CN111265511A - 苯溴马隆作为fxr激动剂的应用 - Google Patents

苯溴马隆作为fxr激动剂的应用 Download PDF

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CN111265511A
CN111265511A CN202010263730.6A CN202010263730A CN111265511A CN 111265511 A CN111265511 A CN 111265511A CN 202010263730 A CN202010263730 A CN 202010263730A CN 111265511 A CN111265511 A CN 111265511A
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fxr
benzbromarone
application
fxr agonist
experiments
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陈卫东
赵世振
王艳东
聂小博
周云
徐晓
李新萍
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Henan University
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    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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Abstract

本发明属于医药新用途技术领域，尤其涉及苯溴马隆作为FXR激动剂的应用。通过均相时间分辨荧光实验、转录激活试验及定量PCR试验证实苯溴马隆可有效激动FXR受体，可用于治疗FXR受体介导的胆汁堵塞、胆结石、糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症的药物中的应用。

Description

苯溴马隆作为FXR激动剂的应用
技术领域
本发明属药物新用途术领域，具体为苯溴马隆作为FXR激动剂的应用。
背景技术
胆汁酸受体法尼酯X受体(FXR，NR1H4)在1995年作为一种孤儿核受体被发现，其主要在肝脏、肠、肾脏以及肾上腺的器官中表达。目前，FXR已被证实能够在多种代谢途径中起到调节作用，包括胆固醇、胆汁酸、脂类以及葡糖糖代谢。通常，FXR以单体或者与维甲酸X受体(RXR)形成异源二聚体来调节一系列靶基因的表达。例如：FXR对胆汁酸结合蛋白(humanintestinal bile acid binding protein,IBABP)、小异源二聚体伴侣分子(smallheterodimer partner,SHP),胆汁盐输出泵(bile salt export pump,BSEP)和磷脂转移蛋白(phospholipid transfer protein,PLTP)等基因进行调节。
胆汁酸(Bile acid，BA)是FXR的内源性配体，可以在生理浓度下激活FXR；鹅脱氧胆酸(CDCA)是目前发现最强的FXR内源性配体，也是目前唯一用于临床的FXR激动剂；奥贝胆酸(OCA)为Intercept制药公司开发的CDCA的类似物，FXR激动活性较高，现已针对NAFLD/NASH的治疗已进入Ⅲ期临床阶段，是该领域最受关注的在研新药之一，其Ⅱ期临床数据表明，45％的患者在使用后，脂肪肝指数得到了明显的改善。葛兰素史克公司根据FXR与RXR具有较高的同源性，对RXR配体进行筛选，并经结构修饰得到了对FXR具有较高激动活性的3,5-二取代异噁唑类化合物GW4064。吉利德和诺华制药公司分别在其结构基础之上进行结构优化分别得到Px-104和Tropifexor(LJN452)，二者均已进入二期临床研究阶段，主要用于治疗NAFLD/NASH。
但是，CDCA、OCA等甾体类化合物对核受体的选择性较差，易引起毒副反应；GW-4064结构中二苯乙烯基具有潜在的毒性，且该化合物利用度较低，限制了临床开发。因此，继续以FXR为靶点寻找合适的配体化合物或对其进行优化、设计和开发具有重要的应用价值。
苯溴马隆又叫苯溴香豆素(Benzybromaron)是一种苯并呋喃类化合物，适用于绝大多数痛风和高尿酸血症患者。该药能够选择性抑制URAT1和GLUT9对尿酸的重吸收，作用于尿酸在肾小管跨膜转运的全过程。
发明内容
针对现有技术存在的问题，本发明的目的在于提供一种新的非甾体类FXR激动剂，具体为苯溴马隆作为FXR激动剂的应用。
为实现上述目的，本发明提供的技术方案为苯溴马隆作为FXR激动剂的应用。
所述苯溴马隆的结构式如下。
Figure BDA0002440418340000021
所述苯溴马隆具有完全不同于抗尿酸血症的新用途,HTRF、转录激活试验以及定量PCR实验结果均显示苯溴马隆具有明显的FXR激动活性。本发明首次提出苯溴马隆为FXR受体激动剂，能够调节糖、脂和胆固醇的代谢，有效减低动物模型血清中糖、脂和胆固醇的水平，改善相应的症状，还可以通过FXR受体介导抑制LPS诱导的炎症反应。因此，临床上可以将苯溴马隆用于治疗代谢性相关疾病的药物，以及用于治疗FXR受体介导的胆汁堵塞、胆结石、糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症的药物中的应用。
附图说明
图1为均相时间分辨荧光(HTRF)实验。
图2为苯溴马隆转录激活试验。
图3为苯溴马隆对FXR相关靶基因表达的影响。
图4为苯溴马隆抑制LPS诱导的炎症反应。
具体实施方式
下面结合具体实验对本发明的技术方案进行详细的说明。
实施例1：HTRF均相时间分辨荧光技术。
在384孔板的测试体系中包含10nM GST-FXRαLBD,100nM biotin-SRC1,0.83nMEu-labeled anti-GST(Cisbio)和41.75nM Streptavidin-XL665(Cisbio)，测试Buffer由50mMHepes pH 7.0,125mM KF,0.125％CHAPS和0.05％奶粉组成，加入不同浓度的待测化合物，室温孵育1小时后，用多功能酶标仪检测620nm和665nm下的荧光值，所测数值用(665nm/620nm*10000)表示，每个化合物设三个复孔，以50μM CDCA为阳性对照药，DMSO作为空白对照，均相时间分辨荧光(HTRF)实验结果见图1。
实施例2：转录激活试验。
HEK293T细胞接种于24孔板中，过夜生长至50％-60％密度时，将培养基换成无血清的DMEM培养基。先将报告基因质粒pCDNA3.l-FXR(400ng/孔)、pCDNA3.1-RXRα(400ng/孔)、pGL3-FXRE-luc(400ng/孔)和PRL-SV40(100ng/孔)与CaCl2(20μL/孔)混匀，然后再将混合物加入到BBS(20μL/孔)中混匀放置15-30分钟，最后40μL/孔共转染到细胞中。6h后换液，同时给予20μM的CDCA或者2μM的苯溴马隆处理24h。24h后，去除培养基，用PBS将细胞轻轻地洗两次，然后用稀释后的lysis buffer裂解细胞15-20分钟，用Dual LuciferaseAssay System Kit(Promega)测定荧光素酶活性。如图2结果显示，苯溴马隆在2μM时对FXR的激动活性大于20μM对照药CDCA的活性。
实施例3：qRT-PCR实验检测FXR相关基因的表达水平。
HepG2细胞在待测化合物或对照药存在下孵化8h，收集细胞，采用PBS缓冲液洗涤，然后用于RNA提取。利用总RNA提取试剂盒(Invitrogen,Thermo Fisher Scientific,Carlsbad,CA)在HepG2细胞中提取RNA，再按照逆转录试剂盒(Applied Biosystems,FosterCity,CA)的说明书进行逆转录成cDNA。最后，利用实时定量PCR仪对FXR的相关基因进行分析。结果显示图3，苯溴马隆能够明显增加SHP的表达，降低CYP7A1的含量，与阳性对照药CDCA结果一致，定量PCR实验中所用引物序列见表1。
表1定量PCR实验中所用引物序列。
Figure BDA0002440418340000041
实施例4：苯溴马隆通过FXR介导抑制LPS诱导的炎症反应。
利用C57NL/6N小鼠，腹腔注射苯溴马隆30mg/kg 24h后禁食，再经12h腹腔注射LPS20mg/kg，6h后取血测试ALT含量，取肝测试相关炎症因子的表达情况。附图4结果显示，苯溴马隆能够降低LPS诱导小鼠体内ALT水平，降低IL-1β、IL-10、INF-γ等炎症因子的水平。

Claims (3)

1.苯溴马隆作为FXR激动剂的应用。
2.如权利要求1所述的苯溴马隆作为FXR激动剂，其特征在于，所述的FXR激动剂用于调节糖、脂和胆固醇的代谢。
3.如权利要求1所述的苯溴马隆作为FXR激动剂，其特征在于，所述的FXR介导抑制LPS诱导的炎症反应。
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Cited By (1)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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