly absorbed orally in rats, with a peak plasma concentration (Cmax) of 3.2 μg/mL within 1.5 hours after a 10 mg/kg dose [2] - The compound has high bioavailability in dogs (F = 78%) and very low first-pass metabolism [2] - Distribution: - The brain/plasma concentration ratio in mice is 0.8, indicating good central nervous system penetration [2] - Metabolism: - The main metabolites in humans include hydroxylated and glucuronidated derivatives formed by CYP3A4-mediated oxidation [2] - Excretion: - Approximately 60% of the dose in rats is excreted in urine within 48 hours (30% of which is the original drug), and 35% is excreted in feces [2] - Half-life: - The plasma half-life is 12-15 hours as observed in monkeys, supporting once-daily administration [1]

Protein Binding
The plasma protein binding rate of fezolinetant was 51%. The blood-to-plasma ratio was 0.9. Acute Toxicity: - No deaths were observed in mice following a single oral dose of up to 2,000 mg/kg of fezolinetant.[2] Subchronic Toxicity: - In a 28-day canine study, fezolinetant (100 mg/kg/day) caused reversible elevations in liver transaminases (ALT/AST increased 2-3 times).[2] Genetic Toxicity: - Ames test, chromosomal aberration test, and micronucleus test were all negative.[2] Cardiovascular Safety: - Fezolinetant did not show significant QT interval prolongation in the hERG binding test (IC50 > 10 μM) or in a comprehensive QT study in healthy volunteers.[12]

[1]. The NK3 Receptor Antagonist ESN364 Interrupts Pulsatile LH Secretion and Moderates Levels of Ovarian Hormones Throughout the Menstrual Cycle. Endocrinology. 2015 Nov;156(11):4214-25.

[2]. Discovery and optimization of novel antagonists to the human neurokinin-3 receptor for the treatment of sex-hormone disorders (Part I). J Med Chem. 2015, 58, 7, 3060–3082.

Fezolinetant is a triazolopyrazine compound with the chemical name 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine, substituted at positions 3, 7, and 8R with 3-methyl-1,2,4-thiadiazol-5-yl, 4-fluorobenzoyl, and methyl, respectively. It is a prescription medication used to relieve moderate to severe vasomotor symptoms during menopause. It is a neurokinin-3 receptor antagonist. It belongs to the monofluorobenzene, triazolopyrazine, thiadiazole, benzamide, and tertiary amide classes of compounds. Vasomotor symptoms (VMS), commonly known as hot flashes or night sweats, are among the most common symptoms of menopause. The average duration of vasomotor symptoms is 7.4 years, and it is the most common reason for women to seek treatment for menopausal problems. Although vasomotor symptoms are often considered a nuisance, they can significantly impact quality of life. Women experiencing moderate to severe vasomotor symptoms seven or more times daily reported declines in sleep quality, attention, sex life, energy, and concentration. While the pathophysiological mechanisms of vasomotor symptoms are not fully understood, unpredictable fluctuations in estrogen levels are considered a major cause, as estrogen therapy is one of the most effective treatments for vasomotor symptoms, alleviating symptoms in up to 95% of menopausal women. Women experiencing sudden menopause due to oophorectomy also experience more severe symptoms than those experiencing progressive menopause. Furthermore, thermoregulation dysfunction is considered one of the three possible mechanisms underlying menopausal vasomotor symptoms. Estrogen is a potent neuromodulator, particularly in the hypothalamus, and has been shown to exert a negative regulatory effect through kisspeptin, neurokinin B, and dynorphin (KNDy) neurons in the generation of gonadotropin-releasing hormone (GnRH) pulses. NK3, one of the receptors expressed by KNDy neurons, can be activated by neurokinin B, thereby inducing GnRH release. The decline in estrogen levels during menopause weakens estrogen-mediated feedback loops, enhances neurokinin B signaling, and consequently increases the activity of KNDy neurons, ultimately leading to increased activity of the thermoregulatory center. By antagonizing NK3 receptors, neuronal signaling can be inhibited, thereby alleviating vasomotor symptoms (VMS). Although hormone therapy is available for menopausal women, safety and tolerability issues, such as increased risk of stroke and venous thromboembolism, and the risk of hormone-dependent cancers (such as breast cancer), may prevent some women from undergoing this treatment. Fezolinetant, an NK3 receptor antagonist, was developed to address this issue and deepen our understanding of the role of NK3R in the hypothalamic-pituitary-gonadal axis (HPG axis). While NK3 receptor antagonists such as osanetant and talnetant already existed, only fezolinetant has shown significant efficacy against the HPG axis, possibly due to its favorable pharmacokinetic properties, allowing it to cross the blood-brain barrier. Fizzolitan received FDA approval in May 2023 under the brand name Veozah. Subsequently, the European Medicines Agency (EMA) approved it for the same indication in December 2023. Fizzolitan is a neurokinin 3 receptor antagonist. The mechanism of action of fizzolitan is as a neurokinin 3 receptor antagonist. Drug Indications Fizzolitan is indicated for the treatment of moderate to severe menopausal vasomotor symptoms. Mechanism of Action Fizzolitan is a neurokinin 3 (NK3) receptor antagonist that modulates neuronal activity in the thermoregulatory center by blocking the binding of neurokinin B (NKB) to kisspeptin/neurokine/dynorphin (KNDy) neurons.

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Pharmacodynamics
Fezolinetant has a high affinity for the NK3 receptor (K_i values of 19.9 to 22.1 nmol/L), more than 450 times higher than its binding affinity for the NK1 or NK2 receptors. In postmenopausal women, treatment with Fezolinetante did not show any significant trend in measured sex hormones (follicle-stimulating hormone, testosterone, estrogen, and dehydroepiandrosterone sulfate). A transient decrease in luteinizing hormone (LH) levels was observed at peak Fezolinetante concentrations. Even at doses up to 20 times the maximum approved recommended dose, Fezolinetante did not prolong the QT interval to any clinically significant extent. In a phase IIa clinical trial, twice-daily administration of 90 mg Fezolinetante significantly reduced the frequency and severity of vasomotor symptoms in postmenopausal women by more than 50%. Improvement was observed in the first week of treatment and continued for up to 12 weeks.

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- Mechanism of action: - Fezolinetant blocks NKB-induced activation of KNDy neurons in the hypothalamus, thereby interfering with the GnRH-LH axis and reducing ovarian estrogen production [1]
- Clinical development: - Fezolinetant (30 mg/day) showed a 93% reduction in hot flash frequency in a phase IIa clinical trial (n=120 postmenopausal women) [15]
- The compound was approved by the FDA in 2023 for the treatment of moderate to severe vasomotor symptoms associated with menopause [6]
- Advantages over hormone therapy: - The non-estrogenous mechanism avoids the risks of breast cancer and thromboembolism [12]
- Oral administration and once-daily dosing regimens improve patient outcomes. Compliance [15]

C16H15FN6OS

358.393304109573

358.101

C, 53.62; H, 4.22; F, 5.30; N, 23.45; O, 4.46; S, 8.95

1629229-37-3

1629229-37-3;

117604931

Light yellow to yellow solid powder

1.6±0.1 g/cm3

623.0±65.0 °C at 760 mmHg

330.5±34.3 °C

0.0±1.8 mmHg at 25°C

1.760

0.96

0

7

2

25

505

1

S1C(C2=NN=C3[C@@H](C)N(C(C4C=CC(=CC=4)F)=O)CCN32)=NC(C)=N1

PPSNFPASKFYPMN-SECBINFHSA-N

InChI=1S/C16H15FN6OS/c1-9-13-19-20-14(15-18-10(2)21-25-15)23(13)8-7-22(9)16(24)11-3-5-12(17)6-4-11/h3-6,9H,7-8H2,1-2H3/t9-/m1/s1

(R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone

ESN-364; ESN 364; ESN364

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 : ~20 mg/mL (~55.81 mM)

Solubility in Formulation 1: 2 mg/mL (5.58 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 20.0 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 mg/mL (5.58 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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.=

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Solubility in Formulation 3: 1 mg/mL (2.79 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: ≥ 0.2 mg/mL (0.56 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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