Neurokinin-1 receptor

Netupitant (CID-6451149) 以高亲和力结合人类 NK1 受体 (pKi=9.0)，选择性比 NK2 和 NK3 受体高 1000 倍以上（两个位点的 pKi=5.8）[2]。 Netupitant (1, 10, 100 nM) 浓度依赖性地拮抗 P 物质 (SP) 的刺激作用，在 CHO NK1 细胞中表现出难以克服的拮抗作用 (pKB=8.87)[2]。

Netupitant（CID-6451149；1-10 mg/kg；腹膜内注射）剂量依赖性地抑制 SP 引发的小鼠典型的抓、咬和舔反应。在沙鼠中，脑室内注射 NK1 激动剂引起的足部敲击行为会被腹腔注射 (ID50 1.5mg/kg) 或口服 (ID50 0.5mg/kg) 的 Netupitant 剂量依赖性地抵消[2]。 Netupitant (0.1-3 mg/kg; iv) 对逼尿肌中 SP-甲酯 (SP-OMe) 的反应产生浓度依赖性抑制（平均 pKB=9.24）。 Netupitant 可降低反射性膀胱收缩的频率[3]。

受体结合筛查概况/Receptor binding screening profile[2]
在表达三种人速激肽受体以及50种不同GPCR、单胺转运蛋白和离子通道的CHO细胞膜上进行的受体结合实验中评估了Netupitant。本研究通过CEREP根据合同进行。

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表达人或大鼠速激肽受体的细胞中的钙动员研究[2]
稳定表达NK受体的细胞是T.Costa教授（ISS，Rome，IT，稳定表达大鼠NK1受体的HEK293细胞）、C.Rojas教授（Johns Hopkins University School of Medicine，Baltimore，US，稳定表达人NK1受体）和T.W.Schwartz教授（CHO细胞表达人NK2或NK3受体）实验室的慷慨礼物。CHO细胞在RPMI 1640培养基中维持，该培养基补充了10%胎牛血清、2 mM l-谷氨酰胺、100 U/ml青霉素和100μg/ml链霉素以及200 mg/l G418。HEK293rNK1细胞在补充有10%胎牛血清、2 mM l-谷氨酰胺、100 U/ml青霉素和100μg/ml链霉素和100 mg/l潮霉素的Eagle最低必需培养基中维持。细胞在37°C的5%CO2加湿空气中培养，以50000个细胞/孔的密度接种到96孔的黑色透明底板中。第二天，将细胞与补充有2.5 mM丙磺舒、3μM钙敏感荧光染料Fluo-4 AM和0.01%普朗尼克酸的培养基在37°C下孵育30分钟。随后，吸取加载溶液，并加入100μl/孔的Hank's平衡盐溶液（HBSS），该溶液补充了20 mM HEPES、2.5 mM丙磺舒和500μM亮黑。[2]
SP、NKA、NKB、[Sar9、Met（O2）11]SP、[βAla8]NKA（4-10）和[MePhe7]NKB 1 mM溶解在双蒸馏水中。将1 mM的NK受体拮抗剂（阿普替坦、奈替坦、SR48968、GR159897、SR142801和SB222200）溶解在DMSO中。在HBSS/HEPES（20mM）缓冲液（含0.02%BSA组分V）中进行连续稀释。[2]
将两个板（细胞板和复合板）放入FlexStation II后，在37°C下测量荧光变化。在线添加的体积为50μl/孔。在拮抗剂型实验中，在加入激动剂之前，将所研究的化合物预孵育24分钟。为了促进拮抗剂型实验中药物扩散到孔中，在向孔中注射拮抗剂后立即进行了三个混合循环（每个孔25μl上下移动3次）。荧光的最大变化（以超过基线水平的百分比表示）用于测量激动剂反应。

将细胞在生长培养基（对照）或含有拮抗剂的培养基中于 37°C 预孵育一小时。为了保证受体饱和，拮抗剂浓度必须至少比 Kd 值高 30 倍。为了使仍然附着在受体上的拮抗剂解离，在预孵育后从细胞中除去拮抗剂，并仅用生长培养基冲洗细胞额外一小时。然后，将等渗 HEPES 缓冲液（pH 7.4，20 mM）添加到细胞培养基中。该缓冲液含有以下浓度的 SP 和 NaCl：3 nM 至 1 mM； KCl (2 mM)、MgCl2 (1 mM)、CaCl2 (2 mM)、Fluo-4 乙酰氧基甲基 (AM) 酯 (2 mM)、pluronic 酸 (0.04%) 和 MgCl2 (1 mM)。最后一次孵育在 37°C 下进行一小时。为了将 AM 酯分子隔离到胶束中以供细胞摄取，添加普朗尼克酸作为非离子表面活性剂。

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
Upon oral administration of a single dose of netupitant, netupitant started to be measurable in plasma between 15 minutes and 3 hours after dosing. Plasma concentrations reached Cmax in approximately 5 hours. There was a greater than dose-proportional increase in the systemic exposure with the dose increase from 10 mg to 300 mg and a dose-proportional increase in systemic exposure with a dose increase from 300 mg to 450 mg.
Primarily fecal.
In cancer patients, Vz/F: 1982 ± 906 L (mean ± SD).
Estimated systemic clearance of 20.3 ± 9.2 L/h (mean ± SD).

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Metabolism / Metabolites
Once absorbed, netupitant is extensively metabolized to form three major metabolites: desmethyl derivative, M1; N-oxide derivative, M2; and OH-methyl derivative, M3. Metabolism is mediated primarily by CYP3A4 and to a lesser extent by CYP2C9 and CYP2D6. Metabolites M1, M2 and M3 were shown to bind to the substance P/neurokinin 1 (NK1) receptor.

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Biological Half-Life
96 hours with CV% of 61.

Protein Binding
> 99.5% at drug concentrations ranging from 10-1300 ng/mL.

[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 obtained by formal condensation of the carboxy group of 2-[3,5-bis(trifluoromethyl)phenyl]-2-methylpropanoic acid with the secondary amino group of N-methyl-4-(2-methylphenyl)-6-(4-methylpiperazin-1-yl)pyridin-3-amine; an antiemetic used in combination with palonosetron hydrochloride (under the trade name Akynzeo) to treat nausea and vomiting in patients undergoing cancer chemotherapy. It has a role as an antiemetic and a neurokinin-1 receptor antagonist. It is a monocarboxylic acid amide, an organofluorine compound, an aminopyridine, a member of toluenes, a N-alkylpiperazine and a N-arylpiperazine.
Netupitant is an antiemitic drug approved by the FDA in October 2014 for use in combination with palonosetron for the prevention of acute and delayed vomiting and nausea associated with cancer chemotherapy including highly emetogenic chemotherapy. Netupitant is a neurokinin 1 receptor antagonist. The combination drug is marketed by Eisai Inc. and Helsinn Therapeutics (U.S.) Inc. under the brand Akynzeo.
Netupitant is a Substance P/Neurokinin-1 Receptor Antagonist. The mechanism of action of netupitant is as a Neurokinin 1 Antagonist, and Cytochrome P450 3A4 Inhibitor, and P-Glycoprotein Inhibitor, and Breast Cancer Resistance Protein Inhibitor.
Netupitant is a selective neurokinin 1 (NK1) receptor antagonist with potential antiemetic activity. Netupitant competitively binds to and blocks the activity of the human substance P/NK1 receptors in the central nervous system (CNS), thereby inhibiting NK1-receptor binding of the endogenous tachykinin neuropeptide substance P (SP), which may result in the prevention of chemotherapy-induced nausea and vomiting (CINV). SP is found in neurons of vagal afferent fibers innervating the brain-stem nucleus tractus solitarii and the area postrema, which contains the chemoreceptor trigger zone (CTZ), and may be elevated in response to chemotherapy. The NK-receptor is a G-protein receptor coupled to the inositol phosphate signal-transduction pathway and is found in both the nucleus tractus solitarii and the area postrema.

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Drug Indication
Netupitant is an antiemitic drug approved by the FDA in October 2014 for use in combination with palonosetron for the prevention of acute and delayed vomiting and nausea associated with cancer chemotherapy including highly emetogenic chemotherapy.
FDA Label

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Mechanism of Action
Delayed emesis (vomiting) has been largely associated with the activation of tachykinin family neurokinin 1 (NK1) receptors (broadly distributed in the central and peripheral nervous systems) by substance P. As shown in in vitro and in vivo studies, netupitant 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

39.68

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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