Neurokinin-1 receptor

Netupitant (CID-6451149) exhibits a high affinity for the human NK1 receptor (pKi=9.0), exhibiting selectivity over NK2 and NK3 (pKi=5.8 for both sites) by more than 1000 times[2].
Netupitant (1, 10, 100 nM) concentration-dependently antagonizes substance P's stimulatory effects, exhibiting irreversible antagonism (pKB=8.87) in CHO NK1 cells[2].

Netupitant (CID-6451149; 1–10 mg/kg; ip) inhibits mice's typical scratching, biting, and licking response in a dose-dependent manner. When NK1 agonists are injected intracerebroventricularly, gerbils will tap their feet. Netupitant, administered intraperitoneally (ID50 1.5 mg/kg) or orally (ID50 0.5 mg/kg), will counteract this behavior in a dose-dependent manner[2].
Netupitant (0.1-3 mg/kg; intraperitoneally) inhibits the responses to SP-methylester (SP-OMe) in a concentration-dependent manner (mean pKB=9.24) in the detrusor muscle. Netupitant reduces the frequency of the bladder's reflex contractions[3].

Receptor binding screening profile[2]
Netupitant was assessed in receptor binding experiments performed on membranes of CHO cells expressing the three human tachykinin receptors as well as 50 different GPCR, monoamine transporters and ion channels. This study was performed under contract through CEREP.

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Calcium mobilization studies in cells expressing human or rat tachykinin receptors[2]
Cells stably expressing NK receptors were a generous gift from the laboratories of Prof. T. Costa (ISS, Rome, IT, HEK293 cells stably expressing the rat NK1 receptor), Prof. C. Rojas (Johns Hopkins University School of Medicine, Baltimore, US, CHO cells stably expressing the human NK1 receptor), and Prof. T.W. Schwartz (CHO cells expressing the human NK2 or NK3 receptors). CHO cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and 200 mg/l G418. HEK293rNK1 cells were maintained in Eagle minimum essential medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and 100 mg/l hygromycin. The cells were cultured at 37 °C in 5% CO2 humidified air and seeded at a density of 50,000 cells/well into 96-well black, clear-bottom plates. The following day, the cells were incubated with medium supplemented with 2.5 mM probenecid, 3 μM of the calcium sensitive fluorescent dye Fluo-4 AM and 0.01% pluronic acid, for 30 min at 37 °C. After that time the loading solution was aspirated and 100 μl/well of assay buffer Hank's Balanced Salt Solution (HBSS) supplemented with 20 mM HEPES, 2.5 mM probenecid and 500 μM Brilliant Black was added.[2]
SP, NKA, NKB, [Sar9,Met(O2)11]SP, [βAla8]NKA(4–10) and [MePhe7]NKB 1 mM were dissolved in bidistilled water. NK receptor antagonists (Aprepitant, Netupitant, SR48968, GR159897, SR142801, and SB222200) 1 mM were dissolved in DMSO. Serial dilutions were carried out in HBSS/HEPES (20 mM) buffer (containing 0.02% BSA fraction V).[2]
After placing both plates (cell and compound plate) into the FlexStation II, fluorescence changes were measured at 37 °C. On-line additions were carried out in a volume of 50 μl/well. In antagonist type experiments the compounds under study were preincubated for 24 min before agonist addition. To facilitate drug diffusion into the wells in antagonist type experiments, three cycles of mixing (25 μl from each well moved up and down 3 times) were performed immediately after antagonist injection to the wells. Maximum changes in fluorescence, expressed in percent over baseline levels, were used to measure agonist responses.

Cells are pre-incubated in growth media (control) or media containing antagonists for one hour at 37°C. To guarantee receptor saturation, antagonist concentrations must be at least 30 times higher than the Kd value. In order to allow antagonists that are still attached to receptors to dissociate, antagonists are removed from the cells after preincubation and they are rinsed with growth media alone for an extra hour. Then, isosmotic HEPES buffer (pH 7.4, 20 mM) is added to the cell media. This buffer contains the following concentrations of SP and NaCl: 3 nM to 1 mM; KCl (2 mM), MgCl2 (1 mM), CaCl2 (2 mM), Fluo-4 acetoxymethyl (AM) ester (2 mM), pluronic acid (0.04%), and MgCl2 (1 mM). The last incubation took place at 37 °C for one hour. In order to sequester the AM ester molecules into micelles for cellular uptake, pluronic acid is added as a nonionic surfactant.

SP (0.01–1 nmol) was given intrathecally (i.t.). I.t. injections (5 μl per mouse) were given under light (just sufficient to produce a loss of the righting reflex) isofluorane anesthesia according to the procedure described by Hylden and routinely adopted in our laboratory. Approximately 45 min before i.t. injection, the mice were adapted to an individual plastic cage which served as the observation chamber. The animals were challenged with SP and individually observed for 10 or 15 min. The total time (s) spent by the animal displaying the following behaviors was measured: hindlimb scratching directed toward the flank; biting or licking of the fore and hind paw; and biting or licking of the tail. Netupitant and Aprepitant (1 and 10 mg/kg, i.p.) were administered 30 min before SP (0.1 nmol i.t.). All experiments were started at 9.00 am[2].

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Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA). Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of AA. After a 30 min stabilization period, netupitant (0.1-3 mg/kg, i.v.) or L-733,060 (3-10 mg/kg, i.v.) were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24) of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI) but had no effect on the amplitude of micturition (AM). L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM. Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the micturition reflex circuit and therefore may be useful clinically in treating bladder overactivity symptoms.[3]

Absorption, Distribution and Excretion
Following a single oral dose of netupitant, netupitant becomes detectable in plasma within 15 minutes to 3 hours. Plasma concentrations reach Cmax at approximately 5 hours. Systemic exposure increases dose-proportionately with increasing dose from 10 mg to 300 mg, and further dose-proportionately with increasing dose from 300 mg to 450 mg. It is primarily excreted in feces. In cancer patients, Vz/F: 1982 ± 906 L (mean ± standard deviation). Estimated systemic clearance is 20.3 ± 9.2 L/h (mean ± standard deviation). Metabolism/Metabolites Netupitant is extensively metabolized after absorption, producing three major metabolites: a demethylated derivative M1; an N-oxide derivative M2; and a hydroxymethyl derivative M3. Metabolism is primarily mediated by CYP3A4, with less involvement from CYP2C9 and CYP2D6. Metabolites M1, M2, and M3 have been shown to bind to the substance P/neurokine 1 (NK1) receptor.

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Biological half-life
96 hours, CV% 61%.

Protein Binding
When the drug concentration ranges from 10 to 1300 ng/mL, the protein binding rate is 99.5%.

[1]. Design and synthesis of a novel, achiral class of highly potent and selective, orally active neurokinin-1 receptor antagonists. Bioorg Med Chem Lett. 2006 Mar 1;16(5):1362-5.

[2]. In vitro and in vivo pharmacological characterization of the novel NK1 receptor selective antagonist Netupitant. Peptides. 2012 Sep;37(1):86-97.

[3]. Netupitant, a Potent and Highly Selective NK1 Receptor Antagonist, Alleviates Acetic Acid-Induced Bladder Overactivity in Anesthetized Guinea-Pigs. Front Pharmacol. 2016 Aug 4;7:234.

Netupitant is a monocarboxylic acid amide formed by the condensation of the carboxyl group of 2-[3,5-bis(trifluoromethyl)phenyl]-2-methylpropionic acid with the secondary amino group of N-methyl-4-(2-methylphenyl)-6-(4-methylpiperazin-1-yl)pyridine-3-amine; it is an antiemetic, often used in combination with palonosetron hydrochloride (brand name Akynzeo) to treat nausea and vomiting in patients undergoing cancer chemotherapy. It has both antiemetic and neurokinin-1 receptor antagonistic effects. It is a monocarboxylic acid amide, an organofluorine compound, an aminopyridine, a toluene derivative, an N-alkylpiperazine, and an N-arylpiperazine. Netupitant is an antiemetic approved by the U.S. Food and Drug Administration (FDA) in October 2014 for use in combination with palonosetron to prevent acute and delayed vomiting and nausea caused by cancer chemotherapy (including highly emetogenic chemotherapy). Netupitant is a neurokinin-1 receptor antagonist. This combination formulation is marketed by Eisai Inc. and Helsinn Therapeutics (US) Inc. under the trade name Akynzeo. Netupitan is a substance P/neurokine-1 receptor antagonist. Netupitan's mechanism of action is as a neurokinin 1 receptor antagonist, cytochrome P450 3A4 inhibitor, P-glycoprotein inhibitor, and breast cancer resistance protein inhibitor. Netupitan is a selective neurokinin 1 (NK1) receptor antagonist with potential antiemetic activity. Netupitan competitively binds to and blocks the activity of human substance P/NK1 receptors in the central nervous system (CNS), thereby inhibiting the binding of endogenous tachykinin neurokinin substance P (SP) to NK1 receptors, which may help prevent chemotherapy-induced nausea and vomiting (CINV). SP is present in neurons innervating vagal afferent fibers of the nucleus solitarius and post-traumatic zone (containing the chemoreceptor trigger zone (CTZ)) in the brainstem, and its levels may be elevated due to chemotherapy. NK receptors are G protein-coupled receptors coupled to the inositol phosphate signaling pathway and are located in the nucleus tractus solitarius and the postmedulatic region. Drug Indications Netupitan is an antiemetic approved by the FDA in October 2014 for use in combination with palonosetron to prevent acute and delayed vomiting and nausea induced by cancer chemotherapy (including highly emetogenic chemotherapy). FDA Label Mechanism of Action Delayed vomiting is primarily associated with substance P activation of neurokinin 1 (NK1) receptors (widely distributed in the central and peripheral nervous systems). In vitro and in vivo studies have shown that netupitan inhibits substance P-mediated responses.

C30H32F6N4O

578.59

578.248

C, 62.28; H, 5.57; F, 19.70; N, 9.68; O, 2.77

290297-26-6

Netupitant-d6; 2070015-31-3; Netupitant metabolite N-desmethyl Netupitant; 290296-72-9; N-desmethyl Netupitant-d6; Netupitant metabolite Netupitant N-oxide; 910808-11-6; Netupitant N-oxide-d6; Netupitant metabolite Monohydroxy Netupitant; 910808-12-7; Monohydroxy Netupitant-d6; 290296-54-7 (2HCl); 290297-26-6

6451149

White solid powder

1.3±0.1 g/cm3

597.4±50.0 °C at 760 mmHg

315.1±30.1 °C

0.0±1.7 mmHg at 25°C

1.540

6.39

0

10

5

41

865

0

FC(C1C([H])=C(C(F)(F)F)C([H])=C(C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C(N(C([H])([H])[H])C1=C([H])N=C(C([H])=C1C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])=O)(F)F

WAXQNWCZJDTGBU-UHFFFAOYSA-N

InChI=1S/C30H32F6N4O/c1-19-8-6-7-9-23(19)24-17-26(40-12-10-38(4)11-13-40)37-18-25(24)39(5)27(41)28(2,3)20-14-21(29(31,32)33)16-22(15-20)30(34,35)36/h6-9,14-18H,10-13H2,1-5H3

2-[3,5-bis(trifluoromethyl)phenyl]-N,2-dimethyl-N-[4-(2-methylphenyl)-6-(4-methylpiperazin-1-yl)pyridin-3-yl]propanamide

AGE-94200; Ro67-3189; AGE 94200; Ro 67-3189/000; AGE94200; Ro 67-3189; Ro-67-3189; 2-[3,5-bis(trifluoromethyl)phenyl]-N,2-dimethyl-N-[4-(2-methylphenyl)-6-(4-methylpiperazin-1-yl)pyridin-3-yl]propanamide; 2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamide; Ro-67-3189; Ro-673189000; Ro 67-3189/000; CHEMBL206253; Netupitant

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: 2~9.1 mg/mL (3.5~15.71 mM)
Ethanol: ~100 mg/mL

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.)