WO2023221361A1 - Use of small-molecule inhibitor bay87-2243 in preparing drug for treating hepatitis b - Google Patents
Use of small-molecule inhibitor bay87-2243 in preparing drug for treating hepatitis b Download PDFInfo
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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
- A61K31/395—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
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
Definitions
- the present invention relates to the use of small molecule inhibitor BAY87-2243 in preparing drugs for treating hepatitis B
- Viral hepatitis B (viral hepatitis type B) is an infectious disease caused mainly by liver lesions caused by hepatitis B virus (HBV).
- HBV hepatitis B virus
- the main clinical manifestations are loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue. Some patients may have jaundice, fever and hepatomegaly accompanied by liver function damage. Some patients can become chronic and even develop cirrhosis, and a few can develop liver cancer.
- nucleoside (acid) analogues and interferons have very limited effects in treating chronic HBV infection, and only a few patients can achieve "functional cure.”
- the key to achieving a cure for chronic hepatitis B virus infection is to completely eliminate covalently closed circular DNA (cccDNA) in the host liver cells. It is the template for HBV transcription and replication and the viral gene reservoir. It is also the key to the recurrence of chronic hepatitis B patients after drug withdrawal. inducement.
- cccDNA covalently closed circular DNA
- the small molecule inhibitor BAY87-2243 is a potent and selective hypoxia-inducible factor-1 (HIF-1) inhibitor. There are currently reports that it inhibits tumor cell proliferation by interfering with mitochondrial function and can be used to treat cancers such as lung cancer. There are also reports that it has a positive impact on the intestinal mucosal barrier (IMB) function and can be used to treat sepsis. There are currently no reports of BAY87-2243 being used to treat hepatitis B.
- IMB intestinal mucosal barrier
- the present invention provides the use of the small molecule inhibitor BAY87-2243 in the preparation of drugs for treating hepatitis B; the BAY87-2243 is a specific inhibitor of HIF-1.
- the drug is a drug that reduces the production level of hepatitis B surface antigen.
- the drug is a drug that reduces the production level of hepatitis B e antigen.
- the drug is a drug that inhibits the transcription of hepatitis B virus.
- the drug is a drug that inhibits hepatitis B virus DNA replication.
- the drug is a drug that reduces the expression level of hepatitis B virus cccDNA.
- the drug is a preparation prepared by using the small molecule inhibitor BAY87-2243 as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.
- the preparation is an oral preparation; the oral preparation is granules, solutions, pills, ointments or tablets.
- the small molecule inhibitor BAY87-2243 can significantly inhibit HBV transcription and replication, and effectively reduce the expression level of cccDNA, so as to completely eliminate hepatitis B.
- the patient's virus-carrying status becomes possible, and the goal of antiviral treatment is finally achieved, which has the prospect of practical promotion and application.
- FIG. 1 Statistical chart of research data of BAY87-2243 treatment on HBsAg/HBeAg secretion (A: Effects of different concentrations of BAY87-2243 treatment on HepAD38 cytotoxicity and HBsAg secretion, B: Different concentrations of BAY87-2243 treatment on HepG2.2.15 cytotoxicity and HBsAg secretion) Effect of HBsAg secretion, C: Effect of different concentrations of BAY87-2243 treatment on HepAD38 cytotoxicity and HBeAg secretion, D: Effect of different concentrations of BAY87-2243 treatment on HepG2.2.15 cytotoxicity and HBeAg secretion)
- FIG. 2 Statistical chart of research data on the effects of BAY87-2243 treatment on HBV transcription and replication levels and HBV cccDNA expression levels (A: The effect of BAY87-2243 treatment on the expression level of HBV total RNA in HepAD38 cells, B: The effect of BAY87-2243 treatment on the expression level of HBV in HepAD38 cells Effect of HBVpgRNA expression level in cells, C: Effect of BAY87-2243 treatment on HBVDNA replication level in HepAD38 cells, D: Effect of BAY87-2243 treatment on HBVcccDNA expression level in HepAD38 cells)
- FIG. 3 Statistical diagram of the effect of BAY87-2243 treatment on HBV transcription and replication levels and HBV cccDNA expression levels in HBV-infected human primary hepatocytes (A: Effect of BAY87-2243 treatment on HBsAg secretion, B: BAY87-2243 The effect of treatment on HBeAg secretion, C: The effect of BAY87-2243 treatment on the expression level of HBV total RNA, D: The effect of BAY87-2243 treatment on the expression level of HBVpgRNA, E: The effect of BAY87-2243 treatment on the expression level of intracellular HBV DNA, F: Effect of BAY87-2243 treatment on intracellular HBV cccDNA expression level)
- hepatitis B virus surface antigen diagnostic kit enzyme-linked immunoassay; Shanghai Kehua, SI0910113
- hepatitis B virus E antigen detection kit enzyme-linked immunoassay; Shanghai Kehua, 20163400144
- the specific microplate reader test results and ELISA kit test results are shown in Figure 1A-D.
- BAY87-2243 has little effect on cytotoxicity at a concentration of 100nM, and BAY87-2243 at a lower concentration ( ⁇ 0.1nM) can effectively inhibit the secretion of HBsAg and HBeAg.
- the present invention has proven through a series of experiments that the small molecule inhibitor BAY87-2243 can significantly inhibit HBV transcription and replication, and effectively reduce the expression level of cccDNA, making it possible to completely eliminate the virus-carrying status of hepatitis B patients, and ultimately achieve the goal of antiviral treatment. , with practical promotion and application prospects.
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Abstract
The present invention provides use of a small-molecule inhibitor BAY87-2243 in preparing a drug for treating hepatitis B virus infection. According to the present invention, by using different hepatoma cell lines stably producing hepatitis B virus (HBV) or HBV-infected human primary hepatocytes to be subjected to in-vitro treatment experiments with different concentrations of BAY87-2243, it is proved that BAY87-2243 can significantly inhibit HBV transcription and replication and effectively reduce the expression level of cccDNA.
Description
本发明涉及小分子抑制剂BAY87-2243在制备治疗乙型肝炎的药物中的用途The present invention relates to the use of small molecule inhibitor BAY87-2243 in preparing drugs for treating hepatitis B
乙型病毒性肝炎(viral hepatitis type B)是由乙型肝炎病毒(hepatitis B virus,HBV)引起的以肝脏病变为主的一种传染病。临床上以食欲减退、恶心、上腹部不适、肝区痛、乏力为主要表现。部分患者可有黄疸发热和肝大伴有肝功能损害。有些患者可慢性化，甚至发展成肝硬化，少数可发展为肝癌。Viral hepatitis B (viral hepatitis type B) is an infectious disease caused mainly by liver lesions caused by hepatitis B virus (HBV). The main clinical manifestations are loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue. Some patients may have jaundice, fever and hepatomegaly accompanied by liver function damage. Some patients can become chronic and even develop cirrhosis, and a few can develop liver cancer.
目前核苷(酸)类似物和干扰素治疗慢性HBV感染的效果十分有限，只有少数患者能实现“功能性治愈”。要实现慢性乙肝病毒感染治愈的关键在于必须彻底清除宿主肝细胞内共价闭合环状DNA(cccDNA)，它是HBV转录复制的模板和病毒基因储存库，也是慢乙肝患者停药后复发的关键诱因。目前临床上仍缺乏有效清除cccDNA的药物，因此寻找和开发有效清除乙肝病毒cccDNA的药物具有良好的临床应用前景。Currently, nucleoside (acid) analogues and interferons have very limited effects in treating chronic HBV infection, and only a few patients can achieve "functional cure." The key to achieving a cure for chronic hepatitis B virus infection is to completely eliminate covalently closed circular DNA (cccDNA) in the host liver cells. It is the template for HBV transcription and replication and the viral gene reservoir. It is also the key to the recurrence of chronic hepatitis B patients after drug withdrawal. inducement. There is still a lack of drugs that can effectively clear cccDNA in clinical practice. Therefore, finding and developing drugs that can effectively clear cccDNA of hepatitis B virus has good clinical application prospects.
小分子抑制剂BAY87-2243是一种有效的选择性hypoxia-inducible factor-1(HIF-1)抑制剂。目前有研究报道其通过干扰线粒体功能抑制肿瘤细胞增殖，可用治疗肺癌等癌症，也有报道研究，其对肠道黏膜屏障(IMB)功能有积极影响，可用于治疗脓毒症。目前还没有BAY87-2243用于治疗乙型肝炎的报道。The small molecule inhibitor BAY87-2243 is a potent and selective hypoxia-inducible factor-1 (HIF-1) inhibitor. There are currently reports that it inhibits tumor cell proliferation by interfering with mitochondrial function and can be used to treat cancers such as lung cancer. There are also reports that it has a positive impact on the intestinal mucosal barrier (IMB) function and can be used to treat sepsis. There are currently no reports of BAY87-2243 being used to treat hepatitis B.
发明内容Contents of the invention
为解决上述问题，本发明提供了小分子抑制剂BAY87-2243在制备治疗乙型肝炎的药物中的用途；所述BAY87-2243为HIF-1特异性抑制剂。In order to solve the above problems, the present invention provides the use of the small molecule inhibitor BAY87-2243 in the preparation of drugs for treating hepatitis B; the BAY87-2243 is a specific inhibitor of HIF-1.
进一步地，所述药物是降低乙肝表面抗原产生水平的药物。Further, the drug is a drug that reduces the production level of hepatitis B surface antigen.
进一步地，所述药物是降低乙肝e抗原产生水平的药物。Further, the drug is a drug that reduces the production level of hepatitis B e antigen.
进一步地，所述药物是抑制乙型肝炎病毒转录的药物。Further, the drug is a drug that inhibits the transcription of hepatitis B virus.
进一步地，所述药物是抑制乙型肝炎病毒DNA复制的药物。Further, the drug is a drug that inhibits hepatitis B virus DNA replication.
进一步地，所述药物是降低乙型肝炎病毒cccDNA表达水平的药物。Further, the drug is a drug that reduces the expression level of hepatitis B virus cccDNA.
进一步地，所述药物是以小分子抑制剂BAY87-2243为活性成分，加入药学上可接受的辅料或辅助性成分制备而成的制剂。Further, the drug is a preparation prepared by using the small molecule inhibitor BAY87-2243 as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.
更进一步地，所述制剂为口服制剂；所述口服制剂为颗粒剂、溶液剂、丸剂、膏剂或片剂。Furthermore, the preparation is an oral preparation; the oral preparation is granules, solutions, pills, ointments or tablets.
本发明小分子抑制剂BAY87-2243在制备治疗乙型肝炎的药物中的用途，经试验证明小分子抑制剂BAY87-2243能显著抑制HBV转录和复制，以及有效降低cccDNA表达水平，使彻底消除乙肝患者病毒携带状态成为可能，最终实现抗病毒治疗的目标，具备实际推广应用前景。The use of the small molecule inhibitor BAY87-2243 of the present invention in the preparation of drugs for the treatment of hepatitis B. Experiments have proven that the small molecule inhibitor BAY87-2243 can significantly inhibit HBV transcription and replication, and effectively reduce the expression level of cccDNA, so as to completely eliminate hepatitis B. The patient's virus-carrying status becomes possible, and the goal of antiviral treatment is finally achieved, which has the prospect of practical promotion and application.
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.
图1 BAY87-2243处理对HBsAg/HBeAg分泌的研究数据统计图(A:不同浓度BAY87-2243处理对HepAD38细胞毒性以及分泌HBsAg的影响,B:不同浓度BAY87-2243处理对HepG2.2.15细胞毒性以及分泌HBsAg的影响,C:不同浓度BAY87-2243处理对HepAD38细胞毒性以及分泌HBeAg的影响,D:不同浓度BAY87-2243处理对HepG2.2.15细胞毒性以及分泌HBeAg的影响)Figure 1 Statistical chart of research data of BAY87-2243 treatment on HBsAg/HBeAg secretion (A: Effects of different concentrations of BAY87-2243 treatment on HepAD38 cytotoxicity and HBsAg secretion, B: Different concentrations of BAY87-2243 treatment on HepG2.2.15 cytotoxicity and HBsAg secretion) Effect of HBsAg secretion, C: Effect of different concentrations of BAY87-2243 treatment on HepAD38 cytotoxicity and HBeAg secretion, D: Effect of different concentrations of BAY87-2243 treatment on HepG2.2.15 cytotoxicity and HBeAg secretion)
图2 BAY87-2243处理对HBV转录和复制水平以及HBV cccDNA表达水平影响的研究数据统计图(A:BAY87-2243处理对HepAD38细胞内HBV总RNA表达水平的影响,B:BAY87-2243处理对HepAD38细胞内HBVpgRNA表达水平的影响,C:BAY87-2243处理对HepAD38细胞内HBVDNA复制水平的影响,D:BAY87-2243处理对HepAD38细胞内HBVcccDNA表达水平的影响)Figure 2 Statistical chart of research data on the effects of BAY87-2243 treatment on HBV transcription and replication levels and HBV cccDNA expression levels (A: The effect of BAY87-2243 treatment on the expression level of HBV total RNA in HepAD38 cells, B: The effect of BAY87-2243 treatment on the expression level of HBV in HepAD38 cells Effect of HBVpgRNA expression level in cells, C: Effect of BAY87-2243 treatment on HBVDNA replication level in HepAD38 cells, D: Effect of BAY87-2243 treatment on HBVcccDNA expression level in HepAD38 cells)
图3在HBV感染的人原代肝细胞中，BAY87-2243处理对HBV转录和复制水平以及HBV cccDNA表达水平影响的统计图(A:BAY87-2243处理对HBsAg分泌的影响,B：BAY87-2243处理对HBeAg分泌的影响,C:BAY87-2243处理对HBV总RNA表达水平的影响,D:BAY87-2243处理对HBVpgRNA表达水平的影响,E：BAY87-2243处理对细胞内HBVDNA表达水平的影响,F:BAY87-2243处理对细胞内HBV cccDNA表达水平的影响)Figure 3 Statistical diagram of the effect of BAY87-2243 treatment on HBV transcription and replication levels and HBV cccDNA expression levels in HBV-infected human primary hepatocytes (A: Effect of BAY87-2243 treatment on HBsAg secretion, B: BAY87-2243 The effect of treatment on HBeAg secretion, C: The effect of BAY87-2243 treatment on the expression level of HBV total RNA, D: The effect of BAY87-2243 treatment on the expression level of HBVpgRNA, E: The effect of BAY87-2243 treatment on the expression level of intracellular HBV DNA, F: Effect of BAY87-2243 treatment on intracellular HBV cccDNA expression level)
实施例1 BAY87-2243对稳定产生HBV的肝癌细胞中HBsAg/HBeAg分 泌的影响研究Example 1 Study on the effect of BAY87-2243 on HBsAg/HBeAg secretion in liver cancer cells that stably produce HBV
首先，将8×10
4个/孔HepG2.215和HepAD38细胞分别接种于48孔板。在细胞铺板后第二天，使用不同浓度梯度(0.01-100nM)的BAY87-2243分别处理细胞。在BAY87处理细胞72h后，对48孔板加入Cell Counting Kit-8试剂(MedChemExpress，HY-K0301)，通过多功能酶标仪在450nm波长下检测细胞吸光度的变化。此外，收集细胞上清液使用乙型肝炎病毒表面抗原诊断试剂盒(酶联免疫法；上海科华，SI0910113)和乙型肝炎病毒E抗原检测试剂盒(酶联免疫法；上海科华，20163400144)分别检测细胞上清中分泌的HBsAg和HBeAg水平。具体酶标仪检测结果和ELISA试剂盒检测结果见图1A-D。从图结果可见：BAY87-2243浓度在100nM时内对细胞毒性的影响较小，且较低浓度(<0.1nM)的BAY87-2243能有效抑制HBsAg和HBeAg分泌。
First, 8 × 10 4 cells/well HepG2.215 and HepAD38 cells were seeded in a 48-well plate. On the second day after cell plating, cells were treated with BAY87-2243 at different concentration gradients (0.01-100 nM). After BAY87 treated the cells for 72 hours, Cell Counting Kit-8 reagent (MedChemExpress, HY-K0301) was added to the 48-well plate, and the changes in cell absorbance were detected by a multifunctional microplate reader at a wavelength of 450 nm. In addition, the cell supernatants were collected and used to use hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunoassay; Shanghai Kehua, SI0910113) and hepatitis B virus E antigen detection kit (enzyme-linked immunoassay; Shanghai Kehua, 20163400144 ) were used to detect the secreted HBsAg and HBeAg levels in the cell supernatant. The specific microplate reader test results and ELISA kit test results are shown in Figure 1A-D. It can be seen from the results that BAY87-2243 has little effect on cytotoxicity at a concentration of 100nM, and BAY87-2243 at a lower concentration (<0.1nM) can effectively inhibit the secretion of HBsAg and HBeAg.
实施例2 BAY87-2243对HepAD38细胞中HBV转录和复制水平以及HBV cccDNA表达水平的影响研究Example 2 Study on the Effect of BAY87-2243 on HBV Transcription and Replication Levels and HBV cccDNA Expression Levels in HepAD38 Cells
将3×10
5个/孔HepAD38细胞接种于12孔板，第二天用不同浓度梯度(1、2或4nM)的BAY87处理72h，然后使用Trizol试剂(Invitrogen，15596026)提取细胞内总RNA，使用One-step Realtime RT-qPCR试剂盒[One Step TB
PrimeScript
TM RT-PCR Kit II(Perfect Real Time)，TaKaRa，RR086]检测细胞内HBV total RNA(图2A)和pgRNA(图2B)的表达水平。从图结果可见：不同浓度的BAY87-2243处理能显著降低细胞内HBV总RNA和pgRNA表达水平，说明BAY87-2243能有效抑制细胞内HBV转录。
3×10 5 cells/well HepAD38 cells were seeded in a 12-well plate, and the next day they were treated with BAY87 at different concentration gradients (1, 2 or 4 nM) for 72 h, and then the total intracellular RNA was extracted using Trizol reagent (Invitrogen, 15596026). Use One-step Realtime RT-qPCR Kit [One Step TB PrimeScript TM RT-PCR Kit II (Perfect Real Time), TaKaRa, RR086] to detect the expression levels of intracellular HBV total RNA (Figure 2A) and pgRNA (Figure 2B). It can be seen from the results that BAY87-2243 treatment at different concentrations can significantly reduce the expression levels of total HBV RNA and pgRNA in cells, indicating that BAY87-2243 can effectively inhibit intracellular HBV transcription.
将6×10
5个/孔HepAD38细胞接种于6孔板，第二天用不同浓度梯度(1、2或4nM)的BAY87-2243处理72h，裂解细胞并提取细胞内被核衣壳包裹的HBV DNA，通过Southern blot方法检测BAY87-2243对细胞内HBV DNA复制水平的影响。具体结果见图2C。从图结果可见：不同浓度的BAY87-2243处理均能显著降低细胞内HBV DNA复制水平，提示BAY87-2243能有效抑制细胞内HBVDNA复制。
6 × 10 5 cells/well HepAD38 cells were seeded in a 6-well plate. The next day, they were treated with BAY87-2243 at different concentration gradients (1, 2 or 4 nM) for 72 hours. The cells were lysed and the HBV enveloped by nucleocapsids in the cells was extracted. DNA, the effect of BAY87-2243 on the intracellular HBV DNA replication level was detected by Southern blot method. The specific results are shown in Figure 2C. It can be seen from the results in the figure that BAY87-2243 treatment at different concentrations can significantly reduce the intracellular HBV DNA replication level, suggesting that BAY87-2243 can effectively inhibit intracellular HBV DNA replication.
将6×10
5个/孔HepAD38细胞接种于6孔板，第二天用不同浓度梯度(1、2或4nM)的BAY87-2243处理96h。然后用PBS洗细胞2次，裂解细胞过夜，用酚氯仿抽提DNA，最后将得到的cccDNA用T5核酸外切酶进行酶切反应，用Realtime qPCR(
Probe qPCR Master Mix，promega，A6101)检测细胞内HBV cccDNA表达水平。具体结果见图2D。从图结果可见：不同浓度的BAY87-2243处理均能显著降低细胞内HBV cccDNA表达水平，提示 BAY87-2243可有效降低细胞内HBVcccDNA表达水平。
6×10 5 cells/well HepAD38 cells were seeded in a 6-well plate and treated with BAY87-2243 at different concentration gradients (1, 2 or 4 nM) for 96 hours the next day. Then wash the cells twice with PBS, lyse the cells overnight, extract the DNA with phenol-chloroform, and finally perform an enzyme digestion reaction on the cccDNA obtained with T5 exonuclease, and use Realtime qPCR ( Probe qPCR Master Mix, promega, A6101) detects the expression level of HBV cccDNA in cells. The specific results are shown in Figure 2D. It can be seen from the results that BAY87-2243 at different concentrations can significantly reduce the expression level of HBV cccDNA in cells, suggesting that BAY87-2243 can effectively reduce the expression level of HBVcccDNA in cells.
实施例3 BAY87-2243对HBV感染的人原代肝细胞中HBV转录和复制水平以及HBV cccDNA表达水平影响的研究Example 3 Study on the effects of BAY87-2243 on HBV transcription and replication levels and HBV cccDNA expression levels in HBV-infected human primary hepatocytes
将商品化的人原代细胞按5×10
5个/孔接种于12孔板，在细胞铺板后第二天，将细胞与含有HBV病毒颗粒(MOI：1000)的William's E完全培养基孵育；在HBV感染24h后，将细胞用PBS洗3次，然后用不同浓度梯度(2nM和4nM)的BAY87-2243连续处理6天。收集细胞上清液，然后使用(乙型肝炎病毒表面抗原诊断试剂盒(酶联免疫法)和乙型肝炎病毒E抗原检测试剂盒(酶联免疫法)分别收集细胞上清后检测细胞上清中分泌的HBsAg和HBeAg水平，具体结果见图3A-B；从图结果可见：BAY87-2243能有效抑制HBsAg和HBeAg分泌。
Commercial human primary cells were seeded into a 12-well plate at 5 × 10 5 cells/well. On the second day after cell plating, the cells were incubated with William's E complete medium containing HBV virus particles (MOI: 1000); After 24 h of HBV infection, the cells were washed three times with PBS and then treated with BAY87-2243 at different concentration gradients (2 nM and 4 nM) for 6 consecutive days. Collect the cell supernatant, and then use (Hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunoassay)) and hepatitis B virus E antigen detection kit (enzyme-linked immunoassay) to collect the cell supernatant and then detect the cell supernatant The levels of HBsAg and HBeAg secreted in the drug, the specific results are shown in Figure 3A-B; it can be seen from the results that BAY87-2243 can effectively inhibit the secretion of HBsAg and HBeAg.
对如图3A-B相同处理的细胞用PBS洗细胞2次，然后使用Trizol试剂提取细胞内总RNA，使用One-step Realtime RT-qPCR试剂盒检测细胞内HBV total RNA(图3C)和pgRNA(图3D)的表达水平。从图结果可见：不同浓度的BAY87-2243处理能显著降低肝细胞内HBV总RNA和pgRNA表达水平，说明BAY87-2243能有效抑制人原代肝细胞内HBV转录。The cells treated as shown in Figure 3A-B were washed twice with PBS, then Trizol reagent was used to extract the total RNA in the cells, and the One-step Realtime RT-qPCR kit was used to detect intracellular HBV total RNA (Figure 3C) and pgRNA ( Figure 3D) expression levels. It can be seen from the results that BAY87-2243 treatment at different concentrations can significantly reduce the expression levels of HBV total RNA and pgRNA in hepatocytes, indicating that BAY87-2243 can effectively inhibit HBV transcription in human primary hepatocytes.
对如图3A-B相同处理的细胞用PBS洗细胞2次，裂解细胞并用酚-氯仿抽提细胞内被核衣壳包裹的HBV DNA，通过Realtime qPCR(TB
Premix Ex Taq
TM II(Tli RNaseH Plus)，TaKaRa，RR820)检测BAY87-2243对细胞内HBV DNA表达水平的影响。具体结果见图3E。从图结果可见：BAY87-2243处理能显著降低细胞内HBV DNA表达水平，提示BAY87-2243能有效抑制人原代肝细胞内HBVDNA复制。
The cells treated the same as in Figure 3A-B were washed twice with PBS, lysed, and phenol-chloroform was used to extract the HBV DNA wrapped by the nucleocapsid in the cells. Realtime qPCR (TB Premix Ex Taq TM II (Tli RNaseH Plus), TaKaRa, RR820) was used to detect the effect of BAY87-2243 on intracellular HBV DNA expression levels. The specific results are shown in Figure 3E. It can be seen from the results that BAY87-2243 treatment can significantly reduce the expression level of HBV DNA in cells, suggesting that BAY87-2243 can effectively inhibit HBV DNA replication in human primary hepatocytes.
对如图3A-B相同处理的细胞用PBS洗细胞2次，裂解细胞然后用酚氯仿抽提DNA，最后将得到的cccDNA用T5核酸外切酶进行酶切反应，用Realtime qPCR(
Probe qPCR Master Mix，promega，A6101)检测细胞内HBV cccDNA表达水平。具体结果见图3F。从图结果可见：BAY87-2243处理能显著降低细胞内HBV cccDNA表达水平，提示BAY87-2243可有效降低人原代肝细胞内HBV cccDNA表达水平。
The cells treated as shown in Figure 3A-B were washed twice with PBS, lysed, and DNA extracted with phenol chloroform. Finally, the cccDNA obtained was digested with T5 exonuclease and analyzed with Realtime qPCR ( Probe qPCR Master Mix, promega, A6101) detects the expression level of HBV cccDNA in cells. The specific results are shown in Figure 3F. It can be seen from the results that BAY87-2243 treatment can significantly reduce the expression level of HBV cccDNA in cells, suggesting that BAY87-2243 can effectively reduce the expression level of HBV cccDNA in human primary hepatocytes.
综上，本发明经一系列试验证明小分子抑制剂BAY87-2243能显著抑制HBV转录和复制，以及有效降低cccDNA表达水平，使彻底消除乙肝患者病毒携带状态成为可能，最终实现抗病毒治疗的目标，具备实际推广应用前景。In summary, the present invention has proven through a series of experiments that the small molecule inhibitor BAY87-2243 can significantly inhibit HBV transcription and replication, and effectively reduce the expression level of cccDNA, making it possible to completely eliminate the virus-carrying status of hepatitis B patients, and ultimately achieve the goal of antiviral treatment. , with practical promotion and application prospects.
Claims (9)
- 小分子抑制剂BAY87-2243在制备治疗乙型肝炎的药物中的用途；所述BAY87-2243为HIF-1特异性抑制剂。The use of the small molecule inhibitor BAY87-2243 in the preparation of drugs for the treatment of hepatitis B; the BAY87-2243 is a specific inhibitor of HIF-1.
- 根据权利要求1所述的用途，其特征在于，所述药物是降低乙肝表面抗原产生水平的药物。The use according to claim 1, characterized in that the drug is a drug that reduces the production level of hepatitis B surface antigen.
- 根据权利要求1所述的用途，其特征在于，所述药物是降低乙肝e抗原产生水平的药物。The use according to claim 1, characterized in that the drug is a drug that reduces the production level of hepatitis B e antigen.
- 根据权利要求1所述的用途，其特征在于，所述药物是抑制乙型肝炎病毒转录的药物。The use according to claim 1, characterized in that the drug is a drug that inhibits the transcription of hepatitis B virus.
- 根据权利要求1所述的用途，其特征在于，所述药物是抑制乙型肝炎病毒DNA复制的药物。The use according to claim 1, characterized in that the drug is a drug that inhibits hepatitis B virus DNA replication.
- 根据权利要求1所述的用途，其特征在于，所述药物是降低乙型肝炎病毒cccDNA表达水平的药物。The use according to claim 1, characterized in that the drug is a drug that reduces the expression level of hepatitis B virus cccDNA.
- 根据权利要1～6任一项所述的用途，其特征在于，所述药物是以小分子抑制剂BAY87-2243为活性成分，加入药学上可接受的辅料或辅助性成分制备而成的制剂。The use according to any one of claims 1 to 6, characterized in that the drug is a preparation prepared by using the small molecule inhibitor BAY87-2243 as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients. .
- 根据权利要7所述的用途，其特征在于，所述制剂为口服制剂。The use according to claim 7, characterized in that the preparation is an oral preparation.
- 根据权利要8所述的用途，其特征在于，所述口服制剂为颗粒剂、溶液剂、丸剂、膏剂或片剂。The use according to claim 8, characterized in that the oral preparation is granules, solutions, pills, ointments or tablets.
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| WO2019043193A1 (en) * | 2017-09-01 | 2019-03-07 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | An inhibitor for use for preventing and/or treating an infection with hepatitis b virus |
| CN113337602A (en) * | 2020-03-02 | 2021-09-03 | 苏州亚盛药业有限公司 | Methods of treatment and biomarkers for MDM2 inhibitors |
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