WO2023241179A1 - Use of asiatic acid in preparation of medicament for treating hepatitis b - Google Patents

Use of asiatic acid in preparation of medicament for treating hepatitis b Download PDF

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Publication number
WO2023241179A1
WO2023241179A1 PCT/CN2023/086552 CN2023086552W WO2023241179A1 WO 2023241179 A1 WO2023241179 A1 WO 2023241179A1 CN 2023086552 W CN2023086552 W CN 2023086552W WO 2023241179 A1 WO2023241179 A1 WO 2023241179A1
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hepatitis
asiatic acid
hbv
levels
preparation
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PCT/CN2023/086552
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French (fr)
Chinese (zh)
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陈娟
杨祯
程胜桃
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重庆医科大学检验医学院
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Publication of WO2023241179A1 publication Critical patent/WO2023241179A1/en

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • 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 field of biomedicine technology, and in particular to the use of asiatic acid in the preparation of hepatitis B therapeutic drugs.
  • Hepatitis B virus (HBV) infection is a major public health problem worldwide. According to WHO, approximately 2 billion people worldwide have been infected with HBV, including approximately 257 million patients with chronic hepatitis B infection. my country is an area with high prevalence of HBV. There are about 97 million hepatitis B virus carriers, accounting for 8% to 10% of the country's total population, of which about 20 million are chronic hepatitis B patients. Every year, there are as many as 263,000 deaths due to complications such as cirrhosis and liver cancer caused by chronic hepatitis B. . Therefore, the task of preventing and controlling hepatitis B is very serious.
  • interferon interferon
  • NAs nucleoside analogs
  • both types of drugs have certain shortcomings, such as nucleoside analogues, which take a long time to take, poor patient compliance, and are prone to rebound after drug withdrawal.
  • Interferons are only effective in less than 30% of patients and have many adverse reactions. More importantly, neither type of drugs can effectively cure hepatitis B, so there is an urgent need to develop new anti-HBV drugs.
  • HBV belongs to the hepadnaviridae family, and its genome is a partially double-stranded, relaxed circular DNA (relaxed-circular DNA, rcDNA) of approximately 3.2 kb in length.
  • rcDNA relaxed circular DNA
  • rcDNA is delivered to the nucleus and forms covalently closed circular DNA (cccDNA) with a superhelical structure.
  • cccDNA persists in the nucleus of liver cells in the form of minichromosomes, has a stable structure, and serves as a transcription template for viral RNA. Therefore, cccDNA is the main cause of persistent HBV infection, drug resistance, and HBV reactivation after discontinuation of antiviral drugs.
  • the virus can encode a transactivator protein - HBx, which can hijack DDB1 to relieve the ubiquitination and degradation of Smc5/6 by the E3 ubiquitin ligase CUL4B and activate cccDNA transcription. It can also activate cccDNA transcription through Binding to the cccDNA minichromosome inhibits the recruitment of transcriptional repressors to cccDNA thereby maintaining cccDNA transcription.
  • HBx plays a key role in the transcription process of cccDNA. Inhibiting the function of HBx may inhibit cccDNA transcription. Therefore, HBx is an important target for new therapies to treat chronic HBV infection.
  • the development of new drugs that target and inhibit HBx can provide new directions and theoretical basis for reducing the infection rate of HBV and exploring new drugs for the effective treatment of hepatitis B. .
  • the purpose of the present invention is to provide the use of asiatic acid in the preparation of hepatitis B therapeutic drugs.
  • Asiatic acid can target HBx and provide a new drug for the effective treatment of hepatitis B. and technical means.
  • the first aspect of the present invention provides the use of asiatic acid as an active ingredient in the preparation of hepatitis B therapeutic drugs.
  • hepatitis B treatment drug has at least one of the following functions:
  • Reduce HBsAg levels reduce intracellular total HBV RNAs levels, reduce intracellular HBV 3.5-kb RNA levels, and reduce HBV DNA levels.
  • the hepatitis B treatment drug must include asiatic acid, and use asiatic acid as the aforementioned function. of active ingredients.
  • the active ingredient that performs the aforementioned functions may only be asiatic acid, or may include other molecules that can perform similar functions.
  • the asiatic acid is used as one of the active ingredients or the only active ingredient of the hepatitis B treatment drug.
  • the hepatitis B treatment drug may be a single-component substance or a multi-component substance.
  • the form of the hepatitis B treatment drug is not particularly limited and can be in various material forms such as solid, liquid, gel, semi-liquid, aerosol, etc.
  • hepatitis B therapeutic drugs are mainly targeted at mammals, such as rodents, primates, etc.
  • the second aspect of the present invention provides a pharmaceutical preparation for treating hepatitis B, including a safe and effective dose of asiatic acid.
  • the pharmaceutical preparation for treating hepatitis B includes 10-30 ⁇ M asiatic acid.
  • the dosage of the pharmaceutical preparation for treating hepatitis B is 15-30 mg/kg asiatic acid.
  • the pharmaceutical preparation for treating hepatitis B also includes pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical preparation for treating hepatitis B must include asiatic acid, and use asiatic acid as an active ingredient for the aforementioned functions.
  • the active ingredient that performs the aforementioned functions may only be asiatic acid, or may include other molecules that can perform similar functions.
  • the asiatic acid is used as one of the active ingredients or the only active ingredient of the pharmaceutical preparation for treating hepatitis B.
  • the pharmaceutical preparation for treating hepatitis B may be a single-component substance or a multi-component substance.
  • the form of the pharmaceutical preparation for treating hepatitis B is not particularly limited and can be in various material forms such as solid, liquid, gel, semi-liquid, aerosol, etc.
  • the pharmaceutical preparations for treating hepatitis B are mainly targeted at mammals, such as rodents, primates, etc.
  • a third aspect of the present invention provides a method for treating hepatitis B, which includes administering asiatic acid to a subject.
  • the subject may be a mammal or a mammalian hepatitis B cell.
  • the mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc.
  • the primate is preferably a monkey, ape or human.
  • the hepatitis B cells may be isolated hepatitis B cells.
  • the subject may be a patient suffering from hepatitis B or an individual with hepatitis B who is looking forward to treatment.
  • the subject is hepatitis B cells from a hepatitis B patient or an individual who is expected to treat hepatitis B.
  • the asiatic acid can be administered to the subject before, during and after hepatitis B treatment.
  • the fourth aspect of the present invention provides a hepatitis B combined treatment drug combination, including a safe and effective dose of asiatic acid and at least one other hepatitis B treatment drug, with the balance being pharmaceutically acceptable carriers and/or excipients.
  • hepatitis B combined treatment drug combination can be in any of the following forms:
  • Asiatic acid and other hepatitis B therapeutic drugs are made into independent preparations.
  • the dosage forms of the preparations can be the same or different, and the routes of administration can also be the same or different.
  • hepatitis B therapeutic drugs are antibodies
  • parenteral administration is generally used.
  • the administration form can be relatively rich, and it can be gastrointestinal administration or parenteral administration. It is generally recommended that each chemical drug be administered by a known route of administration.
  • hepatitis B therapeutic drugs are drugs that are known before the filing date of this application and can be used to treat hepatitis B, such as antiviral drugs: interferon (IFN), nucleoside analogs, etc.
  • IFN interferon
  • nucleoside analogs etc.
  • a fifth aspect of the present invention provides a method for treating hepatitis B, which includes administering an effective amount of asiatic acid to a subject and administering an effective amount of other hepatitis B therapeutic drugs to the subject and/or implementing other hepatitis B treatment methods to the subject. .
  • an effective dose of asiatic acid and at least one effective dose of other hepatitis B therapeutic drugs can be administered simultaneously or sequentially.
  • Asiatic acid is the first hepatitis B therapeutic drug discovered by the present invention. When used in combination with other hepatitis B therapeutic drugs other than asiatic acid, it can at least have an additive effect and further enhance the efficacy of hepatitis B. Therapeutic effect.
  • hepatitis B treatment drugs include but are not limited to: antibody drugs, chemical drugs or targeted drugs, etc.
  • the asiatic acid can be administered through the gastrointestinal tract or parenterally, and the other hepatitis B therapeutic drugs can be administered through the gastrointestinal tract or parenterally.
  • parenteral administration is generally used.
  • the sixth aspect of the present invention provides the use of asiatic acid in preparing substances with any one or more of the following effects: use in preparing substances that inhibit HBx protein; use in preparing substances that inhibit HBsAg levels; Use in the preparation of substances that inhibit the level of total HBV RNAs in cells; use in the preparation of substances that inhibit the level of HBV 3.5-kb RNA in cells; use in the preparation of substances that inhibit the level of HBV DNA.
  • asiatic acid of the present invention in the preparation of hepatitis B therapeutic drugs has the following beneficial effects:
  • the present invention found for the first time that asiatic acid can reduce HBx protein levels, inhibit cccDNA transcription activity and thereby reduce intracellular total HBV RNAs, HBV 3.5-kb RNA levels and HBsAg levels in the supernatant, and can effectively reduce serum levels in the recombinant cccDNA mouse model.
  • HBsAg, HBV DNA levels, and liver tissue HBV RNA and HBV DNA levels are potential new drugs targeting HBx.
  • the present invention provides a new direction and theoretical basis for reducing the infection rate of HBV and exploring new drugs for effective treatment of hepatitis B, and has great application prospects in the treatment of hepatitis B.
  • Figure 1 shows the MTT assay analyzing the cytotoxicity of asiatic acid in HepG2-NTCP and HepG2.2.15 cells.
  • Figure 2 shows the Western blot detection of the effect of asiatic acid on the protein levels of 3 ⁇ Flag-HBx and 2 ⁇ HA-HBx.
  • Figure 3 shows the fluorescence quantitative PCR method and Northern blot experiment to detect the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA.
  • Figure 4 shows the fluorescence quantitative PCR method and Southern blot experiment to detect the effect of asiatic acid on HBV DNA.
  • Figure 5 shows the ELISA test of the effect of asiatic acid on HBsAg secretion.
  • Figure 6 shows the Taq Man probe PCR experiment to detect the effect of asiatic acid on cccDNA levels and transcriptional activity.
  • Figure 7 shows the fluorescence quantitative PCR method and Northern blot experiment to detect the effect of asiatic acid on total HBV RNAs and HBV 3.5-kb RNA in HepG2.2.15 cells.
  • Figure 8 shows the fluorescence quantitative PCR method and Southern blot experiment to detect the effect of asiatic acid on HBV DNA in HepG2.2.15 cells.
  • Figure 9 shows the Taq Man probe PCR experiment to detect the effect of asiatic acid on cccDNA in HepG2.2.15 cells.
  • Figure 10 shows a mouse handling flow chart.
  • Figure 11 shows the effects of asiatic acid on HBsAg and HBV DNA levels in mouse serum detected by ELISA and fluorescence quantitative PCR.
  • Figure 12 shows the fluorescence quantitative PCR method to detect the effect of asiatic acid on HBV RNA levels in mouse liver tissue.
  • Figure 13 shows the fluorescence quantitative PCR method to detect the effect of asiatic acid on HBV DNA levels in mouse liver tissue.
  • Asiatic acid often referred to as AA, the Chinese name is Asiatic acid. It has shown antihypertensive, neuroprotective, cardioprotective, antibacterial and antitumor activities in preclinical studies.
  • the present invention's research found that AA can reduce HBx protein levels, inhibit cccDNA transcription activity and thereby reduce intracellular total HBV RNAs, HBV 3.5-kb RNA levels and HBsAg levels in the supernatant, and can effectively reduce HBsAg in serum in the recombinant cccDNA mouse model.
  • HBV DNA levels, and liver tissue HBV RNA and HBV DNA levels which are potential new drugs targeting HBx.
  • the present invention provides the use of asiatic acid as an active ingredient in the preparation of hepatitis B therapeutic drugs.
  • the hepatitis B treatment drug also includes one or more pharmaceutically acceptable carriers or excipients according to the needs of different pharmaceutical dosage forms.
  • “Pharmaceutically acceptable” means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the pharmaceutical dosage form is not particularly limited and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dropping pills, sprays, etc., and can be prepared by conventional methods.
  • the choice of drug dosage form should match the mode of administration.
  • the present invention also provides a drug combination and administration method for combined treatment of hepatitis B.
  • the hepatitis B combined treatment drug combination can be in any of the following forms:
  • Asiatic acid and other hepatitis B therapeutic drugs are made into independent preparations.
  • the dosage forms of the preparations can be the same or different, and the routes of administration can also be the same or different.
  • several medicines can be used at the same time, or several medicines can be used one after another.
  • administering drugs sequentially other drugs should be administered to the body while the first drug is still effective.
  • hepatitis B therapeutic drugs are antibodies, they are generally administered parenterally, such as intravenous injection, intravenous drip, or arterial infusion. Its usage and dosage can refer to the existing technology.
  • hepatitis B treatment drugs are chemical drugs
  • the administration forms can be richer, and they can be gastrointestinal or parenteral. It is generally recommended that each chemical drug be administered by a known route of administration.
  • combination of drugs in the present invention refers to a reasonable combination of drugs, which should be based on the basic principle of improving efficacy and/or reducing adverse reactions.
  • drug interactions should include interactions that affect pharmacokinetics and pharmacodynamics.
  • types of drugs should be reduced as much as possible to reduce adverse drug reactions caused by drug interactions, avoid affecting drug efficacy or increasing toxicity, and avoid producing opposite results.
  • HepG2-NTCP cells were cultured in DMEM medium containing 10% fetal calf serum, 1% penicillin–streptomycin, and 2ug/mL doxycycline, and HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal calf serum, 1% penicillin– Streptomycin and 400ug/mL G418 in DMEM culture medium, placed in a 37°C, 5% CO2 incubator.
  • Asiatic acid (cas no. 464-92-6) was purchased from MCE.
  • prcccDNA and pCMV-KRAB-Cre were gifts from Professor Deng Qiang (Fudan University, Shanghai, China).
  • 3 ⁇ Flag-HBx was constructed by inserting full-length HBx into p3 ⁇ Flag-CMV7.1.
  • 3 ⁇ Flag-HBx was constructed by inserting full-length HBx into p3 ⁇ Flag-CMV7.1.
  • 2 ⁇ HA HBx was a gift from Professor Jieliang Chen (Fudan University, Shanghai, China).
  • Asiatic acid (cas number 464-92-6) was purchased from MCE Company, dissolved in DMSO, prepared into a 20mM storage solution, aliquoted and stored Store at –80°C until use.
  • HepG2-NTCP cells and HepG2.2.15 cells were seeded in a 96-well culture plate at 15 ⁇ 10 4 /ml. After culturing for 24 hours, use DMEM medium to dilute asiatic acid to 8 concentration gradients of 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0uM. Set up 3 replicate wells for each concentration, and set up a normal cell control. After 72 hours, add 10ul MTT reagent, incubate at 37°C for 4 hours, add 100ul DMSO, shake for 10 minutes, detect the OD value at 490nm with a microplate reader, calculate and draw the cell activity curve.
  • HepG2-NTCP cells were seeded in twelve-well plates at 20 ⁇ 10 4 /ml. After 24 hours of culture, 3 ⁇ Flag-HBx and 2 ⁇ HA-HBx were transfected respectively; 24 hours after transfection, asiatic acid was diluted to 4 concentration gradients of 30, 20, 10, and 0uM using DMEM medium, and HepG2- were treated respectively. NTCP cells. After 48 h, total protein was extracted and Western blot was used to detect HBx protein levels.
  • HepG2-NTCP cells were seeded in twelve-well plates at 20 ⁇ 10 4 /ml. After culturing for 24 hours, use DMEM medium to dilute asiatic acid to 4 concentration gradients of 30, 20, 10, and 0uM. HepG2-NTCP cells were treated for 3 days, 6 days, and 9 days respectively. The medium supernatant and cells were collected, and the supernatant was detected by ELISA.
  • HBsAg levels, Real-time PCR and Northern blot were used to detect total HBV RNAs and HBV 3.5-kb RNA levels in cells, and Real-time PCR and Southern blot were used to detect HBV DNA levels in cells.
  • HepG2.2.15 cells were seeded in twelve-well plates at 20 ⁇ 10 4 /ml. After culturing for 24 hours, HepG2.2.15 cells were treated with DMEM medium diluted with asiatic acid to 4 concentration gradients of 30, 20, 10, and 0uM. The cells were collected after 4 days. Real-time PCR and Northern blot were used to detect total HBV RNAs and total HBV RNAs in the cells. HBV 3.5-kb RNA level, Real-time PCR and Southern blot were used to detect HBV DNA level in cells.
  • mice Fifty male C57BL/6 mice, aged 6-8 weeks and weighing 18-20g, were selected. 4 ⁇ g of plasmid prcccDNA and 4 ⁇ g of plasmid pCMV-KRAB-Cre were injected into the tail vein respectively. One week later, the serum HBV DNA copy number was detected by fluorescence quantitative PCR to determine whether the model was successfully established.
  • mice The successfully modeled mice were randomly divided into four groups: negative control, positive control, low-concentration experimental group, and high-concentration experimental group.
  • Six mice in each group were orally administered 0.9% normal saline, 0.02mg/kg entecavir, and 15mg/kg entecavir.
  • the liver tissue was ground to detect the levels of HBV RNAs and HBV DNA.
  • HBV DNA primers F: CCTAGTAGTCAGTTATGTCAAC (SEQ ID NO.1), R: TCTATAAGCTGGAGGAGTGCGA (SEQ ID NO.2 ). Three duplicate wells were set up for each sample, and each set of experiments was repeated three times.
  • Detect HBV RNA cDNA synthesized by reverse transcription RNA, prepare the reaction system and set the reaction conditions according to the instructions of SYBR Green (Bio Rad).
  • the HBV 3.5kb RNA primers are F: CTCTTCCAGCCTTCCTTCCT (SEQ ID NO. 3), R: AGCACTGTGTTGGCGTACAG (SEQ ID NO.4), the total HBV RNAs primer is F:ACCGACCTTGAGGCATACTT (SEQ ID NO. 5), R: GCCTACAGCCTCCTAGTACA (SEQ ID NO. 6).
  • Three duplicate wells were set up for each sample, and each set of experiments was repeated three times.
  • Figure 1 shows the MTT assay analyzing the cytotoxicity of asiatic acid in HepG2-NTCP and HepG2.2.15 cells.
  • the results shown in Figure 1 show that asiatic acid has a CC50 of 74.82 ⁇ M in the HBV-infected cell line HepG2-NTCP and a CC50 of 67.6 ⁇ M in the HBV stable replication model HepG2.2.15.
  • Figure 2 shows the Western blot detection of the effect of asiatic acid on the protein levels of 3 ⁇ Flag-HBx and 2 ⁇ HA-HBx.
  • the Western blot results shown in Figure 2 show that after asiatic acid treatment, the protein levels of exogenous 3 ⁇ Flag-HBx and 2 ⁇ HA-HBx in HepG2-NTCP cells decreased in a concentration gradient-dependent manner, suggesting that asiatic acid can inhibit HBx Express.
  • Figure 3 shows the fluorescence quantitative PCR method and Northern blot experiment to detect the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA.
  • the Real-time PCR results shown in Figure 3 show that compared with the negative control group, asiatic acid significantly reduced the levels of total HBV RNAs and HBV 3.5-kb RNA in a time- and concentration-gradient-dependent manner.
  • Northern blot verified the PCR results .
  • Figure 4 shows the fluorescence quantitative PCR method and Southern blot experiment to detect the effect of asiatic acid on HBV DNA levels.
  • the Real-time PCR results shown in Figure 4 show that compared with the negative control group, asiatic acid significantly reduced the level of intracellular HBV DNA in a time- and concentration-gradient-dependent manner. Southern blot verified the PCR results.
  • Figure 5 shows the ELISA test of the effect of asiatic acid on HBsAg secretion.
  • the ELISA results shown in Figure 5 show that asiatic acid can effectively reduce the level of HBsAg in HepG2-NTCP cells in a time- and concentration gradient-dependent manner.
  • Figure 6 shows the Taq Man probe PCR experiment to detect the effect of asiatic acid on cccDNA levels and transcriptional activity. Because HBx plays a key role in the transcription process of cccDNA. Inhibiting the function of HBx may inhibit cccDNA transcription. Therefore, the effect of asiatic acid on cccDNA transcription was further determined.
  • the Taq Man probe PCR experimental results shown in Figure 6 show that asiatic acid has no significant effect on cccDNA levels. Further analysis of cccDNA transcription activity (total RNAs/cccDNA and pgRNA/cccDNA ratio) found that asiatic acid can significantly inhibit cccDNA transcription activity.
  • Figure 7 shows the detection of the effect of asiatic acid on total HBV RNAs and HBV 3.5-kb RNA in HepG2.2.15 cells.
  • Figure 8 shows the detection of the effect of asiatic acid on HBV DNA in HepG2.2.15 cells. The results showed that asiatic acid could significantly inhibit intracellular HBV RNA ( Figure 7) and HBV DNA levels (Figure 8), but did not affect the cccDNA level ( Figure 9).
  • Figure 10 shows a mouse handling flow chart.
  • Figure 11 shows the effects of asiatic acid on HBsAg and HBV DNA levels in mouse serum detected by ELISA and fluorescence quantitative PCR.
  • Figure 12 shows the fluorescence quantitative PCR method to detect the effect of asiatic acid on HBV RNA levels in mouse liver tissue.
  • Figure 13 shows the fluorescence quantitative PCR method for detecting the effect of asiatic acid on HBV in mouse liver tissue. DNA level effects.

Abstract

The present invention provides use of asiatic acid in the preparation of a medicament for treating hepatitis B. Asiatic acid can reduce the HBx protein level and inhibit cccDNA transcription activity so as to reduce total HBV RNAs and HBV 3.5-kb RNA levels in cells as well as HBsAg levels in the supernatant, and can effectively reduce HBsAg and HBV DNA levels in serum as well as HBV RNA and HBV DNA levels in liver tissue in a recombinant cccDNA mouse model. It is a potential novel medicament targeting HBx, and can be used for treating hepatitis B.

Description

积雪草酸在制备乙型肝炎治疗药物中的用途Use of Asiatic Acid in Preparing Hepatitis B Treatment Drugs 技术领域Technical field
本发明涉及生物医药技术领域，特别是涉及积雪草酸在制备乙型肝炎治疗药物中的用途。The present invention relates to the field of biomedicine technology, and in particular to the use of asiatic acid in the preparation of hepatitis B therapeutic drugs.
背景技术Background technique
乙型肝炎病毒(Hepatitis B virus，HBV)感染是世界范围内的一个重大公共卫生问题。据WHO报道，全球约20亿人曾感染HBV，其中约有2.57亿慢性乙肝感染患者。我国属HBV高流行区。约有9700万乙肝病毒携带者，占全国总人口的8％至10％，其中约2000万为慢乙肝患者，每年因慢乙肝导致的肝硬化、肝癌等并发症死亡患者更是高达26.3万例。因此，乙型肝炎防治任务十分严峻。Hepatitis B virus (HBV) infection is a major public health problem worldwide. According to WHO, approximately 2 billion people worldwide have been infected with HBV, including approximately 257 million patients with chronic hepatitis B infection. my country is an area with high prevalence of HBV. There are about 97 million hepatitis B virus carriers, accounting for 8% to 10% of the country's total population, of which about 20 million are chronic hepatitis B patients. Every year, there are as many as 263,000 deaths due to complications such as cirrhosis and liver cancer caused by chronic hepatitis B. . Therefore, the task of preventing and controlling hepatitis B is very serious.
目前治疗慢乙肝常用的抗病毒药物包括干扰素(IFN)和核苷类似物(NAs)。但这两类药物都存在一定的缺点，如核苷类似物用药时间长、患者依从性差且停药后易反弹，干扰素仅对不到30％的患者效果明显，且不良反应较多。更重要的是，这两类药物均不能有效治愈乙肝，因此需迫切开发新型抗HBV药物。Currently, commonly used antiviral drugs for the treatment of chronic hepatitis B include interferon (IFN) and nucleoside analogs (NAs). However, both types of drugs have certain shortcomings, such as nucleoside analogues, which take a long time to take, poor patient compliance, and are prone to rebound after drug withdrawal. Interferons are only effective in less than 30% of patients and have many adverse reactions. More importantly, neither type of drugs can effectively cure hepatitis B, so there is an urgent need to develop new anti-HBV drugs.
HBV属嗜肝DNA病毒科(hepadnaviridae)，其基因组为长约3.2kb的部分双链、松弛环状DNA(relaxed-circular DNA,rcDNA)。HBV病毒进入宿主肝细胞后，rcDNA被传递至细胞核并形成具有超螺旋结构的共价闭合环状DNA(Covalently closed circular DNA，cccDNA)。cccDNA以微染色体的形式持续存在于肝细胞核中，结构稳定，且是病毒RNA的转录模板。因此cccDNA是HBV持续感染、耐药以及抗病毒药物停用后HBV再激活的最主要原因。HBV belongs to the hepadnaviridae family, and its genome is a partially double-stranded, relaxed circular DNA (relaxed-circular DNA, rcDNA) of approximately 3.2 kb in length. After the HBV virus enters the host liver cells, rcDNA is delivered to the nucleus and forms covalently closed circular DNA (cccDNA) with a superhelical structure. cccDNA persists in the nucleus of liver cells in the form of minichromosomes, has a stable structure, and serves as a transcription template for viral RNA. Therefore, cccDNA is the main cause of persistent HBV infection, drug resistance, and HBV reactivation after discontinuation of antiviral drugs.
而在感染HBV细胞中，病毒可编码一种反式激活蛋白——HBx，其可通过劫持DDB1从而解除E3泛素连接酶CUL4B对Smc5/6的泛素化降解进而活化cccDNA转录，也可通过结合至cccDNA微染色体上抑制转录阻遏物到cccDNA上的募集从而维持cccDNA转录。这些研究均表明HBx在cccDNA的转录过程中发挥关键作用。抑制HBx的功能，则可能抑制cccDNA转录。因此，HBx是治疗慢性HBV感染的新疗法的一个重要靶点，开发新型靶向抑制HBx的药物，可为降低HBV的感染率、探索有效治疗乙型肝炎的新型药物提供新的方向及理论依据。In infected HBV cells, the virus can encode a transactivator protein - HBx, which can hijack DDB1 to relieve the ubiquitination and degradation of Smc5/6 by the E3 ubiquitin ligase CUL4B and activate cccDNA transcription. It can also activate cccDNA transcription through Binding to the cccDNA minichromosome inhibits the recruitment of transcriptional repressors to cccDNA thereby maintaining cccDNA transcription. These studies indicate that HBx plays a key role in the transcription process of cccDNA. Inhibiting the function of HBx may inhibit cccDNA transcription. Therefore, HBx is an important target for new therapies to treat chronic HBV infection. The development of new drugs that target and inhibit HBx can provide new directions and theoretical basis for reducing the infection rate of HBV and exploring new drugs for the effective treatment of hepatitis B. .
发明内容Contents of the invention
鉴于以上所述现有技术的缺点，本发明的目的在于提供积雪草酸在制备乙型肝炎治疗药物中的用途，积雪草酸能靶向抑制HBx，为乙肝的有效治疗提供一种新的药物和技术手段。In view of the above shortcomings of the prior art, the purpose of the present invention is to provide the use of asiatic acid in the preparation of hepatitis B therapeutic drugs. Asiatic acid can target HBx and provide a new drug for the effective treatment of hepatitis B. and technical means.
为实现上述目的及其他相关目的，本发明第一方面提供积雪草酸作为有效成分在制备乙型肝炎治疗药物中的用途。In order to achieve the above objects and other related objects, the first aspect of the present invention provides the use of asiatic acid as an active ingredient in the preparation of hepatitis B therapeutic drugs.
进一步，所述乙型肝炎治疗药物至少具有以下功用之一：Further, the hepatitis B treatment drug has at least one of the following functions:
降低HBsAg水平，降低细胞内total HBV RNAs水平，降低细胞内HBV 3.5-kb RNA水平，降低HBV DNA水平。Reduce HBsAg levels, reduce intracellular total HBV RNAs levels, reduce intracellular HBV 3.5-kb RNA levels, and reduce HBV DNA levels.
进一步，所述乙型肝炎治疗药物必然包括积雪草酸，并以积雪草酸作为前述功用 的有效成分。Furthermore, the hepatitis B treatment drug must include asiatic acid, and use asiatic acid as the aforementioned function. of active ingredients.
进一步，所述乙型肝炎治疗药物中，发挥前述功用的有效成分可仅为积雪草酸，亦可包含其他可起到类似功用的分子。Furthermore, in the hepatitis B treatment drug, the active ingredient that performs the aforementioned functions may only be asiatic acid, or may include other molecules that can perform similar functions.
进一步，所述积雪草酸作为所述乙型肝炎治疗药物的有效成分之一或唯一有效成分。Further, the asiatic acid is used as one of the active ingredients or the only active ingredient of the hepatitis B treatment drug.
进一步，所述乙型肝炎治疗药物可以为单成分物质，亦可为多成分物质。Furthermore, the hepatitis B treatment drug may be a single-component substance or a multi-component substance.
进一步，所述乙型肝炎治疗药物的形式无特殊限制，可以为固体、液体、凝胶、半流质、气雾等各种物质形式。Furthermore, the form of the hepatitis B treatment drug is not particularly limited and can be in various material forms such as solid, liquid, gel, semi-liquid, aerosol, etc.
进一步，所述乙型肝炎治疗药物主要针对的对象为哺乳动物，如啮齿类动物、灵长类动物等。Furthermore, the hepatitis B therapeutic drugs are mainly targeted at mammals, such as rodents, primates, etc.
本发明第二方面提供一种治疗乙型肝炎的药物制剂，包括安全有效剂量的积雪草酸。The second aspect of the present invention provides a pharmaceutical preparation for treating hepatitis B, including a safe and effective dose of asiatic acid.
进一步，所述治疗乙型肝炎的药物制剂包括10～30μM的积雪草酸。Further, the pharmaceutical preparation for treating hepatitis B includes 10-30 μM asiatic acid.
进一步，所述治疗乙型肝炎的药物制剂的给药量为15～30mg/kg积雪草酸。Further, the dosage of the pharmaceutical preparation for treating hepatitis B is 15-30 mg/kg asiatic acid.
进一步，所述治疗乙型肝炎的药物制剂还包括药学上可接受的载体和/或辅料。Furthermore, the pharmaceutical preparation for treating hepatitis B also includes pharmaceutically acceptable carriers and/or excipients.
进一步，所述治疗乙型肝炎的药物制剂中，必然包括积雪草酸，并以积雪草酸作为前述功用的有效成分。Furthermore, the pharmaceutical preparation for treating hepatitis B must include asiatic acid, and use asiatic acid as an active ingredient for the aforementioned functions.
进一步，所述治疗乙型肝炎的药物制剂中，发挥前述功用的有效成分可仅为积雪草酸，亦可包含其他可起到类似功用的分子。Furthermore, in the pharmaceutical preparation for treating hepatitis B, the active ingredient that performs the aforementioned functions may only be asiatic acid, or may include other molecules that can perform similar functions.
进一步，所述积雪草酸作为所述治疗乙型肝炎的药物制剂的有效成分之一或唯一有效成分。Further, the asiatic acid is used as one of the active ingredients or the only active ingredient of the pharmaceutical preparation for treating hepatitis B.
进一步，所述治疗乙型肝炎的药物制剂可以为单成分物质，亦可为多成分物质。Furthermore, the pharmaceutical preparation for treating hepatitis B may be a single-component substance or a multi-component substance.
进一步，所述治疗乙型肝炎的药物制剂的形式无特殊限制，可以为固体、液体、凝胶、半流质、气雾等各种物质形式。Furthermore, the form of the pharmaceutical preparation for treating hepatitis B is not particularly limited and can be in various material forms such as solid, liquid, gel, semi-liquid, aerosol, etc.
进一步，所述治疗乙型肝炎的药物制剂主要针对的对象为哺乳动物，如啮齿类动物、灵长类动物等。Furthermore, the pharmaceutical preparations for treating hepatitis B are mainly targeted at mammals, such as rodents, primates, etc.
本发明第三方面提供一种治疗乙型肝炎的方法，为向对象施用积雪草酸。A third aspect of the present invention provides a method for treating hepatitis B, which includes administering asiatic acid to a subject.
进一步，所述对象可以为哺乳动物或哺乳动物的乙型肝炎细胞。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。所述乙型肝炎细胞可以为离体乙型肝炎细胞。Further, the subject may be a mammal or a mammalian hepatitis B cell. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, ape or human. The hepatitis B cells may be isolated hepatitis B cells.
进一步，所述对象可以是罹患乙型肝炎的患者或者期待治疗的乙型肝炎的个体。或者，所述对象为乙型肝炎患者或者期待治疗乙型肝炎的个体的乙型肝炎细胞。Further, the subject may be a patient suffering from hepatitis B or an individual with hepatitis B who is looking forward to treatment. Alternatively, the subject is hepatitis B cells from a hepatitis B patient or an individual who is expected to treat hepatitis B.
进一步，所述积雪草酸可以在接受乙型肝炎治疗前、中、后向对象施用。Further, the asiatic acid can be administered to the subject before, during and after hepatitis B treatment.
本发明第四方面提供一种乙型肝炎联合治疗药物组合，包括安全有效剂量的积雪草酸和至少一种其他乙型肝炎治疗药物，余量为药学上可接受的载体和/或辅料。The fourth aspect of the present invention provides a hepatitis B combined treatment drug combination, including a safe and effective dose of asiatic acid and at least one other hepatitis B treatment drug, with the balance being pharmaceutically acceptable carriers and/or excipients.
进一步，所述乙型肝炎联合治疗药物组合可以是以下形式中的任意一种：Further, the hepatitis B combined treatment drug combination can be in any of the following forms:
(一)将积雪草酸和其他乙型肝炎治疗药物分别制成独立的制剂，制剂的剂型可相同或不同，给药途径亦可相同或不同。(1) Asiatic acid and other hepatitis B therapeutic drugs are made into independent preparations. The dosage forms of the preparations can be the same or different, and the routes of administration can also be the same or different.
当其他乙型肝炎治疗药物为抗体时，一般采用胃肠外给药型。当其他乙型肝炎治 疗药物为化学药物时，给药形式可以比较丰富，可以是胃肠道给药亦可以是非胃肠道给药。一般推荐针对各化学药物的已知给药途径给药。When other hepatitis B therapeutic drugs are antibodies, parenteral administration is generally used. When other hepatitis B treatments When the therapeutic drug is a chemical drug, the administration form can be relatively rich, and it can be gastrointestinal administration or parenteral administration. It is generally recommended that each chemical drug be administered by a known route of administration.
(二)将积雪草酸和其他乙型肝炎治疗药物配置成复方制剂，在将积雪草酸和其他乙型肝炎治疗药物采用相同给药途径给药并同时施加时，可采用将两者配置成复方制剂的形式。(2) Configure asiatic acid and other hepatitis B therapeutic drugs into a compound preparation. When asiatic acid and other hepatitis B therapeutic drugs are administered by the same route of administration and administered at the same time, they can be formulated into Form of compound preparation.
进一步，所述其他乙型肝炎治疗药物为在本申请申请日以前已知的可用于治疗乙型肝炎的药物，例如，抗病毒药物：干扰素(IFN)、核苷类似物等。Furthermore, the other hepatitis B therapeutic drugs are drugs that are known before the filing date of this application and can be used to treat hepatitis B, such as antiviral drugs: interferon (IFN), nucleoside analogs, etc.
本发明第五方面提供一种治疗乙型肝炎的方法，为向对象施用有效量的积雪草酸以及向对象施用有效量的其他乙型肝炎治疗药物和/或向对象实施其他乙型肝炎治疗手段。A fifth aspect of the present invention provides a method for treating hepatitis B, which includes administering an effective amount of asiatic acid to a subject and administering an effective amount of other hepatitis B therapeutic drugs to the subject and/or implementing other hepatitis B treatment methods to the subject. .
进一步，可以同步地或顺序地给予有效剂量的积雪草酸和至少一种有效剂量的其他乙型肝炎治疗药物。Further, an effective dose of asiatic acid and at least one effective dose of other hepatitis B therapeutic drugs can be administered simultaneously or sequentially.
基于积雪草酸为本发明首次发现的乙型肝炎治疗药物，在与积雪草酸以外的其他乙型肝炎治疗药物联合用药中，至少可以起到疗效相加的效果，进一步增强对于乙型肝炎的治疗作用。Asiatic acid is the first hepatitis B therapeutic drug discovered by the present invention. When used in combination with other hepatitis B therapeutic drugs other than asiatic acid, it can at least have an additive effect and further enhance the efficacy of hepatitis B. Therapeutic effect.
进一步，其他的乙型肝炎治疗药物包括但不限制于：抗体药物、化学药物或靶向型药物等。Further, other hepatitis B treatment drugs include but are not limited to: antibody drugs, chemical drugs or targeted drugs, etc.
进一步，所述积雪草酸可以是胃肠道给药或者胃肠外给药，所述其他乙型肝炎治疗药物可以是胃肠道给药或者胃肠外给药。对于抗体药物，一般采用胃肠外给药。Furthermore, the asiatic acid can be administered through the gastrointestinal tract or parenterally, and the other hepatitis B therapeutic drugs can be administered through the gastrointestinal tract or parenterally. For antibody drugs, parenteral administration is generally used.
本发明第六方面提供了积雪草酸在制备具有以下任一项或多项作用的物质中的用途：用于制备抑制HBx蛋白的物质的用途；用于制备抑制HBsAg水平的物质的用途；用于制备抑制细胞内total HBV RNAs水平的物质的用途；用于制备抑制细胞内HBV 3.5-kb RNA水平的物质的用途；用于制备抑制HBV DNA水平的物质的用途。The sixth aspect of the present invention provides the use of asiatic acid in preparing substances with any one or more of the following effects: use in preparing substances that inhibit HBx protein; use in preparing substances that inhibit HBsAg levels; Use in the preparation of substances that inhibit the level of total HBV RNAs in cells; use in the preparation of substances that inhibit the level of HBV 3.5-kb RNA in cells; use in the preparation of substances that inhibit the level of HBV DNA.
如上所述，本发明的积雪草酸在制备乙型肝炎治疗药物中的用途，具有以下有益效果：As mentioned above, the use of asiatic acid of the present invention in the preparation of hepatitis B therapeutic drugs has the following beneficial effects:
本发明首次发现积雪草酸能够降低HBx蛋白水平，抑制cccDNA转录活性进而降低细胞内total HBV RNAs、HBV 3.5-kb RNA水平以及上清中HBsAg水平，且在重组cccDNA小鼠模型内可有效降低血清中HBsAg、HBV DNA水平以及肝组织HBV RNA及HBV DNA水平，是潜在的新型靶向HBx的药物。本发明为降低HBV的感染率、探索有效治疗乙型肝炎的新型药物提供了新的方向及理论依据，在乙肝治疗方面具有极大的应用前景。The present invention found for the first time that asiatic acid can reduce HBx protein levels, inhibit cccDNA transcription activity and thereby reduce intracellular total HBV RNAs, HBV 3.5-kb RNA levels and HBsAg levels in the supernatant, and can effectively reduce serum levels in the recombinant cccDNA mouse model. HBsAg, HBV DNA levels, and liver tissue HBV RNA and HBV DNA levels are potential new drugs targeting HBx. The present invention provides a new direction and theoretical basis for reducing the infection rate of HBV and exploring new drugs for effective treatment of hepatitis B, and has great application prospects in the treatment of hepatitis B.
附图说明Description of the drawings
图1显示为MTT实验分析积雪草酸在HepG2-NTCP、HepG2.2.15细胞中的细胞毒性。Figure 1 shows the MTT assay analyzing the cytotoxicity of asiatic acid in HepG2-NTCP and HepG2.2.15 cells.
图2显示为Western blot检测积雪草酸对3×Flag-HBx、2×HA-HBx蛋白水平的影响。Figure 2 shows the Western blot detection of the effect of asiatic acid on the protein levels of 3×Flag-HBx and 2×HA-HBx.
图3显示为荧光定量PCR法和Northern blot实验检测积雪草酸对total HBV RNAs、HBV3.5-kb RNA的影响。Figure 3 shows the fluorescence quantitative PCR method and Northern blot experiment to detect the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA.
图4显示为荧光定量PCR法和Southern blot实验检测积雪草酸对HBV DNA的影响。Figure 4 shows the fluorescence quantitative PCR method and Southern blot experiment to detect the effect of asiatic acid on HBV DNA.
图5显示为ELISA检测积雪草酸对HBsAg分泌的影响。 Figure 5 shows the ELISA test of the effect of asiatic acid on HBsAg secretion.
图6显示为Taq Man探针PCR实验检测积雪草酸对cccDNA水平及转录活性的影响。Figure 6 shows the Taq Man probe PCR experiment to detect the effect of asiatic acid on cccDNA levels and transcriptional activity.
图7显示为荧光定量PCR法和Northern blot实验检测积雪草酸在HepG2.2.15细胞中对total HBV RNAs、HBV 3.5-kb RNA的影响。Figure 7 shows the fluorescence quantitative PCR method and Northern blot experiment to detect the effect of asiatic acid on total HBV RNAs and HBV 3.5-kb RNA in HepG2.2.15 cells.
图8显示为荧光定量PCR法和Southern blot实验检测积雪草酸在HepG2.2.15细胞中对HBV DNA的影响。Figure 8 shows the fluorescence quantitative PCR method and Southern blot experiment to detect the effect of asiatic acid on HBV DNA in HepG2.2.15 cells.
图9显示为Taq Man探针PCR实验检测积雪草酸在HepG2.2.15细胞中对cccDNA的影响。Figure 9 shows the Taq Man probe PCR experiment to detect the effect of asiatic acid on cccDNA in HepG2.2.15 cells.
图10显示为小鼠处理流程图。Figure 10 shows a mouse handling flow chart.
图11显示为ELISA和荧光定量PCR法检测积雪草酸对小鼠血清中HBsAg、HBV DNA水平的影响。Figure 11 shows the effects of asiatic acid on HBsAg and HBV DNA levels in mouse serum detected by ELISA and fluorescence quantitative PCR.
图12显示为荧光定量PCR法检测积雪草酸对小鼠肝组织中HBV RNA水平的影响。Figure 12 shows the fluorescence quantitative PCR method to detect the effect of asiatic acid on HBV RNA levels in mouse liver tissue.
图13显示为荧光定量PCR法检测积雪草酸对小鼠肝组织中HBV DNA水平的影响。Figure 13 shows the fluorescence quantitative PCR method to detect the effect of asiatic acid on HBV DNA levels in mouse liver tissue.
具体实施方式Detailed ways
Asiatic acid，常简称AA，中文名为积雪草酸。其在临床前研究中显示出抗高血压、神经保护、心脏保护、抗菌和抗肿瘤活性。本发明研究发现AA能够降低HBx蛋白水平，抑制cccDNA转录活性进而降低细胞内total HBV RNAs、HBV 3.5-kb RNA水平以及上清中HBsAg水平，且在重组cccDNA小鼠模型内可有效降低血清中HBsAg、HBV DNA水平以及肝组织HBV RNA及HBV DNA水平，是潜在的新型靶向HBx的药物。Asiatic acid, often referred to as AA, the Chinese name is Asiatic acid. It has shown antihypertensive, neuroprotective, cardioprotective, antibacterial and antitumor activities in preclinical studies. The present invention's research found that AA can reduce HBx protein levels, inhibit cccDNA transcription activity and thereby reduce intracellular total HBV RNAs, HBV 3.5-kb RNA levels and HBsAg levels in the supernatant, and can effectively reduce HBsAg in serum in the recombinant cccDNA mouse model. , HBV DNA levels, and liver tissue HBV RNA and HBV DNA levels, which are potential new drugs targeting HBx.
基于此，本发明提供以积雪草酸作为有效成分在制备乙型肝炎治疗药物中的用途。Based on this, the present invention provides the use of asiatic acid as an active ingredient in the preparation of hepatitis B therapeutic drugs.
通常，所述乙型肝炎治疗药物除了包括安全有效剂量的积雪草酸外，根据不同药物剂型的需要，还会包括一种或多种药学上可接受的载体或辅料。Usually, in addition to a safe and effective dose of asiatic acid, the hepatitis B treatment drug also includes one or more pharmaceutically acceptable carriers or excipients according to the needs of different pharmaceutical dosage forms.
“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。"Pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
“药学上可接受的载体或辅料”应当与积雪草酸相容，即能与其共混而不会在通常情况下大幅度降低药物组合物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醉、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。The "pharmaceutically acceptable carrier or excipient" should be compatible with asiatic acid, that is, it can be blended with it without significantly reducing the effectiveness of the pharmaceutical composition under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, Ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, etc. These substances are used as needed to aid the stability of the formulation or to help enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.
本发明中，除非特别说明，药物剂型并无特别限定，可以被制成针剂、口服液、片剂、胶囊、滴丸、喷剂等剂型，可通过常规方法进行制备。药物剂型的选择应与给药方式相匹配。In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dropping pills, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the mode of administration.
另外，本发明还提供一种乙型肝炎联合治疗药物组合和施用方法。所述乙型肝炎联合治疗药物组合可以是以下形式中的任意一种： In addition, the present invention also provides a drug combination and administration method for combined treatment of hepatitis B. The hepatitis B combined treatment drug combination can be in any of the following forms:
(一)将积雪草酸和其他乙型肝炎治疗药物分别制成独立的制剂，制剂的剂型可相同或不同，给药途径亦可相同或不同。使用时，可几种药同时使用，也可几种药先后使用。先后给药时，应当在先用药物仍对机体有效的期间内向机体施加其他药物。(1) Asiatic acid and other hepatitis B therapeutic drugs are made into independent preparations. The dosage forms of the preparations can be the same or different, and the routes of administration can also be the same or different. When used, several medicines can be used at the same time, or several medicines can be used one after another. When administering drugs sequentially, other drugs should be administered to the body while the first drug is still effective.
当其他乙型肝炎治疗药物为抗体时，一般采用胃肠外给药型，如静脉注射、静脉滴注或动脉灌注。其用法用量可参考现有技术。When other hepatitis B therapeutic drugs are antibodies, they are generally administered parenterally, such as intravenous injection, intravenous drip, or arterial infusion. Its usage and dosage can refer to the existing technology.
当其他乙型肝炎治疗药物为化学药物时，给药形式可以比较丰富，可以是胃肠道给药亦可以是非胃肠道给药。一般推荐针对各化学药物的已知给药途径给药。When other hepatitis B treatment drugs are chemical drugs, the administration forms can be richer, and they can be gastrointestinal or parenteral. It is generally recommended that each chemical drug be administered by a known route of administration.
(二)将积雪草酸和其他乙型肝炎治疗药物配置成复方制剂，在将积雪草酸和其他乙型肝炎治疗药物采用相同给药途径给药并同时施加时，可采用将两者配置成复方制剂的形式。(2) Configure asiatic acid and other hepatitis B therapeutic drugs into a compound preparation. When asiatic acid and other hepatitis B therapeutic drugs are administered by the same route of administration and administered at the same time, they can be formulated into Form of compound preparation.
需要注意的是，本发明中的联合用药是指合理的联合用药，应以提高疗效和(或)降低不良反应为基本原则。联合用药时，药物的相互作用，应包括影响药动学的相互作用，应包括影响药效学的相互作用。联合用药时，应尽量减少用药种类，减少药物相互作用引起的药物不良反应，避免影响药物疗效或增加毒性，避免产生相反的结果。It should be noted that the combination of drugs in the present invention refers to a reasonable combination of drugs, which should be based on the basic principle of improving efficacy and/or reducing adverse reactions. When drugs are used in combination, drug interactions should include interactions that affect pharmacokinetics and pharmacodynamics. When using drugs in combination, the types of drugs should be reduced as much as possible to reduce adverse drug reactions caused by drug interactions, avoid affecting drug efficacy or increasing toxicity, and avoid producing opposite results.
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, It is not intended to limit the scope of the present invention. Test methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by each manufacturer.
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present invention based on their understanding of the prior art and the description of the present invention. Any methods, equipment and materials similar or equivalent to those in the prior art may be used to implement the present invention.
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology.
实施例1Example 1
一、实验方法1. Experimental methods
1、细胞培养1. Cell culture
HepG2-NTCP细胞培养于含有10％胎牛血清、1％青霉素–链霉素、2ug/mL强力霉素的DMEM培养基中，HepG2.2.15细胞培养于含10％胎牛血清、1％青霉素–链霉素、400ug/mL G418的DMEM培养基中，置于37℃、5％CO2培养箱中培养。积雪草酸(Asiatic acid，cas号464-92-6)购于MCE。HepG2-NTCP cells were cultured in DMEM medium containing 10% fetal calf serum, 1% penicillin–streptomycin, and 2ug/mL doxycycline, and HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal calf serum, 1% penicillin– Streptomycin and 400ug/mL G418 in DMEM culture medium, placed in a 37°C, 5% CO2 incubator. Asiatic acid (cas no. 464-92-6) was purchased from MCE.
2、质粒和药物2. Plasmids and drugs
prcccDNA和pCMV-KRAB-Cre由邓强教授(复旦大学，上海，中国)馈赠。3×Flag-HBx是将全长HBx插入p3×Flag-CMV7.1构建而成的。3×Flag-HBx是将全长HBx插入p3×Flag-CMV7.1构建而成的。2×HA HBx由陈捷亮教授(复旦大学，上海，中国)馈赠。积雪草酸(Asiatic acid，cas号464-92-6)购于MCE公司，溶于DMSO，配制成20mM储存溶液，分装并储 存于–80℃备用。prcccDNA and pCMV-KRAB-Cre were gifts from Professor Deng Qiang (Fudan University, Shanghai, China). 3×Flag-HBx was constructed by inserting full-length HBx into p3×Flag-CMV7.1. 3×Flag-HBx was constructed by inserting full-length HBx into p3×Flag-CMV7.1. 2×HA HBx was a gift from Professor Jieliang Chen (Fudan University, Shanghai, China). Asiatic acid (cas number 464-92-6) was purchased from MCE Company, dissolved in DMSO, prepared into a 20mM storage solution, aliquoted and stored Store at –80°C until use.
3、MTT试验3. MTT test
将HepG2-NTCP细胞、HepG2.2.15细胞以15×104/ml接种于96孔培养板中。培养24h后，用DMEM培养基将积雪草酸稀释至200，100，50，25，12.5，6.25，3.125，0uM 8个浓度梯度，每个浓度设立3复孔，并设正常细胞对照。72小时后，加入10ul MTT试剂，37℃孵育4h，加入100ul DMSO，振荡10min后，酶标仪检测490nm处的OD值，计算并绘制细胞活性曲线。HepG2-NTCP cells and HepG2.2.15 cells were seeded in a 96-well culture plate at 15×10 4 /ml. After culturing for 24 hours, use DMEM medium to dilute asiatic acid to 8 concentration gradients of 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0uM. Set up 3 replicate wells for each concentration, and set up a normal cell control. After 72 hours, add 10ul MTT reagent, incubate at 37°C for 4 hours, add 100ul DMSO, shake for 10 minutes, detect the OD value at 490nm with a microplate reader, calculate and draw the cell activity curve.
4、HBV感染细胞模型HepG2-NTCP中检测积雪草酸对HBx蛋白水平的影响4. Detection of the effect of asiatic acid on HBx protein levels in the HBV infected cell model HepG2-NTCP
将HepG2-NTCP细胞以20×104/ml接种于十二孔板中。培养24h后，分别转染3×Flag-HBx和2×HA-HBx；转染24h后，用DMEM培养基将积雪草酸稀释至30，20，10，0uM 4个浓度梯度，分别处理HepG2-NTCP细胞。48h后提取总蛋白并采用Western blot检测HBx蛋白水平。HepG2-NTCP cells were seeded in twelve-well plates at 20×10 4 /ml. After 24 hours of culture, 3×Flag-HBx and 2×HA-HBx were transfected respectively; 24 hours after transfection, asiatic acid was diluted to 4 concentration gradients of 30, 20, 10, and 0uM using DMEM medium, and HepG2- were treated respectively. NTCP cells. After 48 h, total protein was extracted and Western blot was used to detect HBx protein levels.
5、HBV感染细胞模型HepG2-NTCP中检测积雪草酸对HBsAg、total HBV RNAs、HBV3.5-kb RNA、HBV DNA水平的影响5. Detection of the effect of asiatic acid on the levels of HBsAg, total HBV RNAs, HBV3.5-kb RNA, and HBV DNA in the HBV infected cell model HepG2-NTCP
将HepG2-NTCP细胞以20×104/ml接种于十二孔板中。培养24h后，用DMEM培养基将积雪草酸稀释至30，20，10，0uM 4个浓度梯度，分别处理HepG2-NTCP细胞3d、6d、9d，收集培养基上清和细胞，ELISA检测上清中HBsAg水平，Real-time PCR及Northern blot检测细胞中total HBV RNAs、HBV 3.5-kb RNA水平，Real-time PCR及Southern blot检测细胞中HBV DNA水平。HepG2-NTCP cells were seeded in twelve-well plates at 20×10 4 /ml. After culturing for 24 hours, use DMEM medium to dilute asiatic acid to 4 concentration gradients of 30, 20, 10, and 0uM. HepG2-NTCP cells were treated for 3 days, 6 days, and 9 days respectively. The medium supernatant and cells were collected, and the supernatant was detected by ELISA. HBsAg levels, Real-time PCR and Northern blot were used to detect total HBV RNAs and HBV 3.5-kb RNA levels in cells, and Real-time PCR and Southern blot were used to detect HBV DNA levels in cells.
6、HBV稳定复制模型HepG2.2.15中检测积雪草酸对total HBV RNAs、HBV 3.5-kb RNA、HBV DNA水平的影响6. Detection of the effect of asiatic acid on the levels of total HBV RNAs, HBV 3.5-kb RNA, and HBV DNA in the HBV stable replication model HepG2.2.15
将HepG2.2.15细胞以20×104/ml接种于十二孔板中。培养24h后，用DMEM培养基将积雪草酸稀释至30，20，10，0uM 4个浓度梯度处理HepG2.2.15细胞，4d后收集细胞，Real-time PCR及Northern blot检测细胞中total HBV RNAs、HBV 3.5-kb RNA水平，Real-time PCR及Southern blot检测细胞中HBV DNA水平。HepG2.2.15 cells were seeded in twelve-well plates at 20×10 4 /ml. After culturing for 24 hours, HepG2.2.15 cells were treated with DMEM medium diluted with asiatic acid to 4 concentration gradients of 30, 20, 10, and 0uM. The cells were collected after 4 days. Real-time PCR and Northern blot were used to detect total HBV RNAs and total HBV RNAs in the cells. HBV 3.5-kb RNA level, Real-time PCR and Southern blot were used to detect HBV DNA level in cells.
7、HBV重组cccDNA小鼠模型验证积雪草酸对HBV复制的影响7. HBV recombinant cccDNA mouse model to verify the effect of asiatic acid on HBV replication
选取50只，年龄6-8周，重量18-20g的雄性C57BL/6小鼠。分别尾静脉注射4μg质粒prcccDNA和4μg质粒pCMV-KRAB-Cre。一周后，通过荧光定量PCR检测血清HBV DNA拷贝数，判断模型是否建立成功。Fifty male C57BL/6 mice, aged 6-8 weeks and weighing 18-20g, were selected. 4 μg of plasmid prcccDNA and 4 μg of plasmid pCMV-KRAB-Cre were injected into the tail vein respectively. One week later, the serum HBV DNA copy number was detected by fluorescence quantitative PCR to determine whether the model was successfully established.
将成功建模的小鼠随机分为四组：阴性对照、阳性对照、低浓度实验组、高浓度实验组，每组6只，分别口服给予0.9％生理盐水、0.02mg/kg恩替卡韦、15mg/kg积雪草酸、30mg/kg积雪草酸处理，隔天给药，每四天采血一次，分离血清，检测血清中HBsAg、HBV DNA水平。药物处理24天后，研磨肝组织检测HBV RNAs、HBV DNA水平。The successfully modeled mice were randomly divided into four groups: negative control, positive control, low-concentration experimental group, and high-concentration experimental group. Six mice in each group were orally administered 0.9% normal saline, 0.02mg/kg entecavir, and 15mg/kg entecavir. kg asiatic acid, 30mg/kg asiatic acid treatment, administration every other day, blood collection once every four days, serum separation, detection of HBsAg and HBV DNA levels in the serum. After 24 days of drug treatment, the liver tissue was ground to detect the levels of HBV RNAs and HBV DNA.
8、Real-time PCR8. Real-time PCR
检测HBV DNA：提取HBV DNA后，按照SYBR Green(Roche,Germany)说明书配制反应体系和设置反应条件，HBV DNA引物，F：CCTAGTAGTCAGTTATGTCAAC(SEQ ID NO.1)，R:TCTATAAGCTGGAGGAGTGCGA(SEQ ID NO.2)。每个样品均设3个复孔，每组实验重复3次。Detect HBV DNA: After extracting HBV DNA, prepare the reaction system and set the reaction conditions according to the instructions of SYBR Green (Roche, Germany). HBV DNA primers, F: CCTAGTAGTCAGTTATGTCAAC (SEQ ID NO.1), R: TCTATAAGCTGGAGGAGTGCGA (SEQ ID NO.2 ). Three duplicate wells were set up for each sample, and each set of experiments was repeated three times.
检测HBV RNA:逆转录RNA合成的cDNA，按照SYBR Green(Bio Rad)说明书配制反应体系和设置反应条件，HBV 3.5kb RNA引物为F:CTCTTCCAGCCTTCCTTCCT(SEQ ID NO.3)，R:AGCACTGTGTTGGCGTACAG(SEQ ID NO.4)，total HBV RNAs引物为F:ACCGACCTTGAGGCATACTT (SEQ ID NO.5)，R:GCCTACAGCCTCCTAGTACA(SEQ ID NO.6)。每个样品均设3个复孔，每组实验重复3次。Detect HBV RNA: cDNA synthesized by reverse transcription RNA, prepare the reaction system and set the reaction conditions according to the instructions of SYBR Green (Bio Rad). The HBV 3.5kb RNA primers are F: CTCTTCCAGCCTTCCTTCCT (SEQ ID NO. 3), R: AGCACTGTGTTGGCGTACAG (SEQ ID NO.4), the total HBV RNAs primer is F:ACCGACCTTGAGGCATACTT (SEQ ID NO. 5), R: GCCTACAGCCTCCTAGTACA (SEQ ID NO. 6). Three duplicate wells were set up for each sample, and each set of experiments was repeated three times.
9、统计学方法9. Statistical methods
采用GraphPad Prism统计软件进行统计，两组间比较采用非配对t检验，以P<0.05为差异具有统计学意义。GraphPad Prism statistical software was used for statistics, and unpaired t test was used for comparison between the two groups. P<0.05 was considered as a statistically significant difference.
二、实验结果：2. Experimental results:
1、MTT试验1. MTT test
图1显示为MTT实验分析积雪草酸在HepG2-NTCP、HepG2.2.15细胞中的细胞毒性。图1所示的结果表明，积雪草酸在HBV感染细胞系HepG2-NTCP中CC50＝74.82μM，HBV稳定复制模型HepG2.2.15中CC50＝67.6μM。Figure 1 shows the MTT assay analyzing the cytotoxicity of asiatic acid in HepG2-NTCP and HepG2.2.15 cells. The results shown in Figure 1 show that asiatic acid has a CC50 of 74.82 μM in the HBV-infected cell line HepG2-NTCP and a CC50 of 67.6 μM in the HBV stable replication model HepG2.2.15.
2、HBV感染细胞模型HepG2-NTCP中检测积雪草酸对HBx蛋白水平的影响2. Detection of the effect of asiatic acid on HBx protein levels in the HBV infected cell model HepG2-NTCP
图2显示为Western blot检测积雪草酸对3×Flag-HBx及2×HA-HBx蛋白水平的影响。图2所示的Western blot结果表明，积雪草酸处理后，HepG2-NTCP细胞中外源性3×Flag-HBx及2×HA-HBx蛋白水平以浓度梯度依赖性降低，提示积雪草酸可以抑制HBx表达。Figure 2 shows the Western blot detection of the effect of asiatic acid on the protein levels of 3×Flag-HBx and 2×HA-HBx. The Western blot results shown in Figure 2 show that after asiatic acid treatment, the protein levels of exogenous 3×Flag-HBx and 2×HA-HBx in HepG2-NTCP cells decreased in a concentration gradient-dependent manner, suggesting that asiatic acid can inhibit HBx Express.
3、HBV感染细胞模型HepG2-NTCP中检测积雪草酸对上清中HBsAg、细胞内total HBV RNAs、HBV 3.5-kb RNA、HBV DNA、cccDNA水平的影响3. Detection of the effect of asiatic acid on the levels of HBsAg, intracellular total HBV RNAs, HBV 3.5-kb RNA, HBV DNA, and cccDNA in the supernatant in the HBV infected cell model HepG2-NTCP
图3显示为荧光定量PCR法和Northern blot实验检测积雪草酸对total HBV RNAs、HBV3.5-kb RNA的影响。图3所示的Real-time PCR结果表明，和阴性对照组相比，积雪草酸呈时间、浓度梯度依赖性显著降低了total HBV RNAs、HBV 3.5-kb RNA的水平，Northern blot验证了PCR结果。图4显示为荧光定量PCR法和Southern blot实验检测积雪草酸对HBV DNA水平的影响。图4所示的Real-time PCR结果表明，和阴性对照组相比，积雪草酸呈时间、浓度梯度依赖性显著降低了细胞内HBV DNA的水平，Southern blot验证了PCR结果。Figure 3 shows the fluorescence quantitative PCR method and Northern blot experiment to detect the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA. The Real-time PCR results shown in Figure 3 show that compared with the negative control group, asiatic acid significantly reduced the levels of total HBV RNAs and HBV 3.5-kb RNA in a time- and concentration-gradient-dependent manner. Northern blot verified the PCR results . Figure 4 shows the fluorescence quantitative PCR method and Southern blot experiment to detect the effect of asiatic acid on HBV DNA levels. The Real-time PCR results shown in Figure 4 show that compared with the negative control group, asiatic acid significantly reduced the level of intracellular HBV DNA in a time- and concentration-gradient-dependent manner. Southern blot verified the PCR results.
图5显示为ELISA检测积雪草酸对HBsAg分泌的影响。图5所示的ELISA结果表明，积雪草酸在HepG2-NTCP细胞中能有效降低HBsAg的水平，并呈时间、浓度梯度依赖性。Figure 5 shows the ELISA test of the effect of asiatic acid on HBsAg secretion. The ELISA results shown in Figure 5 show that asiatic acid can effectively reduce the level of HBsAg in HepG2-NTCP cells in a time- and concentration gradient-dependent manner.
图6显示为Taq Man探针PCR实验检测积雪草酸对cccDNA水平及转录活性的影响。由于HBx在cccDNA的转录过程中发挥关键作用。抑制HBx的功能，则可能抑制cccDNA转录。因此进一步确定积雪草酸对cccDNA转录的影响。图6所示的Taq Man探针PCR实验结果表明积雪草酸对cccDNA水平无明显影响。进一步分析cccDNA转录活性(total RNAs/cccDNA和pgRNA/cccDNA比值)，发现积雪草酸可显著抑制cccDNA转录活性。Figure 6 shows the Taq Man probe PCR experiment to detect the effect of asiatic acid on cccDNA levels and transcriptional activity. Because HBx plays a key role in the transcription process of cccDNA. Inhibiting the function of HBx may inhibit cccDNA transcription. Therefore, the effect of asiatic acid on cccDNA transcription was further determined. The Taq Man probe PCR experimental results shown in Figure 6 show that asiatic acid has no significant effect on cccDNA levels. Further analysis of cccDNA transcription activity (total RNAs/cccDNA and pgRNA/cccDNA ratio) found that asiatic acid can significantly inhibit cccDNA transcription activity.
4、HBV稳定复制模型HepG2.2.15中检测积雪草酸对total HBV RNAs、HBV 3.5-kb RNA、HBV DNA水平的影响4. Detection of the effect of asiatic acid on the levels of total HBV RNAs, HBV 3.5-kb RNA, and HBV DNA in the HBV stable replication model HepG2.2.15
图7显示为HepG2.2.15细胞中检测积雪草酸对total HBV RNAs、HBV 3.5-kb RNA的影响。图8显示为HepG2.2.15细胞中检测积雪草酸对HBV DNA的影响。结果显示积雪草酸可以显著抑制细胞内HBV RNA(图7)、HBV DNA水平(图8)，但不影响cccDNA水平(图9)。Figure 7 shows the detection of the effect of asiatic acid on total HBV RNAs and HBV 3.5-kb RNA in HepG2.2.15 cells. Figure 8 shows the detection of the effect of asiatic acid on HBV DNA in HepG2.2.15 cells. The results showed that asiatic acid could significantly inhibit intracellular HBV RNA (Figure 7) and HBV DNA levels (Figure 8), but did not affect the cccDNA level (Figure 9).
5、HBV重组cccDNA小鼠模型验证积雪草酸对HBV复制的影响5. HBV recombinant cccDNA mouse model to verify the effect of asiatic acid on HBV replication
图10显示为小鼠处理流程图。图11显示为ELISA和荧光定量PCR法检测积雪草酸对小鼠血清中HBsAg、HBV DNA水平的影响。图12显示为荧光定量PCR法检测积雪草酸对小鼠肝组织中HBV RNA水平的影响。图13显示为荧光定量PCR法检测积雪草酸对小鼠肝组织中HBV  DNA水平的影响。Figure 10 shows a mouse handling flow chart. Figure 11 shows the effects of asiatic acid on HBsAg and HBV DNA levels in mouse serum detected by ELISA and fluorescence quantitative PCR. Figure 12 shows the fluorescence quantitative PCR method to detect the effect of asiatic acid on HBV RNA levels in mouse liver tissue. Figure 13 shows the fluorescence quantitative PCR method for detecting the effect of asiatic acid on HBV in mouse liver tissue. DNA level effects.
分离小鼠血清检测HBsAg、HBV DNA。结果表明，与对照组相比，积雪草酸处理组可显著抑制血清HBsAg及HBV DNA水平(图11)。药物处理24天后，研磨肝组织进一步检测肝组织HBV RNA及HBV DNA水平，实验结果表明积雪草酸处理组可显著抑制肝组织HBV RNA(图12)及HBV DNA(图13)水平。Isolate mouse serum to detect HBsAg and HBV DNA. The results showed that compared with the control group, the asiatic acid-treated group could significantly inhibit serum HBsAg and HBV DNA levels (Figure 11). After 24 days of drug treatment, the liver tissue was ground to further detect the levels of HBV RNA and HBV DNA in the liver tissue. The experimental results showed that the asiatic acid treatment group could significantly inhibit the levels of HBV RNA (Figure 12) and HBV DNA (Figure 13) in the liver tissue.
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。 The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (10)

  1. 积雪草酸作为有效成分在制备乙型肝炎治疗药物中的用途。Use of asiatic acid as an active ingredient in the preparation of hepatitis B therapeutic drugs.
  2. 根据权利要求1所述的用途，其特征在于：所述乙型肝炎治疗药物至少具有以下功用之一：The use according to claim 1, characterized in that: the hepatitis B treatment drug has at least one of the following functions:
    降低HBx蛋白水平，降低HBsAg水平，降低细胞内total HBV RNAs水平，降低细胞内HBV 3.5-kb RNA水平，降低HBV DNA水平。Reduce HBx protein levels, reduce HBsAg levels, reduce intracellular total HBV RNAs levels, reduce intracellular HBV 3.5-kb RNA levels, and reduce HBV DNA levels.
  3. 根据权利要求2所述的用途，其特征在于：所述乙型肝炎治疗药物必然包括积雪草酸，并以积雪草酸作为前述功用的有效成分。The use according to claim 2, characterized in that the hepatitis B treatment drug must include asiatic acid, and asiatic acid is used as an active ingredient for the aforementioned functions.
  4. 根据权利要求1所述的用途，其特征在于：所述积雪草酸作为所述乙型肝炎治疗药物的有效成分之一或唯一有效成分。The use according to claim 1, characterized in that the asiatic acid is one of the active ingredients or the only active ingredient of the hepatitis B treatment drug.
  5. 根据权利要求1所述的用途，其特征在于：所述乙型肝炎治疗药物的形式选自固体、液体、凝胶、半流质、气雾。The use according to claim 1, characterized in that: the form of the hepatitis B treatment drug is selected from the group consisting of solid, liquid, gel, semi-liquid, and aerosol.
  6. 一种治疗乙型肝炎的药物制剂，其特征在于：包括安全有效剂量的积雪草酸。A pharmaceutical preparation for treating hepatitis B, which is characterized in that it includes a safe and effective dose of asiatic acid.
  7. 根据权利要求6所述的治疗乙型肝炎的药物制剂，其特征在于：所述积雪草酸作为所述治疗乙型肝炎的药物制剂的有效成分之一或唯一有效成分。The pharmaceutical preparation for treating hepatitis B according to claim 6, characterized in that the asiatic acid is one of the active ingredients or the only active ingredient of the pharmaceutical preparation for treating hepatitis B.
  8. 一种乙型肝炎联合治疗药物组合，其特征在于：包括安全有效剂量的积雪草酸和至少一种其他乙型肝炎治疗药物，余量为药学上可接受的载体和/或辅料。A combination of drugs for the treatment of hepatitis B, characterized by: including a safe and effective dose of asiatic acid and at least one other drug for the treatment of hepatitis B, with the balance being pharmaceutically acceptable carriers and/or excipients.
  9. 根据权利要求8所述的乙型肝炎联合治疗药物组合，其特征在于：所述乙型肝炎联合治疗药物组合可以是以下形式中的任意一种：The drug combination for combined treatment of hepatitis B according to claim 8, characterized in that: the drug combination for combined treatment of hepatitis B can be any one of the following forms:
    (一)将积雪草酸和其他乙型肝炎治疗药物分别制成独立的制剂，制剂的剂型可相同或不同，给药途径亦可相同或不同；(1) Make asiatic acid and other hepatitis B therapeutic drugs into independent preparations. The dosage forms of the preparations can be the same or different, and the routes of administration can also be the same or different;
    (二)将积雪草酸和其他乙型肝炎治疗药物配置成复方制剂，在将积雪草酸和其他乙型肝炎治疗药物采用相同给药途径给药并同时施加时，可采用将两者配置成复方制剂的形式。(2) Configure asiatic acid and other hepatitis B therapeutic drugs into a compound preparation. When asiatic acid and other hepatitis B therapeutic drugs are administered by the same route of administration and administered at the same time, they can be formulated into Form of compound preparation.
  10. 积雪草酸在制备具有以下任一项或多项作用的物质中的用途：The use of asiatic acid in the preparation of substances with any one or more of the following effects:
    用于制备抑制HBx蛋白的物质的用途；Use for preparing substances that inhibit HBx protein;
    用于制备抑制HBsAg水平的物质的用途；Use for the preparation of substances that inhibit HBsAg levels;
    用于制备抑制细胞内total HBV RNAs水平的物质的用途；Use for preparing substances that inhibit intracellular levels of total HBV RNAs;
    用于制备抑制细胞内HBV 3.5-kb RNA水平的物质的用途；Use for the preparation of substances that inhibit intracellular HBV 3.5-kb RNA levels;
    用于制备抑制HBV DNA水平的物质的用途。 Use for the preparation of substances that inhibit HBV DNA levels.
PCT/CN2023/086552 2022-06-17 2023-04-06 Use of asiatic acid in preparation of medicament for treating hepatitis b WO2023241179A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1559463A (en) * 2004-02-18 2005-01-05 刚 刘 Medicine for treating viral hepatitis B
CN104784186A (en) * 2015-05-06 2015-07-22 中国人民解放军第二军医大学 Application of asiatic acid in preparation of medicine for preventing hepatitis virus
CN114796233A (en) * 2022-06-17 2022-07-29 重庆医科大学国际体外诊断研究院 Application of asiatic acid in preparing medicine for treating hepatitis B

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107056874A (en) * 2016-12-28 2017-08-18 福建广生堂药业股份有限公司 A kind of compound of asiatic acid tenofovir dipivoxil and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1559463A (en) * 2004-02-18 2005-01-05 刚 刘 Medicine for treating viral hepatitis B
CN104784186A (en) * 2015-05-06 2015-07-22 中国人民解放军第二军医大学 Application of asiatic acid in preparation of medicine for preventing hepatitis virus
CN114796233A (en) * 2022-06-17 2022-07-29 重庆医科大学国际体外诊断研究院 Application of asiatic acid in preparing medicine for treating hepatitis B

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANG MEI-JUAN, LI YU-HU: "Inhibitory Effect of Alcoholic Extracts of Chinese Medicine on HBV", YIXUE XINXI - MEDICAL INFORMATION, ZHONGGUO RENMIN JIEFANGJUN LANZHOU JUNQU KONGJUN YIXUE KEXUE JISHU WEIYUANHUI, CN, vol. 24, no. 08, 31 August 2011 (2011-08-31), CN , pages 4910 - 4912, XP009551775, ISSN: 1006-1959 *
LV. SHUJUAN; HUANG QUAN-FANG; LIN XING: "Inhibitory effect of asiaticoside from Hydrocotyle sibthorpioides on duck hepatitis B virus effect", CHINESE JOURNAL OF PUBLIC HEALTH, vol. 31, no. 05, 31 May 2015 (2015-05-31), pages 600 - 603, XP009551252, ISSN: 1001-0580 *
SHEN QIAN; YU LU-SHAN; ZHOU HUI; ZENG SU: "Simultaneous determination of made cassoside, asiaticoside and their aglycones in Centella asiatica (L.) urban extracts by RP-HPLC", ZHEJIANG DAXUE XUEBAO (YIXUE BAN) =ZHEJIANG UNIVERSITY. JOURNAL (MEDICAL SCIENCES EDITION), ZHEJIANG DAXUE CHUBANSHE,, CH, vol. 43, no. 02, 31 March 2014 (2014-03-31), CH , pages 135 - 140, XP009551255, ISSN: 1008-9292 *

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