CN116509868A - Vs6766联合bay293的应用及药物组合物 - Google Patents
Vs6766联合bay293的应用及药物组合物 Download PDFInfo
- Publication number
- CN116509868A CN116509868A CN202310808713.XA CN202310808713A CN116509868A CN 116509868 A CN116509868 A CN 116509868A CN 202310808713 A CN202310808713 A CN 202310808713A CN 116509868 A CN116509868 A CN 116509868A
- Authority
- CN
- China
- Prior art keywords
- bay293
- kras
- group
- drug
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- WEGLOYDTDILXDA-OAHLLOKOSA-N CNCc1ccccc1-c1csc(c1)[C@@H](C)Nc1nc(C)nc2cc(OC)c(OC)cc12 Chemical compound CNCc1ccccc1-c1csc(c1)[C@@H](C)Nc1nc(C)nc2cc(OC)c(OC)cc12 WEGLOYDTDILXDA-OAHLLOKOSA-N 0.000 title claims abstract description 100
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 74
- 206010009944 Colon cancer Diseases 0.000 claims description 36
- 238000011282 treatment Methods 0.000 claims description 27
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 102100030708 GTPase KRas Human genes 0.000 abstract description 71
- 101000584612 Homo sapiens GTPase KRas Proteins 0.000 abstract description 71
- 230000000694 effects Effects 0.000 abstract description 44
- 239000003112 inhibitor Substances 0.000 abstract description 39
- 208000005016 Intestinal Neoplasms Diseases 0.000 abstract description 21
- 201000002313 intestinal cancer Diseases 0.000 abstract description 21
- 230000035755 proliferation Effects 0.000 abstract description 20
- 108091054455 MAP kinase family Proteins 0.000 abstract description 15
- 102000043136 MAP kinase family Human genes 0.000 abstract description 15
- 230000004083 survival effect Effects 0.000 abstract description 11
- 230000002195 synergetic effect Effects 0.000 abstract description 11
- 230000000259 anti-tumor effect Effects 0.000 abstract description 7
- 230000011664 signaling Effects 0.000 abstract description 6
- 230000009466 transformation Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 76
- 229940079593 drug Drugs 0.000 description 64
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 34
- XVFDNRYZXDHTHT-PXAZEXFGSA-N BI-3406 Chemical compound COc1cc2nc(C)nc(N[C@H](C)c3cc(N)cc(c3)C(F)(F)F)c2cc1O[C@H]1CCOC1 XVFDNRYZXDHTHT-PXAZEXFGSA-N 0.000 description 27
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 22
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 22
- 208000029742 colonic neoplasm Diseases 0.000 description 22
- 238000002474 experimental method Methods 0.000 description 20
- YGUFCDOEKKVKJK-UHFFFAOYSA-N 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine Chemical compound NC1(CCN(CC1)C1=CN=C(C(=N1)N)C1=C(C(=CC=C1)Cl)Cl)C YGUFCDOEKKVKJK-UHFFFAOYSA-N 0.000 description 16
- 230000037361 pathway Effects 0.000 description 15
- 102200006532 rs112445441 Human genes 0.000 description 15
- 102200006531 rs121913529 Human genes 0.000 description 15
- 230000004663 cell proliferation Effects 0.000 description 14
- 239000002609 medium Substances 0.000 description 9
- 101150086096 Eif2ak3 gene Proteins 0.000 description 8
- 238000001262 western blot Methods 0.000 description 8
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 7
- 108091007960 PI3Ks Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 206010069755 K-ras gene mutation Diseases 0.000 description 6
- 238000001516 cell proliferation assay Methods 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 230000003698 anagen phase Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000006311 Cyclin D1 Human genes 0.000 description 2
- 108010058546 Cyclin D1 Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229940126271 SOS1 inhibitor Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 102200006538 rs121913530 Human genes 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 101100404726 Arabidopsis thaliana NHX7 gene Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101150024075 Mapk1 gene Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000057028 SOS1 Human genes 0.000 description 1
- 108700022176 SOS1 Proteins 0.000 description 1
- 101100197320 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) RPL35A gene Proteins 0.000 description 1
- 101150100839 Sos1 gene Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 230000005959 oncogenic signaling Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 102200006539 rs121913529 Human genes 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明提供了VS6766联合BAY293的应用及药物组合物,涉及生物医药技术领域。与RAF/MEK双靶点抑制剂VS6766单独使用或KRAS活性抑制剂BAY293单独使用相比,KRAS活性抑制剂BAY293联合RAF/MEK双靶点抑制剂VS6766的方案可有效抑制KRAS突变型肠癌细胞的增殖与存活,更有效地抑制KRAS下游MAPK通路与P13K通路的信号传导,二者具有协同抗肿瘤作用。二者可直接联合用药,也可制成组合制剂,用于治疗KRAS突变型肠癌,具有良好的临床转化前景。
Description
技术领域
本发明涉及生物医药技术领域,尤其是涉及VS6766联合BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用及药物组合物。
背景技术
结直肠癌是全球范围内常见的恶性肿瘤之一,约30-50%的结直肠癌患者携带KRAS突变,KRAS突变患者预后不佳,导致EGFR单抗耐药,会降低EGFR靶向药的疗效,预后极差。结直肠癌患者中的KRAS突变以G12D(37%)、G12V(30%)、G13D(15%)最常见,其他突变如G12C等少于10%。不同型KRAS突变型蛋白的生化特征各异,目前仅有KRAS G12C抑制剂的研究取得突破。
KRAS突变主要通过激活下游RAF-MEK-ERK(MAPK)、PI3K-AKT-mTOR通路进行信号传导,控制细胞增殖、凋亡、转移等。KRAS突变蛋白可持续激活下游MAPK、PI3K通路,导致肿瘤发生。然而现有针对KRAS突变型结直肠癌的靶向治疗策略是有限的,因此,提供能够抑制其下游MAPK及PI3K通路的信号传导是靶向KRAS的重要治疗策略。
发明内容
本发明的目的之一在于提供VS6766联合BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用。
本发明的目的之二在于提供一种药物组合物。
为实现上述目的,本发明提供了以下技术方案。
第一方面,本发明提供的VS6766联合BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用。
第二方面,本发明还提供了一种药物组合物,所述药物组合物包括VS6766和BAY293。
基于上述技术方案,本发明的VS6766联合BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用及药物组合物至少具有如下有益效果:
本发明提供了RAF/MEK双靶点抑制剂VS6766联合KRAS活性抑制剂BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用,与RAF/MEK双靶点抑制剂VS6766单独使用或KRAS活性抑制剂BAY293单独使用相比,RAF/MEK双靶点抑制剂VS6766联合KRAS活性抑制剂BAY293的方案可有效抑制KRAS突变型肠癌细胞的增殖与存活,更有效地抑制KRAS下游MAPK通路与PI3K通路的信号传导,二者具有协同抗肿瘤作用。二者可直接联合用药,也可制成组合制剂,用于治疗KRAS突变型肠癌,具有良好的临床转化前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是VS6766与BAY293的结构式及其协同作用机理的示意图;其中,图1a为RAF/MEK双靶点抑制剂VS6766的分子结构式,图1b为KRAS-SOS1抑制剂BAY293的分子结构式,图1c为VS6766与BAY293的协同作用机理示意图;
图2是VS6766联合BAY293的CCK8细胞增殖实验结果图,其中,图2a、图2b和图2c分别为HCT116-KRAS G13D的结肠癌细胞在不同浓度的各药物组的CCK8细胞增殖结果图;图2d、图2e和2f分别为SW480-KRAS G12V的结肠癌细胞在不同浓度的各药物组的CCK8细胞增殖结果图;
图3是VS6766联合BAY293的CCK8细胞增殖实验的协同指数结果图;其中,图3a为HCT116-KRAS G13D的结肠癌细胞的CCK8细胞增殖实验中VS6766联合BAY293组三种不同药物浓度下协同指数(CI)图,图3b为SW480-KRAS G12V的结肠癌细胞的CCK8细胞增殖实验中VS6766联合BAY293组三种不同药物浓度下协同指数(CI)图;
图4是VS6766联合BAY293的克隆形成实验结果图;其中,图4a为HCT116-KRAS G13D的结肠癌细胞在各组药物处理后的细胞克隆数量结果图,图4b为SW480-KRAS G12V的结肠癌细胞在各组药物处理后的细胞克隆数量结果图;
图5是VS6766联合BAY293的蛋白免疫印迹实验结果图;其中,图5a为HCT116-KRASG13D的结肠癌细胞在各组药物处理后的蛋白免疫印迹结果图,图5b为SW480-KRAS G12V的结肠癌细胞在各组药物处理后的蛋白免疫印迹结果图;
图6是VS6766、BAY293、BI3406及SHP099单独使用的CCK8细胞增殖实验结果图;其中,图6a为HCT116-KRAS G13D的结肠癌细胞在不同浓度的各药物组的CCK8细胞增殖结果图;图6b为SW480-KRAS G12V的结肠癌细胞在不同浓度的各药物组的CCK8细胞增殖结果图;
图7是VS6766联合BAY293、BI3406的CCK8细胞增殖实验结果图;图7a和图7b为HCT116-KRAS G13D的结肠癌细胞在不同药物组处理后的细胞克隆数量结果图,图7c和图7d为SW480-KRAS G12V的结肠癌细胞在不同药物处理后的细胞克隆数量结果图;
图8是VS6766联合BAY293、BI3406、SHP099的克隆形成实验结果图,其中,图8a为HCT116-KRAS G13D的结肠癌细胞在各组药物处理后的蛋白免疫印迹结果图,图8b为SW480-KRAS G12V的结肠癌细胞在各组药物处理后的蛋白免疫印迹结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
申请人在研究过程中发现:KRAS突变主要通过激活下游RAF-MEK-ERK(MAPK)、PI3K-AKT-mTOR信号通路控制肿瘤细胞的增殖、分化,是其致癌信号传导的关键途径。抑制KRAS下游MAPK通路的信号传导是间接阻断KRAS的重要研究策略。VS6766,其结构式如图1a所示,VS6766是一种新型的RAF/MEK双靶点抑制剂,可直接与MEK结合并形成稳定的RAF/MEK抑制性复合物。VS6766对多种KRAS突变型肿瘤有效,其客观缓解率达27%,显示出极大的治疗潜力,且该药半衰期长,间歇给药方案能同时保证药效和安全性,降低毒副作用。然而,RAF/MEK双靶点抑制剂VS6766单药治疗KRAS突变型结直肠癌时,VS6766单药治疗只能短时间有效抑制MAPK通路pMEK、pERK等蛋白的激活,且对PI3K-AKT-mTOR通路的作用较弱。在KRAS突变型结直肠癌的治疗中,大多数耐药机制导致MAPK通路重新激活,突出了KRAS突变肿瘤细胞对MAPK信号传导的强烈依赖性。短时间VS6766作用可同时抑制pMEK、pERK的活性,但长时间VS6766单药治疗仍存在pMEK、pERK反弹。
BAY293是新型KRAS活性抑制剂,其结构式如图1b所示,通过破坏KRAS-SOS1相互作用而抑制KRAS活性的物质。KRAS蛋白是一种小GTP酶,当它与GTP结合时处于激活状态,而与GDP结合时处于失活状态。BAY293能够阻断KRAS-SOS1相互作用,可抑制SOS1将KRAS-GDP(失活)转换为KRAS-GTP(激活),从而有效抑制KRAS活性。然而,在KRAS突变型结直肠癌细胞中,BAY293单药的治疗效果不佳,BAY293单药对KRAS突变型结直肠癌细胞增殖、存活的抑制作用有限。
在KRAS突变型结直肠癌的治疗中,如何实现持久有效地抑制其下游MAPK通路、PI3K通路的信号传导仍然是重要的科学问题。基于此,本发明提供一种RAF/MEK双靶点抑制剂VS6766联合KRAS活性抑制剂BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用。
实施例1:
RAF/MEK双靶点抑制剂VS6766联合KRAS活性抑制剂BAY293能够有效抑制KRAS突变肠癌细胞增殖和存活。
1、所涉及的肿瘤细胞及药物:
KRAS突变的人结肠癌细胞:HCT116(KRAS G13D)、SW480(KRAS G12V)(四川大学华西医院生物治疗国家重点实验室)。
RAF/MEK双靶点抑制剂:VS6766(Selleckchem,美国)。
KRAS活性抑制剂:BAY293(Selleckchem,美国)。
2、实验方法:
实验分组说明:
①对照组:使用DMSO(二甲基亚砜,Biofroxx,德国)处理细胞;
②VS6766组:单独使用VS6766处理细胞;
③BAY293组:单独使用BAY293处理细胞;
④VS6766联合BAY293组:同时使用VS6766和BAY293处理细胞。VS6766和BAY293使用时用DMSO分别配制成溶液,联合组是使用2种溶液同时处理细胞。
2.1 CCK8细胞增殖实验:
制备新鲜完全培养基用于细胞培养:DMEM培养基(Gibco,美国)+10%胎牛血清(Gibco,美国)。将处于对数生长期的结肠癌细胞HCT116(KRAS G13D)、SW480(KRAS G12V)均匀接种于96孔板,每个药物浓度3个重复孔。细胞贴壁后加入不同组药物处理。具体分组为:
①对照组:加入DMSO;
②VS6766组:分别加入浓度为0.5μM、1μM和2μM的VS6766;
③BAY293组:分别加入浓度为1μM、2μM和4μM的BAY293;
④VS6766+BAY293组:加入浓度为0.5μM的VS6766和1μM的BAY293、加入浓度为1μM的VS6766和2μM的BAY293和加入浓度为2μM的VS6766和4μM的BAY293(VS6766和BAY293的摩尔比为1:2)。
用新鲜完全培养基配制各组药物,每孔的反应体系的终体积相同(100μL)。继续培养72h后,加入CCK8试剂(Target Mol,美国)、酶标仪测定吸光度。Chou-Talalay公式计算协同指数(CI),CI<1说明两种药物具有协同增效作用,CI指数结果用Compusyn软件呈现。
2.2 克隆形成实验:
将对数生长期的结肠癌细胞均匀接种于12孔板中,细胞贴壁后再培养3-4天,然后加入不同组药物处理。具体分组为:
①对照组:加入DMSO;
②VS6766组:加入浓度为1μM的VS6766;
③BAY293组:加入浓度为5μM的BAY293;
④VS6766+BAY293组:HCT116细胞中加入浓度为1μM的VS6766和5μM的BAY293;SW480细胞中加入1μM的VS6766和3μM的BAY293;用新鲜完全培养基配制各组药物,每孔的反应体系的终体积相同(1mL)。加入相应药物后继续培养5-10天(期间更换2-3次新鲜培养基及药物)。集落形成后,固定、染色,观察细胞克隆数量。
2.3 蛋白免疫印迹实验:
将对数生长期的结肠癌细胞HCT116(KRAS G13D)、SW480(KRAS G12V)均匀接种于6孔板中,细胞贴壁后加入不同药物进行处理。具体分组为:
①对照组:加入DMSO;
②VS6766组:HCT116细胞中加入浓度为0.5μM的VS6766,SW480细胞中加入浓度为0.1μM的VS6766;
③BAY293组:加入浓度为5μM的BAY293;
④VS6766+BAY293组:HCT116细胞中加入浓度为0.5μM的VS6766和5μM的BAY293(VS6766和BAY293的摩尔比为1:10),SW480细胞中加入浓度为0.1μM的VS6766和5μM的BAY293(VS6766和BAY293的摩尔比为1:50);用新鲜完全培养基配制各组药物,每孔的反应体系的终体积相同(2mL)。加入药物24h后提取细胞总蛋白,进行蛋白免疫印迹实验,检测pMEK1(S218/S222,Abcam)、pERK(Erk1(pT202/pY204)+Erk2(pT185/pY187),Abcam)、pAKT1(S473,ABclonal)、pmTOR(S2448,Abcam)、CyclinD1(HUABIO)、GAPDH(Santa Cruz)等蛋白的表达水平。
实验结果如下:
图2和图3为VS6766联合BAY293的CCK8细胞增殖实验结果图。CCK8试剂可快速灵敏地检测细胞增殖和细胞毒性,通过OD值间接反映活细胞的数量。协同指数(combinedindex,CI)可判断两种药物的协同性,CI=1表示相加作用, CI>1表示拮抗作用,CI<1表示协同作用,其中:0.8≤CI<0.9为低度协同作用,0.6≤CI<0.8为中度协同作用,0.4≤CI<0.6为高度协同作用,0.2≤CI<0.4为强协同作用。由图2可知单药VS6766组和单药BAY293组均可有效抑制KRAS突变型肠癌细胞增殖,而VS6766+BAY293组的相对细胞增殖比例均显著低于单药VS6766组和单药BAY293组。并且由图3可知,VS6766+BAY293组的CI值均小于1,说明VS6766联合BAY293方案对于抑制KRAS突变肠癌细胞的增殖具有协同增效作用。更进一步,两药的CI指数<0.6(位于0.2与0.6之间),说明VS6766联合BAY293具有高度协同作用。
图4为VS6766联合BAY293的克隆形成实验结果图。克隆形成是评估细胞存活的实验,用来评估单个细胞经药物处理后增殖并形成克隆的能力。由图4可知,单药VS6766组可有效抑制KRAS突变型肠癌细胞增殖与存活,单药BAY293组对KRAS突变型肠癌细胞克隆形成的抑制作用较弱,而VS6766+BAY293组的细胞克隆数均明显少于单药VS6766组和单药BAY293组。说明与单药VS6766组和单药BAY293组相比,VS6766与BAY293联合组可更有效地抑制KRAS突变型肠癌细胞的增殖与存活。
图5为VS6766联合BAY293的蛋白免疫印迹实验结果图。KRAS突变主要通过激活下游RAF-MEK-ERK(MAPK)、PI3K-AKT-mTOR信号通路控制肿瘤细胞的增殖、分化。KRAS通过级联磷酸化激活pMEK、pERK和pAKT、pmTOR来传导致癌信号。Cyclin D1是细胞周期的关键调控因子,其主要功能是促进细胞增殖,可作为细胞增殖的标记物。由图5可知,单药VS6766组可有效抑制pMEK、pERK活性,但对pAKT、pmTOR的抑制作用较弱。单药BAY293组可有效抑制pAKT、pmTOR活性,部分抑制pMEK、pERK的活性。与单药VS6766组或单药BAY293组相比,VS6766与BAY293联合组可更有效抑制MAPK通路pMEK、pERK的活性、PI3K通路pAKT、pmTOR的活性,且两药联合组的CyclinD1蛋白表达显著低于单药组。说明与单药组相比,VS6766与BAY293联合组可更有效地抑制KRAS下游的MAPK与PI3K通路活性,更有效抑制KRAS突变型肠癌细胞增殖,二者具有协同抗肿瘤作用。与CCK8和克隆形成实验的结果一致。
由上述结果可知,与VS6766单独使用或BAY293单独使用相比,VS6766联合BAY293的方案可有效抑制KRAS突变型肠癌细胞的增殖与存活,更有效地抑制KRAS下游MAPK通路与PI3K通路的信号转导,二者有协同抗肿瘤作用。
实施例2:
RAF/MEK双靶点抑制剂VS6766联合KRAS活性抑制剂BAY293、BI3406或SHP099对KRAS突变肠癌细胞增殖和存活的效果比较。
1、所涉及的肿瘤细胞及药物:
KRAS突变的人结肠癌细胞:HCT116(KRAS G13D)、SW480(KRAS G12V)(四川大学华西医院生物治疗国家重点实验室)。
RAF/MEK双靶点抑制剂:VS6766(Selleckchem,美国)。
KRAS活性抑制剂:BAY293(Selleckchem,美国)、BI3406(KRAS-SOS1抑制剂,Selleckchem,美国)、SHP099(SHP2抑制剂,Selleckchem,美国)。
2、实验方法:
实验分组:
①对照组:使用DMSO(二甲基亚砜,Biofroxx,德国)处理细胞;
②VS6766组:单独使用VS6766处理细胞;
③KRAS活性抑制剂组:单独使用KRAS活性抑制剂处理细胞;
④VS6766联合KRAS活性抑制剂组:使用VS6766和KRAS活性抑制剂同时处理细胞。VS6766和KRAS活性抑制剂使用时用DMSO分别配制成不同浓度的溶液,联合组使用2种溶液同时处理细胞。
2.1通过CCK8细胞增殖实验探究VS6766及KRAS活性抑制剂单药对肠癌细胞的抑制效果:
将对数生长期的结肠癌细胞:HCT116(KRAS G13D)、SW480(KRAS G12V)均匀接种于96孔板,每个药物浓度3个重复孔。细胞贴壁后加入不同药物处理。具体分组为:
①对照组:加入DMSO;
②VS6766组:分别加入浓度为0.05μM、0.1μM、1μM和2μM的VS6766;
③BAY293组:分别加入浓度为0.1μM、1μM、5μM和10μM的BAY293;
④BI3406组:分别加入浓度为0.1μM、1μM、5μM和10μM的BI3406;
⑤SHP099组:分别加入浓度为0.1μM、1μM、5μM和10μM的SHP099;
用新鲜完全培养基配制各组药物,每孔的反应体系的终体积相同(100μL)。继续培养72h后,加入CCK8试剂、酶标仪测定吸光度。
2.2通过CCK8细胞增殖实验探究VS6766联合BAY293、BI3406的协同抗肿瘤作用:
将对数生长期的结肠癌细胞HCT116(KRAS G13D)、SW480(KRAS G12V)均匀接种于96孔板,每个药物浓度3个重复孔。细胞贴壁后加入不同药物处理。具体分组为:
①对照组:加入DMSO;
②VS6766组:分别加入浓度为0.1μM、0.5μM、1μM和2μM的VS6766;
③BAY293组:分别加入浓度为2μM的BAY293;
BI3406组:分别加入浓度为5μM的BI3406;
④VS6766+BAY293组:加入浓度为0.1μM的VS6766和2μM的BAY293,或加入浓度为0.5μM的VS6766和2μM的BAY293,或加入浓度为1μM的VS6766和2μM的BAY293,或加入浓度为2μM的VS6766和2μM的BAY293;VS6766+BI3406组:加入浓度为0.1μM的VS6766和5μM的BI3406,或加入浓度为0.5μM的VS6766和5μM的BI3406,或加入浓度为1μM的VS6766和5μM的BI3406,或加入浓度为2μM的VS6766和5μM的BI3406。用新鲜完全培养基配制各组药物,每孔的反应体系的终体积相同(100μL)。继续培养72h后,加入CCK8试剂、酶标仪测定吸光度。
2.3克隆形成实验:
将对数生长期的结肠癌细胞HCT116(KRAS G13D)、SW480(KRAS G12V)均匀接种于12孔板中,细胞贴壁后再培养3-4天,然后加入不同组药物处理。具体分组为:
①对照组:加入DMSO;
②VS6766组:加入浓度为1μM的VS6766;
③BAY293组:加入浓度为5μM的BAY293;
BI3406组:加入浓度为10μM的BI3406;
SHP099组:加入浓度为10μM的SHP099;
④VS6766+BAY293组:加入浓度为1μM的VS6766和浓度为5μM的BAY293;VS6766+BI3406组:加入浓度为1μM的VS6766和浓度为10μM的BAY293;VS6766+SHP099组:加入浓度为1μM的VS6766和浓度为10μM的SHP099。用新鲜完全培养基配制各组药物,每孔的反应体系的终体积相同(1mL)。加入相应药物后继续培养5-10天(期间更换2-3次新鲜培养基及药物)。集落形成后,固定、染色,观察细胞克隆数量。
实验结果:
图6为VS6766、BAY293、BI3406及SHP099单独使用的CCK8细胞增殖实验结果图。由图6可知:VS6766单药可有效抑制KRAS突变型肠癌细胞增殖,其抑制效果呈浓度依赖;而KRAS活性抑制剂BI3406、SHP099抑制KRAS突变型肠癌细胞增殖的作用较弱;与其他KRAS活性抑制剂BI3406、SHP099相比,BAY293在同等浓度下对KRAS突变型肠癌细胞的抑制作用更强。
图7为VS6766联合BAY293、BI3406的CCK8细胞增殖实验结果图。由图7可知:VS6766+BAY293组、VS6766+BI3406组的相对细胞增殖比例均显著低于各单药组;与BI3406相比,VS6766联合BAY293方案对于抑制KRAS突变肠癌细胞的增殖效果更优。
图8为VS6766联合BAY293、BI3406、SHP099的克隆形成实验结果图。由图8可知:VS6766+BAY293组、VS6766+BI3406组、VS6766+SHP099组的细胞克隆数均明显少于各单药组;且VS6766+BAY293组效果更优,可更有效地抑制KRAS突变型肠癌细胞的增殖与存活,与CCK8细胞增殖实验的结果一致。
本发明研究表明RAF/MEK双靶点抑制剂VS6766与KRAS活性抑制剂BAY293、BI3406或SHP099联用可有效抑制KRAS突变型肠癌细胞的增殖和存活,且上述方案中,VS6766和BAY293的联合协同抗肿瘤效果更佳,其效果明显优于单独使用VS6766或BAY293,两者具有协同抗肿瘤效果。二者可直接联合用药,也可制成组合制剂,用于治疗KRAS突变型肠癌。VS6766是FDA已批准可用于临床治疗的“突破性药物”,KRAS活性抑制剂BAY293在相关临床试验中也显示出可靠的安全性,该新方案具有良好的临床转化前景。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (2)
1.VS6766联合BAY293用于制备治疗KRAS突变型结直肠癌的药物中的应用。
2.一种药物组合物,其特征在于,所述药物组合物包括VS6766和BAY293。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310808713.XA CN116509868B (zh) | 2023-07-04 | 2023-07-04 | Vs6766联合bay293的应用及药物组合物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310808713.XA CN116509868B (zh) | 2023-07-04 | 2023-07-04 | Vs6766联合bay293的应用及药物组合物 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116509868A true CN116509868A (zh) | 2023-08-01 |
CN116509868B CN116509868B (zh) | 2023-10-20 |
Family
ID=87405013
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310808713.XA Active CN116509868B (zh) | 2023-07-04 | 2023-07-04 | Vs6766联合bay293的应用及药物组合物 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116509868B (zh) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007091736A1 (ja) * | 2006-02-09 | 2007-08-16 | Chugai Seiyaku Kabushiki Kaisha | 抗腫瘍活性を有する新規クマリン誘導体 |
US20220105185A1 (en) * | 2020-09-15 | 2022-04-07 | Revolution Medicines, Inc. | Ras inhibitors |
TW202227427A (zh) * | 2020-09-16 | 2022-07-16 | 美商拜歐斯瑞克斯公司 | Sos1蛋白降解劑、其醫藥組合物及其治療應用 |
WO2022170060A1 (en) * | 2021-02-05 | 2022-08-11 | Verastem, Inc. | Combination therapy for treating abnormal cell growth |
TW202304452A (zh) * | 2021-04-09 | 2023-02-01 | 美商銳新醫藥公司 | Sos1抑制劑與ras抑制劑於治療癌症之用途 |
WO2023034720A1 (en) * | 2021-08-28 | 2023-03-09 | Cornell University | Compositions and methods for cell reprogramming |
-
2023
- 2023-07-04 CN CN202310808713.XA patent/CN116509868B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007091736A1 (ja) * | 2006-02-09 | 2007-08-16 | Chugai Seiyaku Kabushiki Kaisha | 抗腫瘍活性を有する新規クマリン誘導体 |
CN101384577A (zh) * | 2006-02-09 | 2009-03-11 | 中外制药株式会社 | 具有抗肿瘤活性的新型香豆素衍生物 |
US20220105185A1 (en) * | 2020-09-15 | 2022-04-07 | Revolution Medicines, Inc. | Ras inhibitors |
TW202227427A (zh) * | 2020-09-16 | 2022-07-16 | 美商拜歐斯瑞克斯公司 | Sos1蛋白降解劑、其醫藥組合物及其治療應用 |
WO2022170060A1 (en) * | 2021-02-05 | 2022-08-11 | Verastem, Inc. | Combination therapy for treating abnormal cell growth |
TW202304452A (zh) * | 2021-04-09 | 2023-02-01 | 美商銳新醫藥公司 | Sos1抑制劑與ras抑制劑於治療癌症之用途 |
WO2023034720A1 (en) * | 2021-08-28 | 2023-03-09 | Cornell University | Compositions and methods for cell reprogramming |
Non-Patent Citations (5)
Also Published As
Publication number | Publication date |
---|---|
CN116509868B (zh) | 2023-10-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer | |
Mazzoletti et al. | Combination of PI3K/mTOR inhibitors: antitumor activity and molecular correlates | |
Chen et al. | Significance of interleukin-6 signaling in the resistance of pharyngeal cancer to irradiation and the epidermal growth factor receptor inhibitor | |
Asokan et al. | CXCL8 signaling in the tumor microenvironment | |
Chen et al. | Deacetylation of β-catenin by SIRT1 regulates self-renewal and oncogenesis of liver cancer stem cells | |
Crompton et al. | High-throughput tyrosine kinase activity profiling identifies FAK as a candidate therapeutic target in Ewing sarcoma | |
Lu et al. | Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR | |
Tobío et al. | Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production | |
Trousil et al. | Phenformin enhances the efficacy of ERK inhibition in NF1-mutant melanoma | |
Xu et al. | Compound C620-0696, a new potent inhibitor targeting BPTF, the chromatin-remodeling factor in non-small-cell lung cancer | |
Chen et al. | Blocking IL-6/GP130 signaling inhibits cell viability/proliferation, glycolysis, and colony forming activity in human pancreatic cancer cells | |
Song et al. | Magnolin targeting of ERK1/2 inhibits cell proliferation and colony growth by induction of cellular senescence in ovarian cancer cells | |
Wang et al. | The antiparasitic drug, potassium antimony tartrate, inhibits tumor angiogenesis and tumor growth in nonsmall-cell lung cancer | |
Barbieri et al. | Receptor tyrosine kinase inhibitors and cytotoxic drugs affect pleural mesothelioma cell proliferation: insight into EGFR and ERK1/2 as antitumor targets | |
Cosenza et al. | Citarinostat and Momelotinib co-target HDAC6 and JAK2/STAT3 in lymphoid malignant cell lines: a potential new therapeutic combination | |
Liu et al. | The role of CCL20-CCR6 axis in ovarian cancer metastasis | |
Jiang et al. | Effective targeting of the ubiquitin-like modifier NEDD8 for lung adenocarcinoma treatment | |
de Miguel et al. | Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer | |
Lv et al. | Cancer cell-autonomous cGAS-STING response confers drug resistance | |
Yang et al. | Nigericin exerts anticancer effects through inhibition of the SRC/STAT3/BCL-2 in osteosarcoma | |
CN116509868B (zh) | Vs6766联合bay293的应用及药物组合物 | |
CN116570599B (zh) | Vs6766联合ly3009120的应用及药物组合物 | |
Cioce et al. | Interrogating colorectal cancer metastasis to liver: a search for clinically viable compounds and mechanistic insights in colorectal cancer Patient Derived Organoids | |
CN116531389B (zh) | Vs6766联合雷公藤红素的应用及药物组合物 | |
Patel et al. | Targeting non-small cell lung cancer with epidermal growth factor tyrosine kinase inhibitors: where do we stand, where do we go |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |