WO2007012285A1 - Desoxynucleosides monocatenaires resistant aux infections virales - Google Patents

Desoxynucleosides monocatenaires resistant aux infections virales Download PDF

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WO2007012285A1
WO2007012285A1 PCT/CN2006/001891 CN2006001891W WO2007012285A1 WO 2007012285 A1 WO2007012285 A1 WO 2007012285A1 CN 2006001891 W CN2006001891 W CN 2006001891W WO 2007012285 A1 WO2007012285 A1 WO 2007012285A1
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virus
hepatitis
modification
deoxynucleotide
stranded
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Chinese (zh)
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Li-Ying Wang
Mu-Sheng Bao
Yong-Li Yu
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Changchun Huapu Biotechnology Co Ltd
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Changchun Huapu Biotechnology Co Ltd
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Priority claimed from CNA2005100855636A external-priority patent/CN1904046A/zh
Priority claimed from CN2005100855621A external-priority patent/CN1903870B/zh
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Publication of WO2007012285A1 publication Critical patent/WO2007012285A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids

Definitions

  • the present invention relates to a single-stranded deoxynucleotide which has a therapeutic effect on viral infectious diseases.
  • oligo allophone, hereinafter referred to as oligo.
  • viruses including, but not limited to, hepatitis B virus, HIV, and thus viral infectious diseases including but not limited to Hepatitis B caused by hepatitis B virus and diseases caused by immunodeficiency virus such as HIV infection have therapeutic effects.
  • viruses including, but not limited to, hepatitis B virus, HIV, and thus viral infectious diseases including but not limited to Hepatitis B caused by hepatitis B virus and diseases caused by immunodeficiency virus such as HIV infection have therapeutic effects.
  • a virus is a type of non-cellular microorganism that is small in size and relatively simple in structure and can only proliferate in living cells.
  • the structure of the virus includes nucleic acids, capsids and envelopes.
  • the nucleic acid of a virus is the core of the virus and determines the inheritance, variation and replication of the virus.
  • the capsid and membrane act to protect the core of the virus and are antigenic.
  • the virus is classified into a DNA virus and an RNA virus.
  • DNA viruses include double-stranded DNA viruses and single-stranded DNA viruses;
  • RNA viruses include single-stranded RNA viruses, single-negative-strand RNA viruses and double-stranded RNA viruses, and retroviruses.
  • DNA viruses include, but are not limited to, hepatitis B virus, TTV virus, adenovirus, papilloma virus, herpes zoster virus, variola virus, and vaccinia virus.
  • RNA viruses include, but are not limited to, influenza virus, swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus And immunodeficiency viruses such as HIV.
  • HIV Acquired Immunodeficiency Syndrome
  • the human immunodeficiency virus has a circular shape with a diameter of 100nm-120nm. Under the electron microscope, a dense conical core is visible, containing viral RNA molecules and enzymes (reverse transcriptase, integrase and protease), and the outer membrane of the virus is double-layered. A protein membrane, in which gpl20 protein and gp41 protein are embedded, which constitute the spike and transmembrane proteins of the virus, respectively.
  • the inner surface of the capsule is a capsid composed of P17 protein.
  • the viral center consists of a core protein (P24) and viral RNA.
  • the HIV genome is about 9. 2 kb in length and contains three structural genes of gag, pol and env and at least six regulatory genes (Tat Rev, Nef, Vif, VPU, Vpr) and each of the 5' and 3' ends of the genome. End repeats.
  • Gag The gene encodes viral core proteins such as p24, p9, p6, pl7, etc.; the pol gene encodes a variety of enzymes, including reverse transcriptase; the er gene encodes a large viral precursor glycoprotein g P 160, gpl60 after translation Degraded to gpl20 and gp41 (STANLEY A. SCHWARTZ AND MADHAVAN PN NAIR. Current Concepts in Human Immunodeficiency Virus Infection and AIDS. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1999, p. 295-305.).
  • HIV-infected people In the weeks after HIV infection, viremia caused by HIV occurs in infected people. At the same time, HIV-specific immune responses occur in most infected people, and the result is a reduction in HIV viremia. After that, a typical infected person will enter a stable, asymptomatic period that can last for years or longer. In this period, the patient's HIV viremia was lighter and there was no significant reduction in the number of CD4+ cells, mainly due to HIV replication and clearance, and the destruction and complementation of infected CD4+ cells was in a dynamic equilibrium (STANLEY A. SCHWARTZ AND MADHAVAN PN NAIR. Current Concepts in Human Immunodeficiency Virus Infection and AIDS. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1999, P. 295 - 305. Over time, if the level of HIV in the peripheral blood continues to rise, HIV-infected people will develop AIDS.
  • the development of anti-HIV drugs and preparations is the key to the treatment of AIDS, and the evaluation of the therapeutic effect of anti-AIDS drugs is the focus of anti-AIDS drugs.
  • the detection of HIV infection mainly includes detection of viral nucleic acid, p24 antigen and anti-HIV antibody.
  • Peripheral blood HIV levels are important indicators of disease progression and treatment outcomes in HIV-infected individuals.
  • Quantitative detection of HIV p24 antigen and HIV RNA is an important method for detecting HIV in human peripheral blood (STANLEY A. SCHWARTZ AND MADHAVAN PN NAIR. Current Concepts in Human Immunodeficiency Virus Infection and AIDS. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1999, p. 295 - 305. ).
  • P24 is the core protein of HIV encoded by the structural gene gag of the HIV virus (Iweala 01. HIV diagnostic tests: an overview. Contraception 2004, Aug; 70 (2): 141-7.). Studies have shown that antiviral drugs can lower the level of p24 antigen in the blood. Therefore, the level of p24 antigen in the blood is a very useful marker for evaluating the antiviral effect of antiviral agents (Spector, SA, et al. The antiviral effect). Of zidovudine and ribavirin in clinical trials and the use of p24 antigen levels as a virologic marker. J. Infect. Ms. 1989,
  • Viral hepatitis is an infectious liver inflammatory disease caused by hepatitis virus (H-printed atitis virus).
  • H-printed atitis virus There are seven types of viruses that cause viral hepatitis, namely Hepatitis A virus (HAV), Hepatitis B virus (HB), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Hepatitis E virus (Hepatitis E virus), Hepatitis G virus (HGV), and sputum (transfusion-transmitted viral hepatitis virus), which can Causes A, B, C, D, E, G and TTV hepatitis.
  • HAV Hepatitis A virus
  • HB Hepatitis B virus
  • HCV Hepatitis C virus
  • HDV Hepatitis D virus
  • Hepatitis E virus Hepatitis E virus
  • HGV Hepatitis G virus
  • sputum transfusion-transmitted viral hepatitis virus
  • Hepatitis A virus is a spherical, non-enveloped, single-stranded RNA virus. Hepatitis A is referred to as Hepatitis A, an intestinal infectious disease caused by HAV (Atmar, RL, et al. Detection of enteric viruses in oyster by using the polymerase chain reaction. Appl. Environ. Microbiol. 1993, 59 : 631 - 635. ). Symptoms of acute hepatitis A include fever, loss of appetite, weakness, hepatomegaly, and abnormal liver function. Only a small number of patients with acute hepatitis A have jaundice. Hepatitis A virus generally does not turn into a chronic and pathogen-carrying state.
  • Hepatitis A patients are most contagious from the latent stage to the 10 days after onset. Hepatitis A usually occurs in children and adolescents and is rare in adults. The general incubation period is 2 to 6 weeks. The winter and spring seasons are the peak of the onset of hepatitis A (JENNIFER A. CUTHBERT et al. Hepatitis A: Old and New. CLINICAL MICROBIOLOGY REVIEWS, Jan. 2001, p. 38-58).
  • Hepatitis C virus is a genus of flavivirus, a single-stranded positive-strand RNA virus, having a diameter of about 50 to 60 nm and having a lipid envelope. Hepatitis C is mainly transmitted through blood and blood products. According to the data, 90% of hepatitis infected by blood transfusion is hepatitis C (JANICE M. MATTHEWS-GREER, et al. Comparison of Hepatitis C Viral Loads in Patients with or without Human Immunodeficiency Virus. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 2001, p.
  • Hepatitis D virus is a defective virus whose nucleic acid is a single negative stranded circular RNA (Theo Heller and Jay H. Hoofnagle. Denying the wolf access to sheep's clothing. J. Clin. Invest. 2003, 112:319-321 ). As a defective virus, HDV requires the help of a hepadnavirus (such as HBV) to complete its life cycle. Hepatitis D virus is transmitted in a similar manner to hepatitis B virus, with vertical transmission, sexual contact and body fluid transmission as the main modes of transmission (Rizzetto, M., et al. 1980. Transmission of the hepatitis B virus-associated delta antigen To chimpanzees. J.
  • Hepatitis E virus is a spherical, non-enveloped virus with a diameter of 32–34 nm. Its nucleic acid is a single positive-stranded RNA containing three reading frames (Reyes, GR, et al. Isolation of a cDNA from the virus responsible for Enterically transmitted non - A, non-B hepatitis. Science. 1990: 247 : 1335 - 1339; Reyes, GR , et al. Hepatitis E virus (HEV): the novel agent responsible for enterically transmitted non- A, non - B hepatitis .
  • Hepatitis E like hepatitis A, is transmitted through the faecal-oral route, because the water source is contaminated and causes large outbreaks and epidemics. The pathological damage caused by hepatitis E is more obvious than that of hepatitis A, and it recovers slowly.
  • Hepatitis G virus is a enveloped, single-stranded positive-strand RNA virus of the Flaviviridae family that replicates mainly in the liver, mainly through blood products, intravenous drugs, sexual contact, and vertical transmission of mother and child. Hepatitis G virus can cause acute and chronic hepatitis, and is even associated with the occurrence of liver cancer and cirrhosis (Schmidt, B., K. Korn, and B. Fleckenstein. Molecular evidence for transmission of hepatitis G virus by blood transfusion. Lancet. 1996 , 347 : 909; Iban ⁇ ez, A. , M. Gime' nez-Barcons, et al.
  • Transfusion transmitted virus is a non-enveloped, single-stranded awakening virus (C.-L. lin, et al. Fecal Excretion of a Novel Human Circovirus, TT Virus, in Healthy Children. Clinical and diagnostic laboratory immunology, 2000, p. 960 - 963 ).
  • the genome of the TTV virus is a circular DNA ( Isa k. Mushahwar, et al. Molecular and biophysical characterization of TT virus: Evidence for a new virus family infecting humans. Proc. Natl. Acad. Sci. USA Vol. 96, pp 3177 - 3182, March 1999 . Microbiology) .
  • TTV virus can be transmitted through sexual contact, vertical transmission, etc.
  • TTV TT Virus
  • TTV-Like Minivirus in Cervical Swabs. JOURNAL OF CLINICAL MICROBIOLOGY , 2001, p. 2022 - 2024 ).
  • Hepatitis B virus is a DNA virus of the Department of Hepatic DNA.
  • the complete HBV consists of an envelope and a core.
  • Hepatitis B surface antigen (HBsAg) is the main component of the envelope.
  • HBsAg is synthesized in hepatocytes and released into the blood circulation in a large amount, which is not contagious in itself.
  • the core contains incompletely circular double-stranded DNA, DNA polymerase, core antigen (HBcAg) and e antigen (HBeAg), which are infectious.
  • Hepatitis B virus is a inflammatory disease of the liver caused by hepatitis B virus that is a serious threat to human health.
  • HepG2. 15 cells are hepatoma cells (HepG2 cells) stably transfected with the HBV genome, which are capable of stably secreting HBsAg, HBeAg and Dane particles. HepG2 cells can maintain many functions of normal hepatocytes during long-term subculture, such as ensuring normal replication of HBV, expressing multiple receptors such as interferon and Tol l-like receptors (Volker Mersch-Sundermann.
  • Interferon induces the production of interferon regulatory factor 1 (IFN-regulated factor 1, IFN-1), RNase L by complex intracellular signal transduction after acting on the corresponding receptor on the surface of He P G2.
  • IFN-regulated factor 1 IFN-1
  • RNase L double-stranded RNA activated protein kinase
  • PSR protein kinase
  • nitrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor 1.
  • Circula tion, 1997, 96 585-591; Pitha, PM , et al. Role of the interferon regulatory factors in virus-mediated signaling and Regulation of cell growth.
  • HepG2.2.15 cells are a good cell model for studying anti-HBV drugs and preparations.
  • PBMC peripheral blood mononuclear cell
  • the invention relates to a synthetic single-stranded deoxynucleotide for use in antiviral infection having the structural formula shown in 1-5 below:
  • a synthetic single-stranded deoxynucleotide comprising a sequence formula of TTCGTCG, preferably,
  • the synthetic single-stranded deoxynucleotides of the invention have the sequence shown below:
  • Oligol Seq No 89 5'-TCgTCgggTgCgATCgCAgggggg-3'
  • Oligo20 seq No 66 5'-gggggCgTCgTTTTCgTCgACgAATT-3'
  • the artificially synthesized single-chain deoxynucleotides of the present invention can be produced by a known method, for example, by a solid phase phosphoramidite triester method.
  • the single-stranded deoxynucleotides described in the present invention may be modified by various chemicals. These chemical modifications include, but are not limited to, phosphate backbone modifications, sugar ring modifications, and base modifications.
  • the modification of the phosphate backbone can be a thio modification.
  • Thio modification refers to the substitution of a non-bridged oxygen atom in a phosphodiester bond by a sulfur atom in a nucleotide fragment (Ekambar R. Kandimalla et al.
  • Mixed backbone antisense oligonucleotides design, biochemical and biological properties of oligonucleotides containing 2 -5 - Ribo - and 3 - 5 -deoxyribonucleotide segments.
  • this substitution may occur in part or all of the phosphodiester bond of the deoxynucleotide.
  • Modification of the phosphate backbone can be accomplished by the formation of other types of phosphate linkages including, but not limited to, methyl phosphates, selenophosphates, boryl phosphates, and phosphorodithioates.
  • the sugar ring modification can be a modification of the 2' position of the pentose sugar.
  • the 2'-position modification of pentose sugars includes, but is not limited to, methoxy, methoxyethoxy, propyleneoxy, and fluoro modifications (Ernst Urban, Christian R. Noe.
  • Base modification mainly refers to the inclusion of rare bases in single-stranded deoxynucleotides (Xiaolan Chen, Nancy Dudgeon, Long Shen and Jui H. Wang. Drug Discovery Target, 22005, 10 (8): 587-593), rare bases Refers to some bases other than A, G, C, U, including dihydrouracil (DHU), pseudouridine, and methylated ⁇ (mG, mA).
  • a typical pyridinium nucleoside is linked to a C- linkage of a sugar ring by N- 1 on a heterocyclic ring, and a pseudouridine nucleoside is linked to a C-oxime of a sugar ring by C-5 on the heterocyclic ring.
  • the addition of the single-stranded deoxynucleotide base means a single-stranded deoxynucleotide formed by adding 1 to 10 bases at one or both ends of the single-stranded deoxynucleotide provided by the present invention.
  • the cleavage at both ends refers to a single-stranded deoxynucleotide formed by cleavage of one or several bases at one or both ends of a single-stranded deoxynucleotide provided by the present invention.
  • a change in a single-stranded deoxynucleotide base refers to a base change of a single-stranded deoxynucleotide provided by the present invention.
  • the invention features a composition comprising the single-stranded deoxynucleotide for use in treating a disease caused by a viral infection.
  • the invention provides a kit for treating a disease caused by a viral infection comprising a single-stranded deoxynucleotide of the invention.
  • the invention relates to the use of said single chain deoxynucleotide for the manufacture of a medicament for the treatment of a disease caused by a viral infection.
  • the invention provides a method of treating a disease caused by a viral infection, comprising providing a therapeutically effective amount of a single chain deoxynucleotide of the invention to a subject, the effective amount being Words can be easily determined.
  • the application of synthetic single-stranded deoxynucleotides includes mucosal surfaces (including respiratory, digestive tract, and genitourinary mucosa) applications, subcutaneous, intramuscular injection, gastrointestinal applications, intraperitoneal applications, intravenous injections, and the like.
  • Viruses causing these viral infectious diseases include, but are not limited to, hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, variola virus and vaccinia virus, influenza virus, swine fever virus, hepatitis A virus, Hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus and human immunodeficiency virus.
  • the disease caused by the viral infection is hepatitis B caused by hepatitis B virus infection and AIDS caused by human immunodeficiency virus infection.
  • DNA chemical synthesis is different from enzymatic DNA synthesis.
  • the enzymatic DNA synthesis process extends from the 5'-3' direction, while DNA chemical synthesis starts from the 3' end.
  • the specific reaction steps are as follows:
  • the protective group dimethoxytrityl (DMT) of the nucleotide attached to the controlled-controlled porous glass was removed with Trichloroacetic Acid (TCA) to obtain a free 5'- The hydroxyl end is used for the next condensation reaction.
  • DMT dimethoxytrityl
  • the phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but the 5'-end is still affected by DMT Protected), this intermediate will undergo a condensation reaction with the deprotected nucleotide on the controlled porous glass.
  • the terminal hydroxyl group is often blocked by acetylation, and the general acetylating reagent is acetic anhydride and N. - Formed by the mixing of methyl imidazole.
  • the nucleoside monomer is linked to the oligonucleotide attached to the controlled porous glass through the phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by acid or alkali. At this time, iodine tetrahydrofuran is commonly used.
  • the solution converts the phosphorous amide to a phosphate triester to give a stable oligonucleotide. .
  • a deoxynucleotide is attached to the nucleotide of the controlled porous glass, and the protective group on the newly added deoxynucleotide 5'-hydroxyl group is also removed by trichloroacetic acid.
  • the above activation, ligation, blocking, and oxidation processes are repeated to obtain a crude DNA fragment.
  • Unvulcanized synthetic single-stranded deoxynucleotides were synthesized on an ABI 3900 DNA synthesizer; the synthesis of fully vulcanized and partially sulfurized CpG single-stranded deoxyoligonucleotides was synthesized on an ABI 394 DNA synthesizer using a displacement method.
  • SEQ ID NO: 1-190 was synthesized by the above method.
  • the sequences of the single-stranded deoxynucleotides (Oligo) exemplified in the following examples are as follows:
  • Oligo 1 Seq No 89 5'-TCgTCgggTgCgATCgCAgggggg-3'
  • Oligo 11 seq No 107 5'-TCgTCgggTgCgACgTCgCAgggggg-3'
  • Oligo 13 seq No 102 5'-TCgTCgggTgCgACgTCgCAg-3'
  • Oligo 14 seq No 103 5'-TCgTCgggTgCgACgTCgCAgg-3'
  • Oligo 15 seq No 104 5'-TCgTCgggTgCgACgTCgCAggg-3' 01igol6 seq No 105: 5'-TCgTCgggTgCgACgTCgCAgggg-3'
  • 01igol7 seq No 106 5'-TCgTCgggTgCgACgTCgCAggggg-3'
  • 01igol9 seq No 1 5'-ggggTCgTTCgTCgTTgggggg-3'
  • Oligo20 seq No 66 5'-gggggCgTCgTTTTCgTCgACgAATT-3'
  • Example 2 Single-chain deoxynucleotide stimulating human PBMC culture supernatant inhibits the secretion of HBsAg by HepG2, 2.15 cells
  • Equipment and equipment low temperature refrigerator, carbon dioxide incubator, ultra clean bench, inverted microscope, liquid nitrogen tank, distilled water, vacuum pump, cell culture flask, bacteria filter, filter bottle, various specifications of straw, plus Samples, droppers, blood cell counting plates, horizontal centrifuges, etc.
  • Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun.
  • Lymphocyte stratification solution in which sucrose-diatrizoate: specific gravity 1. 077 ⁇ 0, 001, purchased from Beijing Dingguo Biotechnology Co., Ltd.
  • RPMI 1640 medium L-glutamine RPMI1640 (GIBC0BRL) 10. g, sodium bicarbonate 2, 0 g, gentamicin 100,000 units, add three distilled water to 1000 ml, 0.22 micron filter Filtering and sterilizing and dispensing.
  • PBMC lymphocytes
  • FBS Longzhou Institute of Biological Products
  • IMM Gibco
  • PBMC peripheral blood mononuclear cell
  • 2ral/well Add each single-stranded deoxynucleotide to a final concentration of 6 g/ml, and set 3 replicate wells.
  • the 12-well plate was placed in a 37 ° C 5% CO 2 incubator for 48 h, and the culture supernatant was collected. The culture supernatants collected from the three replicate wells were mixed together, and stored at a temperature of 20 Torr.
  • the culture supernatants were collected at 48h, 72h, and 96h after the culture, and the HBsAg content was determined by the hepatitis B virus e antigen diagnostic kit (Suzhou Xinbo Biotechnology Co., Ltd.). The HBsAg content was determined according to the kit. Instructions for operation.
  • Single-stranded deoxynucleotide-stimulated human PBMC culture supernatants were effective in inhibiting the secretion of HBsAg by HepG2. 2.15 cells (Table 1).
  • Single-stranded deoxynucleotide-stimulated human PBMC culture supernatants are capable of inhibiting HBV replication, and thus the single-stranded deoxynucleotides provided by the present invention have antiviral, particularly anti-HBV biological activities.
  • Example 3. Single-chain deoxynucleotide stimulates human PBMC culture supernatant to inhibit the secretion of HBeAg by HepG2. 2. 15 cells.
  • Step 1-2 is the same as step 1-2 of the embodiment 2.
  • Steps 1 to 2 are the same as steps 1 and 2 of Embodiment 2.
  • HBV DNA quantitative PCR kit (Shanghai Shenyou Biotechnology Co., Ltd.), HBV DNA. The determination of the content is carried out according to the instructions provided in the kit.
  • Inhibition rate (control well log (copy number) - test well log (copy number) / control well log (copy number) X 100%
  • Each single-stranded deoxynucleotide stimulates human PBMC culture supernatant to effectively inhibit at 1:64 dilution
  • Single-stranded deoxynucleotides stimulate human PBMC culture supernatants to inhibit HBV replication, and thus the single-stranded deoxynucleotides provided by the present invention have antiviral, particularly anti-HBV, biological activities.
  • Example 5 Anti-HIV effect of single-chain deoxynucleotides
  • PBMC Human peripheral blood mononuclear cells
  • fetal bovine serum Trianjin Yuyang (TBD) Biotechnology Co., Ltd.
  • streptomycin GIBCO
  • glutamine GIBCO
  • 10 units of interleukin 2 per ml The RPMI (GIBCOBRL) of the NIH AIDS Research and Reference Reagent Program was used to culture PBMC at a cell concentration of 10 ⁇ 10 6 cells/ml.
  • the HIV virus is provided by the NIH AIDS Research and Reference Reagent Program.
  • 96-well plate was added at a concentration of 10X10 6 cells / ml PBMC (3X 10 6 cells / ml 200 ⁇ 1 / well), each sample provided triplicates.
  • PBMCs were infected with HIV virus (0.0005 TCID50/cell), and after 6 hours of incubation (37 ° C, 5% CO 2 incubator), cells were washed with PBS 3 After the second time, the cells were cultured, and the level of the p24 antigen in the culture supernatant was measured on the fifth day. Control wells without single-stranded deoxynucleotides were placed.
  • P24 polyclonal antibody coated ELISA plate [BioMerieux BioMerieux, Human Immunodeficiency Virus Antigen Antibody (ELISA) (Aalto Bioreagents, Dublin, Eire), polyclonal antibody and p24
  • the antigen binds, and the p24 antigen is then combined with the added anti-p24 monoclonal antibody (provided by the kit).
  • the anti-p24 monoclonal antibody binds to alkaline phosphatase and produces color after the addition of biphenyl diamine.
  • the p24 antigen was obtained by cleavage of viral particles with a detergent. Specific measurement steps:
  • the single-stranded deoxynucleotide of the present invention is capable of inhibiting the replication of HIV virus and thereby suppressing AIDS caused by HIV infection.
  • Oligo 1 (1:64 dilution) 78.26 86.45 92.18
  • Oligo 2 (1:64 dilution) 67.98 79.54 89.78
  • Oligo 3 (1:64 dilution) 76.34 88.09 90, 12
  • Oligo 4 (1:64 dilution) 69.22 76.56 87.98
  • Oligo 5 (1:64 dilution) 74.59 87.06 93.03
  • Oligo 6 (1:64 dilution) 77.34 83.98 90.89
  • Oligo 7 (1:64 dilution) 65.90 73.13 88.09
  • Oligo 8 (1:64 dilution) 66.09 74.34 89.01
  • Oligo 9 (1:64 dilution) 63.89 72.41 85.67
  • Oligo 11 (1:64 dilution) 60.78 69.45 76.35
  • Oligo 12 (1:64 dilution) 70.32 79.46 85.78
  • Oligo 13 (1:64 dilution) 67.90 78.09 83.42
  • Oligo 14 (1:64 dilution) 70.12 78.53 89.21
  • Oligo 15 (1:64 dilution) 60.74 67.90 78.41
  • Oligo 16 (1:64 dilution) 64.09 • 69.97 • -79 ⁇ .56
  • Oligo 17 (1:64 dilution) 69.00 78.00 85.31
  • Oligo 18 (1:64 dilution) 72.87 79.99 91.78
  • Oligo 19 (1:64 dilution) 71.78 82.34 94.07
  • Oligo 20 (1:64 dilution) 77.11 89.44 90.01 2 Inhibition of HepG2.2.15 cells secreting HBeAg by single-chain deoxynucleotide-stimulated human PBMC supernatant
  • Oligo 1 (1:64 dilution) 72.26 88.45 93.18
  • Oligo 2 (1:64 dilution) 66.90 80.51 90. 01
  • Oligo 3 (1:64 dilution) 76.12 88.21 91.12
  • Oligo 4 (1:64 dilution) 70.22 75.66
  • Oligo 5 (1:64 dilution) .73.69 86.96 94.03
  • Oligo 8 (1:64 dilution) 70.09 80.00 89.45
  • Oligo 9 (1:64 dilution) 65.09 70.90 89.12
  • Oligo 10 (1:64 dilution) 61.90 79.34 87.96
  • Oligo 11 (1:64 dilution) 72.34 79.91 , 88.06
  • Oligo 12 (1:64 dilution) 62.09 74.45 8O O7.65
  • Oligo 13 (1:64 dilution) 69.34 79.76 90.01
  • Oligo 14 (1:64 dilution) 66.11 78.54 89.00
  • Oligo 15 (1:64 dilution) 70.00 79.32 88.11
  • Oligo 16 (1:64 dilution) 63.90 76.31 86.11
  • Oligo 17 (1:64 dilution) 70.55 79.66 87.43
  • Oligo 19 (1:64 dilution) 70.01 78.48 87.56
  • Oligo 1 (1:64 dilution) 67.24 79.14 88.09
  • Oligo 2 (1:64 dilution) 69.18 80.01 92.06
  • Oligo 3 (1:64 dilution) 71.11 86.32 91.37
  • Oligo 4 (1:64 dilution) 72.89 85. '32 90.97
  • Oligo 5 (1:64 dilution) 70.65 84.36 90.06
  • Oligo 6 (1:64 dilution) 69.78 78.35 89.76
  • Oligo 8 (1:64 dilution) 71.19 81.00 89.75
  • Oligo 9 (1:64 dilution) 64.09 71.02 84.12
  • Oligo 10 (1:64 dilution) 64.90 79.44 88.96
  • Oligo 11 (1:64 dilution) 70.24 79.44 86.36
  • Oligo 12 (1:64 dilution) 63.19 74.55 86.69
  • Oligo 13 (1:64 dilution) 70.24 81.12 90.01
  • Oligo 15 (1:64 dilution) 71.00 79.42 88.71
  • Oligo 16 (1:64 dilution) 64.32 76.31 85.17
  • Oligo 17 (1:64 dilution) 71.55 79.86 87.88
  • Oligo 19 (1:64 dilution) 71.01 78.48 87.56
  • Oligo 20 (1:64 dilution) 65.55 79.21 91.01
  • Table 4 Levels of p24 antigen production when different single-stranded deoxynucleotides were added

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Abstract

La présente invention se rapporte à des désoxynucléosides monocaténaires utiles pour le traitement des maladies infectieuses virales. Ces désoxynucléosides monocaténaires peuvent stimuler les cellules humaines pour produire une substance antivirale qui peut inhiber la réplication du virus. L'invention se rapporte également à la composition contenant ces désoxynucléosides, à une trousse les contenant et à son utilisation pour le traitement de maladies infectieuses virales.
PCT/CN2006/001891 2005-07-28 2006-07-28 Desoxynucleosides monocatenaires resistant aux infections virales Ceased WO2007012285A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CNA2005100855636A CN1904046A (zh) 2005-07-28 2005-07-28 抗人免疫缺陷病毒的单链脱氧核苷酸
CN200510085563.6 2005-07-28
CN200510085562.1 2005-07-28
CN2005100855621A CN1903870B (zh) 2005-07-28 2005-07-28 对病毒感染性疾病有治疗作用的单链脱氧核苷酸

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1235609A (zh) * 1996-10-30 1999-11-17 艾奥华大学研究基金会 免疫刺激性核酸分子
CN1271733A (zh) * 2000-04-04 2000-11-01 中国预防医学科学院病毒学研究所 对人免疫细胞有特异免疫刺激活性的CpG寡核酸
CN1513559A (zh) * 2003-08-06 2004-07-21 复旦大学 一种可增强基因枪接种dna疫苗诱生细胞免疫应答的方法
CN1526719A (zh) * 2003-03-05 2004-09-08 长春华普生物技术有限公司 增强蛋白类疫苗免疫效果的含CpG单链脱氧寡核苷酸
CN1526718A (zh) * 2003-03-05 2004-09-08 \ 抗病毒和抗肿瘤的含CpG单链脱氧寡核苷酸
CN1810970A (zh) * 2005-01-27 2006-08-02 长春华普生物技术有限公司 含CpG的单链脱氧核苷酸与其疫苗组合物及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1235609A (zh) * 1996-10-30 1999-11-17 艾奥华大学研究基金会 免疫刺激性核酸分子
CN1271733A (zh) * 2000-04-04 2000-11-01 中国预防医学科学院病毒学研究所 对人免疫细胞有特异免疫刺激活性的CpG寡核酸
CN1526719A (zh) * 2003-03-05 2004-09-08 长春华普生物技术有限公司 增强蛋白类疫苗免疫效果的含CpG单链脱氧寡核苷酸
CN1526718A (zh) * 2003-03-05 2004-09-08 \ 抗病毒和抗肿瘤的含CpG单链脱氧寡核苷酸
CN1513559A (zh) * 2003-08-06 2004-07-21 复旦大学 一种可增强基因枪接种dna疫苗诱生细胞免疫应答的方法
CN1810970A (zh) * 2005-01-27 2006-08-02 长春华普生物技术有限公司 含CpG的单链脱氧核苷酸与其疫苗组合物及其应用

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