WO2006108358A1 - UTILISATION ANTIVIRALE D’OLIGODESOXYNUCLEOTIDES ARTIFICIELS A UN SEUL BRIN CONTENANT UN CpG EN COMBINAISON AVEC LA RIBAVIRINE - Google Patents

UTILISATION ANTIVIRALE D’OLIGODESOXYNUCLEOTIDES ARTIFICIELS A UN SEUL BRIN CONTENANT UN CpG EN COMBINAISON AVEC LA RIBAVIRINE Download PDF

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WO2006108358A1
WO2006108358A1 PCT/CN2006/000667 CN2006000667W WO2006108358A1 WO 2006108358 A1 WO2006108358 A1 WO 2006108358A1 CN 2006000667 W CN2006000667 W CN 2006000667W WO 2006108358 A1 WO2006108358 A1 WO 2006108358A1
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virus
seq
stranded
ribavirin
deoxynucleotide
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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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    • 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
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    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
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    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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Definitions

  • the present invention relates to a synthetic CpG-containing single-stranded deoxynucleotide and ribavirin, in particular to a combination of artificially synthesized single-stranded deoxynucleotides and ribavirin, the artificial labor involved in the present invention
  • the combined single-stranded deoxynucleotide and ribavirin can be used in the treatment and prevention of diseases associated with viral infections and viral infections.
  • the viruses referred to in the present invention include, but are not limited to, influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, and papillomavirus.
  • the invention also provides sequences of synthetic single-stranded deoxynucleotides. Background of the invention
  • CpG ODN is a synthetic deoxyoligonucleotide single-stranded DNA containing one or more CpG dinucleotides, wherein CpG is a dinucleotide formed by cytosine and guanine linked by phosphate, and C represents a cell. Pyrimidine, G represents guanine, p represents phosphoric acid, and cytosine is located at the 5' end.
  • CpG ODNs can take many forms due to differences in sequences, especially on both sides of CpG. Some CpG ODNs have clear immunomodulatory effects and show good clinical value (Weiner GJ. The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides.
  • CpG ODN CpG ODN
  • Oligodeoxynucleotide for Activating Primate Immune Responses In Vitro and In Vivol The Journal of Immunology, 2000, 164: 1617-1624.
  • single-chain deoxynucleotides containing CpG are classified into three types: A-type single-stranded deoxynucleotides (CpG-A ODN), and B-type single-stranded deoxynucleotides (CpG-B ODN). And C-type single-stranded deoxynucleotides (CpG-C ODN).
  • the single-stranded deoxynucleotides that act to activate dendritic cells, natural killer cells, and stimulate dendritic cells to secrete interferon activity are classified as type A single-stranded deoxynucleotides.
  • Type A single-stranded deoxynucleotides are expected to be useful in the treatment of tumors, viral infectious diseases, and other diseases treated with interferon. The main manifestation is activation of B lymphocytes, and single-stranded deoxynucleotides that enhance humoral immune response activity are classified as type B single-stranded deoxynucleotides.
  • Type B single-chain deoxynucleotides are a class of molecular adjuvants that enhance vaccine immunity. Effect.
  • Single-stranded deoxynucleotides having both A-type single-stranded deoxynucleotides and B-type single-stranded deoxynucleotides active are classified as C-type single-stranded deoxynucleotides.
  • Influenza virus is the causative agent of human influenza. In 1918-1919, 20 million people worldwide died of the flu virus (Patterson, KD & Pyle, GF (1991) Bull. Hist. Med. 65, 4-212). Vaccination and the use of antiviral drugs are the two main methods of combating influenza viruses (Bridges, CB et al. (2001) Morbid. Mortal. Wkly. Rep. 50, 1-44), but it turns out that both methods The flu epidemic cannot be effectively controlled (Webby, RJ & Webster, RG (2001) Philos. Trans. R. Soc. London 356, 1817-1828).
  • Hepatitis C virus is the causative agent of hepatitis C and has infected more than 170 million people worldwide. Liver cancer and cirrhosis are two serious complications caused by HCV infection. Worldwide, the combination of alpha-interferon and ribavirin is a method of treating hepatitis C virus infection with an effective rate of approximately 40% [Jon Cohen. Science, 1999, 285: 26-30]. Hepatitis C virus is an enveloped single-stranded positive-strand RNA virus belonging to the Flaviviridae virus. The other two viruses of the Flaviviridae family are the dengue virus (Wang WK, et al. J Virol.
  • Dengue virus and Japanese encephalitis virus are also single-stranded RNA viruses. Since no cell and animal models of hepatitis C virus have been studied, the results obtained by experiments using dengue virus and Japanese encephalitis virus can represent the results obtained by cell experiments with hepatitis C virus to a considerable extent.
  • Foot-and-mouth disease is a potent infectious disease caused by foot-and-mouth disease virus. Pigs, cows, and sheep can be infected with foot-and-mouth disease virus and develop foot-and-mouth disease. In light, blisters, ulcers, and plaques appear in the mouth, tongue, lips, hooves, and breasts.
  • Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is a member of the genus Aphthovims of the family Picornaviridae. Seven serotypes, namely 0, A, C, have been discovered. Type (called European type), SAT1, SAT2, SAT3 (South African 1, 2, 3, also known as African) and Asianl (Asian I, Asian).
  • Sputum encephalitis is caused by the H encephalitis virus of the genus Flavivirus. Most people do not develop symptoms after infection with Japanese encephalitis virus. Most of the patients have mild clinical symptoms, such as fever, headache, nausea, burnout, abdominal pain or dysfunction and mental symptoms. In severe cases, sudden onset of high fever, headache, vomiting, followed by meningeal irritation, 3-5 days of paralysis, abnormal behavior, muscle rigidity, significant changes in consciousness, and even coma, death.
  • Hepatitis B virus is the causative agent of hepatitis B and is a DNA virus.
  • the complete hepatitis B virus particle is 42 nm in diameter and can be divided into two parts: the envelope and the core.
  • the protein on the envelope is called hepatitis B surface antigen (HBsAg), which is synthesized in hepatocytes and then released into the blood circulation, which is not contagious in itself.
  • the core contains cyclic double-stranded DNA, DNA polymerase, core antigen (HBcAg) and e antigen (HBeAg), which are the mainstay of viral replication and are infectious.
  • AIDS is a fatal infection caused by infection with human immunodeficiency virus (HIV).
  • HIV mainly destroys the body's immune system, causing the body to gradually lose its defense ability and not be able to resist various pathogens from the outside world.
  • HIV is an enveloped RNA retrovirus that is classified as a retroviridae. HIV is spherical or ovoid, 100-130 nm in diameter, consisting of two parts, the envelope and the core. Envelope proteins include the outer membrane glycoprotein (gpl20) and the transmembrane glycoprotein (gp41). The core is composed of nucleocapsid protein, two identical copies of nucleic acid genomic RNA, and enzymes.
  • the papillomavirus belongs to the genus Papovaviridae of the papavirus family and includes papavirus and human papillomavirus (HPV) of various animals.
  • HPV is a small DNA virus with a diameter of 45 ⁇ 55nm.
  • the capsid is icosahedral in stereo symmetry, containing 72 shell particles and no capsule.
  • the HPV genome is a closed-loop, double-stranded DNA with a molecular weight of 5 x 106 Daltons. According to the function, it can be divided into three areas: early zone (E zone), late zone (L zone) and non-coding zone (NCR).
  • E is divided into E1 ⁇ E7 ⁇ reading frames, which mainly encode proteins involved in viral replication, transcription, regulation and cell transformation.
  • L distinguishes between L1 and L2, encoding the major capsid protein and the minor capsid protein, respectively.
  • NCR is a DNA fragment of -6.4 to 1.0 bp in the E region and the L region, and is responsible for the regulation of transcription and replication. According to epidemiological survey data, the global infection rate of papillomavirus is about 9-13%, that is, about 630 million people are infected with papillomavirus every year. Papillomavirus is closely related to many skin and mucous membrane diseases.
  • papillomavirus infection is closely related to cervical cancer, penile cancer and anal cancer (L Koutsky. Am J Med, May 5, 1997; 102(5 A): 3-8).
  • herpes simplex virus type II and human papillomavirus are important biological factors leading to cervical cancer and the most common pathogens of reproductive system infection.
  • the incidence of cervical cancer in women infected with cervical simple sore type 2 virus is 8 times higher than that in healthy women.
  • Human papillomavirus is more likely to cause cervical cancer than herpes simplex type II virus. More than 100 types of human papillomavirus (HPV) have been identified (Brown DR. et al. J Clin icrohiol, 1999, 37(10): 3316-3322). Summary of the invention
  • One of the objects of the present invention is to provide a composition comprising a synthetic single-stranded deoxynucleotide and a ribavirin which is composed of one or more CpG-containing oligos
  • the nucleotide single-stranded DNA molecule has the following structural formula (i)-(v;).
  • the artificially synthesized CpG-containing single-stranded deoxynucleotide of the present invention has the following structural formula: (1) (G)n(L)n X 1 X 2 CGYiY 2 (M)n (G)n
  • the synthetic CpG-containing single-stranded deoxynucleotide of the present invention has the sequence shown below
  • CpG ODN 8 5 '-tcgtcgggtgcgacgtcgcagggggg-3 ' SEQ ID NO 102
  • CpG ODN 11 5 ' -tcgtgcgacgtcgcagatgat-3 ' SEQ ID NO 169
  • CpG ODN 12 5 ' -tcgtatgcatcgatgcatagggagg-3 ' SEQ ID NO 95
  • CpG ODN 17 5'-gtcgttttcgtcgacgaattgggggggg-3' SEQ ID NO 17
  • the artificially synthesized single-stranded deoxynucleotides of the present invention can be produced by a known method, for example, by a solid phase phosphoramidite triester method.
  • a method for producing the artificially synthesized CpG-containing single-stranded deoxyoligonucleotide of the present invention is exemplified in detail in the following examples.
  • One of the objects of the present invention is to provide a composition for treating or preventing a viral infection and a related disease caused by a viral infection, the composition comprising ribavirin and a synthetic CpG-containing single-stranded deoxynucleotide.
  • the artificially synthesized single-stranded deoxynucleotide is composed of an oligonucleotide single-stranded DNA molecule containing one or more CpGs, and has a sequence contained in one of the formulas (i) to (v).
  • the phosphodiester bond of a synthetic CpG-containing single-stranded deoxynucleotide is non-vulcanized, partially vulcanized or fully vulcanized, and can be chemically modified.
  • a second object of the present invention is to provide a method for the combined use of artificially synthesized CpG-containing single-stranded deoxynucleotides and ribavirin for treating or preventing diseases caused by viral infections and viral infections, and artificial synthetic
  • the phosphodiester bond of the single-stranded deoxynucleotide of CpG is non-vulcanized, partially vulcanized or fully vulcanized and can be chemically modified.
  • a third object of the present invention is to provide a method of treating a viral infection and a viral infection-associated disease comprising administering to a patient in need of such treatment a therapeutically effective amount of a human having a sequence of structural formula (i)-(v) Synthetic single-stranded deoxynucleotides and ribavirin. Both can be administered simultaneously, sequentially or in separate doses.
  • the virus refers to RA virus, DNA virus and retrovirus, including but not limited to influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus and nipple Tumor virus.
  • These synthetic single-stranded deoxynucleotides are administered at a single dose of 1-5000 micrograms in the prevention and treatment of diseases caused by viral infections and viral infections.
  • a single application dose of ribavirin is 300-10000 mg.
  • the application of synthetic single-stranded deoxynucleotides and ribavirin includes mucosal surfaces (including respiratory, digestive tract and genitourinary mucosa) applications, subcutaneous, intramuscular injection, gastrointestinal applications, abdominal applications, intravenous injections, etc. Way to apply.
  • a fourth object of the present invention is to provide a method of treating or preventing a viral infection and a related disease caused by a viral infection comprising administering to a patient in need of such treatment a therapeutically effective amount of the above composition.
  • a fifth object of the present invention is to provide use of the above composition for the preparation of a medicament for treating or preventing a viral infection and a disease associated with viral infection.
  • the phosphodiester bond of the artificially synthesized single-stranded deoxynucleotide in the present invention may be non-vulcanized, partially vulcanized, or fully vulcanized.
  • Synthetic single-stranded deoxynucleotides can be chemically modified. The chemical modifications described therein are well known to those of ordinary skill in the art.
  • the virus includes, but is not limited to, influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, and papillomavirus.
  • influenza virus foot-and-mouth disease virus
  • dengue virus Japanese encephalitis virus
  • hepatitis C virus hepatitis B virus
  • human immunodeficiency virus papillomavirus
  • the protecting group dimethoxytrityl (DMT) of the nucleotide attached to the controlled glass was removed by Trichloroacetic Acid (TC A) to obtain a free 5'. - a hydroxyl end for the next condensation reaction.
  • 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 5'-hydroxy group undergoes an affinity reaction, condenses and removes the tetrazole, and the synthesized oligonucleotide chain is extended by one base forward.
  • 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.
  • 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 sulfurized and partially sulfurized CpG single-stranded deoxyoligonucleotides was synthesized on an ABI 394 DNA synthesizer using a displacement method.
  • Example 2 Example 3, Example 4, Example 5
  • the artificially synthesized single-chain deoxynucleotide used in Example 6 was CpG ODN 1-20 described above.
  • EXAMPLES 2 Comparison of anti-follicular inflammatory virus effects of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides in combination with ribavirin
  • VSV follicular inflammatory virus
  • Equipment and materials low temperature refrigerator, carbon dioxide incubator, ultra clean bench, inverted microscope, liquid nitrogen tank, distilled water, vacuum pump, cell culture flask, pipette of various specifications, pipette, dropper, etc.
  • IMDM medium IMDM (GIBCOBRL) containing L-glutamine 10.4 g, 2.0 g of sodium bicarbonate, 100,000 units of gentamicin, plus three distilled water to a volume of 1000 ml.
  • the 0.22 micron filter vacuum pump was used for filtration and sterilization.
  • Vero E6 cells purchased from Shanghai Cell Institute
  • IMDM medium containing 10% calf serum (Invitrogen, treated at 56 C for 30 minutes).
  • VSV follicular inflammatory virus
  • TCID50 follicular inflammatory virus
  • Set no virus control After about 24-48 hours, all of the Vero E6 cells infected with VSV developed lesions, and the diseased cells and supernatant were collected and allowed to stand at 20 ° C overnight. The next morning, melt it, slam it with a straw, and centrifuge to collect the supernatant. After the TCID50 was measured, the supernatant was stored at 20 ° C for use.
  • IMDM medium containing 10% calf serum 10 ml of calf serum (Invitrogen, treated at 56 ° C for 30 minutes), 90 ml of IMDM medium.
  • Crystal violet dye solution Crystal violet 0.5 g, NaCl 0.85 g, dissolved in 50 ml of anhydrous ether. Add 3 ml of formaldehyde and 47 ml of distilled water.
  • Crystal violet decolorizing solution 50 ml of ethylene glycol monomethyl ether is mixed with 50 ml of distilled water.
  • the culture solution was aspirated, and 200 ⁇ l of 0.5% crystal violet dye solution was added to each well at 37 ° C for 15 minutes. The running water washes away the crystal violet dye solution. Add 200 ⁇ l of crystal violet decolorizing solution to each well, shake the oscillator to completely remove the dye from the cells, and use a spectrophotometer at 540 nm wavelength (Cytokines. A practical approach, second edition, edited by RR. BAL WILL. The practical approach series.). The reciprocal X 10 of the virus dilution with 50% cytopathic effect was the TCID50 I ml value for this viral solution.
  • 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.
  • IMDM medium Same as above.
  • PBMC peripheral blood mononuclear cells
  • CpGODN group, ribavirin group, CpG ODN + ribavirin group, IMDM control group were established. Vero E6 cells in good growth were seeded in 96-well plates at 1.3 X 10 4 cells per well, 37 ° C, 5% After 24 hours of C0 2 culture, the CpGODN group was supplemented with a 1:100 dilution of CpG ODN-stimulated human PBMC culture supernatant, and the ribavirin group was added with ribavirin (to a final concentration of 10 ⁇ mol/l), CpGODN + ribavi The forest group was supplemented with 1:100 diluted CpG ODN-stimulated human PBMC culture supernatant and ribavirin (to a final concentration of 10 ⁇ mol/1) (Zhejiang Chengyi Pharmaceutical Co., Ltd.), and the IMDM group only added the same amount of IMDM.
  • TCID50 I ml of VSV virus was added, and a normal VERO cell control group was set and cultured for 44 hours.
  • the degree of cytopathic effect was determined by staining with crystal violet. Aspirate the culture solution. Add 200 ⁇ l of 0.5% crystal violet stain to each well and incubate for 15 minutes at 37 °C. The running water washes away the crystal violet dye solution. 200 microliters of crystal violet decolorizing solution was added to each well, and the shaker was shaken to completely remove the dye from the cells, and the spectrophotometric value was measured by a spectrophotometer at a wavelength of 540 nm.
  • Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun.
  • Polysucrose - diatrizoate Specific gravity 1.077 ⁇ 0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd.
  • DMEM medium 1000 ml containing 100,000 units of gentamicin. The 0.22 micron filter was sterilized by filtration and dispensed.
  • Mononuclear cells of human peripheral blood were isolated using a sucrose-diatrizoate lymphocyte stratified solution. The volume ratio of the stratified fluid to the heparin anticoagulated peripheral blood is about 2:1. Centrifuge horizontally (1,000 X g, 20 min). Pipette the liquid containing the mononuclear cells with a pipette and place in a separate centrifuge tube. Add an equal volume of serum-free medium. 1,000 g Centrifugal for 15 min, discard the supernatant. Repeat the wash twice. Discard the supernatant, resuspend the cells in 2 ml of culture medium, and perform cell counting.
  • CpGODN group, ribavirin group, CpGODN+ribavirin group and DMEM control group were established.
  • concentration of VeroE6 cells in a well-growth state was adjusted to 3 ⁇ 10 5 /ml with 0.3% BSADMEM.
  • the cells were seeded in 96-well flat-bottomed plates at 1.3 ⁇ 10 4 cells per well, cultured at 37° C. and 5% CO 2 for 24 hours, and the CpG group was stimulated with a 1:100 dilution of synthetic single-stranded deoxynucleotides.
  • ribavirin group Human PBMC culture supernatant, ribavirin group was added to ribavirin (to a final concentration of 10 ⁇ 1/1), and CpG+ribavirin group was supplemented with 1:100 dilution of synthetic single-stranded deoxynucleotide-stimulated human PBMC.
  • the supernatant and ribavirin were cultured (to a final concentration of 10 ⁇ mol/1) (Zhejiang Chengyi Pharmaceutical Co., Ltd.), and only DMEM was added to the DMEM group.
  • BSA DMEM 4 wg/ml trypsin diluted influenza virus solution (Changchun Institute of Biological Products) was added to each well 200 ⁇ l, and a normal VERO cell control was set.
  • the cells were cultured in a 37 ° C, 5% CO 2 incubator for 44 hours, and the degree of cytopathic effect was determined by crystal violet staining. Aspirate the culture solution. Add 200 ⁇ l of 0.5% crystal violet dye solution to each well at 37 ° C for 15 minutes. The running water washes away the crystal violet dye solution. 200 microliters of crystal violet decolorizing solution was added to each well, and the shaker was shaken to completely remove the dye from the cells, and the spectrophotometric value was measured by a spectrophotometer at a wavelength of 540 nM.
  • mice Normal mouse control, model control group (negative control group), ribavirin group, CpGODN group, ribavirin plus CpG ODN group were established.
  • the CpGODN group was injected subcutaneously with artificially synthesized single-stranded deoxynucleotide 80Pg (CpG ODN 1-20).
  • the influenza virus was diluted to 0.3 LD 5 Q with 0.3% BSA DMEM, except for the normal mouse control group.
  • other groups of mice were anesthetized with ether, nasally given 20 ⁇ 1 of 10 LD 5Q flu virus.
  • mice in the ribavirin group were administered by gavage, 50 mg ribavirin/kg body weight, 0.2-0.4 mI per mouse, 3 times daily.
  • 80 wg CpG ODN was administered to mice in the CpGODN group by subcutaneous injection, once every two days.
  • Ribavirin plus CpGODN group, ribavirin and CpGODN were administered at the same dose and administration as the ribavirin group and the CpGODN group.
  • the mice were dissected, the lungs were taken, weighed, and the lung lesion index was determined. Pulmonary Lesion Index (Biochemical and Biophysical Research Communications 279, 158-161 (2000). Inhibitory Effects of an Antisense Oligonucleotide in an Experimentally Infected Mouse Model of Influenza A Virus.)
  • Foot-and-mouth disease virus type 0 is provided by Inner Mongolia Biopharmaceutical Factory, and anti-virus experiment is carried out at Inner Mongolia Bio-Pharmaceutical Factory.
  • Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun.
  • Polysucrose - diatrizoate specific weight 1.077 ⁇ 0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd.
  • IMDM medium 1000 ml containing 100,000 units of gentamicin. The 0.22 micron filter was sterilized by filtration and dispensed.
  • Mediator IMDM culture liquid containing 10% fetal bovine peripheral blood mononuclear cells at a final concentration 3x l0 6 cells / ml. Add this cell suspension to a 12-well culture plate at 2 ml per well. Add synthetic single-stranded deoxynucleotides to a final concentration of 6 ⁇ ⁇ / ⁇ 1. The 3TC, 5 % carbon dioxide incubator was cultured for 48 hours, and the supernatant was collected and tested for activity against foot-and-mouth disease virus.
  • CpGODN group, ribavirin group, CpG ODN + ribavirin group, IMDM control group were established.
  • concentration of BHK cells with good growth was adjusted to 3 x 105/ml with 5% fetal bovine serum IMDM.
  • the cells were seeded in a 96-well flat-bottomed plate, 1.3 ⁇ 10 4 cells per well, cultured at 37 ° C, 5% CO 2 for 24 hours, and the CpGODN group was spiked with a 1:100 dilution of synthetic single-stranded deoxynucleotide.
  • the final concentration of human peripheral blood mononuclear cells was adjusted to 3 x 106/ml with IMDM medium containing 10% calf serum.
  • the cell suspension was added to a 12-well culture plate, and 2 mL per well was added with synthetic single-stranded deoxynucleotides to a final concentration of 6 g/ml. Incubate for 48 hours at 37 ° C, 5 % CO 2 incubator, and collect the supernatant.
  • CpGODN group, ribavirin group, CpG ODN + ribavirin group, IMDM control group were established.
  • BHK cells Inner Mongolia Biopharmaceutical Factory
  • CpGODN group was diluted 1:100.
  • CpG ODN stimulated human PBMC culture supernatant ribavirin group added ribavirin (to a final concentration of 10 ⁇ ⁇ )
  • CpGODN + ribavirin group added 1: 100 dilution of synthetic single-stranded deoxynucleotide Stimulated human PBMC culture supernatant and ribavirin (Zhejiang Chengyi Pharmaceutical Co., Ltd.)
  • IMDM group only added the same amount of IMDM.
  • 100 TCID50 / ml of Japanese encephalitis virus was added, and a normal BHK cell control was set and cultured for 44 hours. Under the microscope, the cytopathic effect (CPE) was observed.
  • CPE cytopathic effect
  • the cytopathic effect of the ODN and ribavirin combination group was significantly lower than that of the CpG ODN group. This result indicates that the synthetic single-stranded deoxynucleotide has a significant anti-Japanese encephalitis virus effect, and the synthetic single-chain deoxygenation. The combination of nucleotides and ribavirin has a more pronounced anti-Japanese encephalitis virus effect. A comparison of cytopathic effects between the two groups is shown in Table 5.
  • the above experimental results show that the artificial single-stranded deoxynucleotide of the present invention has an obvious anti-Japanese encephalitis virus effect, and the synthetic single-stranded deoxynucleotide and ribavirin have a more significant anti-drug application.
  • the role of Japanese encephalitis virus Hepatitis C virus is the same as a single-stranded positive-strand RNA virus like dengue virus and Japanese encephalitis virus. Therefore, synthetic single-stranded deoxynucleotides can also be used in combination with ribavirin.
  • the role of anti-hepatitis C virus Schedule section

Abstract

La présente invention procure une composition contenant de la Ribavirine et des oligodésoxynucléotides artificiels à un seul brin CpG ou une trousse qui peut empêcher ou traiter une infection virale et des maladies infectieuses provoquées par un virus, et leur utilisation. L’invention procure aussi un procédé pour la prévention ou le traitement d’une infection virale et de maladies infectieuses causées par un virus.
PCT/CN2006/000667 2005-04-13 2006-04-13 UTILISATION ANTIVIRALE D’OLIGODESOXYNUCLEOTIDES ARTIFICIELS A UN SEUL BRIN CONTENANT UN CpG EN COMBINAISON AVEC LA RIBAVIRINE WO2006108358A1 (fr)

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WO2009069447A1 (fr) * 2007-11-28 2009-06-04 Toray Industries, Inc. Adjuvant pour vaccin contre la grippe et vaccin contre la grippe
WO2009069682A1 (fr) * 2007-11-28 2009-06-04 Toray Industries, Inc. Agent thérapeutique ou agent préventif de l'hépatite
US7615539B2 (en) * 2003-09-25 2009-11-10 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
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US7998492B2 (en) 2002-10-29 2011-08-16 Coley Pharmaceutical Group, Inc. Methods and products related to treatment and prevention of hepatitis C virus infection
US7615539B2 (en) * 2003-09-25 2009-11-10 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
WO2009069448A1 (fr) * 2007-11-28 2009-06-04 Toray Industries, Inc. Adjuvant pour vaccin contre encéphalite b japonaise et vaccin contre l'encéphalite b japonaise
WO2009069447A1 (fr) * 2007-11-28 2009-06-04 Toray Industries, Inc. Adjuvant pour vaccin contre la grippe et vaccin contre la grippe
WO2009069682A1 (fr) * 2007-11-28 2009-06-04 Toray Industries, Inc. Agent thérapeutique ou agent préventif de l'hépatite
JP5359883B2 (ja) * 2007-11-28 2013-12-04 東レ株式会社 肝炎の治療剤又は予防剤

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