kappa opioid receptor

HSK21542是一种外周限制性Kappa阿片受体激动剂[2]
为了揭示HSK21542在KOR中的药理学特征，我们进行了[3H]二丙诺啡结合实验，研究HSK21542对[3H]二丙诺啡竞争结合的抑制作用，并确定未标记的HSK21542的结合动力学。阳性对照U69593明显阻止[3H]二丙诺啡与KOR结合，IC50值为14.72 nM （95% CI: 9.08 ~ 22.38 nM）。正如预期的那样，HSK21542显著抑制[3H]二丙诺啡与KOR的结合，IC50值为0.54 nM (95% CI: 0.38-0.75 nM)，而CR845的IC50值为1.16 nM （95% CI: 0.85-1.57 nM，图2A）。结合动力学研究结果显示，HSK21542和CR845与KOR结合的K d值分别为0.068 nM （95% CI: 0.028-0.092 nM）和0.23 nM (95% CI: 0.17-0.26 nM)（图2B，C）。HSK21542的t1 /2值为90.6 min (95% CI: 53.6 ~ 292.7 min)，比CR845的t1 /2值（42.0 min, 95% CI: 28.6 ~ 79.4 min）长。另一方面，HSK21542在稳定表达人κ阿片受体的HEK-293细胞中显著抑制福斯克林诱导的cAMP积累，EC50值为2.41 pM (95% CI: 1.43-4.67 pM)，分别比CR845和U69593低12.4倍和747倍（图2D）。

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为了研究HSK21542的特异性，我们在体外观察了HSK21542对多种受体、离子通道、转运体和酶的作用，包括MOR和DOR。在10 μM浓度下，HSK21542与大麻素CB1受体的结合抑制率为47%，其余85个靶点无明显活性（补充表S1）。

由于Kappa阿片受体（KOR）激动剂具有缓解疼痛和治疗顽固性瘙痒的潜力，因此一直是有希望的治疗候选者。虽然缺乏吗啡样中枢神经系统（CNS）的作用，但KOR激动剂确实引起镇静、烦躁和利尿，严重阻碍了它们的发展。外周限制性KOR激动剂渗透到CNS系统的能力较差，因此可以改善甚至消除CNS相关的不良反应。然而，唯一批准的外周限制性KOR激动剂CR845仍然存在一些常见的中枢神经系统不良事件。在本研究中，我们旨在研究HSK21542的药理学特征，期望为患有疼痛和瘙痒的患者提供一种安全有效的替代药物。体外实验结果表明，HSK21542是一种选择性强效的KOR激动剂，其效价高于CR845，脑/血浆浓度比为0.001，表明其具有外周选择性。在疼痛动物模型中，HSK21542显著抑制醋酸、后爪切开或慢性收缩损伤引起的疼痛相关行为，给药后15分钟的疗效与CR845相当。HSK21542具有持久的镇痛作用，药后24 h扭体试验中位有效剂量为1.48 mg/kg。同时，HSK21542的抗痛觉活性被一种KOR拮抗剂非双萘哌胺有效逆转。此外，HSK21542在化合物48/80致痒模型中具有较强的止痒活性。另一方面，HSK21542在热板实验中产生中枢抗伤害感受作用的能力较弱，对小鼠运动活性的影响较小。HSK21542对小鼠呼吸速率无影响。因此，HSK21542可能是一种安全有效的KOR激动剂，是治疗疼痛和瘙痒的有希望的候选药物。[2]

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HSK21542能产生有效的抗伤害感受作用。HSK21542具有显著的抗变动力作用。HSK21542减轻化合物48/80诱导的瘙痒。 HSK21542对中枢神经系统的副作用较少。

体外SafetyScreen Panel：使用SafetyScreen Panel （target selectivity Panel）评估Anrikefon (HSK21542)在86个靶点上的体外脱靶药理活性，相应的方法可在https://www.eurofinsdiscoveryservices.com/上找到。[2]

[3H]二丙诺啡结合实验：HEK-293细胞（ATCC）保存在含有10% FBS的Eagle’s Minimum Essential Medium中，在含5% CO2的湿空气中37°C孵育。我们在实验室建立了稳定表达人κ阿片受体的HEK-293细胞，并用于本实验。用50 mM Tris-HCl缓冲液（pH 7.4）制备细胞膜。将相当于30 μg的膜与化合物和0.6 nM [3 H]二丙诺啡（一种阿片拮抗剂）在25°C下孵育60分钟（抑制效果）或多个时间点（结合动力学）。在10 μM纳洛酮存在时，估计非特异性结合。结合馏分和游离馏分通过GF/B过滤器真空过滤分离，该过滤器用0.3%聚醚酰亚胺预处理。用冰冷的缓冲液洗涤滤片，然后计数以确定结合的放射性配体（Olianas等人，2006）。[3H]二丙诺啡结合的抑制百分比计算公式为：抑制率(%)= (CPMtotal−CPMcompound)/(CPMtotal−cpmnonspecific) × 100，其中CPMtotal =总[3H]二丙诺啡结合（膜+0.6 nM [3H]二丙诺啡），CPMnon-specific =非特异性[3H]二丙诺啡结合（膜+0.6 nM [3H]二丙诺啡+ 10 μM纳洛酮）。对于未标记的化合物，通过使用Motulsky和Mahan （Motulsky和Mahan， 1984）描述的方程拟合数据来计算缔合/离解常数。[2]

For in vivo experiments, all the test compounds (e.g. Anrikefon (HSK21542)) were solubilized in normal saline, and intravenously administered with a volume of 10 μL/g, except for morphine and nor-binaltorphimine (subcutaneously) when the animals were not under anesthesia and were awake. All other reagents used were of analytical grade unless otherwise stated.

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In vivo Brain/Plasma Distribution of Anrikefon (HSK21542)in Rats[2]
SD rats (half male and half female) were intravenously given a single dose of 0.3 mg/kg Anrikefon (HSK21542). The samples were collected at 0.083, 0.5, 1.5 and 4 h after dosing. The rats were anesthetized with isoflurane and then sacrificed by taking blood from the abdominal aorta. The whole brains were rapidly removed from the crania. The plasma (∼100 μL) was separated from the blood by centrifugating at 2000 ×g for 10 min at 4°C. The brains were rinsed with ice-cold normal saline, blotted dry, weighted and placed into a plastic tube. For 1.0 g of brain sample, 4 ml of acetonitrile-ultrapure water solution (1:4, v/v) was added to the tube. The brain samples were then homogenized for 120 s at 50 Hz and ultrasound was performed for 5 min. The plasma and the brain samples were analyzed using a LC-MS/MS assay as detailed in supplementary materials.

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LC-MS/MS Assay[2]
The plasma or brain homogenate was ice thawed. After 30 μL of plasma or brain homogenate was transferred into a centrifuge tube, 50 μL of internal standard (D4-HSK21542, 50 ng/ml) and 120 μL of acetonitrile were added. The mixture was vortexed for 10 min and centrifuged at 2000 ×g for 10 min at 4°C. The collected supernatant (150 μL) was placed in a 96-well plate and dried under nitrogen. The residue was reconstituted with 150 μL of ultrapure water and vortexed for 10 min. The resulting solution was then analyzed to determine the concentrations of Anrikefon (HSK21542) on a LC-MS/MS system, which consisted of a DGU-20A5R degasser, a LC-30AD pump, a SIL-30AC autosampler, a CTO-20A column oven (Shimadzu, Japan) and an AB Sciex Triple Quad 5500 mass spectrometer (Sciex, Canada). The LC system was coupled to mass spectrometer by using an electro-spray ionization (ESI) source (Yang et al., 2011; Dong et al., 2018). Chromatographic separation was performed on a reverse phase column (Venusil ASB C18, 4.6 mm × 50 mm) under a ternary gradient elution. The temperatures of autosampler and column were maintained at 4 and 40°C, respectively. The mobile phase A consisted of 0.3% formic acid in 2 mM acetic acid solution and the mobile phase B consisted of 0.2% formic acid in acetonitrile. The flow rate was held constant (0.7 ml/min) and the injection volume was set to 20 μL. Quantification was conducted in positive ion mode. The MRM transition of m/z 704.4→295.2 was used to quantify HSK21542.

Furthermore, it was extremely hard for HSK21542 to penetrate into the brain tissues with a brain/plasma concentration ratio of 0.001 (Supplementary Figure S2).[2]

[1]. WHO Drug Information, Vol. 36, No. 2, 2022. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO.

[2]. Antinociceptive and Antipruritic Effects of HSK21542, a Peripherally-Restricted Kappa Opioid Receptor Agonist, in Animal Models of Pain and Itch. Front Pharmacol . 2021 Nov 16:12:773204..

Kappa opioid receptor (KOR) agonists have been promising therapeutic candidates, owing to their potential for relieving pain and treating intractable pruritus. Although lacking morphine-like central nervous system (CNS) effects, KOR agonists do elicit sedation, dysphoria and diuresis which seriously impede their development. Peripherally-restricted KOR agonists have a poor ability to penetrate into the CNS system, so that CNS-related adverse effects can be ameliorated or even abolished. However, the only approved peripherally-restricted KOR agonist CR845 remains some frequent CNS adverse events. In the present study, we aim to address pharmacological profiles of HSK21542, with an expectation to provide a safe and effective alternative for patients who are suffering from pain and pruritus. The in vitro experimental results showed that HSK21542 was a selective and potent KOR agonist with higher potency than CR845, and had a brain/plasma concentration ratio of 0.001, indicating its peripheral selectivity. [2]

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In conclusion, the in vitro findings revealed that HSK21542 is a selective KOR agonist with a higher potency than CR845. The brain/plasma distribution study showed that HSK21542 has an extremely poor ability to penetrate into the CNS system. The in vivo pharmacological activities supported the translational potential of HSK21542 as a safe and effective analgesic and antipruritic candidate. Generally, HSK21542 has the ability to avoid adverse CNS effects that are associated with centrally penetrating KOR agonists and MOR agonist, and might provide an effective alternative for treating patients with pain or pruritus.[2]

C39H57N7O5

703.913789510727

703.442

C, 66.54; H, 8.16; N, 13.93; O, 11.36

2269511-95-5

Anrikefon acetate;2584931-05-3

137433038

Typically exists as solid at room temperature

2.6

180

5

7

17

51

1150

4

CC(C)C[C@H](C(=O)N[C@H](CCCCN)C(=O)N1CCC2(CC1)CN(C2)C(=O)C)NC(=O)[C@@H](CC3=CC=CC=C3)NC(=O)[C@@H](CC4=CC=CC=C4)N

VWXDZAZSCZVCPV-YFRBGRBWSA-N

InChI=1S/C39H57N7O5/c1-27(2)22-33(36(49)42-32(16-10-11-19-40)38(51)45-20-17-39(18-21-45)25-46(26-39)28(3)47)44-37(50)34(24-30-14-8-5-9-15-30)43-35(48)31(41)23-29-12-6-4-7-13-29/h4-9,12-15,27,31-34H,10-11,16-26,40-41H2,1-3H3,(H,42,49)(H,43,48)(H,44,50)/t31-,32-,33-,34-/m1/s1

(2R)-N-[(2R)-1-(2-acetyl-2,7-diazaspiro[3.5]nonan-7-yl)-6-amino-1-oxohexan-2-yl]-2-[[(2R)-2-[[(2R)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanamide

Anrikefon; Anrikefon [INN]; 22SCY98BXV; 2269511-95-5; UNII-22SCY98BXV; 1-(7-(D-Phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2,7-diazaspiro(3.5)non-2-yl)ethanone; 1-(7-(D-Phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)- 2,7-diazaspiro(3.5)nonan-2-yl)ethan-1-one; Ethanone, 1-(7-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2,7-diazaspiro(3.5)non-2-yl)-;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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));
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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；
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