CN111686116A - 孕甾烷衍生物在制备破骨细胞分化抑制剂中的应用 - Google Patents
孕甾烷衍生物在制备破骨细胞分化抑制剂中的应用 Download PDFInfo
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Abstract
本发明公开了孕甾烷衍生物在制备破骨细胞分化抑制剂中的应用。孕甾烷衍生物在制备破骨细胞分化抑制剂中的应用，所述的孕甾烷衍生物，其结构式如式1所示。本发明通过试验发现，孕甾烷衍生物对破骨细胞的分化具有直接而显著地抑制作用，因此可以作为破骨细胞分化抑制剂类药物，用于治疗破骨细胞分化引起的各类疾病，如骨质疏松症。
Description
技术领域
本发明属于生物医药领域，具体涉及孕甾烷衍生物在制备破骨细胞分化抑制剂中的应用。
背景技术
骨质疏松是一种全身性骨代谢疾病，随着人类寿命的延长以及人口老龄化速度的加快，骨质疏松症发病率逐年升高，已成为影响人们生活质量的重要慢性病之一，成为全球公共健康问题。据2014年美国骨矿盐研究学会(ASBMR)年会上统计，骨质疏松性骨折是21世纪重要的慢性病之一，全球1/2的老年女性和1/3的老年男性将会发生骨质疏松性骨折。因此，开发有效的抗骨质疏松药物对于提高人类特别是老年人生活质量，减轻社会经济压力具有重要意义。
骨质疏松症是一种以骨量减少、骨组织微结构破坏、骨脆性增加和易于骨折为特征的代谢性骨病。目前抗骨质疏松药物治疗的作用机制主要分为两大类：抗骨吸收和促进骨形成。抗骨吸收涉及的靶点或通路主要有OPG/RANKL/RANK信号通路、组织蛋白酶K、C-src激酶。促进骨形成的相关靶点或通路有Wnt/β-catenin信号通路、骨形成蛋白、Activin信号通路以及甲状旁腺激素PTH。目前临床上抗骨吸收药物主要有双膦酸盐类(阿仑膦酸钠、伊班膦酸钠等)，雌激素受体调节剂(雷洛昔芬)，RANKL抑制剂(地诺单抗)，组织蛋白酶K抑制剂(奥当卡替)，src激酶抑制剂(Saracatinib)。促骨形成的药物主要有甲状旁腺激素类似物(特立帕肽PTH1-34)，Wnt信号通路调控剂(AMG785、BHQ880)等。这些药物虽然能在一定程度上提高骨质疏松患者的骨密度，但是不能显著降低非典型性骨折的风险，同时存在各种副作用，如双膦酸盐类药物会引起下颌骨坏死，雷洛昔芬会引起静脉栓塞。因此，我们亟需研究更有效、副作用更小的抗骨质疏松症治疗药物。
OPG/RANKL/RANK信号系统被确认是骨代谢中一个重要的信号通路，RANKL(receptor activator ofNFκB ligand)与破骨细胞前体细胞或破骨细胞表面上的RANK结合，促进破骨细胞的分化和激活，并抑制其凋亡；骨保护素(osteoprotegerin，OPG)阻止RANKL与RANK的结合，起负调节作用。RANKL和OPG比率的动态平衡在骨吸收中起重要的控制作用。OPG-RANKL-RANK信号系统的研究成果是骨分子生物上的一个里程碑，不仅阐明了OPG-RANKL-RANK信号系统与破骨细胞的发育、成熟之间的存在的多种信号偶联，以及破骨细胞骨吸收与成功细胞成骨作用的偶联失衡，而且为骨质疏松症的发生机制奠定了一定的基础，为骨质疏松症的靶向治疗开辟了新的思路。目前针对OPG-RANKL-RANK系统，美国安进公司已开发出单抗药物地诺单抗，用于实体瘤骨转移患者中骨骼相关事件的预防，现在也在研究用于治疗骨质疏松症。随着OPG-RANKL-RANK晶体结构的解析，靶向OPG-RANKL-RANK抗骨质疏松的小分子多肽类药物成为研究热点。因此，靶向OPG-RANKL-RANK已被广泛证实是抗骨质疏松治疗的重要策略。
发明内容：
本发明的目的是提供孕甾烷衍生物或其药用盐在制备破骨细胞分化抑制剂中的应用。
破骨细胞分化活性测试显示化合物孕甾烷衍生物可以剂量依赖抑制破骨细胞的成熟分化，IC50值为1.73μM。细胞毒性测试CC50为106.2μM。说明孕甾烷衍生物具有很好的抑制活性，具有潜在开发抗骨质疏松药物的价值。
因此，可以将化合物孕甾烷衍生物，或其药用盐用于制备破骨细胞分化抑制剂：
所述的孕甾烷衍生物，其结构式如式1所示：
所述的破骨细胞分化抑制剂为抗骨质疏松性药物。
所述的破骨细胞分化抑制剂为口服剂型或外用剂型。
所述的破骨细胞分化抑制剂包括活性成分化合物孕甾烷衍生物和药学上可接受的药用辅料。
本发明通过试验发现，化合物孕甾烷衍生物对破骨细胞的分化具有直接而显著地抑制作用，因此可以作为破骨细胞分化抑制剂类药物，用于治疗破骨细胞分化引起的各类疾病，如骨质疏松症。
附图说明：
图1是孕甾烷衍生物剂量依赖抑制破骨细胞的成熟分化；
图2是孕甾烷衍生物抑制曲线及IC50值；
图3是孕甾烷衍生物细胞毒性测试。
具体实施方式：
实施例1：破骨细胞分化抑制活性测试
1、实验方法
细胞培养：取8周龄大小的C57BL/6小鼠股骨髓细胞，用含有10ng/mL M-CSF的MEM-α完全培养基(含10％胎牛血清(FBS),100U/mL青霉素和100μg/mL链霉素),在二氧化碳培养箱中于37℃条件下培养24h，弃掉培养液，用磷酸盐缓冲液(PBS)缓慢地冲洗细胞1～2次，去除悬浮细胞，然后用4mL MEM-α完全培养基将贴壁细胞轻轻吹下，于1000RPM/min离心10min，获得小鼠骨髓巨噬细胞(BMMs)。
诱导破骨细胞形成：用含10ng/mLM-CSF细胞因子的MEM-α完全培养基将BMMs细胞稀释成密度为1×105个细胞/mL的细胞悬液，并接种于96孔板中，每孔100μL。培养24h贴壁后，弃培养液，用含100ng/mL RANKL和10ng/mL M-CSF细胞因子的MEM-α完全培养基与BMMs细胞共培养4d诱导破骨细胞的形成。每2天换一次培养液。诱导培养结束后在倒置显微镜下观察破骨细胞形成情况并拍片保存。
TRAP染色及破骨细胞数量的测定：用含10ng/mL M-CSF细胞因子的MEM-α完全培养基将BMMs细胞稀释成密度为1×105个细胞/mL的细胞悬液，并接种于96孔板中，每孔100μL。培养24h贴壁后，弃培养液，用含100ng/mL RANKL，10ng/mL M-CSF和含不同终浓度化合物的MEM-α完全培养基与BMMs细胞共培养4d诱导破骨细胞的形成。每2天换一次培养液，每个处理3个重复。在相同条件下，只含有与药物溶液等体积的DMSO培养液的细胞作为诱导对照组。处理结束后，弃培养液，PBS清洗细胞，4％多聚甲醛细胞固定液将细胞固定10min。然后用抗酒石酸酸性磷酸酶(TRAP)染色液对BMMs细胞分化形成的破骨细胞进行染色。倒置相差显微镜下观察，TRAP染色阳性多核细胞(>3个核)计为破骨细胞。培养板中的各孔细胞随机选10个不同视野拍片保存,并用Image-Pro Plus 5.0软件计数TRAP染色阳性细胞数取平均值，计算相应药物浓度下的破骨细胞抑制率。
将药物浓度及其相对应的破骨细胞抑制率数值输入GraphPad Prism 8软件中，以药物浓度的Log值为横坐标，抑制率纵坐标，绘制IC50抑制曲线，选取软件自带的非线性拟合曲线法计算IC50值。
细胞毒性测试方法：取BMMs细胞，用含10ng/mLM-CSF细胞因子的MEM-α完全培养基将细胞密度稀释成1×105个细胞/mL的细胞悬液，并接种于96孔板中，每孔100μL。培养24h后，加入含不同终浓度化合物进行干预，空白对照组用同等体积的DMSO溶液代替，每个处理3个重复。继续培养24h后，弃去培养基，采用MTT法测定活细胞数，以570nm处波长测得的OD值进行评价，计算相应药物浓度下细胞生长抑制率。
将药物浓度及其相对应的细胞生长抑制率数值输入GraphPad Prism 8软件中，以药物浓度的Log值为横坐标，抑制率纵坐标，绘制CC50抑制曲线，选取软件自带的非线性拟合曲线法计算CC50值。
2、实验结果
结果如图1-3所示，从图1-3可以看出，破骨细胞分化活性测试显示孕甾烷衍生物AO-774/41465570可以剂量依赖抑制破骨细胞的成熟分化，IC50值为1.73μM。细胞毒性测试CC50为106.2μM。说明化合物AO-774/41465570具有很好的抑制活性，具有潜在开发抗骨质疏松药物的价值。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.孕甾烷衍生物，或其药用盐在制备破骨细胞分化抑制剂中的应用
所述的孕甾烷衍生物，其结构式如式1所示：
2.根据权利要求1所述的应用，其特征在于，所述的破骨细胞分化抑制剂为抗骨质疏松性药物。
3.根据权利要求1所述的应用，其特征在于，所述的破骨细胞分化抑制剂为口服剂型或外用剂型。
4.根据权利要求1、2或3所述的应用，其特征在于，所述的破骨细胞分化抑制剂包括活性成分化合物孕甾烷衍生物或其药用盐，和药学上可接受的药用辅料。
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| CN106902124A (zh) * | 2017-01-18 | 2017-06-30 | 南京大学 | 齐墩果酸类衍生物的应用 |
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