BAY 41-2272

Guanylate cyclase

体外活性：在体外，BAY 41-2272导致人和兔海绵体的浓度依赖性松弛，EC50 分别为 489.1 nM 和 406.3 nM。激酶测定：BAY 41-2272是一氧化氮敏感鸟苷酸环化酶（NO 敏感 GC）的激活剂，在存在和不存在 100 nmol/L DEA-NO 的情况下，EC50 值为 0.3 μmol/L 和 3 μmol/L，分别。细胞测定：在血小板中，使用 3 μmol/L（次最大有效浓度）的 GSNO 来评估 BAY 41-2272 对 NO 敏感的 GC 可能的致敏作用。在不存在 BAY 41-2272 的情况下，应用该浓度的 NO 产生的 cGMP 响应仅是微不足道的。在 100 μmol/L BAY 41-2272 存在的情况下，用 3 μmol/L GSNO 处理导致 cGMP 快速增加至 1000 pmol/109 血小板。

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背景：一氧化氮最重要的受体是可溶性鸟苷酸环化酶（sGC），一种含血红素的异二聚体。最近，一种与YC-1结构相关的吡唑并吡啶衍生物BAY 41-2272被鉴定为以NO非依赖的方式刺激可溶性鸟苷酸环化酶，从而产生血管舒张和抗血小板活性。这里描述的研究涉及可溶性鸟苷酸环化酶上NO非依赖性位点的鉴定。 结果：我们通过在氚标记化合物中引入叠氮基团，为直接和NO非依赖性可溶性鸟苷酸环化酶（sGC）刺激剂BAY 41-2272开发了一种光亲和标记物（3H-meta-PAL）。合成的光亲和标记直接刺激纯化的sGC，并与NO结合显示出对sGC活性的协同作用。3H-meta-PAL与高纯度sGC一起用紫外光照射，导致与酶的α1亚基共价结合。这种结合被未标记的间PAL、YC-1和BAY 41-2272阻断。为了进一步鉴定NO非依赖性调控位点，3H-meta-PAL标记的sGC被溴化氰消化片段化。3H-meta-PAL与由α1亚基的氨基酸236-290组成的溴化氰片段结合。通过测序该溴化氰片段来确定单个PTH循环的放射性，检测到半胱氨酸238和243是3H-meta-PAL的结合残基。
结论：我们的数据表明，sGCα1亚基中半胱氨酸238和243周围的区域可能在sGC活性的调节中发挥重要作用，并可能成为这种新型sGC刺激剂的靶点。[1]

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通过与sGC中该结合位点的相互作用，BAY 41-2272具有独特的体外和体内药理学特征。苯肾上腺素预收缩（3× 10-8g ml-1）兔主动脉环，BAY 41-2272 诱导浓度依赖性舒张，半最大抑制浓度（IC50）为304 ± 63 nM（n=6），而用作对照的三硝酸甘油和YC-1的IC50为1300 ± 385 nM（n=6）和10035 ± 632 nM（n=26）。
我们和其他人已经证明，YC-1在体外、人血小板、sGC过表达细胞系和平滑肌细胞中激活纯化的sGC并使酶对NO敏感。与BAY 41-2272相比，需要大约100倍的YC-1浓度才能在sGC上产生类似的刺激。BAY 41-2272和YC-1之间的结构相似性可能表明这两种化合物的作用机制相同。然而，与YC-1（参考文献21）相比，BAY 41-2272高达10-5 M、 不具有任何PDE-5抑制活性。[2]

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BAY 41-2272导致人和兔海绵体的浓度依赖性舒张（平均EC50+/-SEM分别为489.1+/-22.5和406.3+/-21.5nM）。该化合物的效力是YC-1的32倍，是精胺NONOate的两倍。ODQ降低了BAY41-2272的效力，使得在30微摩尔的存在下。ODQ, BAY 41-2272诱导的舒张作用的EC50分别为1407.3+/-158.0和1902.7+/-11.0 nM。分别在人和兔组织中。L-NAME还抑制了BAY41-2272在兔组织中引起的舒张作用。在500微米的存在下。L-NAME BAY41-2272诱导的反应的EC50为836.7+/-46.7nM。BAY41-2272的亚阈值浓度为30至50 nM。增强的氮能反应。此外，L-NAME对氮能反应的抑制作用被逆转了0.3至3微摩BAY 41-2272。 结论：我们报告了一种非基于NO的可溶性鸟苷酸环化酶激活剂可以放松人和兔的海绵体，并增强氮能反应。[3]

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在之前的研究中，研究人员发现，鸟苷酸环化酶激动剂5-环丙基-2-[1-（2-氟-苄基）-1H-吡唑并[3,4-b]吡啶-3-基]-嘧啶-4-胺（BAY 41-2272）激活人单核细胞和THP-1细胞系产生超氧阴离子，增加体外杀菌活性，表明该药物可用于调节原发性免疫缺陷患者的免疫功能[4]。

在雌性自发性高血压大鼠中，BAY 41-2272（10 mg/kg，口服）显示出抗血小板作用，可显着降低血压并提高存活率。在白色念珠菌感染的小鼠中，BAY 41-2272（10 mg/kg，腹膜内）除了增强巨噬细胞功能外，还显着增加巨噬细胞依赖性细胞流入腹膜，并降低死亡率。在 db/db-/- II 型糖尿病和肥胖小鼠中，BAY 41-2272 可改善受损的海绵体 (CC) 松弛。

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在本研究中，我们研究了体内注射BAY 41-2272治疗通过腹腔和皮下接种引入的白色念珠菌和金黄色葡萄球菌感染的潜力。我们发现，除了巨噬细胞的功能，如扩散、酵母聚糖颗粒吞噬和一氧化氮以及佛波醇肉豆蔻酸酯醋酸酯刺激的过氧化氢产生外，BAY 41-2272的腹膜内治疗还显著增加了巨噬细胞依赖性细胞流入腹膜。BAY 41-2272治疗在降低腹腔接种白色念珠菌死亡率方面非常有效，但对金黄色葡萄球菌无效。然而，我们发现用BAY 41-2272体外刺激腹腔巨噬细胞显著提高了对这两种病原体的杀菌活性。我们的结果表明，用BAY 41-2272治疗感染白色念珠菌的小鼠预防死亡可能主要是通过巨噬细胞活化调节宿主免疫反应来实现的。[4]

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腹膜细胞内流和细胞募集到淋巴器官-小鼠接受（或不接受）BAY 41-2272（0.3-10mg/kg IP）治疗48小时，之后收获腹膜腔并收集脾脏、BM和LN（图1A）。细胞分布显示，与对照组相比，BAY 41-2272治疗诱导腹膜中细胞总数显著增加（图1B）。该细胞群主要由巨噬细胞组成（图1C），但在用该药物治疗的组中，多形核白细胞（PMNs）的百分比也升高了（图1D）。所有使用的载体（transcutol、Cremophor EL和水溶液以及DMSO）对本研究中进行的本次或其他检测没有影响（数据未显示）。[4]

与未治疗的动物相比，用BAY 41-2272治疗的动物的其他淋巴器官（如脾脏、BM或肠系膜LN）中的细胞数量没有差异（图1E-G）。只有肠系膜LN的细胞数量有增加的趋势，表明细胞被募集到这个引流器官。[4]

角叉菜胶诱导的足垫水肿——为了评估BAY 41-2272对炎症过程的影响，我们使用了角叉菜聚糖诱导的小鼠足水肿模型。小鼠用BAY 41-2272（0.3-10mg/kg，IP）治疗（或不治疗）48小时，之后将角叉菜胶（300μg/爪）注射到脚垫中，每小时测量水肿形成，持续4小时（图2A）。用BAY 41-2272进行腹膜内预处理显著增加了爪子水肿，这在角叉菜胶注射后180和240分钟观察到（图2B）。Con A也有类似的数据。这些结果证实了BAY 41-2272的促炎潜力。[4]

BAY 41-2272诱导的离体巨噬细胞活化-扩散和吞噬作用-关于BAY 41-2172产生的促炎活性，扩散和吞噬被评估为腹腔巨噬细胞活化的标志物。收集用BAY 41-2272（0.3-10mg/kg IP）治疗（或不治疗）48小时的小鼠腹膜腔，将腹膜细胞在载玻片上孵育以测量扩散，或用酵母聚糖孵育以评估吞噬作用（图3A）。与未处理的动物相比，从BAY 41-2272处理的小鼠中获得的巨噬细胞的扩散增加（图3B），这与它们增加的吞噬活性是一致的（图3C）。[4]

NO和H 2 O 2的产生-用BAY 41-2272（0.3-10mg/kg IP）处理（或不处理）小鼠48小时，然后收获腹膜腔，将腹膜细胞与或不与PMA（30nM）一起孵育1小时，以评估H2O2的释放，或孵育48小时以评估NO的产生（图4A）。众所周知，吞噬作用和ROS释放是相关的，并且是许多抗菌反应的原因。然而，在这项研究中，尽管吞噬活性增加，但我们没有观察到自发H2O2释放的变化（图4C）。然而，在BAY 41-2272治疗小鼠的巨噬细胞培养物中添加PMA显著增加了这种代谢物的水平（图4C）。[4]

尽管预处理没有诱导H2O2的自发释放，但与对照组的巨噬细胞相比，BAY 41-2272显著增加了NO的自发产生（图4B）。[4]

BAY 41-2272可提高感染真菌的小鼠的存活率-吞噬作用和杀微生物活性的增加表明BAY 41-2172具有治疗感染的潜力。因此，用白色念珠菌和金黄色葡萄球菌攻击C3H/HePas小鼠，并评估这些动物的存活率。小鼠接种白色念珠菌或金黄色葡萄球菌IP，48小时后，用BAY 41-2272（0.3-10mg/kg IP）或伊曲康唑（20mg/kg）、青霉素G（5KU/kg）和四环素（1mg/kg）治疗（或不治疗）三天。对动物的存活率进行了20天的评估（图5A）。 [4]

结果显示，感染后48小时腹腔注射BAY 41-2272显著提高了感染白色念珠菌的小鼠的存活率（图5B），但对感染金黄色葡萄球菌的小鼠没有影响（图5C）。此外，正如预期的那样，伊曲康唑在控制念珠菌感染方面完全有效，使小鼠存活率保持在100%。 [4]

BAY 41-2272可增加小鼠对局部白色念珠菌的反应，但不会增加金黄色葡萄球菌感染的反应——根据BAY 41-2172可提高白色念珠菌感染小鼠存活率的观察，使用了动物脚垫爪感染相同病原体的模型（图6A）。该方案允许评估BAY 41-2272对感染部位（病灶内药物注射）和全身（腹腔内药物给药）的直接影响。 [4]

病灶内注射BAY 41-2272显著减少了白色念珠菌引起的脚垫肿胀，而腹腔内治疗没有显著效果（图6B，C）。皮下或腹腔BAY 41-2272治疗没有显著改变金黄色葡萄球菌引起的脚垫肿胀（图6D，E）。 [4]

BAY 41-2272增加了对白色念珠菌和金黄色葡萄球菌的体外和离体杀菌活性——对于体内感染模型，BAY 41-2172对白色念珠菌的反应比对金黄色葡萄杆菌的反应更好。因此，通过评估其对这两种病原体的杀菌活性，研究了用BAY 41-2272对腹腔巨噬细胞进行体外或离体治疗的效果。用BAY 41-2272（0.3-10mg/kg IP）治疗（或不治疗）小鼠48小时，然后收获腹膜腔，将腹膜细胞与白色念珠菌或金黄色葡萄球菌一起孵育2小时，以评估杀菌活性（图7A）。[4]

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2型糖尿病（DM2）和肥胖是勃起功能障碍（ED）的主要危险因素。在糖尿病中，氧化应激的增加会导致一氧化氮（NO）生物利用度降低，糖尿病患者对磷酸二酯酶5型抑制剂的常规治疗反应似乎较差。我们研究了可溶性鸟苷酸环化酶刺激剂BAY 41-2272（5-环丙基-2-[1-（2-氟苄基）-1H-吡唑并[3,4-b]吡啶-3-基]嘧啶-4-胺）是否通过减少氧化应激有效改善肥胖DM2小鼠受损的海绵体（CC）松弛。在BAY 41-2272存在或不存在的情况下，使用成年db/db（-/-）小鼠或其瘦db（/+）同窝小鼠评估血管功能、cGMP水平、抗氧化状态、NADPH氧化酶表达和超氧化物形成。结果显示，BAY 41-2272（10（-8）至10（-5）M）以类似的方式有效地放松了db（/+）或db/db（-/-）小鼠的CC。BAY 41-2272显著增强了电场刺激（EFS）诱导的内皮依赖性和氮能舒张，并以浓度依赖的方式改善了糖尿病动物对乙酰胆碱和EFS的受损舒张（10（-8）至10（-7）M）。BAY 41-2272增加了CC中cGMP水平，增强了对外源性NO的舒张反应。db/db（-/-）小鼠CC中的总抗氧化状态降低，而血管NADPH氧化酶亚基（gp91phox、p22phox和p47phox）的表达增加，表明处于氧化应激状态。BAY 41-2272以浓度依赖的方式阻止了这些影响。这些结果表明，BAY 41-2272通过增加cGMP和增强抗氧化状态来改善db/db（-/-）小鼠的CC松弛，使该药物成为治疗ED的潜在新候选药物[5]。

BAY 41-2272 是一种对一氧化氮敏感的鸟苷酸环化酶激活剂（NO 敏感的 GC），在存在和不存在 100 nmol/L DEA-NO 的情况下，EC50 值分别为 3 μmol/L 和 0.3 μmol/L。

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可溶性鸟苷酸环化酶（sGC）的纯化及sGC活性的测定[1]
sGC从杆状病毒/Sf9表达系统中高度纯化，并通过在Mg2+作为二价金属阳离子的存在下，由根据Gerzer修饰的[α-32P]-GTP形成[32P]-cGMP来测量酶活性。在存在和不存在1mM DTT的情况下进行孵育。除非另有说明，否则所有测量均重复进行三次。sGC的比活性计算为每分钟孵育时间每毫克蛋白质形成的nmol cGMP。为了表征不同sGC刺激物，sGC的比活性表示为x倍刺激与比基础活性。试验中DMSO的最高浓度为1%（v/v），本身对cGMP的产生没有任何影响。

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sGC分析[2]
我们使用杆状病毒/Sf9表达系统纯化sGC，并如上所述在Mg2+存在下测量酶活性。

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我们研究了在可溶性鸟苷酸环化酶抑制剂ODQ（1H-[1,2,4]恶二唑[4-3a]喹喔啉-1-酮）或一氧化氮合酶抑制剂L-NAME（N-硝基-L-精氨酸甲酯HCl）存在和不存在的情况下，BAY 41-2272对人和兔海绵条张力和氮能弛豫反应的影响。将BAY 41-2272的效力与另一种可溶性鸟苷酸环化酶激活剂YC-1和释放NO的化合物精胺NONOate（N-2-氨基乙基-N-2-羟基-2-亚硝基肼基-1,2-乙二胺）的效力进行了比较。3.

当评估 3 μmol/L（次最大有效浓度）的 GSNO 时，BAY 41-2272 可能对血小板中 NO 敏感的 GC 具有敏化作用。在没有 BAY 41-2272 的情况下应用此浓度的 NO 产生的 cGMP 响应仅是微不足道的。在 100 μmol/L BAY 41-2272 存在下，用 3 μmol/L GSNO 处理后，观察到 cGMP 快速上升至 1000 pmol/109 血小板。

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血小板聚集[2]
我们按照所述制备了洗涤过的人血小板14，并通过浊度法测量了血小板聚集。将血小板悬浮液与试验化合物在37℃下预孵育 10°C 胶原蛋白（0.1-2µg ml-1）诱导血小板聚集。

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血小板聚集[2]
我们按照所述制备了洗涤过的人血小板14，并通过浊度法测量了血小板聚集。将血小板悬浮液与试验化合物在37℃下预孵育 10°C 胶原蛋白（0.1-2µg ml-1）诱导血小板聚集。

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扩散试验[4]
根据Rabinovitch等人（1977）进行了扩散试验。将含有2×106个细胞的腹膜细胞悬浮液离心，并悬浮在1 mL 5 mM葡萄糖的PBS中。将50微升细胞悬浮液分层放在玻璃盖玻片上，在37℃下孵育1小时。盖玻片在PBS中轻轻冲洗，玻璃粘附细胞在2.5%戊二醛中固定，用相差显微镜在400倍放大倍数下检查。计数200个巨噬细胞，并将其分为圆形或扩散型。然后计算巨噬细胞扩散指数（SI）如下：SI=扩散巨噬细胞数量×100）/200，即SI=扩散的巨噬细胞百分比。

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酵母聚糖吞噬试验[4]
根据Pinello等人（2006）进行吞噬试验。将含有2×106个细胞的腹膜细胞悬浮液离心并悬浮在1 mL RPMI培养基中。将细胞分配到六孔平底微量测试板中的圆形玻璃盖玻片（20 mm）上，并在37℃下孵育培养物20分钟。孵育后，吸出培养上清液并去除非贴壁细胞。用PBS冲洗粘附的单层。随后，向培养物中加入1mL含有5%热灭活胎牛血清的RPMI-1640培养基。在1mg/L酿酒酵母酵母聚糖（Sigma）存在下，将培养物在37℃下保持1小时。然后用冷PBS洗涤培养物以去除未内化的颗粒。然后用0.5%戊二醛固定细胞。使用相差显微镜对平均200个巨噬细胞进行计数，以确定吞噬百分比。吞噬指数（PI）计算如下：PI=具有吞噬活性的巨噬细胞数量×100）/200个计数的贴壁细胞，即PI=至少有两个吞噬酵母聚糖颗粒的巨噬细胞的百分比。

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H 2 O 2释放和一氧化氮（NO）产生[4]
使用先前描述的方法在单个巨噬细胞样品中测定H2O2释放和NO产生（Cruz等人，2007）。为了评估H2O2的释放，使用了HRP依赖性酚红氧化微量测定法（Pick&Mizel 1981）。对于该试验，将2.0 x 106个腹膜细胞悬浮在1 mL新鲜制备的酚红溶液中[含有5.5 mM葡萄糖、0.56 mM酚红和8.5 U/mL HRP II型的冰冷PBS]。将100微升细胞悬浮液加入每个孔中，在37℃、5%CO2潮湿的气氛中与或不与PMA（30 nM）一起孵育1小时。将板以150g离心一次3分钟，然后将上清液转移到另一个板上。用10μL氢氧化钠停止反应。用微孔板读数器在620nm处测量吸光度。通过与在RMPI培养基中稀释的已知浓度的H2O2（5-40μM）获得的标准曲线进行比较，将吸光度转换为μM的H2O2（Pick&Keisari 1980）。

此后，用PBS洗涤含有细胞的板三次，将剩余的粘附巨噬细胞在37℃、5%CO2潮湿气氛中在100μL RPMI-1640培养基（补充有10 mM HEPES、11 mM碳酸氢钠、100 U/mL青霉素、100μg/mL链霉素、2 mM L-谷氨酰胺、23 mM L-天冬酰胺、1 mM叶酸、0.1 mM丙酮酸和5%胎牛血清）中培养48小时。孵育后，收集50μL上清液，在室温（RT）下与等体积的Griess试剂（1%磺胺/0.1%萘二胺二盐酸盐/2.5%磷酸）孵育10分钟，以定量亚硝酸盐的积累（Ding等人，1988）。在550nm处测定吸光度。通过与在RPMI培养基中稀释的已知浓度（5-60μM）的亚硝酸钠获得的标准曲线进行比较，将吸光度转换为NO的μM。

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离体和体外腹腔巨噬细胞杀菌活性[4]
为了评估杀菌活性，在细胞与细菌或真菌孵育后使用MTT氧化微量测定法。对于该试验，使用两种方案处理腹腔巨噬细胞，如下所示：（i）在未经处理的动物体外刺激驻留的腹腔细胞（体外），以及（ii）从如本方案中所述处理的动物体内采集细胞（离体）。制备后，将2.5×105个细胞悬浮在200µL RPMI-1640（不含补充剂）中，并分布在96孔板中。然后，金黄色葡萄球菌以10:1（病原体：巨噬细胞）的比例添加病原体，白色念珠菌以2:1的比例添加。共培养物在37℃和5%CO2下孵育2小时。孵育后，将平板离心，收集上清液并储存在-80℃下，用于随后的细胞因子剂量测定。用PBS洗涤细胞颗粒两次，以去除未吞噬的病原体。洗涤后，在室温下加入Triton X-100（1.5%）10分钟，以裂解巨噬细胞并释放病原体。然后用PBS洗涤细胞两次以去除Triton X-100，加入100µL MTT（0.5mg/mL），在室温下避光孵育2小时。孵育后，加入100µL DMSO，再孵育30分钟，将甲氮沉淀释放到上清液中。孵育后，将平板离心（300g，3分钟），并将上清液转移到新的平板上。用酶标仪在λ=570 nm处测定吸光度。从吸光度到细胞死亡百分比的转换是通过以下方程式实现的：1-（样品OD-90%杀灭OD）/（0%杀灭OD-90%杀伤OD）×100。该计算是基于与细胞一起孵育的病原体总数的100-10%的病原体浓度进行的。

Animal treatments [4]
For the in vivo experiments, BAY 41-2272 was diluted in a transcutol, Cremophor-EL and water solution (10/20/70 ratio, vol/vol/vol) to a final concentration of 1 mg/mL, as previously described (Bischoff et al. 2003). The animals were then weighed and the drug doses were adjusted to 0.3, 1.0, 3.0 and 10.0 mg/kg. For in vitro stimulation, BAY 41-2272 diluted in a 0.7% DMSO solution was used at concentrations of 1.0 and 3.0 µM, according to Bischoff et al. (2003). Treatment with BAY 41-2272 was administered intraperitoneally (IP) for 48 h. A negative control group was injected with a saline solution and a positive control group was treated with 4% thioglycolate or Con A (0.5 mg/kg) (Sigma). An additional control group was treated with a dilution solution only.
The ex vivo experiments were performed using resident macrophages or macrophages obtained from mice treated with BAY 41-2272. Treatments with penicillin G (5 kU/kg) and tetracycline (1 mg/kg) or itraconazole (20 mg/kg) were also administered in the infection assays. To evaluate hydrogen peroxide (H2O2) production, an additional in vitro treatment with PMA (30 nM) was performed. Other reagents, treatments and models are described in the following specific methodologies.

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Footpad oedema induction [4]
Animals were anaesthetised and injected subcutaneously (SC) with carrageenan (300 μg/paw in saline) into the right paw. Differences in the sizes of the injected vs. un-injected paws were used as an indicator of inflammation (paw oedema) (Winter et al. 1962). The properties of BAY 41-2272 were assessed by injecting various doses of this drug (0.01-1.0 mg kg-1) IP at 48 h before the administration of carrageenan. Control mice were injected with same volume of a solvent (0.5 mL olive oil). Con A (100 mg kg-1) served as a positive control. Inflammation was assessed at 60-min intervals during a 4-h period.

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Resistance of mice to C. albicans and S. aureus infections [4]
To assess the resistance of the BAY 41-2272-treated animals to C. albicans (ATCC 90028) and S. aureus (ATCC 25923), two models of infection were used as follows: (i) the inoculation of pathogens in the peritoneal cavity followed by survival rate evaluation and (ii) the inoculation of pathogens SC into the footpad of the animals. For the first model, the animals were inoculated IP with 0.5 × 106 C. albicans blastospores or 5 × 106 colony-forming units of S. aureus. Forty-eight hours from inoculation to the establishment of infection, the animals were also treated daily IP with BAY 41-2272 (1 or 3 mg/kg) or itraconazole (20 mg/kg) or penicillin G (5 KU/kg) and tetracycline (1 mg/kg) for three days. The survival rate of the animals was evaluated for 20 days from the first day of inoculation. We attempted to perform survival experiments in mice with less than 12 days of infection, but the results were reliable only for those infected for 20 days. For the second model, the animals were inoculated SC with the same concentrations of pathogens into the footpad of the left paw and the right paw served as the control. At 48 h from inoculation to the establishment of infection, the animals were treated daily IP or intralesionally (inoculated paw) with BAY 41-2272 (1 or 3 mg/kg) or itraconazole (20 mg/kg) or penicillin G (5 KU/kg) and tetracycline (1 mg/eg) for three days. Paw thickness was then measured after seven days from inoculation to assess the development of the lesion or infection.

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Dissolved in 10/20/70 (v/v/v) Transcutol/Cremophor EL/water; 1 mg/kg; p.o.

Female spontaneously hypertensive rats

[1]. BMC Pharmacol . 2001:1:13.

[2]. Nature . 2001 Mar 8;410(6825):212-5.

[3]. J Urol . 2003 Feb;169(2):761-6.

[4]. Mem Inst Oswaldo Cruz . 2015 Feb;110(1):75-85.

[5]. J Pharmacol Exp Ther . 2015 May;353(2):330-9.

BAY 41-2272 is a pyrazolopyridine that is 1H-pyrazolo[3,4-b]pyridine which is substituted by a 2-fluorobenzyl group at position 1 and by a 4-amino-5-cyclopropylpyrimidin-2-yl group at position 3. It is an activator of soluble guanylate cyclase. It has a role as a soluble guanylate cyclase activator, a platelet aggregation inhibitor, a vasodilator agent and an antihypertensive agent. It is a pyrazolopyridine, a member of monofluorobenzenes, an aminopyrimidine and a member of cyclopropanes.

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In summary, using photoaffinity labelling, we identified the region of the cysteines 238 and 243 in the α1 subunit of sGC as the target for NO-dependent sGC stimulators. However, the relevance of the identified region as a regulatory unit remains to be confirmed by mutational analysis and co-crystallization studies.[1]

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In summary, the effects of BAY 41-2272on sGC and the photoaffinity labelling studies suggest the existence of a new NO-independent regulatory site on sGC in the Cys 238 and Cys 243 region of the α1-subunit that modulates the catalytic rate and the responsiveness towards the haem ligand. Our data offer both an approach to understanding the regulation of sGC and a potent new stimulator of sGC, BAY 41-2272, which induces vasodilation without developing nitrate tolerance, antiplatelet activity, and finally reduces mortality. [2]

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Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases. [2]

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Purpose: In cavernous smooth muscle nitric oxide (NO) activates soluble guanylate cyclase, which catalyzes the synthesis of cyclic guanosine 3',5'-monophosphate, leading to smooth muscle relaxation, increased blood flow and penile erection. The pyrazolopyridine derivative BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) was identified and found to stimulate soluble guanylate cyclase in a NO independent manner. We investigated the effect of BAY41-2272 on human and rabbit corpus cavernosum. [3]

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In the in vivo models of infection, BAY 41-2272 was more effective in responding to the fungi compared to the bacteria. Thus, we investigated the in vitro and ex vivo microbicidal activities of peritoneal macrophages against the same pathogens. Our results showed that the in vitro treatment enhanced the microbicidal activities of the peritoneal macrophages against C. albicans and S. aureus and these increases were even more significant ex vivo. These results confirm the potential of BAY 41-2272 for treating fungal infections, specifically C. albicans. We also show that this treatment is effective in promoting S. aureus killing. These data support our hypothesis that the apparent non-resolution of S. aureus infection in vivo involves the maintenance of inflammation generated by the pathogen and potentiated by BAY 41-2272.

This increase in microbicidal activity is probably related to the oxidative burst, reactive nitrogen production and phagocytosis. However, we cannot exclude the possible involvement of other processes, such as phagosome pH acidification and lysosomal/granular enzyme release (Sokolovska et al. 2012), in addition to the participation of other cells. Importantly, the extensiveness of the ex vivo response indicates the relevance of chemical mediators and cells present in the physiological environment to the activation and modulation of phagocyte responses. It is likely that the action of BAY 41-2272 on other immune cells creates an environment with significantly more stimuli for macrophage activation. These data, considering a complex physiological system, provide new evidence in support of the notion that BAY 41-2272, or its pathway (sGC-cGMP), can be used as a treatment for some infections, especially in immunocompromised patients. It is important to emphasise that the cardiovascular effects of BAY 41-2272 (Thorsen et al. 2010, Joshi et al. 2011) did not limit its in vivo application.

We conclude that BAY 41-2272 causes a pro-inflammatory effect, activating mononuclear phagocytes (peritoneal macrophages). Moreover, treatment with BAY 41-2272 significantly increases mouse responses to C. albicans (in vivo and in vitro) and S. aureus (in vitro), improving peritoneal macrophage microbicidal activities against these pathogens. Our group is actively investigating the pharmacological aspects of BAY 41-2272, aiming to clarify its signalling pathways and elucidate its effects on mononuclear phagocytes. With this information, we intend to develop novel treatments to increase the quality of life of patients susceptible to infections, especially those with PID. [4]

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BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) is an sGC stimulator that has been shown to produce antiproliferative and vasodilatory effects (Evgenov et al., 2006), as well as to potentiate erectile responses (Bischoff et al., 2003) and relax the CC of humans and animals (Baracat et al., 2003; Kalsi et al., 2003; Claudino et al., 2011). This compound was suggested to have a high potency and no PDE inhibitory activity (Stasch et al., 2001). In a NO-deficient rat model, long-term oral treatment with BAY 41-2272 improved the impaired cavernosal relaxation (Claudino et al., 2011). In a previous investigation of the effects of BAY 41-2272 in mice CC, our group showed that this compound reverses the increased NADPH oxidase-dependent superoxide generation by decreasing protein expression of its subunits gp91phox and p22phox (Teixeira et al., 2007).

BAY 41-2272, but not a PDE-5 inhibitor, enhances the nitrergic relaxation response in anococcygeus and retractor penile muscle (Kalsi et al., 2004) (ideal tissues to study nitrergic neurotransmission), which are impaired in streptozotocin-induced diabetic rats (Cheah et al., 2002). These data suggest that endogenous NO from nitrergic nerves is decreased in diabetes and show that sGC stimulators are more effective than PDE-5 inhibitors in the treatment of diabetes-induced ED.

To the best of our knowledge, there are no previous studies investigating the action of BAY 41-2272 on diabetic CC. Additionally, few studies have been performed using db/db−/− mice to investigate ED, even though these animals have shown altered vasoreactivity consistent with impaired cavernosal relaxation and penile veno-occlusive disorder. The db/db−/− mice lack leptin receptors, and this deficiency contributes to the development of both diabetes and obesity. Therefore, these mice are widely considered an appropriate model for DM2, which has been used for the study of DM2-associated ED (Luttrell et al., 2008). In addition, db/db−/− mice develop hyperglycemia and hyperinsulinemia, the latter of which raises resting sympathetic output and contributes to impaired cavernosal relaxation (Anderson et al., 1991).

In this study, we examine the effect of BAY 41-2272 on relaxation of the CC from db/db−/− obese DM2 mice and their lean db/+ counterparts in response to vasodilatory agonists and the effects of the drug on markers of oxidative stress in these animals.

Our data showed that in diabetic, obese (db/db−/−) mice BAY 41-2272 ameliorated impaired endothelial and nitrergic cavernosal relaxation by elevating the intracellular cGMP concentration, preventing elevated expression of NADPH oxidase enzyme subunits, and decreasing superoxide formation. Although the pathogenesis of ED in diabetes is multifactorial, vascular dysfunction is a major contributor to the high incidence of ED in men with diabetes (Chu and Edelman, 2002). [5]

C20H17FN6

360.39

360.149

C, 66.65; H, 4.75; F, 5.27; N, 23.32

256376-24-6

9798973

White to off-white solid powder

1.5±0.1 g/cm3

496.1±45.0 °C at 760 mmHg

253.8±28.7 °C

0.0±1.3 mmHg at 25°C

1.767

1.99

83.24

1

6

4

27

517

0

FC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])N1C2=C(C([H])=C([H])C([H])=N2)C(C2=NC([H])=C(C(N([H])[H])=N2)C2([H])C([H])([H])C2([H])[H])=N1

ATOAHNRJAXSBOR-UHFFFAOYSA-N

InChI=1S/C20H17FN6/c21-16-6-2-1-4-13(16)11-27-20-14(5-3-9-23-20)17(26-27)19-24-10-15(12-7-8-12)18(22)25-19/h1-6,9-10,12H,7-8,11H2,(H2,22,24,25)

5-cyclopropyl-2-[1-[(2-fluorophenyl)methyl]pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-amine

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: ≥ 1.75 mg/mL (4.86 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 17.5 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.75 mg/mL (4.86 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 17.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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: ≥ 1.75 mg/mL (4.86 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 17.5 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。