Neurokinin-3 receptor (NK3R)

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].

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].

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].

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.

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

Absorption, Distribution and Excretion
In healthy women, fezolinetant Cmax and AUC increased proportionally over a dosage range from 20 to 60 mg once daily (0.44 to 1.33 times the approved recommended dosage). Steady-state plasma concentrations of fezolinetant were reached after two once-daily doses, with minimal fezolinetant accumulation. The median (range) time to reach fezolinetant Cmax is 1.5 (1 to 4) hours in healthy women.
Following oral administration of fezolinetant, 76.9% of the dose was excreted in urine (1.1% unchanged) and 14.7% in feces (0.1% unchanged).
The mean apparent volume of distribution (Vz/F) of fezolinetant is 189 L.
The apparent clearance at steady state of fezolinetant is 10.8 L/h.

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Metabolism / Metabolites
Fezolinetant is primarily metabolized by CYP1A2 and to a lesser extent by CYP2C9 and CYP2C19. A major metabolite of fezolinetant, ES259564, was identified in plasma. ES259564 is approximately 20-fold less potent than the parent. The metabolite-to-parent ratio ranges from 0.7 to 1.8.

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Biological Half-Life
The effective half-life (t_1/2) of fezolinetant is 9.6 hours in women with vasomotor symptoms.

Protein Binding
The plasma protein binding of fezolinetant is 51%. The blood-to-plasma ratio is 0.9.

[1]. Fraser GL, et al. 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]. J. Med. Chem. 2015, 58, 7, 3060–3082

Fezolinetant is a triazolopyrazine that is 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine substituted by 3-methyl-1,2,4-thiadiazol-5-yl, 4-fluorobenzoyl and methyl groups at positions 3, 7, and 8R, respectively. It is a prescription medicine used to reduce moderate to severe vasomotor symptoms due to menopause. It has a role as a neurokinin-3 receptor antagonist. It is a member of monofluorobenzenes, a triazolopyrazine, a member of thiadiazoles, a member of benzamides and a tertiary carboxamide.
Vasomotor symptoms (VMS), more colloquially known as hot flashes or night sweats, are some of the most common symptoms in menopause. With a median duration of 7.4 years, vasomotor symptoms are also the most common reasons why women seek treatments for menopausal issues. Although mostly considered a nuisance, vasomotor symptoms can significantly affect quality of life, as women with 7 or more moderate to severe VMS daily have reported a decline in sleep quality, concentration, sexual activity, energy, and concentration. Although the pathophysiology of VMS is not fully understood, unpredictable fluctuation in estrogen levels is thought to be the main cause of VMS, as estrogen therapy has been one of the most effective treatments for VMS by relieving symptoms in as many as 95% of menopausal women. Women undergoing abrupt menopause due to oophorectomy also experienced more severe symptoms than those going through a gradual transition. Additionally, thermoregulatory dysfunction has been proposed as one of the three possible mechanisms for VMS in menopause. Estrogen is a potent neuromodulator, particularly in the hypothalamus, and has been shown to be involved as a negative regulator in generating Gonadotropin-releasing hormone (GnRH) pulse through the kisspeptin, neurokinin B, and dynorphin (KNDy) neurons. NK3, one of the receptors expressed in KNDy neurons, is activated by neurokinin B and can thus induce the release of GnRH. Lower estrogen levels during menopause will decrease the estrogen-mediated feedback loop and increase neurokinin B signalling, increasing the activity of KNDy neurons and therefore the activity of the temperature control center. By antagonizing NK3 receptors, neuronal signalling can be dampened to reduce VMS. Although hormone therapy is available for menopausal women, safety and tolerability concerns, such as an increased risk of stroke and venous thromboembolism or hormone-dependent cancer like breast cancer, can prevent some women from receiving this treatment. Fezolinetant, an NK3 receptor antagonist, was developed in response to this issue as well as more understanding of the role of NK3R in the hypothalamic-pituitary-gonadal (HPG) axis. Although previous NK3 receptor antagonists exist, such as osanetant and talnetant, only fezolinetant showed tangible effects on the HPC axis, potentially due to its favorable pharmacokinetics profile to cross the blood-brain barrier. Fezolinetant was approved by the FDA in May 2023 under the brand name Veozah. It was subsequently approved by the EMA in December 2023 for the same indication.
Fezolinetant is a Neurokinin 3 Receptor Antagonist. The mechanism of action of fezolinetant is as a Neurokinin 3 Receptor Antagonist.

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Drug Indication
Fezolinetant is indicated for the treatment of moderate to severe vasomotor symptoms due to menopause.

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Mechanism of Action
Fezolinetant is a neurokinin 3 (NK3) receptor antagonist that blocks neurokinin B (NKB) binding on the kisspeptin/neurokinin B/dynorphin (KNDy) neuron to modulate neuronal activity in the thermoregulatory center.

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Pharmacodynamics
Fezolinetant has a high affinity for the NK3 receptor (K_i value of 19.9 to 22.1 nmol/L), which is more than 450-fold higher than the binding affinity for NK1 or NK2 receptors. Treatment with fezolinetant did not show any clear trends in sex hormones measured (follicle-stimulating hormone, testosterone, estrogen, and dehydroepiandrosterone sulfate) in menopausal women. A transient decrease of luteinizing hormone (LH) levels was observed at peak concentrations of fezolinetant. At a dose 20 times the maximum approved recommended dose, fezolinetant does not prolong the QT interval to any clinically relevant extent. In a phase 2a clinical trial, fezolinetant 90 mg BID significantly reduce the frequency and severity of vasomotor symptoms in postmenopausal women by more than 50%. The improvement was observed as early as in the first week of treatment and was maintained throughout the 12 weeks of treatment.

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

929016-98-8 (HCl);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.)