WO1996003500A1 - Substance a activite antivirale - Google Patents

Substance a activite antivirale Download PDF

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Publication number
WO1996003500A1
WO1996003500A1 PCT/JP1995/001472 JP9501472W WO9603500A1 WO 1996003500 A1 WO1996003500 A1 WO 1996003500A1 JP 9501472 W JP9501472 W JP 9501472W WO 9603500 A1 WO9603500 A1 WO 9603500A1
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Prior art keywords
sequence
seq
type
compound
topology
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PCT/JP1995/001472
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English (en)
Japanese (ja)
Inventor
Yoko Shoji
Jingoro Shimada
Yutaka Mizushima
Wakao Iwatani
Nobuya Tamura
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Ltt Institute Co., Ltd.
Kaken Pharmaceutical Co., Ltd.
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Priority to AU29912/95A priority Critical patent/AU2991295A/en
Publication of WO1996003500A1 publication Critical patent/WO1996003500A1/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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1133—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against herpetoviridae, e.g. HSV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to a substance that suppresses the growth of a virus, and more particularly, to a modified oligodoxyribonucleotide having a virus growth-inhibiting effect.
  • nucleoside analogs acyclovir (hereinafter referred to as “Acv”), ganciclovir, and blobavir are known as chemotherapeutic agents for herpes virus infections.
  • Acv acyclovir
  • ganciclovir ganciclovir
  • blobavir chemotherapeutic agents for herpes virus infections.
  • These drugs undergo triphosphorylation in the virus-infected cells by the virus's thymidine kinase and the host cell's kinase, and the resulting phosphorylated form inhibits the viral DNA polymerase, thereby causing virus infection. Blocks DNA replication and suppresses virus growth.
  • the above-mentioned ACV is clinically widely used for the treatment of herpes virus infections.
  • Oligonucleotides (hereinafter referred to as "antisense oligonucleotides”) are known. Recently, as the number of immunodeficient patients due to human immunodeficiency virus (hereinafter referred to as “HIV”) infections and immunosuppression due to organ transplantation has increased, the health of humans as opportunistic infections has increased. Infection ⁇ is increasing. Furthermore, isolation of a strain of Herpesvirus that is resistant to ACV has also been reported.
  • HIV human immunodeficiency virus
  • WO 94/08053 discloses that an oligonucleotide containing one GGG G (G is a guanine-containing nucleotide or an analog thereof) sequence or two GGG sequences in its base sequence is a simple herpes virus. (Hereinafter referred to as “HSV”), which have been disclosed to have antiviral activity against viruses such as HIV. Further, as a preferred specific example, it is disclosed that a bond between nucleosides comprises a phosphorothioate bond. Purpose of the invention
  • the present invention has a chemical structure different from that of nucleoside analogs such as ACV, which is an anti-herpes virus agent currently found, and also has a different mechanism of action, not only HSV but also varicella's shingles virus, Providing a substance that exhibits high antiviral activity against viruses such as human cytomegalovirus, Ebsteinbar virus, human herpesvirus 16 and HIV, and a therapeutic agent for viral infections containing it as an active ingredient The purpose is to do. Disclosure of the invention
  • the present inventors have conducted intensive studies to find an antisense oligonucleotide that is more suitable as a drug from the viewpoints of solubility in water, transferability into cells, antiviral activity and sustained activity. When they were overlapped, they found that phosphorothioate oligodeoxyliponucleotides having a specific base sequence in their sequences, even if they were not antisense oligonucleotides, showed high antiviral activity, and the present invention was completed.
  • At least one nucleotide sequence : '' one GXGGG— '' (wherein G represents guanine and X represents any of adenine, thymine, guanine, and cytosine) is contained in the nucleotide sequence. And the bond between each nucleoside is
  • R 1 — ⁇ 1 P— 0— R2 ' (In the formula, R 1 Dokishiribosu residue 5 '- indicates the carbon atom, R 2 is Dokishiribo - scan residues 3 3 - represents a position of carbon.)
  • a first aspect is a phosphorothioate oligodeoxyribonucleotide having 5 or more bases comprising a phosphorothioate bond represented by or a salt thereof and a chemically modified product thereof.
  • the present invention relates to at least one of the phosphorothio oligodeoxyribonucleotides or the salts thereof and the chemically modified products thereof (hereinafter, referred to as “the compound of the present invention”) which is the first gist of the present invention as an active ingredient.
  • Antiviral agents, including seeds, are the second gist.
  • the third aspect of the present invention provides a method for treating a viral disease, which comprises administering the compound of the present invention.
  • FIG. 1 shows HSV-I (hereinafter referred to as "HSV-I") immediate early precursor mRNAs 4 and 5, and HSV-II (hereinafter referred to as "HSV-II").
  • FIG. 3 is a diagram showing the nucleotide sequence of the splicing ceptor region of the mid-early precursor mRNAs 5 and 4.
  • FIG. 2 is a view showing a melting curve of a duplex formed by a compound of the present invention and a deoxyoligonucleotide having a nucleotide sequence of the sceptor region of the immediate early precursor mRN A5 of HSV-; .
  • FIG. 3 is a graph showing the relationship between the concentration of the compound of the present invention and the amount of viable virus by the plaque method.
  • FIG. 4 is a graph showing the time-dependent changes in MIC values in the virus inoculation-compound simultaneous addition group (a) and the compound addition group after virus infection was established (b).
  • FIG. 5 is a photograph showing an electrophoresis pattern (A) of Compound 1 of the present invention and an electrophoresis pattern (B) of Compound 7.
  • the compounds of the present invention does not necessarily have a sequence complementary to mRNA from viruses, in its sequence 5 '- GXGGG- 3' (in the sequence, G and X are as defined above (Hereinafter referred to as “essential sequence”), the anti-viral activity is recognized. That is, it is clear from the test examples described below that the so-called antisense effect not only inhibits the translation stage of viral mRNAs.
  • FIG. 1 shows the nucleotide sequences of the splicing region of the immediate early precursor mRNAs 4 and 5 of HSV-I and the immediate early mRNAs 4 and 5 of HSV-II.
  • the HSV-II immediate early precursor mRNAs 4 and 5 do not have a site corresponding to the essential sequence of the compound of the present invention. "Shows virus growth inhibitory effect not only on 15-1 but also on HSV-II, and on HIV as well.
  • the compound of the present invention is composed of a phosphorothioate bond in which an atom bonded to a phosphorus atom of a phosphate involved in a bond between nucleosides is replaced with an oxygen atom to a sulfur atom. .
  • it is less susceptible to hydrolysis in vivo, and the persistence of antiviral activity is significantly increased.
  • the compound of the present invention does not show toxicity by the compound itself to host cells at a concentration (dose) within a range that can suppress virus growth, and is highly safe for living organisms.
  • the compound of the present invention exhibits a virus growth inhibitory effect even when administered at the beginning of or before virus infection, and even after infection, and is effective when administered at any time.
  • those having 1 to 4 essential sequences in their base sequence and having 5 to 30 bases, particularly 6 to 30 bases exhibit a high virus growth inhibitory effect.
  • those having a sequence complementary to the except region of splicing of immediate early precursor mRNA 4 or 5 of HSV-I have a particularly high virus growth inhibitory effect.
  • Such compounds of the present invention include:
  • the immediate early precursor mRNA4 of HSV-I which does not have a sequence complementary to the spider pig region of splicing 5, has a particularly high virus growth inhibitory effect.
  • Such compounds of the present invention include:
  • the compound of the present invention also shows excellent anti-HIV activity against HIV as compared with conventional antiviral agents.
  • those showing particularly excellent anti-HIV activity include:
  • T represents thymine
  • C represents cytosine
  • G represents guanine
  • A represents adenine.
  • the compound of the present invention can be produced by various methods, for example, the production by an automatic nucleic acid synthesizer by the phosphoramidite method shown in Example 1 (Production method 1), and the following methods (Production methods 2 and 3) can do.
  • phosphorothioate oligodeoxyribonucleotide (hereinafter, referred to as “phosphorothioate oligo DNA”), which is the desired compound of the present invention, was converted into a phosphoramidite using a nucleic acid synthesizer (AB 1394, manufactured by Applied Biosystems).
  • the synthesis was performed by the method [Aliquot Biosystems recommended method (Applied Biosystems DNA / RNA Synthesizers user's manual)].
  • the present synthesis method will be described in detail.
  • a guanine-SNAP ⁇ C PG column (manufactured by Applied Biosystems), which is a solid support in which deoxyguanosine has been introduced in advance, is treated with a trichloro mouth acetic acid-dichloromethane solution (3:97), and is treated with deoxyguanosine.
  • the dimethoxytrityl group which is the protecting group for the 5, 1-terminal hydroxyl group of guanosine, was removed.
  • the amide was reacted with the released 5'-terminal hydroxyl group at room temperature for 25 seconds.
  • a tetraethylthiuram disulfide'acetonitrile solution (15:85) was allowed to act on the resulting coupling reaction product at room temperature for 15 minutes to oxidize the phosphite triester with sulfur.
  • the 5′-trityl group which is a protecting group for the 5′-terminal hydroxyl group of the added second nucleotide, was removed by treatment with a trichloroacetic acid-dichloromethane solution (2:98).
  • chain extension was performed repeatedly by sequentially linking nucleoside phosphoramidites corresponding to the target base sequence in accordance with the above procedure.
  • the phosphorothionucleotide oligonucleotide was treated with 26% aqueous ammonia and separated from the CPG column and collected. Further, in order to remove the protecting group, the mixture is heated at 55 ° C. for 8 hours in the above-mentioned aqueous ammonia to obtain the target phosphorothioate oligo DNA of the present invention.
  • the 5'-terminal trityl group of the generated dinucleoside H-phosphonate is removed by treatment with dichloroacetic acid, and the generated 5'-terminal hydroxyl group and 3'-H-phosphonate.nucleoside are further reacted to form trinucleoside H-phosphonate. To form In this manner, the force-pulling reaction is repeated until the target sequence is completed. Finally, the H-phosphonate linkage is oxidized by sulfur in carbon disulfide and converted to a phosphorothioate linkage. After completion of the chain elongation reaction, the target phosphorothioate oligo DNA of the present invention is subjected to ammonia treatment from the solid phase. Can be recovered.
  • the compound of the present invention can also be synthesized according to the method of Stec et al., Nuc. Acids Res. 19: 5883-5888 (1991), the outline of which is as follows.
  • 5'-O-Dimethoxytritylated 3,10- (2_thio-1,3,2) oxoxathia-phosphorane nucleoside and 5-, 1-terminal hydroxyl group of nucleoside bound to solid phase via linker are coupled with acetonitrile in the presence of 1,8-diazobicyclo [5.4.0] indene 7-ene to form dinucleosides.
  • the 5'-terminal hydroxyl group of the unreacted carrier nucleotide that failed to elongate was inactivated by acetylation with acetic anhydride in the presence of 2,6-lutidine and 1-methylimidazole to inactivate the side reaction. To prevent.
  • the trityl group at the 5 'end of the resulting dinucleotide is removed by dichloroacetic acid treatment. Then, the resulting 5'-terminal hydroxyl group is reacted with 5,10-dimethoxytritylated 3,1o- (2-thio-1,3,2) -oxoxathia-monophosphorane nucleoside to form a trinucleoside. Is formed. Thus, the coupling reaction is repeated until the target sequence is completed. After completion of the chain elongation reaction, the target phosphorothioate oligo DNA of the present invention can be recovered from the solid phase by ammonia treatment.
  • the compound of the present invention can be used in the form of a salt.
  • a salt examples include salts of alkali metals such as sodium and potassium; salts of alkaline earth metals such as calcium and magnesium; Salts of basic amino acids such as lysine, arginine and histidine; and inorganic or organic salts such as salts of alkylamines such as triethylamine.
  • Pharmaceutical preparations of the compounds of the present invention as antiviral agents can be prepared by methods known in the art.
  • the dosage form of the compound of the present invention includes, for example, tablet administration, granules, capsules, powders and the like, or parenteral administration such as injections, drops, suppositories, ocular infusions, eye drops and the like.
  • the compound of the present invention may be subjected to further chemical modification in order to improve the translocation to a viral disease site, and may be subjected to various chemical modifications according to the disease situation.
  • the chemical modification include, for example, a lipid, an oligopeptide which is an organic functional group or a biological component having an affinity for a virus-infected cell at the 5, 1 or 3 'one end of the oligo DNA strand of the compound of the present invention, or a nucleic acid base moiety. , A protein or a sugar, and the like.
  • the compound of the present invention may be used alone as an active ingredient, or may be used in combination of two or more kinds. Further, the compound of the present invention may be used alone as an active ingredient, or may be used as a mixture with an agent effective for treating other viral diseases.
  • Example 1 The compound of the present invention may be used alone as an active ingredient, or may be used in combination of two or more kinds. Further, the compound of the present invention may be used alone as an active ingredient, or may be used as a mixture with an agent effective for treating other viral diseases.
  • Trivalent phosphoric acid produced by the coupling reaction was acidified by tetraethylthiuram disulphide and converted to a phosphorothioate bond.
  • the above reaction cycle is repeated according to the number of bases.
  • the oligomer is separated from the CPG column by treatment with 26% aqueous ammonia, collected and further removed at 55 ° C in the above-mentioned ammonia water to remove the protecting group. For 8 hours.
  • the crude 5′-triphosphoryl phosphorothioate oligonucleotide having a trityl group at the end was synthesized using a reversed-phase column (PRP-3, 150 X 4. Lmm l. D.
  • the product was purified by high performance liquid chromatography (Shimadzu Corporation, LC-1 OA D) equipped with Ruton Corporation. After injecting the product into the column, add 0.1 M triethylamine / acetate buffer (pH 7.0) containing 5-40% acetonitrile (1.4% increase in acetonitrile per minute (gradient)). Elution was performed at a flow rate of 1 ml / min, and a fraction showing oligonucleotide peaks was collected from the eluate ( purified 5, phosphorothioate oligonucleotide having a trityl group at one end was purified). The trityl group was cleaved by leaving the mixture in an 80% aqueous acetic acid solution for 20 minutes at room temperature, and the cleaved trityl group was removed by extraction with dimethyl ether.
  • Each of the compounds of the present invention synthesized and purified as described above was detected as one sharp peak when analyzed by high performance liquid chromatography, and was also detected by electrophoresis using a 17% polyacrylamide gel. It was observed as a band.
  • the analysis was performed by the following method.
  • polyacrylamide gel electrophoresis analysis a 17% polyacrylamide gel containing 7M urea was prepared according to the usual Sanger sequencing gel preparation method, and tris 'borate' EDTA buffer at 750 V. Electrophoresis was performed.
  • R 1 represents a carbon at the 5′-position of a deoxyribose residue
  • R 2 represents a carbon at the 3-position of a deoxyribose residue.
  • the HSV proliferation inhibitory effect of the compound of the present invention was examined using the cytopathic effect on host cells caused by virus (hereinafter referred to as “CPE”) as an index.
  • CPE cytopathic effect on host cells caused by virus
  • the strength of the virus CPE depends on the amount of virus growth. Therefore, the test compound
  • the CPE-suppressing effect of the product indicates the effect of suppressing the growth of the virus.
  • the viruses used in the test were giant cell-forming HSV-I, Miyama strain (hereinafter referred to as "GG strain") and non-giant cell-forming HSV-I, Miyama strain (hereinafter referred to as “KP strain”). And HSV-II, UW268 strain, and African green kidney cells (hereinafter referred to as “Vero cells”) were used as host cells.
  • MEM Eagle MEM
  • FCS non-mobilized fetal calf serum
  • NIPRO non-mobilized fetal calf serum
  • Nucleotide sequence of the compound of the present invention used in this test SEQ ID NO (Id. No.), number of nucleotides (mer), and submerging of immediate early precursor mRN A5 of HSV-I Table 1 shows whether the sequence is complementary to the sequence (Co .: “Primarily complementary,” N “indicates non-complementary). And the bond between the nucleosides is a phosphorothioate bond, also shown in Table 2, and the comparison compound in which the bond between the nucleosides is a phosphodiester bond is also shown in Table 3. Show.
  • test compound Various concentrations of the test compound were added to the prepared cell culture, and HSV was simultaneously inoculated.
  • ISIS 1080 SEQ ID NO: 66
  • ISIS 1082 SEQ ID NO: 67
  • ISIS 1080 SEQ ID NO: 66
  • ISIS 1082 SEQ ID NO: 67
  • ISIS 5320 SEQ ID NO: 208
  • Acyclovir ACV is a currently widely used treatment for HSV infection, which inhibits the viral DNA polymerase. Is a nucleoside analog that suppresses replication of viral DNA.
  • the amount of virus inoculated is the amount that will infect the virus in the absence of the drug, and after 48 hours of culture, the amount of virus CPE that can be reliably induced on the entire surface of the monolayer cells (100 TC ID
  • the virus growth inhibitory effect was expressed as the minimum inhibitory concentration (hereinafter referred to as “MIC”) that suppressed the virus CPE by 100% at the time of culture for 72 hours after virus inoculation.
  • Table 4 shows the results of this test for the compound of the present invention containing an essential sequence
  • Table 5 shows the results of this test for a comparative compound which does not contain an essential sequence and has a phosphorothioate bond.
  • Table 6 shows the results of this test, although the bond between the nucleosides was a phosphodiester bond.
  • Table 1 Compounds of the present invention (Part 1) o — Bti system ij—5 leu no. Mer # Co.
  • the compound of the present invention is extremely effective for HSV diseases caused by HSV-I and II.
  • the immediate early precursor mRNA 4 or 5 has a considerably different nucleotide sequence between HSV-I and II.
  • the compound of the present invention containing the sequence is considered to exhibit a virus growth inhibitory effect against all kinds of viruses.
  • the compounds 1, 13, 14, 17, 21, 25, 26, 31, 46, 47, and 47 of the present invention having a nucleotide sequence complementary to the immediate early precursor mRNA 4 or 5 of HSV-I 48, 49, 50, 51, 52, 55, 58 and 62 show an extremely high virus growth inhibitory effect of 4 to 32 times as high as ACV, an anti-herbal drug widely used in clinical practice. In comparison with ISIS 1080, 1082 and 5320, it showed 16-64 times better effect.
  • the compounds of the present invention 45, 53, 107, 119, 124, 125, 131, 134, 135, 144, 146, 182, 242, which do not have a nucleotide sequence complementary to the HSV-I immediate early precursor mRNA.
  • 243, 245, 246, 247, 252, 253, 254, 258, 260, 261, 262 and 263 also showed an excellent virus growth inhibitory effect as compared with the comparative compound.
  • Compound 7 (SEQ ID NO: 8), which is an oligo DNA having the same nucleotide sequence as that of Compound 1 of the present invention and whose nucleoside bond is composed of a phosphodiester bond, completely inhibits virus growth against any of the virus strains. No effect was shown.
  • the virus growth inhibitory effect of the compound of the present invention is affected by differences in host cell types. We examined whether or not it was possible.
  • Egret corneal cells (hereinafter referred to as "SIRC") were used as host cells, and HSV-I, GG strain was used as a virus.
  • test was performed in the same manner as in Test Example 2, except that the type of host cell was changed to SIRC.
  • Table 7 shows the results of the compounds of the present invention, and Table 8 shows the results of the comparative compounds.
  • the antiviral growth inhibitory effect of the compound of the present invention is hardly affected by the cell type. Therefore, it is presumed that the compound of the present invention exerts its effects at various viral disease sites in humans.
  • the virus growth inhibitory effect of the compound of the present invention was examined by a plaque assay for quantifying the amount of surviving virus.
  • test conditions were the same as in Test Example 2, except that various concentrations of the present compound were added to the host cells (Vero cells) and simultaneously the virus (HSV-I, GG strain, 9.5 x 10 PFU / well, 100 TC ID 5). ) was inoculated and cultured for 72 hours.
  • the culture solution containing the infected cells was once freeze-thawed and diluted appropriately. This diluted solution was added to fresh Vero cells that had been grown in a monolayer state in advance, and cultured for 2 hours to allow the virus to colonize.
  • the cells were overlaid with MEM containing 1% agar and cultured for another 72 hours. After culture for 72 hours, virus plaque formation was detected by trypan blue staining, and the amount of virus (number) was determined from the number of plaques.
  • Figure 3 shows the amount of virus detected from cell suspensions cultured for 72 hours after adding various concentrations of compounds.
  • test compound was added to the group to which the test compound was added simultaneously with the HSV-I and GG strain inoculations (simultaneous addition group), and to the Vero cells cultured for 2 hours after the infection was established, that is, 2 hours after the virus inoculation.
  • addition group after infection was established
  • the MIC value was measured every 24 hours after the start of culture up to 96 hours, and the fluctuation was examined.
  • Fig. 4-1a shows the results of the simultaneous addition group
  • Fig. 4-1b shows the results of the addition group after infection was established. From the graphs in FIGS. 4A and 4B, it can be seen that the effects of the present compounds 1, 13 and AVC on the effect of inhibiting the virus growth are affected by the timing of addition. In other words, for each compound, the post-infection addition group showed a higher MIC value than the simultaneous addition group. MIC values during the 72- and 96-hour incubation periods after the addition of the test compound increased to the same extent for each compound, but compounds 1 and 13 of the present invention were still approximately 8-fold higher than ACV It showed a virus growth inhibitory effect.
  • ACV increased the MIC value by a factor of 16 to 32 over time during the culture period from 24 hours to 72 hours after addition, whereas the compounds of the present invention 1 and 1 In the case of 3, the increase was only about 2 to 4 times 48 hours after the addition, and did not fluctuate thereafter.
  • the compound of the present invention exerts a potent virucidal activity at a lower concentration than ACV and during a short culture period, that is, in a short time. It is also suggested that the compounds of the present invention may act prophylactically, early or before viral infection, and therapeutically after viral infection.
  • the test is carried out by the following method.
  • comparative compound 7 was almost degraded by nuclease 1 hour after addition, and oligo DNA having less than 20 bases was detected (photograph in FIG. 5B).
  • Compound 1 of the present invention was hardly degraded by nuclease even 24 hours after the addition (FIG. 5-A).
  • Compound 1 of the present invention which is a phosphorothioate-type oligo DNA, is extremely stable against nuclease existing inside and outside of cells as compared with the phosphodiester-type oligo DNA.
  • HIV-infected MT-4 cells (2.5 ⁇ 10 / we 11, MOI: 0.01) were added immediately after the infection together with various concentrations of the test substance. 37 ° in C0 2 incubator (: After culturing 5 days, the MTT method to measure the number of viable cells.
  • the antiviral activity was represented by a concentration (EC: 50% effective concentration) that protects the cell damage by HIV infection by 50%.
  • Table 9 shows the test results of the compound of the present invention, and Table 10 shows the test results of the comparative compound.
  • the compounds of the present invention 1, 11, 1, 12, 13, 14, 17, 17, 18, 21, 23, 25, 26, 29, 30, 31, 45, 46, 47, 48, 49, 50, 51, 52, 53, 109, 124 and 145 are found to have particularly excellent anti-HIV activity. From the above results, the compounds of the present invention are effective for viral diseases caused by HSV-I, HSV-II and HIV.
  • the present invention relates to a compound having low toxicity to host cells, high safety to the living body, and a remarkable anti-virus growth effect irrespective of the administration time, and furthermore, prevention and treatment of viral infectious diseases containing the compound as an active ingredient.
  • An agent can be provided.
  • Organism name (ORGANISM): simple virus type I
  • Organism name (ORGANISM): simple herpes virus type I
  • Organism name (ORGANISM): simple herpes virus I type I
  • Organism name (ORGANISM): simple virus I type I
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between the nucleosides is a phosphodiester bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • Antisense (ANTI-SENSE): Yes Sequence features (FEATURE):
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • HYPOTHETICAL array No Antisense (ANTI-SENSE): Yes
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • Sequence type SEQUENCE TYPE: Nucleic acid Number of chains (STRANDENDNESS): single
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • This nucleotide sequence is complementary to the immediate early virus type I immediate early precursor mRNA4.
  • the bond between each nucleoside is a phosphorothioate bond.
  • This nucleotide sequence is complementary to the simple early virus type I immediate early precursor mRNA4.
  • the bond between each nucleoside is a phosphorothioate bond.
  • This nucleotide sequence is complementary to the simple early virus type I immediate early precursor mRNA4.
  • Sequence type SEQUENCE TYPE: Nucleic acid Number of chains (STRA DENDNESS): single strand
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • Sequence type SEQUENCE TYPE: Nucleic acid Number of chains (STRANDENDNESS): single
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.
  • Sequence length SEQUENCE LENGTH: 20 Sequence type (SEQUENCE TYPE): Nucleic acid
  • the bond between each nucleoside is a phosphorothioate bond.
  • the bond between each nucleoside is a phosphorothioate bond.

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Abstract

Oligodésoxyribonucléotide de phosphorothioate qui possède 5 à 30 unités de base et contient au moins une séquence de base ?5'-GXGGG-3'¿ (dans laquelle G représente guanine et X représente adénine, thymine, guanine ou cytosine) et dans lequel la liaison entre les nucléosides est une liaison phosphorothioate, sel dudit oligodésoxyribonucléotide et modifications chimiques de ce dernier, ainsi qu'un agent antiviral contenant cet oligodésoxyribonucléotide. Ledit composé possède une forte activité antivirale contre le virus de l'herpès simplex et le virus de l'immunodéficience humain (VIH) et est utile pour traiter ou prévenir diverses infections virales.
PCT/JP1995/001472 1994-07-26 1995-07-25 Substance a activite antivirale WO1996003500A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU29912/95A AU2991295A (en) 1994-07-26 1995-07-25 Sustance with antiviral activity

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP6/173862 1994-07-26
JP17386294 1994-07-26
JP6/268603 1994-11-01
JP26860394 1994-11-01

Publications (1)

Publication Number Publication Date
WO1996003500A1 true WO1996003500A1 (fr) 1996-02-08

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PCT/JP1995/001472 WO1996003500A1 (fr) 1994-07-26 1995-07-25 Substance a activite antivirale

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AU (1) AU2991295A (fr)
WO (1) WO1996003500A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11820985B2 (en) 2019-03-26 2023-11-21 University Of Massachusetts Modified oligonucleotides with increased stability
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US12024706B2 (en) 2019-08-09 2024-07-02 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
US12077755B2 (en) 2015-08-14 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01503302A (ja) * 1987-03-25 1989-11-09 アメリカ合衆国 レトロウイルスの複製および腫瘍遺伝子産生物の発現の阻害剤
JPH05503016A (ja) * 1990-02-26 1993-05-27 アイシス・ファーマシュティカルス・インコーポレーテッド ヘルペスウイルスの効果を変調するオリゴヌクレオチド治療法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01503302A (ja) * 1987-03-25 1989-11-09 アメリカ合衆国 レトロウイルスの複製および腫瘍遺伝子産生物の発現の阻害剤
JPH05503016A (ja) * 1990-02-26 1993-05-27 アイシス・ファーマシュティカルス・インコーポレーテッド ヘルペスウイルスの効果を変調するオリゴヌクレオチド治療法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANTIMICROB. AGENTS. CHEMOTHER., (1990), Vol. 34, No. 5, GAO W. et al., "Inhibition of Herpes Simplex Virus Type 2 Growth by Phosphorothioate Oligodeoxynucleotides", pages 808-812. *
ANTIMICROB. AGENTS. CHEMOTHER., (1992), Vol. 36, No. 3, CROOKE R.M. et al., "In Vitro Toxicological Evalucation of ISIS 1082, a Phosphorothioate Oligonucleotide Inhibitor of Herpes Simplex Virus", pages 527-532. *
ANTIVIRAL. RES., (1991), Vol. 16, No. 2, KMETZ M.E. et al., "Vmw65 Phosphorothioate Oligonucleotides Inhibit HSV KOS Replication and Vmw65 Protein Synthesis", pages 173-184. *
NUCLEIC. ACIDS. RES., (1988), Vol. 16, No. 8, STEIN C.A. et al., "Physicochemical Properties of Phosphorothioate Oligodeoxynucleotides", pages 3209-3221. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12077755B2 (en) 2015-08-14 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
US11820985B2 (en) 2019-03-26 2023-11-21 University Of Massachusetts Modified oligonucleotides with increased stability
US12024706B2 (en) 2019-08-09 2024-07-02 University Of Massachusetts Modified oligonucleotides targeting SNPs

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