云南地不容中异喹啉类生物碱镇痛抗炎构效关系及其经皮给药增效减毒研究

Most of isoquinoline alkaloids displays remarkable activities of analgesia and anti-inflammation, which are related to the mechanism of acting on multi-ion channel and synaptic transmission，and are the prospective lead compounds for developing analgesics and anti-inflammatory drugs with non-addictive and low side-effect. Based on our preliminary study, a series of isoquinoline alkaloids extracted from Stephania yunnanensis H. S. Lo and their structure-modification products (sinoacutine , crebanine, 2Br- crebanine, stephanine, etc.) were applied to compare the activities of analgesia and anti-inflammation, and to characterise the their structure-activity/acute toxicity relationship by using animal models of outer peripheral/central pain, inflammation and LD50 test; Then, 1~2 compounds are screened to investigate the mechanism by taking animal tests of anti-inflammatory and analgesia and by determing the related biochemical factors (BDNF, 5-HT, β-EP, PGE2 etc.) and iNOS expression. By adapting pharmacokinetics-pharmacodynamics model, the differences of the onset /duration time, the effect intensity of anti-inflammatory and analgesia, and the animal pharmacokinetic features of the screened compounds between transdermal and oral administration will be compared, the possible principle of toxicity and activity due to defferent concentration of issue drug when administrated by different routes (local / systemic) will be speculated. The purpose is to provide experimental and theoretical basis for screening anti-inflammatory and analgesic drug candidates from isoquinoline alkaloids, analyzing the relationship and mechanism of analgesic efficacy and toxicity(false positive), providing reasonalbe drug delivery system for enhancing of efficacy and reducing of toxicity.

多种异喹啉类生物碱具有显著镇痛与抗炎活性，与其具有影响突触传递与多种离子通道等机制有关，是有前景的非成瘾低副作用镇痛抗炎先导化合物。其镇痛作用与可能的中枢抑制毒性反应的关系也值得探究。本课题利用前期获得的云南地不容异喹啉类生物碱及其结构修饰产物（青风藤碱、克班宁、2Br-克班宁、千金藤碱等），采用中枢/外周疼痛、炎症动物模型与LD50测试，分析其结构与镇痛抗炎活性/毒性关系；筛选高效低毒成分通过疼痛与炎症动物实验、相关因子（BDNF、5-HT、β-EP、PGE2等）及iNOS表达检测，探讨作用机制；通过药动/药效结合模型，比较该成分经皮与口服给药镇痛抗炎起效/持续时间与强度差异，结合血浆及局部组织药物动力学特征，探究局部与全身给药时不同组织药量对药效/毒性作用的规律。为在异喹啉类生物碱中筛选镇痛抗炎药物，探讨镇痛与毒性（假阳性）的关系及机制，选择合理的给药系统增效减毒提供实验与理论依据。

对从云南地不容与临沧地不容中提取（结构修饰）的3类型8个异喹啉生物碱：阿朴菲型克班宁Cre及其衍生物2Br-Cre与3Br-Cre、千金藤碱Step、异可利定Isoc、荷包牡丹碱Dic，吗啡烷型青风藤碱Sino、原小檗碱型四氢巴马汀L-Thp，进行镇痛抗炎活性筛选、作用机制与经皮给药增效减毒研究。① Cre、Sino、Dic、Isoc灌胃LD50 (mg/kg)分别为108、598、633、276 ②以青藤碱、L-Thp为对照，采用二甲苯致耳肿胀、棉球肉芽肿、角叉菜致足趾肿胀、热板法、醋酸扭体法等动物模型，比较上述8个生物碱的镇痛与抗炎活性，结果显示Cre、2Br-Cre、Sino、Step具有显著活性，并分析结构与活性/毒性关系；③采用CFA致大鼠关节炎、甲醛致痛、坐骨神经缩窄损伤神经疼痛等模型，验证了Cre和Sino贴剂经皮给药有显著镇痛抗炎活性；Sino对小鼠急性肺损伤有显著改善作用；④检测了该4个成分在模型动物血浆、关节与肺组织、LPS 诱导RAW264.7 细胞中炎性与疼痛因子NO、IL-6、IL-1β、TNF-α、MPO、BDNF、5-HT、PGE2等水平，确证Sino与2Br-Cre抗炎镇痛活性高且毒性低，Cre与Step活性高但毒性也较高。选择Sino与Cre深入研究； ⑤对iNOS、NF-κB 和MAPK 信号通路影响的研究，提示Sino抗炎作用与抑制PI3K/Akt/NF-κB通路和MAPK 中JNK 信号通路有关； ⑥Sino静注与经皮给药药动学研究显示符合二室模型，AUC0-∞与t1/2β具性别差异（P<0.01），雄鼠消除更快；大鼠肝、肾组织中分布较高，蛋白结合率高(80%)，原型经尿排泄少(<5%)且雌鼠比雄鼠高(P<0.01)。⑦微透析研究Cre贴剂皮下局部药动学为非房室模型，MRT0-t为13.0h。⑧大鼠佐剂性关节炎模型的血浆PK-PD模型研究结果显示Cre经皮和灌胃给药的模型参数E0 为291.0和277.3 , IC50为0.084和0.155（ng/L）；经皮给药t1/2β为19.3h，灌胃t1/2为2.8h，经皮给药显著延长持续时间，提高生物利用度；综上Sino 与Cre是十分值得深入研究开发的经皮给药抗炎镇痛活性成分。研究为从异喹啉生物碱中筛选镇痛抗炎药物，探讨毒性与镇痛关系及机制、经皮给药系统增效减毒提供实验与理论依据。

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