Motilin Receptor ( pEC50 = 7.9 )

GSK962040 盐酸盐，也称为盐酸camecicinal，对生长素释放肽和其他受体、离子通道和酶的影响可忽略不计。当给予Camicinal盐酸盐（GSK962040盐酸盐）300 nmol L 1-10 μmol L 1时，兔胃窦胆碱能介导的收缩幅度延长，并在3 μmol L 1时达到最大值248±47%。在样品中发现：胃动素、红霉素和盐酸Camicinal（GSK962040盐酸盐）的值为10.4±0.01（n=770）、7.3±0.29（n=4）和7.9±0.09（n=17）[1]。狗胃动素受体激活是由Camicinal盐酸盐（GSK962040盐酸盐）引起的（pEC50 5.79；相对于[Nle13]-胃动素的内在活性0.72）[2]。由于其对 CYP3A4 的初始 IC50 值远大于我们推荐的 10 μM 阈值 [3]，因此优选盐酸Camicinal 盐酸盐（GSK962040 盐酸盐）。

Camicinal (GSK962040) (5 mg 游离碱·kg 1) 给药两小时后粪便总重量也增加 (21.2 ± 4.5 g; P < 0.05)。 [1]。 Camicinal (GSK962040) 导致平均血浆浓度>1.14 μmol L 1 ，从而引起剂量相关的阶段性收缩，分别持续 48 和 173 分钟，剂量为 3 mg kg 1 和 6 mg kg 1 。当 Camicinal (GSK962040) 的作用减弱时，迁移运动复合体 (MMC) 活动会恢复。 3 mg kg 1 Camicinal (GSK962040)对运动复合体的迁移恢复没有影响；然而，在 6 mg kg 1 剂量下，MMC 在分娩后 253 分钟恢复，而生理盐水后 101 分钟恢复（各 n = 5）。[2]。 Camicinal (GSK962040) 的口服生物利用度 (Fpo) 为 48 (13%)。与短期效应相比，camicinal (GSK962040) 表现出长期效应 (T1/2) 46.9（3 μM 时 5.0 分钟）[Nle13]。胃动素 (T1/2) 11.4（0.3 μM 时）持续 1.5 分钟[ 3]。化合物camicinal (GSK962040) 在胃底和小肠中活性较低，但显着增加胃窦中的胆碱能活性[4]。

胃动素受体激动剂FLIPR测定[3]
使用荧光成像板阅读器（FLIPR）和稳定表达人胃动素受体的中国仓鼠卵巢（CHO-K1）细胞研究了靶化合物对人胃动蛋白受体的效力和功效。简而言之，将人胃动素受体克隆（从人基因组DNA进行PCR）到载体中的pCDNA3.1中，亚克隆到pENTR/D-TOPO中（使用pENTR-D-TOPO定向克隆试剂盒），然后重组到pCIN1GW载体（LR克隆酶网关反应试剂盒）中，得到pCIN1胃动素接收器。然后用pCIN1胃动素受体质粒稳定转染CHO-K1细胞（ATCC编号CCl-61）。这些细胞在添加了10%v/v FBS、2 mM GlutaMAX和1 mg/mL Geneticin的DMEM/HamF-12中作为单层生长。用胃动素刺激该细胞系会引起细胞内信号传导，导致细胞内钙增加，这是使用钙敏感荧光染料测量的，并使用FLIPR定量。简而言之，将细胞接种到384孔黑壁、透明底部微量滴定板上（10000个细胞/孔）并孵育24小时。在测定当天，使用细胞洗涤器从细胞板中吸出培养基（留下10μL培养基）。立即用加载缓冲液（Tyrodes（Elga水+145 mM NaCl+5 mM KCl+20 mM HEPES+10 mM葡萄糖+1 mM MgCl2）+1.5 mM CaCl2+0.714 mg/mL Probenicid（预溶于1 M NaOH）+0.5 mM亮黑色+2.5μM Fluo 4染料）加载细胞，并在37.5°C下孵育1小时。在100%DMSO中制备主复合板。使用3mM的最高浓度（在测定中得到12μM的最终浓度），并将其连续稀释1比4。然后将1μL的母板转移到子板上，向其中加入50μL的化合物稀释缓冲液（Tyrodes+1 mg/mL BSA+1.5 mM CaCl2）。然后使用FLIPR 3钙成像仪将化合物板中的10μL立即加入细胞板中，并在1分钟内测量荧光变化。荧光相对于基线的最大变化用于确定激动剂反应，并使用四参数逻辑斯谛方程构建浓度反应曲线。目标化合物的内在活性是通过使用其浓度-反应曲线的最大渐近线相对于胃动素浓度-反应关系曲线的最大渐近线来计算的。

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CYP 3A4时间依赖性抑制（TDI）测定[3]
通过量化CYP3A4特异性荧光探针底物二乙氧基荧光素（DEF）与大肠杆菌中异源表达的CYP3A4（Cypex）和受试化合物或阳性对照（曲拉度霉素）孵育后荧光代谢物的产生来确定抑制作用。NADPH再生系统（辅因子）的制备如下：在2%w/v碳酸氢钠溶液中制备7.8mg/mL葡萄糖6-磷酸（27.65mM）、1.7mg/mL NADP（2.22mM）和6个酶单位/mL的葡萄糖6-磷酸脱氢酶。制备含有酶、探针底物和50 mM磷酸钾缓冲液（pH 7.4）的孵育混合物，并向96孔板的每个孔中加入220μL。加入5μL连续稀释的试验化合物，并在37°C下孵育10分钟。为了开始反应，加入25μL辅因子。然后在37°C下，在30分钟的时间范围内每分钟测量一次荧光的产生。表5给出了测定条件的总结。

迫切需要一种安全、有效的胃排空刺激剂；目前的治疗方法包括红霉素（一种具有额外特性的抗生素，可以防止长期使用）和胃复安（一种与运动障碍相关的5-羟色胺4型受体激动剂和脑D2受体拮抗剂）。为了摆脱红霉素复杂的胃动素结构，鉴定并表征了一种小分子胃动素受体激动剂GSK962040。使用重组人受体、已知对胃动素有反应的兔和人分离胃制剂以及体内，通过测量其增加清醒兔排便的能力，对该化合物进行了评估。在人胃动素受体上，Camicinal (GSK962040)和红霉素作为激动剂的pEC50（EC50值的负对数，以10为底，产生50%最大反应的激动剂浓度）值分别为7.9和7.3；GSK962040在一系列其他受体（包括胃饥饿素）、离子通道和酶上没有显著活性。在兔胃窦中，GSK962040 300 nmol L（-1）-10微摩尔L（-1”）导致胆碱能介导的收缩幅度延长，在3微摩尔L时最大可达248+/-47%。在人类离体胃中，GSK962040 10微摩尔L（-1）、红霉素10微摩尔L（-1）和[Nle13]-胃动素100 nmol L（-1”）均引起类似幅度的肌肉收缩。在清醒的兔子中，静脉注射5 mg kg（-1）GSK962040或10 mg kg（-1）红霉素在2小时内显著增加了粪便排出量。这些数据共同表明，GSK962040是一种非胃动素结构，选择性地激活胃动素受体。简化激活该受体的结构要求极大地促进了胃轻瘫潜在新药的设计[1]。

Rat Pharmacokinetic Studies[3]
The pharmacokinetics and oral bioavailability of the HCl salt of compound 12/Camicinal (GSK962040) were investigated in the male Sprague−Dawley rat (n = 3). The study was carried out on 2 study days with a period of 2 days between each study day. On study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a target concentration of 0.2 mg of free base/mL. Compound 12 was administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three rats to achieve a target dose of 1 mg of free base/kg. Serial blood samples were taken from each rat up to 12 h after the start of the infusion. On study day 2, compound 12 was suspended in 1% (w/v) methylcellulose at a target concentration of 1 mg of free base/mL. Three rats received an oral gavage dose of compound 12 administered at 5 mL/kg to achieve a target dose of 5 mg of free base/kg. Serial blood samples were taken from each rat up to 12 h after dosing. Diluted blood samples were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 μM).
The systemic exposure of the HCl salt of compound 16 following oral suspension administration was investigated the male Sprague−Dawley rat. Compound 16 was dosed to three rats orally by gavage at a target dose of 5 mg of free base/kg. Compound 16 was prepared on the day of dosing in 1% (w/v) aqueous methylcellulose at a concentration of 1 mg of free base/mL and administered at 5 mL/kg. Serial blood samples were taken from each rat up to 8 h after dose administration. Diluted blood samples were analyzed for parent compound by LC/MS/MS.

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Dog Pharmacokinetic Studies[3]
The pharmacokinetics and oral bioavailability of the HCl salt of compound 12/Camicinal (GSK962040) were investigated in the male beagle dog (n = 3). The study was carried out on 2 study days with a period of 7 days between each study day. On study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a concentration of 0.4 mg of free base/mL. Compound 12 was administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three dogs to achieve a target dose of 2 mg of free base/kg. Serial blood samples were taken from each dog up to 30 h after the start of the infusion. On study day 2, compound 12 was suspended in 1% (w/v) methylcellulose at a concentration of 1 mg of free base/mL. The same three dogs each received an oral gavage dose of compound 12 administered at 5 mL/kg to achieve a target dose of 5 mg of free base/kg. Serial blood samples were taken from each dog up to 30 h after dosing. Diluted blood samples were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 μM).

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Background: GSK962040, a small molecule motilin receptor agonist, was identified to address the need for a safe, efficacious gastric prokinetic agent. However, as laboratory rodents lack a functional motilin system, studies in vivo have been limited to a single dose, which increased defecation in rabbits. Motilin agonists do not usually increase human colonic motility, so gastric prokinetic activity needs to be demonstrated.
Methods: The effect of intravenous GSK962040 on gastro-duodenal motility was assessed in fasted dogs implanted with strain gauges. Activity was correlated with blood plasma concentrations of GSK962040 (measured by HPLC-MS/MS) and potency of GSK962040 at the dog recombinant receptor [using a Fluorometric Imaging Plate Reader (Molecular Devices, Wokingham, UK) after expression in HEK293 cells].
Key results: GSK962040 activated the dog motilin receptor (pEC(50) 5.79; intrinsic activity 0.72, compared with [Nle(13) ]-motilin). In vivo, GSK962040 induced phasic contractions, the duration of which was dose-related (48 and 173 min for 3 and 6 mg kg(-1) ), driven by mean plasma concentrations >1.14 μmol L(-1) . After the effects of GSK962040 faded, migrating motor complex (MMC) activity returned. Migrating motor complex restoration was unaffected by 3 mg kg(-1) GSK962040 but at 6 mg kg(-1) , MMCs returned 253 min after dosing, compared with 101 min after saline (n=5 each).
Conclusions & inferences: The results are consistent with lower potency for agonists at the dog motilin receptor, compared with humans. They also define the doses of GSK962040 which stimulate gastric motility. Correlation of in vivo and in vitro data in the same species, together with plasma concentrations, guides further studies and translation to other species.[2]

It was found that the presence of the more polar cyano group was detrimental to the oral pharmacokinetics of compound 16. In the male Sprague−Dawley rat, 16 showed low and variable oral exposure whereas the fluoro analogue 12 gave higher and more consistent levels. Following determination of its intravenous pharmacokinetics, the oral bioavailability (Fpo) of 12 was found to be 48 ± 13%. This highly promising result led us to determine the pharmacokinetic profile of 12 in the male beagle dog, and we were pleased to find that its oral bioavailability was 51 ± 16%.[1]
Compound 12 has also been assessed for its inhibition of the other major human CYP isoforms, and it was found to possess a favorable profile (1A2, 2C19, 2C9 IC50 > 100 μM, 2D6 IC50 = 34 μM). Furthermore, there were no TDI liabilities at any of these isoforms or at CYP3A4 with 7BQ as the substrate. Selectivity at the closely related human ghrelin acceptor was high (pEC50 < 6.0), and there were no liabilities at the hERG channel (binding assay pIC50 = 4.8). In vitro plasma protein binding levels were acceptable (human 83%, rat 63%), and solubility in water and a range of simulated gastrointestinal fluids was high (HCl salt, >1 mg/mL).[1]
Additionally, the duration of action of 12 in the rabbit gastric antrum native tissue assay has been determined. This will be reported in full elsewhere, but in summary, 12 shows a long lasting effect (T1/2 = 46.9 ± 5.0 min at 3 μM) when compared with the short-lived effect of [Nle13]motilin (T1/2 = 11.4 ± 1.5 min at 0.3 μM). Its duration of action is also longer than that of erythromycin 1 (T1/2 = 24.0 ± 5.6 min at 3 μM), which is used successfully in the clinic to improve gastric emptying when dosed repeatedly at a low level. Therefore, these data may indicate a low potential for 12 to cause tachyphylaxis when dosed appropriately in vivo. General selectivity, efficacy in human native stomach tissue, and prokinetic activity in a rabbit model of whole gut transit have also been determined for 12, and these data will also be reported in full elsewhere. [1]

[1]. GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility. Neurogastroenterol Motil, 2009. 21(6): p. 657-64, e30-1.

[2]. GSK962040: a small molecule motilin receptor agonist which increases gastrointestinal motility in conscious dogs. Neurogastroenterol Motil, 2011. 23(10): p. 958-e410.

[3]. Discovery of N-(3-fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-pi peridinamine (GSK962040), the first small molecule motilin receptor agonist clinical candidate. J Med Chem, 2009. 52(4): p. 1180-9.

[4]. Regional- and agonist-dependent facilitation of human neurogastrointestinal functions by motilin receptor agonists. Br J Pharmacol, 2012. 167(4): p. 763-74.

Camicinal is a member of acetamides. Camicinal has been used in trials studying the treatment of Gastroparesis.

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N-(3-fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-piperidinamine 12 (GSK962040) is a novel small molecule motilin receptor agonist. It possesses excellent activity at the recombinant human motilin receptor and also at the native rabbit motilin receptor where its agonist activity results in potentiation of the amplitude of neuronal-mediated contractions of isolated gastric antrum tissue. Compound 12 also possesses highly promising pharmacokinetic profiles in both rat and dog, and these results, in combination with further profiling in human native tissue and an in vivo model of gastrointestinal transit in the rabbit, have led to its selection as a candidate for further development.[1]

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In summary, compound 12/In summary, compound 12 demonstrated a highly favorable overall profile and has been selected as a candidate for further development. Thus, compound 12 represents a novel and selective small molecule motilin receptor agonist (molecular weight, 424) developed via an extensive SAR program based on activity at the recombinant human motilin receptor and is the first such compound to progress to phase I clinical trials for potential use as a therapeutic agent for conditions associated with delayed gastric emptying. demonstrated a highly favorable overall profile and has been selected as a candidate for further development. Thus, compound 12 represents a novel and selective small molecule motilin receptor agonist (molecular weight, 424) developed via an extensive SAR program based on activity at the recombinant human motilin receptor and is the first such compound to progress to phase I clinical trials for potential use as a therapeutic agent for conditions associated with delayed gastric emptying.[1]

C25H33N4OF.HCL

461.01506

460.24

C, 65.13; H, 7.43; Cl, 7.69; F, 4.12; N, 12.15; O, 3.47

923565-22-4

Camicinal;923565-21-3

25222897

Typically exists as solid at room temperature

3.542

47.61

3

5

6

32

560

1

O=C(N1CCC(NC2=CC=CC(F)=C2)CC1)CC3=CC=C(CN4C[C@H](C)NCC4)C=C3.Cl

MZEVMNJFEUATKJ-FYZYNONXSA-N

InChI=1S/C25H33FN4O.ClH/c1-19-17-29(14-11-27-19)18-21-7-5-20(6-8-21)15-25(31)30-12-9-23(10-13-30)28-24-4-2-3-22(26)16-24;/h2-8,16,19,23,27-28H,9-15,17-18H2,1H3;1H/t19-;/m0./s1

1-[4-(3-fluoroanilino)piperidin-1-yl]-2-[4-[[(3S)-3-methylpiperazin-1-yl]methyl]phenyl]ethanone;hydrochloride

Camicinal hydrochloride; 923565-22-4; GSK962040 (HCl salt); GSK962040 (hydrochloride); GSK962040 hydrochloride; 1-[4-(3-fluoroanilino)piperidin-1-yl]-2-[4-[[(3S)-3-methylpiperazin-1-yl]methyl]phenyl]ethanone;hydrochloride; CHEMBL489679;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~216.91 mM)
DMSO : ~100 mg/mL (~216.91 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.42 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.42 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.42 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 100 mg/mL (216.91 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。