Guanylate cyclase ( EC50 = 15 nM )
In the intermittent setting, cinaciguat hydrochloride (BAY 58-2667) is a potent GC activator (EC50 15 nM), but the maximum effect is around 1% of NO. The concentration response curve of cinaciguat hydrochloride in the presence of sildenafil in the absence of ODQ, with an EC50 of 18 nM, and in the presence of ODQ, there was no significant difference in the efficacy of cinaciguat hydrochloride, with an EC50 of 13 nM. The efficacy of cinaguat hydrochloride in the distal end (EC50 15 nM) was very similar to the estimate achieved for sealed recombinant GC. Cinaciguat hydrochloride at a maximum effective concentration of 1 μM stimulated control GC activity to approximately 25% of the activity observed with NO, and compared with stimulation with NO, this activity remained at lower levels as the proportion of ODQ-cut GC increased. Change[1].

在间歇中，盐酸西那西呱（BAY 58-2667）是一种有效的GC激活剂（EC50 15 nM），但最大作用为NO的1%左右。盐酸西那西呱的浓度响应曲线是在存在西地那非在没有 ODQ 的情况下，EC50 为 18 nM，在 ODQ 的情况下，盐酸西那呱的功效没有显着差异，EC50 为 13 nM。盐酸西那呱在远端中的功效（ EC50 15 nM) 与对密封的重组 GC 完成的估计非常相似。最大有效浓度为 1 μM 的盐酸西那西呱对照 GC 活性刺激到用 NO 观察到的活性的约 25%，并且与 NO 的刺激相比，随着 ODQ 削减 GC 的比例增加，该活性保持水平不变[1]。

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在纯化的牛肺sGC上，BAY 58-2667 刺激对照（模拟处理）酶的活性达到约10 µM DEA/NO观察到的活性的25%。这种活性归因于制剂中存在无血红素蛋白。当血红素被完全去除（使用Tween-20预处理）后，BAY 58-2667 几乎能像对照条件下的NO一样有效地激活该酶。相比之下，用血红素氧化剂ODQ预处理并不能增强BAY 58-2667 对纯化酶的刺激作用。
在完整的大鼠血小板中，BAY 58-2667 是sGC的有效激活剂 (EC50 ~15 nM)。然而，其最大活性仅约为饱和NO浓度 (50 nM) 可达到的峰值活性的1%。用ODQ (10 µM) 预处理血小板以氧化血红素，使BAY 58-2667 的最大功效提高了约22倍，表明在这些条件下无血红素sGC的比例显著增加。
即使由BAY 58-2667 单独产生的低水平cGMP（最大NO效应的1%）也足以在血小板中引起血管舒张刺激磷蛋白 (VASP，cGMP依赖性蛋白激酶的下游靶标) 的显著磷酸化。[1]

在apo-sGC小鼠和WT小鼠中，给予盐酸西那呱可降低血压并增加心率。盐酸西那呱在apo-sGC小鼠中的降压作用比在WT小鼠中显着着且持续时间更长。另外，盐酸西那呱在不影响WT 小鼠血糖的浓度下降低了apo-sGC 小鼠的血压。另外，盐酸西那呱诱发的预收缩主动脉离体收缩的IC50 值在apo-sGC 小鼠中比WT 小鼠低三倍 (IC50=0.2 nM 和 0.7 nM，分别)。总之，我们的结果表明像 Cinaciguat 盐酸盐这样的 sGC 激活剂是像 BAY 41-2272 这样的 sGC 刺激剂激活 apo-sGC。另外，观察到盐酸西那西呱可以调节WT小鼠的血管舒张和血压表明，即使在健康小鼠中，可用sGC池的一个子集也清晰血红素并且对sGC激活剂有反应[2]。

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在 apo‑sGC 小鼠中，静脉注射 cinaciguat（300 µg·kg⁻¹）可降低平均动脉压并增加心率。其降压效果在 apo‑sGC 小鼠中比野生型小鼠更显著、更持久。[2]
Cinaciguat 也能降低野生型小鼠的血压，提示即使在健康小鼠中，也有一部分 sGC 处于血红素缺失状态并对 sGC 激活剂有反应。[2]
在 TNF 诱导的全身性休克模型中，cinaciguat 在适当的时间窗内给药可改善器官功能并提高存活率。[2]

在添加精胺 NONOate 或 Cinaciguat 之前和之后定期取出血小板悬浮液等分试样。然后将它们迅速转移到灭活缓冲液（50 mM Tris、4 mM EDTA、pH 7.4）中，并在 100°C 下保存至少 10 分钟。使用时，在添加精胺 NONOate 之前 15 分钟向血小板中添加 10 μM ODQ。用 DEA/NO (10 μM) 或 Cinaciguat (1 μM) 刺激后，并像血小板一样进行酶灭活，在测定缓冲液 (50 mM Tris) 中以 50 ng/mL 的终浓度测量纯化的 GC 活性、100 μM EDTA、1 mM NaGTP、1.3 mM MgCl₂、50 μg/mL BSA，pH 7.4）。通过使用放射免疫测定法，可以测定 cGMP 水平并表示与蛋白质含量的关系。每个实验由三次独立运行组成，结果显示为平均值±semean[1]。

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使用纯化的牛肺鸟苷酸环化酶 (GC) 测量其活性。将酶 (5 µg/ml) 稀释在含有Tris、二硫苏糖醇和BSA的缓冲液中。对于某些实验，酶在37°C下用10 µM ODQ（血红素氧化形式）或0.5% Tween-20（无血红素形式）预处理15分钟。对照酶接受溶剂 (0.5% DMSO)。预处理后以及可选地与对照酶混合后，样品通过尺寸排阻过滤器离心以去除小分子。
在含有Tris、EDTA、GTP和MgCl₂的测定缓冲液中，以50 ng/ml的最终酶浓度测量GC活性。反应在37°C下用NO供体DEA/NO (10 µM) 或BAY 58-2667 (1 µM) 刺激2分钟，然后通过热失活终止。生成的cGMP水平通过放射免疫分析法进行定量。[1]
为了研究血红素丢失动力学，将纯化的GC在37°C下与底物 (GTP) 和1 µM BAY 58-2667（含或不含10 µM ODQ）一起孵育。在不同时间点测量cGMP。[1]

本研究未采用传统的基于细胞培养的实验。主要的细胞模型是新分离的大鼠血小板。在这些完整细胞中评估了功能性sGC活性和下游信号传导。[1]
血小板cGMP测定: 将大鼠血小板悬浮并孵育。在添加BAY 58-2667 或NO供体后的不同时间点取出等分试样，并立即转移至热灭活缓冲液中。通过放射免疫分析法测量cGMP水平，并相对于蛋白质含量表示。为了消除cGMP降解带来的复杂性，实验通常在磷酸二酯酶-5抑制剂西地那非 (100 µM) 存在下进行。分析cGMP积累曲线以提取sGC活性（合成速率）和脱敏的动力学参数。[1]
VASP磷酸化测定: 将血小板与测试化合物（例如BAY 58-2667、DEA/NO）在有或无西地那非或ODQ的情况下孵育2分钟。通过在Laemmli缓冲液中煮沸来停止反应。通过SDS-PAGE分离蛋白质，转移到膜上，并用针对Ser²³⁹位点磷酸化VASP的特异性单克隆抗体进行检测。通过增强化学发光法检测条带，并通过光密度法进行定量。[1]

Mice: The tail tip of 12 week old male and female mice is removed with a sharp razor blade, leaving 0.5 cm of tail tip exposed. The tail tip is then placed into freshly prepared, 37°C pre-warmed PBS. By using a chronometer and visual scoring, an observer who is blind to the genotype records the latency until the tail stops bleeding. With an awake mouse tail-cuff method, Hatteras MC4000 Blood Pressure Analysis System measures systolic blood pressure (SBP) and heart rate (HR) non-invasively. The BP measuring apparatus takes seven days to acclimate mice to. Mice are measured for five days, with 15 consecutive cycles per day, in order to establish basal BP. The following are administered intraperitoneally: L-NAME (100 mg/kg), SNP (1.5 mg/kg), DETA-NO (60 mg/kg), BAY 41-2772 (4 mg/kg), Cinaciguat (30 μg/kg), and the vehicle controls (PBS or, in the case of the BAY-compounds, 20% Cremophor+20% diethylene-glycol-monoethylether in PBS). SBP measurements are taken 5 to 15 minutes after SNP injection; L-NAME and DETA-NO recordings are taken 1 to 25 minutes after injection; and BAY-compounds recordings are taken 10 to 25 minutes after injection.

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For telemetric blood‑pressure measurements, mice were implanted with a PA‑C10 transmitter in the carotid artery. After 7 days recovery, cinaciguat (300 µg·kg⁻¹) or vehicle (20% Cremophor + 20% diethylene‑glycol‑monoethyl‑ether in PBS) was administered intravenously. Blood pressure and heart rate were recorded continuously before and after injection. [2]
For aortic‑ring relaxation studies, thoracic aortas from mice were cut into 2‑mm rings and mounted in an organ bath containing Krebs‑Ringer bicarbonate solution at 37 °C. After equilibration and pre‑contraction with phenylephrine, cumulative concentrations of cinaciguat (1 nmol·L⁻¹ to 1 µmol·L⁻¹) were added, and isometric tension was recorded. [2]

[1]. Probing the presence of the ligand-binding haem in cellular nitric oxide receptors. Br J Pharmacol. 2008 Apr;153(7):1495-504.

[2]. Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice. Nat Commun. 2015 Oct 7;6:8482.

BAY 58-2667 (cinaciguat) is a prototype compound of a novel class of sGC activators. Its mechanism of action is distinct from NO; it does not require the haem group for activation. Instead, it binds with high affinity to the haem-free form of sGC, acting as a haem mimetic to restore catalytic activity.
The study demonstrates that under normal physiological conditions in rat platelets, only a small fraction (~2%) of sGC exists in a haem-free state accessible to BAY 58-2667. Haem oxidation by ODQ dramatically increases (22-fold) the compound's efficacy, not by acting on the oxidized haem itself, but by promoting haem loss, thereby increasing the pool of haem-free sGC.
Rapid desensitization of NO-evoked sGC activity (85% loss) in platelets was not associated with a change in the proportion of haem-free enzyme, indicating that haem loss is not the mechanism for this form of desensitization.
The research highlights the utility of BAY 58-2667 as a pharmacological tool to probe the haem occupancy status of sGC in cells. Its enhanced effectiveness under conditions of oxidative stress (which promotes haem loss) suggests therapeutic potential in pathologies where sGC haem deficiency occurs. [1]

C36H40CLNO5

602.1595

601.259

C, 71.81; H, 6.70; Cl, 5.89; N, 2.33; O, 13.28

646995-35-9

Cinaciguat; 329773-35-5

10031513

White to off-white solid powder

87.1

3

6

17

43

767

0

Cl[H].O(C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])C([H])([H])N(C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1C(=O)O[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(=O)O[H]

LLHMBJOVHATVSP-UHFFFAOYSA-N

InChI=1S/C36H39NO5.ClH/c38-35(39)12-6-7-24-37(26-30-19-21-33(22-20-30)36(40)41)25-23-32-10-4-5-11-34(32)42-27-31-17-15-29(16-18-31)14-13-28-8-2-1-3-9-28;/h1-5,8-11,15-22H,6-7,12-14,23-27H2,(H,38,39)(H,40,41);1H

4-[[4-carboxybutyl-[2-[2-[[4-(2-phenylethyl)phenyl]methoxy]phenyl]ethyl]amino]methyl]benzoic acid;hydrochloride

BAY 582667 HCl; BAY-582667 HCl; BAY582667 HCl; BAY 58-2667; BAY-58-2667; BAY58-2667; Cinaciguat HCl; Cinaciguat hydrochloride;

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，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)

DMSO: ~110 mg/mL (~182.7 mM)
H2O: < 0.1 mg/mL

配方 1 中的溶解度: ≥ 2.75 mg/mL (4.57 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 27.5 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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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。