Icilin

TRPM8/transient receptor potential M8

Icilin 诱发的 TRPM8 电流表现出非常不同的延迟和显着的脱敏。它们还具有可变的激活动力学和 Ca2+ 依赖性[1]。

大鼠和小鼠的行为被 iscilin 激活，iscilin 是一种瞬时受体电位阳离子通道亚家族 M (TRPM8) 激动剂。当给小鼠注射icin（3 mg/kg；皮下注射）时，会给予小鼠“湿狗奶昔”[2]。

TRPM8是瞬态受体电位离子通道家族的成员，在寒冷时会使体感神经元去极化。TRPM8也被冷却剂薄荷醇和西林激活。当暴露于薄荷醇或寒冷时，TRPM8表现得像许多配体门控通道一样，表现出快速激活，随后是适度的钙依赖性适应。相比之下，如果存在钙，西林会以极不稳定的潜伏期激活TRPM8，随后进行广泛的脱敏。在这里，我们表明，为了达到完全的疗效，氨苄青霉素需要通过TRPM8通道的渗透或细胞内储存的释放同时提高细胞溶质Ca2+。因此，必须将两个刺激配对以引发全通道激活，这说明了TRP通道进行重合检测的可能性。西林敏感性的决定因素映射到TRPM8的一个区域，该区域对应于有害热受体TRPV1上的辣椒素结合位点，这表明化学激动剂对这些热敏通道的门控具有保守的分子逻辑[1]。

细胞活力测定[1]
细胞类型：表达 TRPM8 的卵母细胞或 HEK293 细胞
测试浓度： 10 μM
孵育时间： 表达TRPM8的卵母细胞3分钟； HEK293 细胞 1 分钟
实验结果：在表达电压钳制 TRPM8 的卵母细胞或 HEK293 细胞中，激活的膜电流具有不同的起始延迟。

Animal/Disease Models: The C57BL/ 6 mice 9–10 weeks (adult, 26-30 g) or 24 months (aged, 35-42g)[2].
Doses: 3 mg/kg
Route of Administration: Injected sc
Experimental Results: Produced vivid and quantifiable shaking behaviors (“wet- dog shakes”), which were TRPM8-dependent.

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To determine if “compound 5” effectively inhibits TRPM8 activity in live mice, a WDS assay was performed. In this assay, a potent TRPM8 agonist (icilin 3 mg/kg s.c.) was injected in mice treated with “compound 5” or vehicle. Icilin injections in mice produce vivid and quantifiable shaking behaviors (“wet-dog shakes”), which are TRPM8-dependent (18–22)(2, 3). Accordingly, effective inhibition of TRPM8 is predicted to result in decreased number of icilin-induced behavioral events. “Compound 5” or vehicle was administered 60 minutes prior to icilin. The number of “wet-dog shakes” was counted over a 5-minute period starting 10 minutes after icilin injection.Experimental protocols (summarized in Table 1): In protocol 1, used to establish a method for long-lasting and sustained hypothermia through TRPV1 activation, DHC (2–4 mg/kg) or vehicle (20% DMSO in saline) was infused subcutaneously through the PE-10 line in a conscious mouse. The full dose was delivered in two bolus injections 30 minutes apart (Figure 1B) or as continuous infusion with or without an initial bolus injection (Figure 2). Core temperature was recorded by the thermocouple implanted in the abdomen; a second thermocouple recorded cage ambient temperature (22–24 °C). Temperature was measured at a frequency of 1 sample/min for up to 8 hours after the first infusion.[2]

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Protocol 3 was used to determine the effectiveness of TRPV1 agonists in aged subjects. Young (9–10 weeks) or aged (24 months) mice were injected with a single bolus of DHC (1.25 mg/kg s.c.). Temperature was measured by telemeter for 2 hours after injection (Figure 4). In protocol 4, used to determine the hypothermic effect of TRPM8 inhibition in mildly subneutral and cold environments, “compound 5” (20 mg/kg s.c.) or vehicle was administered to telemeter implanted mice (Figure 5). “Compound 5” is a selective TRPM8 inhibitor (see Drugs section). Core temperature was recorded for 30 minutes starting 30 minutes after the injection, at which point the mice were transferred to a cold room for 2 hours and measured at 20 minute intervals. The individual cages were fitted with a plastic mesh top to allow heat exchange with the atmosphere. Temperature in the cage averaged 8 °C.[2]

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Protocol 5 was used to determine if TRPM8 inhibition potentiates the hypothermic effect of TRPV1 agonists. TRPM8 antagonist “compound 5” (30 mg/kg i.p.) or vehicle was injected in mice with implanted telemeter (Figure 6). After 60 minutes, DHC (0.6, 1.25, or 2.5 mg/kg i.p.) was injected in both groups. Temperature was recorded for 5.5 hours (330 minutes) from the time of “compound 5” or vehicle injection, with a measurement taken every 10 minutes. The depth of hypothermia was determined by calculating the minimum value (nadir) of Tcore with each treatment. The duration of hypothermia was determined by measuring the time from DHC injection until recovery of Tcore to ≥34 °C to the nearest 10 minutes. The 0.6 mg/kg DHC dose failed to reliably drop Tcore below 34 °C during the experiment and was therefore excluded from Tcore recovery analysis. In the two groups treated with higher doses of DHC and pretreated with “compound 5”, the Tcore occasionally failed to return to 34 °C within the 330 minute measurement period. In these cases (6 out of 16 mice), the maximum time value (330 min) was assigned for the recovery time. All mice, however, fully recovered Tcore by the next morning. In addition, in a single mouse in the vehicle group treated with 2.5 mg/kg DHC, Tcore did not drop below 34 °C. This mouse was assigned the minimum time value (0 minutes) for the recovery time.[2]

[1]. The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel. Neuron. 2004 Sep 16;43(6):859-69.

[2]. Transient receptor potential melastatin 8 channel inhibition potentiates the hypothermic response to transient receptor potential vanilloid 1 activation in the conscious mouse. Crit Care Med. 2014 May;42(5):e355-63.

[3]. The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response. FASEB J. 2018 Mar;32(3):1236-1249.

3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-1,4-dihydropyrimidin-2-one is a C-nitro compound.

C16H13N3O4

311.29

311.09

C, 61.73; H, 4.21; N, 13.50; O, 20.56

36945-98-9

161930

Typically exists as Light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

594.8±50.0 °C at 760 mmHg

200-208ºC

313.5±30.1 °C

0.0±1.7 mmHg at 25°C

1.672

2.73

98.39

2

4

2

23

502

0

O=C1N([H])C(C2C([H])=C([H])C([H])=C(C=2[H])[N+](=O)[O-])=C([H])C([H])([H])N1C1=C([H])C([H])=C([H])C([H])=C1O[H]

RCEFMOGVOYEGJN-UHFFFAOYSA-N

InChI=1S/C16H13N3O4/c20-15-7-2-1-6-14(15)18-9-8-13(17-16(18)21)11-4-3-5-12(10-11)19(22)23/h1-8,10,20H,9H2,(H,17,21)

3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-1,4-dihydropyrimidin-2-one

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.03 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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。