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DrugDosage/
g·kg-1/x(day)
Change in
body weight/g
Prolonged
survival/%
Whole prescription5.7/9-1.14.0
Aloe + Indigo7.7/10-1.517.0
Aloe7.6/10+0.33.8
Indigo5.6/10+0.716.3
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 Anticancer activity of different compositions for L7212 leukemia

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DrugDosage/
g·kg-1/x(day)
Change in
body weight/g
Prolonged
survival/%
Whole prescription5.7/9-1.14.0
Aloe + Indigo7.7/10-1.517.0
Aloe7.6/10+0.33.8
Indigo5.6/10+0.716.3
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TumorDosage
time
Body
wt/g
Tumor wt or survival dayInhibition/%
ExperimentControl
S-18010+1.01.29 g1.47 g12.1
W-2567+14.96.56 g7.14 g8.1
EAC10+6.022.1 days19.8 days11.7
L-6156+0.57.3 days7.3 days0
L-721210+0.712.1 days10.4 days16.3 (P < 0.01)
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 Inhibitory effect of indigo on various tumor models

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TumorDosage
time
Body
wt/g
Tumor wt or survival dayInhibition/%
ExperimentControl
S-18010+1.01.29 g1.47 g12.1
W-2567+14.96.56 g7.14 g8.1
EAC10+6.022.1 days19.8 days11.7
L-6156+0.57.3 days7.3 days0
L-721210+0.712.1 days10.4 days16.3 (P < 0.01)
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Drug sampleDosage
/g·kg-1
Change in
body wt/g
Prolonged
survival life/day
P
value
FI (hot water ext)15.5-1.60-
RI (water ext)28.5-1.20-
RI (alc. ext)22.9-0.45.0-
Indigo (powder)5.6-0.214.6< 0.01
Indigo (water ext)30.4+1.70-
Indigo (alc. ext)28.5+0.71.9-
Indigo (hot acid
water ext)
2.7018.7< 0.01
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 Inhibitory effect of various extraction part of indigo on mouse leukemia 7212

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Drug sampleDosage
/g·kg-1
Change in
body wt/g
Prolonged
survival life/day
P
value
FI (hot water ext)15.5-1.60-
RI (water ext)28.5-1.20-
RI (alc. ext)22.9-0.45.0-
Indigo (powder)5.6-0.214.6< 0.01
Indigo (water ext)30.4+1.70-
Indigo (alc. ext)28.5+0.71.9-
Indigo (hot acid
water ext)
2.7018.7< 0.01
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Compd.RDosage mg·kg-1/x (days)TWI/%
1H100/945.4
3CH3105/941.5
4C2H5110/957.9
5n-C3H7116/953.9
6n-C4H9121/941.1
7n-C5H11134/915.7
8n-C10H21150/919.8
9n-C14H29174/925.2
10n-C18H37195/912.7
11121/9-6.0
12COOC2H5127/948.7
13CH2COOH20/9 (ip)38.7
14Benzyl136/935.1
15Acetyl116/947.7
16Formyl111/929.0
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 Structure and activity of N1′-substituted indirubins

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Compd.RDosage mg·kg-1/x (days)TWI/%
1H100/945.4
3CH3105/941.5
4C2H5110/957.9
5n-C3H7116/953.9
6n-C4H9121/941.1
7n-C5H11134/915.7
8n-C10H21150/919.8
9n-C14H29174/925.2
10n-C18H37195/912.7
11121/9-6.0
12COOC2H5127/948.7
13CH2COOH20/9 (ip)38.7
14Benzyl136/935.1
15Acetyl116/947.7
16Formyl111/929.0
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Compd.RR′Inhibition/%Compd.RR′Inhibition/%
W-256aLLCbW-256aLLCb
3HCH341.550.020CH3H51.538.2
4HC2H557.938.221C2H5H54.644.1
5Hn-C3H753.927.522n-C3H7H15.0-
17CH3H15.0-23CH3H67.650.0
18C2H5H16.3-24C2H5H48.538.2
19CH3CH346.942.925n-C3H7H12.5-
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Structure and activity of compounds of substituted indirubin, indigotin and isoindigotin. a: Walker 256 carcinosarcoma; b: Lewis lung carcinoma

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Compd.RR′Inhibition/%Compd.RR′Inhibition/%
W-256aLLCbW-256aLLCb
3HCH341.550.020CH3H51.538.2
4HC2H557.938.221C2H5H54.644.1
5Hn-C3H753.927.522n-C3H7H15.0-
17CH3H15.0-23CH3H67.650.0
18C2H5H16.3-24C2H5H48.538.2
19CH3CH346.942.925n-C3H7H12.5-
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由当归芦荟丸研制的靛玉红和甲异靛
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郭宗儒
药学学报 | 新药发现与研究实例简析 2024,59(6): 1894-1896
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药学学报 | 新药发现与研究实例简析 2024, 59(6): 1894-1896
由当归芦荟丸研制的靛玉红和甲异靛
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郭宗儒
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  • 中国医学科学院、北京协和医学院药物研究所, 北京 100050
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出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2020-0769
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郭宗儒. 由当归芦荟丸研制的靛玉红和甲异靛. 药学学报, 2024 , 59 (6) : 1894 -1896 . DOI: 10.16438/j.0513-4870.2020-0769
. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1894 -1896 . DOI: 10.16438/j.0513-4870.2020-0769
新药创制是复杂的智力活动, 涉及科学研究、技术创造、产品开发和医疗效果等多维科技活动。每个药物都有自身的研发轨迹, 而构建化学结构是最重要的环节, 因为它涵盖了药效、药代、安全性和生物药剂学等性质。本栏目以药物化学视角, 对有代表性的药物的成功构建, 加以剖析和解读。
甲异靛是我国研制的1.1类化学创新药物, 也是一个由中药复方研制新分子实体(NME) 的成功范例。本文简明叙述了由10味中药组成的当归芦荟丸出发, 根据中医药治疗发现对白血病的治疗效果, 然后通过拆解方剂组成, 发现了其中的青黛是治疗白血病药效成分所在, 进而在青黛中发现靛玉红是有效的单体分子, 并且临床证明了靛玉红治疗白血病的有效性而批准上市。为了克服靛玉红的药代和物化性质的缺点, 对双吲哚啉酮类结构骨架进行了修饰改造, 从而研制出治疗慢性粒细胞白血病新药甲异靛。尽管研制过程中不清楚靛玉红和甲异靛的分子靶标和作用机制, 但用细胞表型变化和动物肿瘤模型实验, 确实发现了非细胞毒性作用的抗白血病药物。本世纪的深入研究揭示了靛玉红和甲异靛对激酶作用的分子机制。
(编者按)
当归芦荟丸为中药古方, 是由大黄、黄柏、黄芩、黄连、栀子、龙胆草、芦荟、青黛、当归和木香等10味药炼制而成, 主治肝经实火, 头晕目眩。近代临床发现当归芦荟丸具有消炎杀菌和抗肿瘤的功效。中国医学科学院血液内科用当归芦荟丸治疗慢性粒细胞型白血病显示有一定疗效, 遂进行了深入研究。
研究者为了简化组方和浓集有效成分, 考虑到方剂中的芦荟和青黛有抗菌消炎的作用, 设计实验分组如下: 全方(whole prescription, WP)、芦荟+青黛(Aloe+Indigo, A+I)、芦荟(A)、青黛(I) 等4组, 经灌胃途径给药荷瘤试验动物(小鼠白血病L7212模型), 日给一剂, 连续9或10日, 评价抑制肿瘤生长作用。表 1列出了实验结果。
表 1结果提示, 芦荟+青黛对L7212白血病有一定疗效, 生存时间延长了17%, 然而单用芦荟组无效。而青黛组呈现疗效生存时间延长16.3%, 说明芦荟+青黛组的效果来自青黛。全方组未能呈现效果, 可能是由于其中青黛含量较少, 剂量不足所致。
为了探索对青黛敏感的瘤株, 选用了小鼠肉瘤180 (S-180)、艾氏腹水癌(EAC)、Walker256癌肉瘤(W-256)、小鼠白血病615 (L-615) 和小鼠白血病7212 (L-7212) 等移植性肿瘤模型, 每日灌胃1次, 剂量均为5 g·kg-1, 结果列于表 2表 2数据提示, 青黛灌胃小鼠只对白血病L-7212有抑制作用, 对其他瘤株未呈现活性。
青黛是由大青叶(Folium isatidis, FI) 和板蓝根(Radix isatidis, RI) 与石灰混合发酵而制得, 为了确定有效成分, 研究了其原料大青叶、板蓝根及青黛的不同萃取方法对活性影响。包括有沸水萃取(hot water ext)、常温水萃取(water ext)、乙醇萃取(alc. ext)、或酸性热水萃取(acid water ext) 等萃取物, 灌胃给L-7212小鼠, 观测体重变化和存活的延长时间, 结果列于表 3表 3的结果表明, 只有热酸性水萃取物冷却后析出的深色固体呈现活性, 而大青叶或板蓝根或青黛用水或醇萃取物都没有活性。提示活性成分应是难溶于水和醇的有机物, 可溶于酸性水液中。化学结构应有共轭系统和生色团, 后经分析证明是靛玉红(1, indirubin) (中国医学科学院分院六室. 当归芦荟丸抗白血病实验研究的初步报告. 中草药, 1984, 15: 534-536)。
中药青黛可由不同科属的大青叶和板蓝根炮制加工而得, 其性味咸寒, 有清热解毒的功效。陈棣华等从蓼科植物蓼蓝(Polygonum tinctorium) 炮制加工的青黛分离得到靛玉红(1, indirubin)、靛蓝(2, indigotin)、N-苯基-2-萘胺、β-谷甾醇(β-sitesterol) 和虫漆蜡醇(laccerol) 等多种单体天然化合物(陈棣华, 谢晶曦. 中药青黛的化学成分. 中草药, 1984, 15: 6-8)。
靛玉红是两个吲哚啉酮经2, 3′双键偶联形成的二聚物, 分子呈平面的共轭系统, 为暗红色针状结晶。经药理实验研究, 表明对多种移植性动物肿瘤有抑制作用, 破环白血病细胞。深入研究作用靶标为DNA聚合酶, 抑制了DNA的聚合, 对蛋白质的合成没有直接影响, 此外, 靛玉红还可增强动物单核巨噬细胞的吞噬能力。靛玉红可口服吸收, 生物利用度F = 46%。后经临床研究, 靛玉红可治疗慢性粒细胞白血病, 有效率为87%。主要不良反应是胃肠道的刺激与不适, 对骨髓有轻度影响(中国医学科学院血液学研究所, 分院附属医院, 基础医学研究所. 靛玉红治疗慢性粒细胞白血病的临床于实验研究. 中华内科杂志, 1979, 18: 83-87)。
后来证明靛玉红是细胞周期依赖激酶(CDKs) 抑制剂, 引起细胞凋亡, 可抑制白血病细胞等8种肿瘤细胞。靛玉红与CDK复合物单晶衍射分析提示, 分子定位于CDK激酶的ATP结合位点, 含有3个氢键结合(Hoessel R, Leclerc S, Eedicott JA, et al. Indirubin, the active constituent of a Chinese antileukemia medicine, inhibits cyclin-dependant kinases. Nat Cell Biol, 1999, 1: 60-67)。
靛玉红的水溶性很低, 限制了胃肠道吸收。水溶性低的原因应是在共轭平面结构的分子间形成氢键, 导致晶格能高, 这可由靛玉红的熔点高达365 ℃得到印证。由于靛玉红的N1上氢原子可与2′的羰基氧形成分子内氢键, 不是高晶格能的起因, 很可能是N1′的氢原子与另一靛玉红形成分子间氢键。因而在N1′上作烷基取代, 合成的化合物列于表 4。用移植Walker癌肉瘤大鼠以同等摩尔体质量灌胃给药连续9天, 测定化合物对肿瘤生长的抑制率(TWI)。
化合物3为N′-甲基取代(mp 296 ℃) 和N′-乙基(4) 的mp 196 ℃, 熔点显著低于靛玉红, 提示降低了晶格能, 因而溶解度提高, 4的抑瘤活性强于1, 当R大于正丙基时活性降低, N1′-酰化或提高极性的基团都没有提高活性的趋势(籍秀娟, 张福荣. 靛玉红类化合物的抗肿瘤作用和构效关系的研究. 药学学报, 1985, 20: 137-139; 吴克美, 张曼云, 方政等. 抗白血病药物靛玉红乙基靛蓝和异靛蓝衍生物的合成. 药学学报, 1985, 20: 821-826)。
青黛中含有的靛玉红和靛蓝, 推测是大青叶中的化学物在碱性环境中缩合产生的, 靛蓝是2, 2′偶联的对称性分子, 溶解性更低, 可能是活性低下的原因。基于引入低碳烷基可提高靛玉红抗肿瘤活性的事实, 设计合成了靛蓝和异靛蓝(17, isoindigotin) 为骨架的改构物, 列于表 5中。
用等摩尔剂量灌胃大鼠Walker 256癌肉瘤和Lewis肺癌模型评价化合物活性, 结果表明: ①只在靛玉红的N1作取代的化合物1718活性很低, 甚至低于靛玉红, 这可解释只消除靛玉红的分子内氢键对药效是不利的; N1和N1′都被甲基取代的化合物19与靛玉红活性相近。②靛蓝的N1甲基或乙基取代(2021) 对两种肿瘤模型的活性与相应的靛玉红活性相近, 但正丙基取代物的活性降低。③异靛蓝的N1甲基或乙基取代(2324) 的活性比相应的靛玉红和靛蓝化合物抑制Walker 256癌肉瘤的活性明显提高。
进一步对高活性化合物进行药代动力学和安全性实验研究, 全面比较高活性化合物的药效、药代和物化性质, 确定了化合物23为优选的候选物, 定名为甲异靛(meisoindigo), 经系统的临床前研究和III期临床试验研究, 表明甲异靛是口服治疗慢性粒细胞白血病的有效药物, 于1992年经我国卫生部批准上市。
甲异靛研制于上世纪80年代, 当时的肿瘤细胞/分子生物学尚处开始阶段。本世纪以来的研究表明, 甲异靛可诱导急性髓性白血病(AML) 的髓细胞分化和凋亡, 使细胞增殖停止在G0/G1期。可下调抗凋亡的Bcl-2蛋白表达, 而上调前凋亡蛋白Bak和Bax, 以及和细胞周期相关的蛋白p21和p27。甲异靛还下调端粒酶逆转录酶的表达, 因而可提高常规化疗药物的细胞毒作用(Lee CC, Lin CP, Lee YL, et al. Meisoindigo is a promising agent with in vitro and in vivo activity againt human acute myeloid leukemia. Leuk Lymphoma, 2010, 51: 897-905)。此外还发现甲异靛可抑制人结直肠癌细胞-29, 并证明是抑制了糖原合成酶激酶(GSK)-3β磷酸化的环节(Zuo MX, Li Y, Wang HB, et al. The antitumour activity of meisoindigo against human colorectal cancer HT-29 cells in vitro and in vivo. J Chemother, 2008, 20: 728-733)。
2024年第59卷第6期
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doi: 10.16438/j.0513-4870.2020-0769
  • 首发时间:2025-11-26
  • 出版时间:2024-06-12
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多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
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