P2Y1 Receptor (IC50 = 51.6 nM)

当添加2-MeSADP时，MRS2279抑制火鸡红细胞膜中磷酸肌醇的产生，pKb值为7.75[2]。在1321N1人星形细胞瘤细胞中，MRS2279对人P2Y1受体表现出高亲和力竞争性拮抗作用，pKb值为8.10[2]。人 P2Y2、P2Y4、P2Y6 和 P2Y11 受体同源激动剂激活不受 MRS2279 的影响，但它对 P2Y1 受体有明显的影响 [2]。尚未表明 MRS2279 可以通过血小板 Gi/腺苷酸环化酶连接的 P2Y 受体阻碍 ADP，从而防止环化 AMP 的积聚 [2]。

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MRS2279以竞争动力学拮抗2MeSADP刺激的火鸡红细胞膜中肌醇磷酸的形成（pKB=7.75）。在1321N1人星形细胞瘤细胞中稳定表达的人P2Y1受体（pKB=8.10）上也观察到MRS2279的高亲和力竞争性拮抗作用。拮抗作用对P2Y1受体具有特异性，因为MRS2279对其同源激动剂激活人P2Y2、P2Y4、P2Y6或P2Y11受体没有影响。 MRS2279也没有阻断ADP通过血小板的Gi/腺苷酸环化酶连接的P2Y受体抑制环AMP积聚的能力。 相比之下，已知P2Y1受体在ADP诱导的血小板聚集过程中是必需的，MRS2279竞争性抑制ADP促进的血小板聚集，其明显的亲和力（pKB=8.05）与1321N1细胞中异源表达的人P2Y1受体相似。 综上所述，这些结果说明了非核苷酸分子对P2Y1受体的选择性高亲和力拮抗作用，这应被证明对各种组织中该受体的药理学描述有用[2]。

在接受高压通气的小鼠中，MRS2279（2 μL，1 nM；脑室内注射；机械通气前 30 分钟）可以减轻机械通气带来的脑损伤[3]。

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抑制P2Y1受体激活可改善机械通气诱导的小鼠脑损伤[3]
为了验证机械通气是否通过激活小鼠海马中的P2Y1受体诱导脑损伤，实验小鼠在机械通气前30分钟侧脑室注射P2Y1拮抗剂MRS2279，同时注射ACSF作为对照（图5a）。小鼠的潜伏期较短（HVT组：63.61±4.49秒；HVT+ACSF组：64.25±5.81秒，HVT+MRS2279组：37.17±3.50秒），游泳距离较短（HVT组：756.53±30.03厘米；HVT+ACSF组：762.83±38.06厘米，HVT+MRS2279组：559.0±37.63厘米），在目标象限停留的时间较长（HVT组合：10.45±0.72秒；HVT/ACSF组合：10.48±0.67秒，HVT/MRS2279组合：13.63±0.54秒）抑制P2Y1受体激活后（p<0.05，图5b）。同时，神经元数量增加（p<0.05，图5c），而海马中的dysbinding-1蛋白水平降低（p<0.05，见图5d），海马中TNF-α、IL-1β、IL-6和DA的水平均降低（p<0.05），见图5e。总的来说，我们的研究结果表明，抑制P2Y1受体的激活可以减轻机械通气引起的小鼠脑损伤。

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阻断DA受体减轻了机械通气通过P2Y1受体激活介导的DA释放增加引起的小鼠脑损伤[3]
同样，为了验证P2Y1受体激活是否通过增加DA表达模式诱导脑损伤DA神经传导，实验小鼠在机械通气前30分钟腹腔注射DA受体拮抗剂氟哌啶醇（图5a）。与MRS2279的功能相似，注射氟哌啶醇的小鼠在机械通气后的认知能力有了显著改善（p<0.05，图6a），潜伏期更短（HVT组：63.61±4.49秒；HVT+盐水组：62.71±5.54秒，HVT+氟哌啶醇组：31.53±4.82秒），游泳距离更短（HVT-组：756.53±30.03厘米；HVT+saline组：753.46±34.13厘米，HVT+halopidolization组：565.85±45.98厘米），在目标象限停留的时间更长（HVT+10.45±0.72秒）；HVT+生理盐水组：10.36±0.63秒，HVT+氟哌啶醇组：12.34±0.67秒）。同时，海马神经元数量增加（p<0.05，图6b），DA和dysbinding-1蛋白水平没有显著变化（p>0.05，图6c-d），而TNF-α、IL-1β和IL-6水平降低（p<0.05，表6d）。总的来说，我们的研究结果表明，抑制DA受体可以减轻由机械通气通过P2Y1受体激活介导的DA释放增加引起的小鼠脑损伤。

洗涤血小板的制备[2]
静脉血取自健康志愿者，并与最终体积的20%的酸/柠檬酸盐/葡萄糖混合。将血液离心180×g 20分钟，取出富含血小板的血浆，在1 mM阿司匹林的存在下孵育1小时。在1000×g下离心血小板，并将其重新悬浮在含有0.2%BSA和0.05 U ml−1 apyrase的HEPES缓冲Tyrode溶液中，密度为2×108个血小板ml−l。

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人血小板中环AMP积聚的测定[2]
如前所述测量环AMP积累（Meeker&Harden，1982）。简而言之，从50毫升血液中分离出的血小板在37°C下用1μCi ml−1[3H]-腺嘌呤标记1小时。然后洗涤血小板并重新悬浮在（mM）：NaCl 137中，KCl 2.7、MgCl2 1、NaH2PO4 3、葡萄糖5和HEPES 10，pH 7.4。在200μM 3-异丁基-1-甲基黄嘌呤存在下孵育10分钟，用10%三氯乙酸停止反应。在Dowex和氧化铝柱上色谱后定量[3H]-环AMP。

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血小板聚集[2]
使用四通道Chrono-Log聚集仪测量血小板聚集。简而言之，在37°C下搅拌500μl洗涤过的血小板，补充2 mM CaCl2和1 mg ml-1纤维蛋白原，加入指定浓度的ADP，并在8分钟的孵育过程中监测聚集情况。通过在添加ADP之前将血小板与P2Y1拮抗剂预孵育2分钟，研究了MRS2279的拮抗作用。使用500μl HEPES缓冲Tyrode溶液设定聚集反应的基线。

P2Y1受体促进火鸡红细胞膜肌醇脂质水解的检测[2]
如我们所述，在火鸡红细胞膜中研究了P2Y1受体促进的磷脂酶C激活（Boyer等人，19891996a）。简而言之，红细胞在37°C、95%空气/5%CO2的加湿气氛中，在含有0.5 mCi 2-[3H]-肌醇（20 Ci/mmol）的无肌醇培养基中孵育18-24小时。制备膜，并在25μl 3H-肌醇标记的膜（约175μg蛋白质，每次测定200-500000 c.p.m.）在含有（mM）：CaCl2 0.424、MgSO4 0.91、EGTA 2、KCl 115、KH2PO4 5和HEPES 10的培养基中，pH 7.0。测定（最终体积200μl）含有1μm GTPγS和指定浓度的核苷酸类似物。将膜在30°C下孵育5分钟，通过阴离子交换色谱法定量总[3H]-肌醇磷酸盐。肌醇磷酸盐积累的典型值约为200-300 c.p.m.（基础），2000-3000 c.p.m.（仅1μm GTPγS）和15000-20000 c.p.m。三次重复值的范围在平均值的10%以内。

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表达P2Y受体的1321N1细胞中肌醇磷酸积累的测定[2]
如前所述，使用逆转录病毒载体在1321N1人星形细胞瘤细胞中稳定表达P2Y1、P2Y2、P2Y4、P2Y6或P2Y11受体（Schachter等人，1996）。用[3H]-肌醇标记细胞过夜，在10mM LiCl和每种受体的同源激动剂存在下孵育10分钟后，定量激动剂促进的[3H]--肌醇磷酸积累。使用的激动剂是2MeSADP或ADP，用于表达P2Y1受体的细胞，UTP，UTP用于表达P2Y4受体的细胞、UDP用于表达P2YH受体的细胞和ATP用于表达P2Y11受体的细胞。在每种受体存在最大有效浓度的同源激动剂的情况下，肌醇磷酸盐积累的典型值为基础500-700 c.p.m.和4000-10000 c.p.m。三次重复值的范围在平均值的10%以内。

C57BL6 mice aged 8–12 weeks were housed in specific pathogen-free animal rooms with ad libitum access to food and water under 12/12 h light-dark cycles. The spontaneously breathing mice (sham group) received the same sedation as the mice in other groups: low-pressure ventilation (LVT group) [peak inspiratory pressure (PIP) of 12 cm H2O; positive end-expiratory pressure of 2 cm H2O; respiratory rate of 100 breaths/min] or high-pressure ventilation (HVT group) (PIP of 20 cm H2O; positive end-expiratory pressure of 0 cm H2O; respiratory rate of 50 breaths/min) for 90 min, followed by an array of 330-min long-term ventilation experiments under high-pressure ventilation. High-pressure ventilated mice were randomly selected and intraperitoneally injected with the DA receptor antagonist haloperidol (0.5 mg/kg in 0.2 mL saline) 30 min prior to mechanical ventilation with the mice injected with an equivalent amount of normal saline as controls, or simultaneously intracerebroventricularly (coordinates with respect to bregma: AP = 0.4 mm; L = 0.95 mm) injected with 2 μL of the P2Y1 receptor antagonist MRS2279 (1 nmol, 96% purity; Tocris Bioscience, Abingdon, UK) 30 min prior to mechanical ventilation with mice injected with an equivalent amount of artificial cerebrospinal fluid (ACSF) as controls. Animals were assigned into the following groups with 12 mice in each group (total 96): 1. the sham group, spontaneous breathing; 2. the LVT group, low tidal volume; 3. the HVT group, high tidal volume; 4. the long term group, mechanical ventilation for 330 min under high tidal volume; 5. the HVT + ACSF group, high tidal volume mechanical ventilation was performed 30 min after intracerebroventricular injection of ACSF; 6. the HVT + MRS2279 group, high tidal volume mechanical ventilation was performed 30 min after lateral ventricular injection of MRS2279 ; 7. the HVT + saline group, normal saline was injected intraperitoneally 30 min before mechanical ventilation; 8. the HVT + haloperidol group, haloperidol was injected intraperitoneally 30 min before mechanical ventilation. All ventilated mice were euthanized (intraperitoneal administration of 200 mg/kg pentobarbital sodium) after conducting the Morris water maze test. Hippocampus and lung tissues of mice were harvested for subsequent experimentation. The tissues were randomly selected from 6 mice in each group for pathological examination while the tissues of the remaining mice were homogenized for protein expression detection.[3]

[1]. Synthesis, biological activity, and molecular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y(1) receptor ligands. J Med Chem. 2000;43(5):829-842.

[2]. Boyer JL, et al, Ravi RG, Jacobson KA, Harden TK. 2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist. Br J Pharmacol. 2002;135(8):2004-2010.

[3]. Mechanical ventilation induces lung and brain injury through ATP production, P2Y1 receptor activation and dopamine release. Bioengineered. 2022 Feb;13(2):2346-2359.

We investigated the structure-activity relationship of adenosine-3',5'-diphosphate as a P2Y(1) receptor antagonist, revealing the enhancing effect of the N(6)-methyl group and the possibility of ribose partial substitution (Nandanan et al., J. Med. Chem. 1999, 42, 1625-1638). We introduced a confined carbocyclic ring (to explore the role of sugar ring wrinkling), a non-glycosidic bond linked to the adenine moiety, and phosphate group transfer. The bioactivity of each analog on the P2Y(1) receptor was characterized by measuring its ability to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and its ability to inhibit phospholipase C stimulation induced by 30 nM 2-methylthioadenosine-5'-diphosphate (antagonist effect). In some cases, the introduction of the N(6)-methyl group can convert a pure agonist into an antagonist. One carbocyclic N(6)-methyl-2'-deoxyadenosine diphosphate analog is a pure P2Y(1) receptor antagonist with potency comparable to the riboside analog (MRS 2179). Among a series of ring-bound methoxycarbon derivatives, the fused cyclopropane moiety restricts the pseudoglycosidic ring of the nucleoside to the north (N) or south (S) conformation as defined in the pseudo-rotational cycle, with the 6-NH(2)(N)-analyte being a pure agonist with an EC(50) of 155 nM, exhibiting 86-fold greater potency than the corresponding (S)-isomer. The 2-chloro-N(6)-methyl-(N)-methoxycarbon analog is an antagonist with an IC50 of 51.6 nM. Thus, the riboside ring (N) conformation appears to be more dominant in P2Y(1) receptor recognition. The cyclobutyl analog is an antagonist with an IC50 of 805 nM, while the morpholine ring-containing analog shows almost no activity. Dehydrated hexitol ring-modified diphosphate derivatives exhibit micromolar potency as agonists (6-NH2) or antagonists (N(6)-methyl). Molecular models of the energy-minimizing structure of potent antagonists suggest that the two phosphate groups may occupy a common region. (N)- and (S)-methoxycarbazine agonist analogs were docked to the putative binding sites of previously reported P2Y(1) receptor models. [1] Mechanical ventilation can induce lung injury and exacerbate brain injury due to lung-brain interaction. This study aims to explore the mechanism of lung-brain interaction induced by mechanical ventilation and to provide theoretical guidance for the treatment of ventilator-associated brain injury. Experimental mice were divided into spontaneous breathing group and mechanical ventilation group, and were injected with dopamine (DA) receptor antagonist haloperidol or P2Y1 receptor antagonist MRS2279 before ventilation. In vitro experiments were conducted using lung epithelial cells MLE-12 and hippocampal neurons HT-22. The recognition function and lung injury status of mice were detected, and the state and concentration of hippocampal neurons were observed. We examined the levels of various inflammatory factors, dopamine (DA), adenosine triphosphate (ATP), P2Y1 receptor, and dysbindin-1. Mechanical ventilation can induce lung and brain injury in mice, manifested by elevated levels of inflammatory factors in bronchoalveolar lavage fluid and hippocampus, prolonged escape latency, reduced swimming distance and time in the target quadrant, and decreased hippocampal neuronal density. Our results showed that the expression of ATP and P2Y1 receptor was increased in mechanically ventilated mice and stretched MLE-12 cells. The levels of DA and dysbindin-1 were also increased in mechanically ventilated mice and HT-22 cells treated with the P2Y1 receptor activator MRS2365. Inactivation of P2Y1 receptor or blockade of DA receptor in the hippocampus could alleviate mechanical ventilation-induced brain injury in mice. In summary, this study shows that mechanical ventilation-induced lung injury can exacerbate brain injury in mice by increasing ATP production, activating P2Y1 receptor, thereby promoting DA release. [3]

C13H20CLN5O8P2

469.71132

469.032

367909-40-8

MRS2279 diammonium;2387505-47-5

9847505

Typically exists as solid at room temperature

1.132

208.77

5

12

7

29

740

4

CNC1C2N=CN([C@@H]3[C@H]4C[C@@]4(COP(O)(O)=O)[C@@H](OP(O)(O)=O)C3)C=2N=C(Cl)N=1

LPZJKPSGEADHTQ-HLTSFMKQSA-N

InChI=1S/C13H20ClN5O8P2/c1-15-11-10-12(18-13(14)17-11)19(6-16-10)4-7-2-8(5-26-28(20,21)22)9(3-7)27-29(23,24)25/h6-9H,2-5H2,1H3,(H,15,17,18)(H2,20,21,22)(H2,23,24,25)/t7-,8-,9+/m1/s1

[(1S,2R,4R)-4-[(2-chloro-6-methylaminopurin-9-yl)methyl]-2-(phosphonooxymethyl)cyclopentyl] dihydrogen phosphate

MRS2279 MRS-2279 MRS 2279.

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));
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;
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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。