WO2002080958A1 - Interferon supercompose de recombinaison utilise comme inhibiteur de l'antigene de surface et de l'antigene e de l'hepatite b - Google Patents

Interferon supercompose de recombinaison utilise comme inhibiteur de l'antigene de surface et de l'antigene e de l'hepatite b Download PDF

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WO2002080958A1
WO2002080958A1 PCT/CN2002/000128 CN0200128W WO02080958A1 WO 2002080958 A1 WO2002080958 A1 WO 2002080958A1 CN 0200128 W CN0200128 W CN 0200128W WO 02080958 A1 WO02080958 A1 WO 02080958A1
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interferon
recombinant
hepatitis
efficiency
highly efficient
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PCT/CN2002/000128
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English (en)
French (fr)
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Guangwen Wei
Rongbing Guo
Renhuai Zhang
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Sichuan Biotechnology Research Center
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Priority to DE60234085T priority Critical patent/DE60234085D1/de
Priority to CA002439503A priority patent/CA2439503A1/en
Priority to JP2002578997A priority patent/JP4617058B2/ja
Priority to EP02702211A priority patent/EP1371373B1/en
Priority to AT02702211T priority patent/ATE446104T1/de
Application filed by Sichuan Biotechnology Research Center filed Critical Sichuan Biotechnology Research Center
Publication of WO2002080958A1 publication Critical patent/WO2002080958A1/zh
Priority to US10/650,365 priority patent/US7364724B2/en
Priority to AU2003248419A priority patent/AU2003248419B2/en
Priority to HK04104296.9A priority patent/HK1061201A1/xx
Priority to US10/927,975 priority patent/US20050079579A1/en
Priority to US11/077,813 priority patent/US20060035327A1/en
Priority to US12/105,455 priority patent/US8114395B2/en
Priority to US12/246,153 priority patent/US8551469B2/en
Priority to US12/889,521 priority patent/US20110070195A1/en
Priority to US13/019,044 priority patent/US8425896B2/en
Priority to US13/923,378 priority patent/US20130281667A1/en
Priority to US14/019,519 priority patent/US9944686B2/en
Priority to US15/910,165 priority patent/US20180258151A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • 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
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • 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
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Recombinant high-efficiency complex interferon is used as hepatitis B surface antigen and e-antigen inhibitor
  • the present invention relates to the application of a recombinant and highly efficient interferon by changing the spatial structure of a protein, and is characterized in that as a preparation, it not only inhibits hepatitis B virus DNA, but also inhibits HBsAg and HBeAg. Background technique
  • Recombinant high-efficiency compound interferon is a new type of interferon molecule constructed by genetic engineering from the most common conserved amino acid structure in several natural human ⁇ -interferon subtypes. This type of interferon is described in US Patent Nos. 4,756,623 and 4897471. Interferon has been described, rSIFN-co has been proven to have a broad spectrum of interferon activity, and has strong antiviral and antitumor and natural killer cell activities. US Patent No. 5,372,808 discloses the use of recombinant and highly effective interferon to treat diseases. Methods: Chinese patent 97193506.8 discloses a method for treating hepatitis C again by using a recombinant high-efficiency compound interferon.
  • Chinese patent 98114663.5 publicly discloses a method for preparing the recombinant high-efficiency compound interferon and the use for treating hepatitis B and hepatitis C.
  • the US Food and Drug Administration (FDA) has approved rSIFN-co produced by E. coli in the United States for the treatment of clinical hepatitis C patients at the end of 1997.
  • hepatitis B patients For the diagnosis of hepatitis B patients, when the surface antigen (HBsAg) positive and e antigen (HBeAg) positive are detected, they can be judged as hepatitis B patients.
  • various types of ⁇ -interferon are used in chronic hepatitis B clinically.
  • the treatment mechanism for hepatitis patients is: Interferon binds to special membrane receptors on the cell surface to exert its anti-DNA and RNA effects. This includes the induction of certain enzymes to prevent virus replication in virus-infected cells. However, all interferons can only inhibit viral DNA and RNA, not e-antigen and s-antigen. Summary of the Invention
  • the present invention surprisingly found that a recombinant highly efficient interferon that changes the spatial structure of a protein, as a preparation, not only inhibits hepatitis B virus DNA, but also inhibits HBsAg and HBeAg, thereby proving that the recombinant highly efficient interferon can By inhibiting hepatitis B virus DNA and inhibiting The hepatitis B HBsAg antigen and HBeAg antigen achieve the purpose of treating hepatitis B.
  • the purpose of the present invention is to provide a recombinant and highly effective interferon drug for treating hepatitis B.
  • this drug not only the viral DNA is inhibited, but also the hepatitis B surface antigen and the hepatitis B e antigen are reduced to normal levels. To achieve the purpose of treatment.
  • the invention is achieved by:
  • a recombinant highly efficient interferon having a sequence as shown in FIG. 1 is first prepared by gene recombination technology.
  • the recombination technology of the present invention uses E. coli to preferentially express codons, and to ensure that the amino acid sequence is unchanged, the DNA coding sequence is performed. After redesigning, the full-length cDNA of rSIFN-co was artificially synthesized.
  • the present invention uses recombinant DNA technology to clone the full-length rSIFN-co cDNA sequence into a highly efficient expression vector of E. coli, and then uses the L-arabinose induction / activation expression regulation mechanism to activate the strong P BAD promoter in the vector to mediate downstream rSIFN-co gene is highly expressed.
  • This arabinose induction / activation expression control system has obvious advantages over the temperature control, pH control, and IPTG induction systems commonly used in genetic engineering production: (1) Several control systems commonly used are “de-repressed” The "form releases the inhibitory effect on the functional promoter, so that the promoter can mediate the expression of downstream genes.
  • an arabinose induction / activation system is used to establish an efficient and stable rSIFN-co expressing E. coli engineering strain.
  • the application method of the present invention is to use a recombinant high-efficiency complex interferon to achieve therapeutic purposes for patients.
  • the recombinant high-efficiency complex interferon used by patients can be made into various dosage forms such as tablets, capsules, oral solutions, patches, injections, sprays, Suppositories, solution preparations, the recommended formulation is injection, which can be administered subcutaneously or intravenously.
  • the carrier in the pharmaceutical composition can use any suitable acceptable pharmaceutical carrier. These carriers can be sugars, cellulose products, adhesives Mixtures, disintegrants, lubricants, fillers, solubilizers, buffers, preservatives, thickeners, complexing agents and other adjuvants.
  • Figure 1 DNA coding sequence and deduced amino acid sequence for rSIFN-co
  • the present invention verifies the inhibitory effect of recombinant high-efficiency complex interferon that changes the spatial conformation on hepatitis B virus DNA, surface antigen and e antigen through the following experiments.
  • the experimental method is as follows:
  • Solvent and preparation method When preparing the test drug, add 1 ml of physiological saline to each raw material bottle, and after dissolving, dissolve it in MEM culture solution according to the concentration difference of the different dose groups set. Active use.
  • Control drug The interference energy of IFN-ot2b produced by Schering is a lyophilized preparation. Each 3 ⁇ 10 6 U experiment was prepared with a culture solution into a 3 ⁇ 10 6 IU / ml solution. Ganjin produced by Amgen is an injection. Each 9 ⁇ ⁇ , 0.3ml, equivalent to 9 X 10 6 IU / branch, the culture solution was used to prepare a 9X 10 0 IU / ml solution in the experiment, stored in a refrigerator at 4 ° C, 2.2.15 cells: Hepatitis B virus (HBV ) DNA clone was transfected into 2.2.15 cell line of human hepatoma cell line (Hep G2) and constructed by Mount Sinai Medical Center.
  • HBV Hepatitis B virus
  • HBeAg solid-phase radioimmunoassay kit purchased from Northern Institute of Immunology, China Isotope Company; Kanamycin, a product from North China Pharmaceutical Factory; Lipofectin, a product from Gibco, USA. ,
  • 2.2.15 Cell Culture Add 0.25% trypsin to the culture flask with 2.2.15 cells. 37. C digestion for 3 minutes, pipetted with culture medium, passaged at 1: 3, and grown for 10 days.
  • Drug-to-cytotoxicity test The experiment was divided into a drug-free cell control group and a different drug concentration administration group. Cells were digested and formulated into 100,000 cells per milliliter, inoculated into culture plates, 200 ⁇ l per well in a 96-well plate, and cultured at 37 ° C and 5% CO 2 for 24 hours. The cells were grown into a monolayer for experiments.
  • High-efficiency recombination and high-efficiency complex interferon was prepared into a L8x10 7 IU / ml solution with a culture solution, diluted twice and added to a 96-well cell culture plate, 3 holes per concentration, and the same concentration of drug solution was changed every 4 days, with the observation of cell lesions as indicators, 8 Observe the cytopathic effect under the microscope, the complete destruction is 4; 75% is 3; 50% is 2; 25% is 1; no lesion is 0. Calculate the average degree of cytopathy and% inhibition of each concentration of drug solution. The Reed Muench method was used to calculate the half toxic concentration (TC50) and the maximum non-toxic concentration (TC0).
  • TC50 Antilog (B + —— xC)
  • A log> 50% drug concentration
  • B log ⁇ 50% drug concentration
  • C log dilution factor on HBeAg, HBsAg inhibition test: The test consists of HBeAg, HBsAg positive control group, negative control group, cell control group and different drug concentrations group. 700,000 2.2.15 cells per ml were inoculated into a 6-well cell culture plate, 3 ml per well, and cultured at 37 ° C and 5% CO 2 for 24 hours, and the test solution was diluted 3 times below the non-toxic concentration, and the five dilutions were 4.5 X 10 6 IU / ml, 1.5 X 10 6 IU / ml, 0.5 X 10 6 IU / ml, 0.17
  • HBsAg and HBeAg were determined by the Northern Immune Reagent Research Institute of China Isotope Company, and the solid-phase radioimmunoassay box was used for the determination.
  • Southern blot @ 2.2.15 intracellular 13 ⁇ 4 ⁇ ⁇ 0 ⁇ Extraction: 2.2.15 cells were cultured for 8 days after dosing, the cells were aspirated to collect cells, cell lysate was added, cells were lysed, and equal volume of phenol: chloroform: iso The amyl alcohol was extracted twice, centrifuged at 10,000 g at high speed, the supernatant was taken out, the nucleic acid was precipitated by adding absolute ethanol, vacuum-dried, and redissolved in 20 ⁇ 1 TE buffer. 2 Electrophoresis: Add 6X DNA sample buffer, and add the sample to 1.5% agarose gel for electrophoresis, IV / cm, constant pressure, 14-18 hours.
  • Denaturation and hybridization Immerse the glue in HC1, denaturing solution and neutralizing solution. 4 Transfer the membrane: Transfer the DNA to Hybond-N membrane according to the procedure. Blotting, blotting, and exposure with spot blotting. The scanner scans the light sheet and analyzes the relative density with gel-pro analysis software to calculate the inhibition rate and IC50.
  • Compound interferon has obvious activity of inhibiting hepatitis B surface antigen and e antigen, while the control group interfering energy and Ganfujin do not have the above activities.
  • Clinical cases have also confirmed that patients with chronic active hepatitis B can reduce or return to normal levels of hepatitis B surface antigen and e antigen by taking recombinant high-efficiency compound interferon.
  • the recombinant high-efficiency compound interferon preparation applied by the present invention can be prepared by the following examples:
  • Preparation process Weigh according to the prescription, dissolve with sterile pyrogen-free injection water, sterilize and filter with a 0.22 ⁇ pore size filter membrane, and store at 8 ⁇ 2 ° C. Take a sample for sterility and pyrogen inspection after passing the test. Packed in vials, single-dose 1.0ml bottles. After packaging, place them in a lyophilizer and freeze-dry.
  • Preparation process Weigh according to the prescription, dissolve with sterile pyrogen-free injection water, sterilize and filter with a 0.22 ⁇ m pore size filter membrane, and store at 8 ⁇ 2 ° C. After sampling for sterility and pyrogen inspection, Dispensed in a closed container. Divide into a vial, and store a single dose of 1.0 mL of each bottle in a dark place at 2-10 ° C. Table 1: Test results of rSIFN-co on hepatitis B surface antigen and e-antigen inhibition rate The first batch of experiments
  • E antigen IC50 mean 450.2434 standard deviation 132.315479
  • Surface antigen IC50 mean 649.1894 standard deviation 42.29580

Description

重组高效复合千扰素用作乙型肝炎表面抗原和 e抗原抑制剂 所属技术领域
本发明涉及一种通过改变了蛋白质空间结构的重组高效复合干扰素的应 用， 其特征是它作为一种制剂不仅对乙型肝炎 ^毒 DNA有抑制作用， 而且对 HBsAg、 HBeAg均有抑制作用。 背景技术
重组高效复合干扰素是将几种天然人 α-干扰素亚型中最常见的保守性氨基 酸结构用遗传工程的方法建构而成的一种全新干扰素分子， 美国专利 4695623、 4897471 中对此种干扰素已经有所描述， 已证明 rSIFN-co具有广谱的千扰素活 性， 有较强的抗病毒和抗肿瘤及天然杀伤细胞活性， 美国专利 5372808 公开了 用重组高效复合干扰素治疗疾病的方法， 中国专利 97193506.8公开了应用重组 高效复合干扰素再次治疗丙型肝炎的方法， 中国专利 98114663.5公幵了重组高 效复合干扰素的制备方法和治疗乙型肝炎和丙型肝炎的用途。 美国食品与药品 管理局 (FDA)已于 1997年底批准了美国 Amgen公司用大肠杆菌生产的 rSIFN- co 用于临床丙肝病人的治疗。
对于乙型肝炎病人的诊断， 当检测出表面抗原 （HBsAg) 阳性和 e 抗原 (HBeAg)阳性者即可判定为乙型肝炎患者， 目前临床上采用各种类型的 α-干扰素 对慢性乙型肝炎患者进行治疗， 其作用机理是： 干扰素与细胞表面的特殊膜受 体结合而发挥其抗 DNA和 RNA的作用。 包括对某些酶的诱导作用阻止受病毒 感染细胞中病毒的复制。 但所有的干扰素均只能抑制病毒 DNA和 RNA, 不能 抑制 e抗原与 s抗原。 发明内容
本发明惊奇地发现改变了蛋白质空间结构的重组高效复合干扰素， 作为一 种制剂不仅对乙型肝炎病毒 DNA有抑制作用， 而且对 HBsAg、 HBeAg均有抑 制作用，从而证明重组高效复合干扰素可以通过抑制乙型肝炎病毒 DNA和抑制 乙型肝炎 HBsAg抗原、 HBeAg抗原达到治疗乙型肝炎的目的。
本发明的目的是提供用于治疗乙型肝炎的重组高效复合干扰素药物， 通过 使用这种药物不仅抑制病毒 DNA， 而且抑制乙型肝炎表面抗原和乙型肝炎 e抗 原， 使之降低到正常水平， 达到治疗目的。
本发明是通过以下方式实现的：
本发明首先通过基因重组技术制备出具有如图 1 序列的重组高效复合干扰 素， 本发明的重组技术利用大肠杆菌优先表达密码子， 在保证氨基酸序列不变 的情况下， 对其 DNA编码序列进行了重新设计， 然后人工合成其 rSIFN-co全 长的 cDNA基因。
本发明采用重组 DNA技术将上述 rSIFN-co全长 cDNA序列克隆到大肠杆 菌高效表达载体中去， 然后利用 L-阿拉伯糖诱导 /活化表达调控机制激活载体中 的强 PBAD启动子介导下游的 rSIFN-co基因高效表达。这一阿拉伯糖诱导 /活化表 达调控系统比通常遗传工程生产中所采用的温控、 pH调控、 IPTG诱导等系统 有明显的优点： （1)通常采用的几种调控系统都是以 "去阻遏"的形式解除对功 能启动子的抑制作用， 从而启动子可介导下游基因的表达。 改变温度、 pH值本 身以及加入 IPTG诱导物等都对启动子无直接激活作用。 在我们采用的系统中， 阿拉伯糖与阻遏蛋白 （AraC) 结合后， 不仅解除了对 PBAD启动子的抑制作用， 同时 "阿拉伯糖- AraC复合物"又可直接激活 PBAD启动子介导下游基因的表达。 所以阿拉伯糖诱导 /活化调控系统是一种比其它几种系统更有效的大肠杆菌高效 表达系统； （2)PBAD启动子活化的程度与加入的 L-阿拉伯糖剂量成线性关系。 这 样可以直接通过改变阿拉伯糖浓度而调节外源基因产物的表达量。 这一特性对 改变包涵体的形成等非常有意义。加入阿拉伯糖比改变温度 /pH等更容易直接控 制外源基因产物在大肠杆菌中的表达； （3)L-阿拉伯糖来源丰富， 价廉、 无毒性， 这克服其它诱导物如 IPTG在这方面的缺点。
本发明用阿拉伯糖诱导 /活化系统建立了高效、稳定的 rSIFN-co表达大肠杆 菌工程菌株， 通过对该菌株在适宜的条件下的培养发酵， 收获菌体， 超声破菌 并反复洗涤得包涵体， 通过包涵体的变性、 复性及分离纯化等工艺， 获得了大 量高纯度的改变了空间构象的 rSIFN-co蛋白以用于本发明的研究和临床治疗。 本发明的应用方法是通过患者使用重组高效复合干扰素达到治疗目的， 患 者使用的重组高效复合干扰素可以制成各种剂型如： 片剂、 胶囊、 口服液、 贴 剂、 注射剂、 喷雾剂、 栓剂、 溶液制剂， 推荐的剂型为注射剂， 可皮下或静脉 注射给药， 药物组合物中的载体可使用任何一种适宜的可接受的药物载体， 这 些载体可以是糖类、 纤维素制品、 粘合剂、 崩解剂、 润滑剂、 填充剂、 增溶剂、 缓冲剂、 防腐剂、 增稠剂、 配合剂和其他佐剂。 附图说明
图 1： 为 rSIFN-co的 DNA编码序列以及推断的氨基酸序列 具体实施方式
本发明通过以下实验验证了改变了空间构象的重组高效复合干扰素对乙型 肝炎病毒 DNA， 表面抗原和 e抗原抑制作用， 实验方法如下：
材料
溶剂及配制方法： 受试药物配制时向每支原料瓶内加入 lml生理盐水， 溶解 后， 再根据所设不同剂量组浓度差异用 MEM培养液调配。 现用现配。
对照药品： 先灵公司生产的 IFN-ot2b干扰能为冻干制剂， 每支 3xl06U实验 时用培养液配成 3xl06IU/ml溶液。 安进公司生产的干复津为注射液。 每支 9μ§， 0.3ml, 相当于 9 X 106IU/支， 实验时用培养液配成 9X 100IU/ml溶液， 4°C冰箱 保存， 2.2.15 细胞： 乙型肝炎病毒 (HBV)DNA克隆转染人肝癌细胞 (Hep G2)的 2.2.15细胞系， 美国 Mount Sinai医学中心构建。
试剂： Eagles MEM干粉，美国 Gibco公司产品；胎牛血清，美国 Hyclone Lab 公司产品； G-418(Geneticin), MEM配制， 美国 Gibco公司产品； L-谷氨酰胺， 京科化学试剂公司进口分装； HBSAg， . HBeAg固相放射免疫测定盒， 购自中国 同位素公司北方免疫试剂研究所； 卡那霉素， 华北制药厂产品； Lipofectin, 美 国 Gibco公司产品。 ，
实验用品及仪器： 培养瓶， 丹麦 Tunclon TM; 培养板 96孔板、 24 ?L， 美国 Coming公司产品； 二氧化碳孵箱， 美国 Shel-Lab产品； MEM培养液 lOOmh 含胎牛血清 10%， 3%谷氨酰胺 1%， G418 380 g/ml， 卡 那毒素 50U/ml。
试验方法
2.2.15细胞培养： 在长满 2.2.15细胞的培养瓶内加 0.25%胰酶， 37。C消化 3 分钟， 加培养液吹打， 1:3传代， 10天长满。
药物对细胞毒性试验： 实验分无药物细胞对照组和不同药物浓度给药组。 细胞消化， 配制成每毫升 10万个细胞， 接种培养板， 96孔板每孔 200μ1， 37°C 5%C02培养 24小时， 细胞长成单层后进行实验。高效重组高效复合干扰素用培 养液配制成 L8xl07IU/ml溶液， 2倍稀释加入 96孔细胞培养板， 每浓度 3孔， 每 4天换同浓度药液， 以观察细胞病变为指标， 8天显微镜下观察细胞病变， 完 全破坏为 4; 75%为 3; 50%为 2; 25%为 1 ; 无病变为 0。 计算每浓度药液平均 细胞病变程度和抑制％。按 Reed Muench法计算半数有毒浓度 (TC50)和最大无毒 浓度 （TC0)。
50-B
TC50=Antilog(B+—— xC)
A-B
A=log>50%药物浓度 B=log<50%药物浓度 C=log稀释倍数 对 HBeAg、 HBsAg抑制试验： 试验设 HBeAg、 HBsAg阳性对照组， 阴性对 照组， 细胞对照组及不同药物浓度给药组。 每毫升 70万个 2.2.15细胞接种 6孔 细胞培养板， 每孔 3ml， 37°C5%C02培养 24小时， 加无毒浓度以下 3倍稀释试 验药液， 5个稀释度分别为 4.5 X 106IU/ml、 1.5 X 106IU/ml、 0.5 X 106IU/ml、 0.17
X 106IU/ml、 和 0.056X 106,每浓度 1孔， 37°C5 %C02培养， 每 4天换原浓度药 液培养,第 8天时收获培养液， -20°C冰冻保存。试验重复三批，分别测定 HBsAg 和 HBeAg。 HBsAg和 HBeAg测定采用中国同位素公司北方免疫试剂研究所产 品， 固相放射免疫测定盒测定， 方法见说明书， 用 γ-计数仪测定每孔 cpm值。
药物效果计算： 计算细胞对照及每浓度 cpm均值及标准差， P/N值如抑制百 分率 (％)， 半数有效浓度 (IC )及选择指数 (SI)。
细胞对照 cpm-给药组 cpm
①抗原抑制百分率 (％)= X 100
细胞对照 cpm ②计算药物抑制抗原半数有效浓度 （IC50):
50-B
IC50=Antilog(B+ X C)
A-B
A=log>50 %药物浓度 B=log<50 %药物浓度 Olog稀释倍数
③改变了空间构象的重组高效复合干扰素在 2.2.15 细胞培养内对 HBsAg和 HBeAg的选择指数 (SI)， 按其对细胞毒性指标细胞病变 (SI)计算。 细胞病变毒性 TC50
SI=
IC50
④以 t检验法计算各稀释度 HBsAg、 HBeAg和对照组间 cpm的差别。
Southern blot: @2.2.15细胞内1¾¥~~0^提取： 2.2.15细胞加药后培养 8天， 吸除培养液收取细胞， 加入细胞裂解液， 裂解细胞， 等体积苯酚： 氯仿： 异戊醇抽提 2次，高速 10,000g离心，取上清，加无水乙醇沉淀核酸，真空抽干， 重溶于 20μ1 ΤΕ缓冲液中。②电泳：加入 6X DNA样品缓冲液，将样品加于 1.5% 琼脂糖胶上电泳， IV/cm,恒压， 14-18小时。③变性、杂交： 将胶分别浸于 HC1、 变性液、 中和液中。 ④转膜： 按程序将 DNA转至 Hybond- N膜上。 同斑点杂交 一同进行烤膜、 杂交、 曝光。 扫描仪扫描光片， 以 gel-pro凝胶分析软件分析相 对密度， 计算抑制率及 IC50。
统计与分析方法： 各组计量资料结果用算术平均数 (X)士标准差 (S)表示。 根 据中国 《新药 （西药） 临床前研究指导原则汇编》 的有关新药药效学研究中统 计处理的指导原则， 计数资料用 Fisher精确检验， 计量资料选择 Student t检验进 行各组均数差异显著性。
结果
本发明通过几批实验的结果 （见表一、 表二、 表三） 表明： 样品以最大 无毒浓度加入 2.2.15细胞培养 8天， 高效复合干扰素最大无毒浓度 9.0±0 X 106IU/ml对 HBeAg的平均抑制率为 46.0±5.25% (PO.001 )， IC50为 4.54 ± 1.32 X 106IU/ml， ^择指数 SI为 3.96; 对 HBsAg的平均抑制率为 44.8土 6.6%, IC50 6.49± 0.42 X 106IU/ml， 选择指数 SI为 2.77。 因此， 重组高效 复合干扰素具有明显的抑制乙型肝炎表面抗原和 e抗原活性， 而对照组干扰能 和干复津不具有上述活性。 临床病例也证实了慢性活动性乙型肝炎患者， 通过 服用重组高效复合干扰素使乙型肝炎表面抗原和 e抗原阳性降低或恢复到正常 水平。
本发明应用的重组高效复合干扰素制剂可通过如下实施例制备：
<实施例一>: 冻干注射剂的制备
a. 重组高效复合干扰素 300万 IU;
b. 枸橼酸 ， 0.2 毫克；
c 磷酸氢二钠 bin 2.5毫克；
d. 氯化钠 4.0 毫克；
e. 右旋糖酐 20毫克；
f. 聚氧乙烯失水山梨醇 J 0.1毫升；
g. 注射用水 适量加至 1.0毫升。
制备工艺： 按处方称料， 用无菌无热原注射水溶解， 除菌过滤用 0.22μπι孔 径滤膜除菌过滤， 保存于 8±2°C， 取样作无菌和热原检查合格后分装于西林瓶 中， 单剂量装每瓶 1.0ml。 分装后放置到冻干机中冷冻干燥。
<实施例二>: 水溶液注射剂的制备
a. 重组高效复合干扰素 300万 IU;
b. 枸橼酸 0.2 毫克；
c 磷酸氢二钠 2.5毫克；
d. 氯化钠 4.0 毫克；
e. 右旋糖酐 20毫克；
f. 聚氧乙烯失水山梨醇单油酸酯 0.1毫升；
g. 注射用水 适量加至 1.0毫升。
制备工艺： 按处方称料， 用无菌无热原注射水溶解， 除菌过滤用 0.22μπι孔 径滤膜除菌过滤， 保存于 8 ±2°C， 取样作无菌和热原检査合格后分装于密闭容 器中。分装于西林瓶中， 单剂量装每瓶 l.OmL分装后成品置 2— 10°C下暗处保存。 表一： 测定 rSIFN-co对乙型肝炎表面抗原和 e抗原抑制率的实验结果 第一批实验
Figure imgf000009_0001
Figure imgf000010_0001
续表一： 第三批实验
Figure imgf000011_0001
E抗原： IC50均值 450.2434 标准差 132.315479 表面抗原： IC50均值 649.1894 标准差 42.29580
表二： 干扰能（IFN-a2b)对乙型肝炎表面抗原和 e抗原抑制率的实验结果
Figure imgf000012_0001
表三、 干复津对乙型肝炎表面抗原和 e抗原抑制率的实验结果
Figure imgf000013_0001
续表三
Figure imgf000014_0001
续表三
第三批实验
Figure imgf000015_0001
IC50均值 0标准差
IC50均值 0标准差

Claims

权利要求
1、 一种通过改变了蛋白质空间结构的重组高效复合干扰素的应用， 其特征 是它作为一种制剂不仅对乙型肝炎病毒 DNA 有抑制作用， 而且对 HBsAg、 HBeAg均有抑制作用。
2、 根据权利要求 1所述的重组高效复合干扰素的应用， 其特征是该制剂是 通过一定生产工艺改变了蛋白质空间结构的重组高效复合干扰素， 它具有美国 专利号 4695623和 4897471所描述的干扰素不具备的功效及对其它病毒性疾病， 如艾滋病毒等的治疗作用的应用。
3、 一种权利要求 1或 2中所述的重组高效复合干扰素， 其特征是具有权利 要求 1或 2药理作用的蛋白质二级及高级空间结构。
4、 一种权利要求 1或 2中所述的重组高效复合干扰素， 其特征是它含有应 用特定的启动子构建的重组高效复合干扰素高效表达系统， 包括大肠杆菌、 酵 母菌及 CHO表达系统。
5、 根据权利要求 4所述的重组高效复合干扰素， 其特征是所说的启动子包 括 PBAD°
6、 根据权利要求 4所述的重组高效复合干扰素， 其特征是其中高效复合干 扰素基因是人工合成的 cDNA, 且其序列根搪宿主密码子偏爱性而作相应调整。
7、 一种生产权利要求 1或 2中所述的重组高效复合干扰素的生产工艺。
' 8、 根据权利要求 7所述的工艺， 其特征是它包括从表达产物中提取重组高 效复合千扰素的方法， 包括包涵体的制备、 变性及复性， 所用的层析介质及分 离纯化条件， 试剂及其浓度等。
9、 根据权利要求 7所述的工艺， 其特征是使变性的重组高效复合干扰素恢 复其稳定的具有权利要求 1或 2所述的药理作用的蛋白质空间结构的方法， 包 括所用的试剂、 试剂的浓度等。
10、 根据权利要求 Ί所述的工艺， 其特征是其中包括分离纯化重组高效复 合干扰素的工艺。
11、 根据权利要求 7所述的工艺， 其特征是该工艺还包括将高度纯化的重 组高效复合干扰素制成冻干制剂的配方及工艺。
12、 根据权利要求 7所述的工艺， 其特征是该工艺还包括将高度纯化的重 组高效复合千扰素制成的水溶液制剂的配方及工艺。
13、重组高效复合干扰素制备成抑制乙型肝炎病毒 DNA、 HBsAg及 HBeAg 的药物的应用， 权利要求 1或 2所指为乙型肝炎， 病毒性疾病包括（但不仅限于 此） 甲型肝炎、 乙型肝炎、 丙型肝炎、 其它除了乙型或者丙型肝炎以外的非甲 非乙型肝炎， EB病毒感染、 HIV感染、 疱疹病毒（EB， CML, 单纯疱疹病毒）、 乳头瘤病毒、 痘病毒、 微小 RNA病毒、 腺病毒、 鼻病毒、 人类 T细胞白血病病 毒 I、 人类 T细胞白血病病毒 II及人类轮状病毒。
14、 根据权利要求 1或 2所述的应用， 其特征是重组高效复合干扰素选自 各种 α、 β、 ，Υ型干扰素 （如 la， 2b等） 或其它突变体。
15、 根据权利要求 13所述的应用， 其特征是重组高效复合干扰素是通过口 服、 静脉注射、 肌肉注射、 皮下注射、 鼻内、 粘膜给药等途径给药。
16；根据权利要求 13所述的应用， 其特征是重组高效复合干扰素是通过注 身方式药 9μ§、 15μ§, 每周 3次， 疗程 24周。
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ATE446104T1 (de) 2009-11-15
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US20080305080A1 (en) 2008-12-11
AU2003248419A1 (en) 2003-11-06
AU2003248419B2 (en) 2007-01-04
HK1061201A1 (en) 2004-09-10
EP1371373A4 (en) 2005-05-04
CN1245215C (zh) 2006-03-15
EP1371373B1 (en) 2009-10-21
US20110189128A1 (en) 2011-08-04
US8425896B2 (en) 2013-04-23
CA2439503A1 (en) 2002-10-17
JP2005508848A (ja) 2005-04-07
CN1311035A (zh) 2001-09-05
US20040202641A1 (en) 2004-10-14
US8114395B2 (en) 2012-02-14
US7364724B2 (en) 2008-04-29
DE60234085D1 (de) 2009-12-03

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