WO2002068582A2 - Oligonucleotides a phosphorothioate (p=s) renfermant des nucleotides modifies par des azasucres hexagonaux et leur utilisation dans une therapie contre le sida - Google Patents

Oligonucleotides a phosphorothioate (p=s) renfermant des nucleotides modifies par des azasucres hexagonaux et leur utilisation dans une therapie contre le sida Download PDF

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WO2002068582A2
WO2002068582A2 PCT/KR2002/000325 KR0200325W WO02068582A2 WO 2002068582 A2 WO2002068582 A2 WO 2002068582A2 KR 0200325 W KR0200325 W KR 0200325W WO 02068582 A2 WO02068582 A2 WO 02068582A2
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oligonucleotides
oligonucleotide
seq
hiv
present
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PCT/KR2002/000325
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WO2002068582A3 (fr
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Yong-Soo Bae
Dong-Sung Lee
Hong Lim
Sun-Ok Kim
Kwangjun Lee
Kyeong-Eun Jung
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Dongbu Hannong Chemical Co., Ltd.
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Publication of WO2002068582A2 publication Critical patent/WO2002068582A2/fr
Publication of WO2002068582A3 publication Critical patent/WO2002068582A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12N15/1132—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 retroviridae, e.g. HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • Oligonucleotides of the present invention have low toxicity against cells and are working outside of cells, resulting in inhibition of HIV-1 replication against a wide spectrum of HIV-1 variants by blocking the viral attachment. Therefore, oligonucleotides of the present invention can be used as one of effective AIDS therapeutic agents.
  • B is a wild-type or a modified nucleobase with or without a protecting group
  • X is hydrogen, hydroxyl protecting group, conjugate group or oligonucleotide
  • Y is hydrogen, phosphate, activated phosphate, activated phosphite, solid support, conjugate group or oligonucleotide .
  • the AIDS (acquired immunodeficiency syndrome) patient was first reported in Los Angeles, USA in 1982. Since then, 34,300,000 HIV carriers (Global summary of the HIV/AIDS epidemic, end 1999) have been reported to WHO by the end of 1999. In Korea, since the first American AIDS patient was reported in 1985, the numbers of patients have been steadily increased, with an estimation of 1,173 HIV carriers as of June, 2000 (included 186 AIDS patients, Communicable Disease Monthly Report, July issue of 2000. Korean NIH), which caused serious debate on AIDS therapy. Because of the rapidly growing number of HIV carriers and AIDS patients, USA, France, Canada, Japan and other countries have studied to develop AIDS therapeutic agents.
  • HIV belongs to the Retrovirus Family, which keeps genetic information as a form of RNA. Concerning the structure of the virus particle, viral RNA genome and reverse transcriptase are encapsulated by capsid protein, and lipid membrane is covering the nucleocapsid particle. Glycoproteins, gpl20 and gp41, are located in the lipid membrane and especially, gpl20 is necessary for virus attachment to T-cells and penetration into thereafter. For the treatment of AIDS, various types of therapeutic agents have been tried but not yet been fully successful since HIV virus has the most complicated replication mechanism. Reverse transcriptase inhibitor is known to be the most effective one so far. Reverse transcriptase is essentially required for synthesis of provirus DNA from the viral RNA genome.
  • reverse transcriptase inhibitors have been studied to disrupt HIV-1 replication.
  • Azidothymidine (Glaxo-Wellcome) , 2' , 3' -dideoxyinosine (Bristol Myers-Squibb) , 2' , 3' -dideoxycytidine (Hoffmann-La Roche) , D4T (Bristol Myers-Squibb) and 3TC (Glaxo-Wellcome) have been developed and clinically used as reverse transcriptase inhibitors .
  • those compounds are efficient to inhibit the HIV-1 replication in vitro, they are not so effective in vivo as shown in vitro, resulting in rather prolonging survival time of patients than curing the disease completely. Those compounds also cause serious side effects such as degeneration of platelets and blood stem cells. Besides, many variants showing resistance to those compounds have been pointed out to be another problem.
  • HIV protease inhibitor In order to overcome those problems, another crucial therapeutic agent, HIV protease inhibitor, has been developed. HIV does not synthesize capsid protein and enzyme, respectively, from mRNA, but produce long polyprotein first, such as gag-protein (p55) or gag-pol protein (pl65) , in which capsid proteins and enzymes are all integrated. The capsid proteins, reverse transcriptase and integrase are cleaved by HIV-1 protease residing in the long polyprotein. Thus, protease inhibitor has been developed on the basis of the idea that the HIV-1 replication will be quenched if viral protease responsible for polyprotein processing is inhibited.
  • HIV protease consists of two identical monomers (homodimer) each of which has 99 amino acids (molecular weight of each monomer is 10,793 daltons) . HIV protease is a typical aspartic acid protease characterized by the sequence of aspartate-threonine- glycine on an active site.
  • HIV protease inhibitors As demonstrated in the process of developing inhibitors (especially lenine) against other aspartic acid proteases, HIV protease inhibitors has been developed from selecting the compounds showing high affinity to the viral protease by simulating the transition state of enzyme to form the transition state analogue (TSA) . HIV protease inhibitors developed on the basis of that concept have been reported (Robert, et al., Science, 248, 358, 1990;
  • antisense oligonucleotides which can be hybridized with messenger RNA specifically.
  • messenger RNA For protein synthesis, only one strand of DNA double helix is transcribed into mRNA, which is later translated into protein in the cytoplasm.
  • the antisense oligonucleotides are targeting mRNA to block the translation of mRNA into proteins.
  • the mechanism of antisense oligonucleotides is to cut off the target mRNA or disrupt the ribosome-mediated translation of mRNA into protein, resulting in the lack of the target protein synthesis.
  • oligonucleotides are complementary to the target mRNA sequence, so that the oligonucleotide is named by antisense oligonucleotide and the technique is called antisense technology.
  • Some of the antisense agents of first generation are in the midst of clinical test as anti-viral or anti-cancer agents, and some have already been into the market as anti-viral agents (Bennett, et al . , Biochem . Pharmacol .
  • second-generation antisense oligonucleotides have a modified sugar. Oligonucleotides having methoxy, methoxyethoxy (Martin, et al . , Helv. Chim . Acta . , 186, 584, 1995) or aminoalcoxy group (Griffey, et al., J. Med. Chem . , 39, 5100-5109, 1996) introduced to 2' of ribose, oligonucleotides containing hexose (Herdewijn, et al .
  • oligonucleotides containing 4'- thioribose (Bellon, et al . , 4-Thio RNA: a novel class of sugar-modified B-RNA.
  • 4-Thio RNA a novel class of sugar-modified B-RNA.
  • UV absorbance of a single strand is higher than that of duplex, that is to say, if the amount of single strand increases by temperature rise, UV absorbance also increases resulting in sigmoid changing curve.
  • the temperature giving middle level of UV absorbance in the sigmoid curve is defined as a Tm (melting temperature) .
  • High Tm to mRNA means high affinity (binding capacity) of oligonucleotides to RNA, which is a very important factor as an effective antisense molecule.
  • oligonucleotides with the modified base having methoxy group at 2 ' -site of ribose showed rather high Tm to mRNA, which was likely due to that the introduced electronegative groups such as methoxy and fluorine increased the affinity to RNA (Kawasaki, et al., J. med. Chem . , 36, 831-841, 1993). And also oligonucleotides having alcoxy group such as methoxy at 2' -site showed increased resistance against nucleases comparing to natural DNA.
  • the present inventors have studied to develop antisense oligonucleotides which are more stable • and resistant against nucleases and also have higher affinity to mRNA. As a result, it has been developed and confirmed that newly designed oligonucleotides containing 6-membered azasugars instead of 5-membered ribose have greater affinity and stability, which were filed for a patent (Korea patent application #99-26947).
  • It is an object of this invention to provide oligonucleotides with P S backbone, which contain nucleotide derivatives whose five-membered ribose, a natural nucleotide sugar was substituted with six- membered azasugar.
  • FIG. 1 is photographs observed by light microscopy showing the antiviral activity of oligonucleotides of the present invention against HIV-1 replication. They are showing the cytopathic effect observed on 7 th day after virus infection, A: Control,
  • FIG. 2 is graphs showing the antiviral activity of oligonucleotides of the present invention against HIV-1 replication with varying concentrations in a serum-free medium.
  • FIG. 3 is a graph showing the antiviral effect of oligonucleotides of the SEQ. ID. NO: 2 and 22 of the present invention, on HIV-1 replication in serum-free Opti-MEM medium by measuring the reverse-transcriptase activity of HIV-1.
  • FIG. 4 is a graph showing the antiviral effect of oligonucleotides of the present invention on HIV-1 replication in RPMI 1640 medium supplemented with 10% fetal bovine serum by measuring the reverse- transcriptase activity of HIV-1.
  • FIG. 5 is photographs showing the short-term
  • oligonucleotides of the present invention against nuclease degradation in the supernatant of cell culture medium.
  • A Oligonucleotide of the SEQ. ID. NO: 2
  • B Oligonucleotide of the SEQ. ID. NO: 4
  • C Oligonucleotide of the SEQ. ID. NO: 5
  • D Oligonucleotide of the SEQ. ID. NO: 13
  • FIG. 6 is photographs showing the long-term stability of oligonucleotides of the present invention against nuclease degradation in the fresh RPMI 1640 medium supplemented with 10% fetal bovine serum.
  • FIG. 7 is photographs of the experimental results showing whether the oligonucleotides of the SEQ. ID. NO: 2, 4 and 22 of the present invention are able to inhibit HIV-1 LTR activity in Magi cells (HeLa-CD4 cells containing LTR- ⁇ -gal gene) when they are simply added in the culture medium or when they are transfected into the cells.
  • Magi cells HeLa-CD4 cells containing LTR- ⁇ -gal gene
  • FIG. 8 is the graph showing the inhibition capacity of oligonucleotides of the present invention against the HIV-1 LTR activity when they were transfected into the cells in different concentrations.
  • FIG. 9 is a photograph showing the result of CAT assay in the LTR-CAT-transfected Jurkat-tat cells, which was performed to see whether the oligonucleotides of the SEQ. ID. NO: 2 and 4 were able to pass through cell membrane and inhibit the LTR function in nuclei when they are simply added into the culture medium.
  • Control (cells were transfected with 5 ⁇ g of LTR- CAT without further treatment of oligonucleotide) ,
  • FIG. 10 is a photograph showing the result of CAT assay, which was performed to see if the oligonucleotides of the present invention have sequence-specific anti-viral activity against HIV-1 LTR within the Jurkat-tat cells when they were co- transfected into the cells with LTR-CAT plasmid DNA,
  • Control cells transfected with 5 ⁇ g of LTR-CAT
  • B cells co-transfected with 5 ⁇ g of LTR-CAT and 2 ⁇ g of oligonucleotide SEQ. ID. NO: 2
  • E cells co-transfected with 5 ⁇ g of LTR-CAT and 2 ⁇ g of oligonucleotide SEQ. ID. NO: 11,
  • F cells co-transfected with 5 ⁇ g of LTR-CAT and 2 ⁇ g of oligonucleotide SEQ. ID. NO: 12,
  • G cells co-transfected with 5 ⁇ g of LTR-CAT and 2 ⁇ g of oligonubleotide SEQ. ID. NO: 13
  • FIG. 11 is photographs showing the curing effects of the oligonucleotides of the present invention on the
  • HIV-1-infected Jurkat cells when large amounts of syncytia (giant cells especially appeared in HIV-1 infected cells) were already produced,
  • FIG. 12 is a graph showing the effect of the oligonucleotides of the present invention on the SIV replication
  • FIG. 13 is a graph showing the effect of the oligonucleotides of the present invention on the poliovirus Sabin 1 proliferation.
  • the present invention also provides a therapeutic agent used for the treatment of AIDS containing the above oligonucleotides.
  • n 0 ⁇ 30 , 1
  • B is a .wild-type or a modified nucleobase with or without a protecting group
  • X is hydrogen, hydroxyl protecting group, conjugate group or oligonucleotide
  • Y is hydrogen, phosphate, activated phosphate, activated phosphite, solid support, conjugate group or oligonucleotide .
  • n is 0 ⁇ 30 including nucleotides of both up and down position in the above ⁇ Chemical Formula 1>, and 6 ⁇ 21 is more preferable.
  • the modified monomer of the present invention could be located in any position of oligonucleotides, and oligonucleotides having more than 4 modified monomers are preferable for the use in the treatment of AIDS. Oligonucleotides of the SEQ. ID. NO: 1 ⁇ 29 are preferable for the treatment of AIDS and further those of the SEQ. ID. NO: 2, 3, 11, 17, 18, 19, 20, 21, 22, 23 and 29 are more preferable.
  • Oligonucleotides of the present invention can be prepared in the condition of both liquid and solid phase, but the later is more preferable.
  • the detailed synthetic method of oligonucleotides in the solid phase condition is described in Oligonucleotide Synthesis, A Practical Approach, Gait (ed.), IRL Press, Washington D. C. (1984), Caruthers, et al . , U. S. Pat. No. 4,458,066 and 4,500,707.
  • Oligonucleotides of the present invention have been prepared by condensation reaction using nucleotide monomer of the ⁇ Chemical Formula 2> (Korea patent application #99-26947) shown below.
  • the primary alcohol group bound to nucleotide sugar should be replaced by dimethoxytrityl group and so does the secondary alcohol group by phosphoamidite group. Beside of thymine, nucleobases should be protected by the proper protecting group as well.
  • B is a natural nucleobase or modified nucleobase with or without a protecting group
  • X is hydrogen or hydroxyl protecting group
  • Y is hydrogen, phosphate, activated phosphate, activated phosphite or solid support.
  • nucleotide monomer of the ⁇ Chemical Formula 2> shown above is introduced to the 3' -site of oligonucleotides, this derivative needs to be attached to solid support.
  • This monomer was converted to hemiccinate by using the commercially available CPG
  • the monomer of ⁇ Chemical Formula 2> was introduced to any position except 3' -end of oligonucleotides by standard phosphoamidite method using DNA synthesizer (ex. ABI 392 etc.) .
  • concentration of monomer and its reaction timing with solid support were identical with those in phosphoamidite method using DNA synthesizer, but it was preferable to prolong the condensation time from 60 seconds to 600 seconds when hydrogen was replaced with other functional groups at 2'-site.
  • oligonucleotides should be separated from solid support and protecting group also should be removed. These procedures could be performed simultaneously or one by one.
  • the synthesis of oligonucleotides are complete, they are released from solid support by treating ammonia water at room temperature, and the protecting group is removed by using ammonia water while being heated (generally at 55 ° C for 17 hours) .
  • Dimethoxytrityl group a protecting group for 5 '-hydroxyl group of oligonucleotides is removed at the last step of synthesis using the program installed in DNA synthesizer as well as by an additional treatment with 80% acetic acid, dichloroacetic acid or trichloroacetic acid.
  • isobutyl group is used as a protecting group for the synthesis of oligonucleotides, and then removed by treatment with ammonia solution as used in general DNA synthesis protocol.
  • Monomer having hydrogen at the nitrogen site of azarsugar is protected by fluorenyl group (F-moc) , and is also separated from the protecting group after synthesis of oligonucleotides as described in the general protocols for separation of protecting group.
  • the preparation method of the oligonucleotides of the present invention with OF backbone, which contain nucleotide derivative having the structure of ⁇ Chemical Formula 1> was simplified by attaching nucleobase to 3 ' -carbon site of 6-membered azasugar (piperidine) .
  • nucleobases could be introduced to both alpha and beta position, which required an additional step for separation of isomers .
  • the present inventors introduced a base to carbon site instead of aminal site, which resulted in nucleotides having a base only at beta position.
  • many functional groups were, easily introduced to nitrogen position using azasugar instead of the tedious method of using strong base. Since the oligonucleotides having various groups introduced to nitrogen position show relatively high Tm against RNA, they can be effectively used as antisense agents having strong affinity to mRNA.
  • the oligonucleotides of the present invention can improve the cell membrane permeability.
  • Oligonucleotides containing carbocyclic nucleotides are known to be stable with strong resistance against enzymes and actually those of the present invention were confirmed to be highly resistant against nucleases.
  • the present invention also provides a pharmaceutical composition used for the treatment AIDS containing oligonucleotides of the present invention as an effective ingredient.
  • oligonucleotides of the present invention are superior to AZT (conventional AIDS therapeutic agent) showing long- lasting anti-viral activity for 12 days after a single treatment when tested in the same concentration (see Table 2 and 3) .
  • Anti-HIV-1 activity of the oligonucleotides of the present invention was also examined by measuring reverse transcriptase activity in the HIV-1 infected cells cultured in their presence. It was confirmed therefrom that virus replication was completely inhibited at 0.5uM of oligonucleotides regardless of the serum presence in medium (see Fig. 3 and 4) .
  • oligonucleotides of the present invention were added into a culture supernatant (see Fig. 5) or a fresh medium (see Fig. 6) for an indicated period of time, and then harvested and examined for their integrity on polyacrylamide gel electrophoresis .
  • oligonucleotides of the present invention kept their stability up to 15 days without being decomposed in the serum-containing fresh medium (see Fig. 6) .
  • oligonucleotides of the present invention are able to pass through cell membrane and have anti-viral activity within the cells
  • the expression of the HIV-1 LTR-mediated reporter gene was studied in cell culture in the presence of each oligonuceleotide .
  • cells containing genes for LTR-CAT or LTR- ⁇ -galactosidase, as a reporter system were used for these experiments.
  • Magi cells having LTR- ⁇ -galactosidase were treated or transfected with oligonucleotides of the present invention in order to examine their membrane permeability by measuring the inhibition level of HIV-1 LTR-mediated reporter expression.
  • the number of cells expressing LTR-mediated ⁇ -galactosidase was not reduced when the cells were only treated with oligonucleotides of the present invention in the culture medium (see Fig. 7A) .
  • oligonucleotides still showed potent anti-viral activity regardless of membrane permeability and sequence-specificity, which led us to assume that they might be working outside of the cells probably by blocking viral attachment, resulting in effective shut off of the HIV-1 infection.
  • oligonucleotides of the present invention were added to HIV-infected cells which showed progressed cytopathy (lots of syncytia were observed) , and then the change of cytopathy was observed.
  • the number and the size of syncytia were remarkably reduced in the presence of oligonucleotides of the present invention in the culture.
  • oligonucleotides of the present invention affect the replication of other viruses than HIV-1 by blocking nonspecific cell surface molecules
  • SIV and poliovirus were tested for their replication in the presence of these oligonucleotides.
  • Oligonucleotides of the present invention did not inhibit SIV proliferation regardless of the sequence- specificity to SIV TAR (see Fig. 12).
  • poliovirus replication was also undisturbed by any oligonucleotides of the present invention having complementary to IRES (internal ribosomal entry site) or random sequence (see Fig. 13) .
  • oligonucleotides of the present invention Serum effects on anti-viral activity of the oligonucleotides of the present invention were tested. Oligonucleotides of the present invention were added into media containing different kinds and different concentrations of sera, and the syncytium formation was observed therefrom. Many of the oligonucleotides of the present invention showed rather reduced anti-viral activity in the presence of serum. However, anti-viral activity of a specific oligonucleotide (represented by the SEQ. ID. NO: 22) was not clearly disturbed by serum addition (see Table 5) .
  • Oligonucleotides of the present invention can be administered orally or parenterally .
  • the compounds of the present invention can be prepared for oral or parenteral administration by mixing with generally-used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients.
  • the present invention also includes pharmaceutical formulations in dosage units. This means that the formulations are presented in the form of individual parts, for example tablets, coated tablets, capsules, pills, suppositories and ampules, the active compound content of which corresponds to a fraction or a multiple of an individual dose.
  • the dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
  • An individual dose preferably contains certain amount of active compound, which is administered in one application and which usually corresponds to a whole, one half, one third, or a quarter of a daily dose.
  • Non-toxic inert pharmaceutically suitable excipients are to be understood as solid, semi-solid or liquid diluents, fillers and formulation auxiliaries of all types .
  • Preferred pharmaceutical formulations which may be mentioned are tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, dusting powders and sprays .
  • Solid formulations for oral administration are tablets, pill, dusting powders and capsules .
  • Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the abovementioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin.
  • Tablets, coated tablets, capsules, pills and granules can contain the active compound or compounds in addition to the customary excipients, such as (a) fillers and extenders, for example starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, for example carboxymethylcellulose, alginates, gelatine and polyvinylpyrrolidone, (c) humectants, for example glycerol, (d) disintegrating agents, for example agar-agar, calcium carbonate and sodium carbonate, (e) solution retarders, for example paraffin, and (f) absorption accelerators, for example quaternary ammonium compounds, (g) wetting agents, for example cetyl alcohol and glycerol monostearate, (h) adsorbents, for example kaolin and bentonite, and (i) lubricants, for example talc, calcium stearate, magnesium stearate, and solid polyethylene glyco
  • the tablets, coated tablets, capsules, pills and granules can be provided with the customary coatings and shells, optionally containing opacifying agents, and can also be of a composition such that they release the active compound or compounds only or preferentially in a certain part of the intestinal tract, if appropriate in a delayed manner, examples of embedding compositions which can be used would be polymeric substances and waxes. If appropriate, the active compound or compounds can also be presented in microencapsulated form with one or more of the abovementioned excipients .
  • Formulations for parenteral administration are stirilized aqueous solutions, water- insoluble excipients, suspensions, emulsions, and suppositories.
  • Suppositories can contain, in addition to the active compound or compounds, the customary water-soluble or water-insoluble excipients, for example polyethylene glycols, fats, for example cacao fat, and higher esters (for example C14-alcohol with C16-fatty acid) or mixtures of these substances.
  • Ointments, pastes, creams and gels can contain, in addition to the active compound or compounds, the customary excipients, for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures of these substances .
  • Dusting powders and sprays can contain, in addition to the active compound or compounds, the customary excipients, for example lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain the customary propellants, for example chlorofluorohydrocarbons .
  • Solutions and emulsions can contain, in addition to the active compound or compounds, the customary excipients, such as solvents, solubilizing agents and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahydrofurfuyl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances .
  • solvents such as solvents, solubilizing agents and emulsifiers
  • solvents such as solvents, solubilizing agents and emulsifiers
  • solvents such as solvents, solubilizing agents and emul
  • the solutions and emulsions are also be in a sterile form which is isotonic with blood.
  • Suspensions can contain, in addition to the active compound or compounds, the customary excipients, such as liquid diluents, for example water, ethyl alcohol and propylene glycol, and suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.
  • liquid diluents for example water, ethyl alcohol and propylene glycol
  • suspending agents for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.
  • the formulation forms mentioned can also contain coloring agents, preservatives and additives that improve the smell and taste, for example peppermint oil and eucalyptus oil, and sweeteners, for example saccharin.
  • the abovementioned pharmaceutical formulations can also contain other pharmaceutical active compounds in addition to the compounds according to the present invention.
  • the abovementioned pharmaceutical formulations are prepared in the customary manner by known methods, for example by mixing the active compound or compounds with the excipient or excipients.
  • the therapeutically active compounds should preferably be present in the abovementioned pharmaceutical formulations in a concentration of about 0.1 to 99.5, preferably about 0.5 to 95% by weight of the total mixture.
  • the formulations mentioned can be used on humans and animals orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally or locally (dusting powder, ointment, drops) and for the therapy of infections in hollow spaces and body cavities.
  • Possible suitable formulations are injection solutions, solutions and suspensions for oral therapy and gels, infusiton formulations, emulsions, ointments or drops, ophthalmological and dermatological formulations, silver salts and other salts, eardrops, eye onintments, dusting powders or solutions can be used for local therapy.
  • intake can also be in suitable formulations via the feed or drinking water.
  • Gels, powders, dusting powders, tablets, delayed release tablets, premixes, concentrates, granules, pellets, boli, capsules, aerosols, sprays and inhalants can furthermore be used on humans and animals .
  • the compounds according to the present invention can moreover be incorporated into other carrier materials, such as for example, plastics (chain of plastic for local therapy) , collagen or bone cement.
  • the particular optimum dosage and mode of administration required for the active compounds can be determined by any expert on the basis of his expert knowledge .
  • Example 1 Synthesis of oligonucleotide of the SEQ. ID.
  • oligonucleotides were synthesized trityl-on with 1 ⁇ M scale using ABI 392 DNA/RNA synthesizer (Applied Biosystem) . The method is described in Korea Patent Application No. 99-26947. Time for general condensation is 1 min, whereas was 10 min in case of nucleotides of the present invention containing azasugar with other functional groups than hydrogen at position 4. Solid support and protecting group were removed by heating with ammonium hydroxide for 17 hours at 55 ° C, and this solution was freeze- dried by adding 5 drops of trimethylamine every hour to keep the protecting group, dimethoxytrityl (DMT) .
  • DMT dimethoxytrityl
  • the residue was dissolved in 1 ml of 100 mM triethylammonium acetate (TEAA, pH 7.0), and then purified by reversed phase high-performance liquid chromatography (RP-HPLC, Hamilton PRP-1, 300 mm x 7 mi, 18-28% acetonitrile/100 mM TEAA, pH 7, monitored at 260 nm) .
  • RP-HPLC reversed phase high-performance liquid chromatography
  • the desired fractions were freeze-dried and the residual TEAA was removed by freeze-drying twice after adding 1 l-fi. of distilled water thereto.
  • the residual solid was re-dissolved thoroughly by adding 0.3 ml of 80% acetic acid and vortexing, and dimethoxytrityl group was removed by incubating for 20 minutes at room temperature.
  • Ethanol (0.3 ml ) was added to the above solution to remove excess acetic acid, and then freeze- dried.
  • the sample was re-dissolved by vortexing in 1 ml of distilled water, and then 1 ml of ether was added thereto and vortexed again. After ether layer was removed by pipette, 1 ml of ether was added thereto again. This procedure was repeated twice. Water layer was collected for freeze-drying. Dried residue was dissolved in 1 ml of distilled water, and then quantified by UV absorbance at 260nm at 70 ° C.
  • the extinction coefficients of natural nucleotides (at 260nm) used for calculation were as follows: dAMP, 15200; dCMP, 7700; TMP, 8830; dGMP, 11500.
  • the extinction coefficients of nucleotides having azasugar were considered to be same as those of natural nucleotides.
  • the base composition of the purified oligonucleotides was confirmed by total digestion with nuclease followed by RP-HPLC (Hewlett Packard, ODS hypersil, C-18; 20 mM K 2 HP0 4 , pH 5.6(A), MeOH (B) , 100% A to 40% B in 20 min) and laser desorption mass spectrometry .
  • oligonucleotide of the SEQ. ID. NO: 1 containing adenosine derivative (@) having the structure of ⁇ Chemical Formula 3> at various position in its base sequence was synthesized.
  • oligonucleotides of the SEQ. ID. NO: 2 ⁇ 29 containing the adenosine derivative at various position in its nucleotide sequence were synthesized.
  • Oligonucleotides were synthesized, whose sequences were complementary to TAR sequences of HIV-1 and SIV (simian immunodeficiency virus), IRES (internal ribosomal entry site) of poliovirus, and sd (splicing donor) , LTR and gag of HIV.
  • the oligonucleotides of the present invention include random oligonucleotides (represented by the SEQ. ID. NO: 20 ⁇ 23) having random base sequence without target gene.
  • the oligonucleotide of the SEQ. ID. NO: 29 was designed to have CpG sequence that had been reported to induce strong immune response in vivo (A. M. Crieg, et al .
  • oligonucleotides of the present invention were infected with HXBc2/ ⁇ tat virus (tat-defective HIV-1, NIH AIDS Research and Reference Reagent program, USA) .
  • Antiviral activity of each oligonucleotide was measured by analysis of syncytium formation or reverse transcriptase (RT) activity in the presence or absence of each oligonucleotide in the cultures of the HIV-1 infected cells .
  • RT reverse transcriptase
  • HXBc2/ ⁇ tat virus laboratory strain of tat- defective HIV-1
  • Jurkat-Tat cells Tu-expressing Jurkat cells
  • gpl20 and gp41 HIV-1 envelop glycoproteins
  • anti-HIV-1 activity of each oligonucleotide of the present invention was examined by the relative amounts of syncytia formed in the culture of infected cells in the presence of each oligonucleotide.
  • Jurkat- Tat cells were washed twice with PBS, and then infected with HXBc2/ ⁇ tat virus (TCID 50 50/lxlO 5 cells) in a serum-free medium for 1 hour at 37 ° C, C0 2 incubator. After infection, cells were washed with PBS buffer to remove uninfected virus, and then cultured in the 24- well plate (2xl0 5 cells/well) .
  • oligonucleotides (0.1 ⁇ M) of the present invention synthesized in example 1 were added at the beginning of culture.
  • the infected cells were cultured in the presence of each oligonucleotide and the dates of syncytium formation and the number of syncytium were observed for 12 days after a single treatment.
  • oligonucleotides of the SEQ. ID. NO: 2, 3, 11, 17-23 and 29 showed higher anti-viral activity than that of AZT .
  • oligonucleotides of the SEQ. ID. NO: 17, 20 and 22 showed remarkable anti-viral activity.
  • oligonucleotides of the SEQ. ID. NO: 20-23 and 29 showed strong anti-HIV-1 activity though they had no sequence specificity to HIV-1 gene.
  • Both of the oligonucleotides of the SEQ. ID. NO: 4 and 5 seemed to accelerate HIV proliferation in the infected cells at the concentration of 0.1 ⁇ M.
  • anti-viral activity of the oligonucleotides of the present invention seemed to depend on the position or number of azasugar nucleotides in the sequence, and the oligonucleotides containing more than 4 azasugar nucleotides seemed to be much more efficient to inhibit HIV-1 replication.
  • the oligonucleotides of the SEQ. ID. NOs: 2, 11, 17, 22 and 29 inhibited HIV-1 proliferation efficiently even at the final concentration of 0.06 ⁇ M. While, the oligonucleotide of the SEQ. ID. NO: 4 showed no antiviral activity in the same condition of serum-free medium even at the concentration of 0.1 ⁇ M (Fig. 2) .
  • oligonucleotides of the SEQ. ID. NOs: 2, 11 and 17 was markedly reduced in the serum-containing medium
  • the oligonucleotides of the SEQ. ID. NOs: 22 and 29 was merely or not affected for their anti-viral activity by the existence of serum.
  • the 5 oligonucleotides mentioned above were much more efficient than AZT for a long-lasting anti-HIV-1 activity even in serum-existing condition.
  • Anti-viral activity of each oligonucleotide was measured at varying concentrations in serum-containing medium and summarized in Table 4.
  • Anti-HIV-1 activity of oligonucleotides treated with different concentrations in serum-existing condition Anti-HIV-1 activity of oligonucleotides treated with different concentrations in serum-existing condition.
  • HXBc2/ ⁇ tat-infected Jurkat-Tat cells were cultured in the presence of oligonucleotides at varying concentrations in a medium supplemented with FBS (RPMI1640-10% FBS) .
  • SEQ. ID. NO: 17 for the inhibition of virus proliferation.
  • oligonucleotide of the SEQ. ID. NO: 2 was similar to that of the oligonucleotide of the SEQ. ID. NO: 22 for their anti-viral activity at 0.1 ⁇ M.
  • anti-viral activity of the oligonucleotide of the SEQ. ID. NO: 2 was significantly attenuated, while that of oligonucleotide 22 was not.
  • the oligonucleotides of the SEQ. ID. NO: 22 and 29 were merely affected for its anti-HIV-1 activity by the existence of serum, while anti-viral activity of the oligonucleotide of the SEQ. ID. NO: 2 was markedly reduced when serum was added in comparison with those of other oligonucleotides. It means that the oligonucleotides of the present invention, such as SEQ. ID. NO: 20, 22, and 29, can be used as an AIDS therapeutic drug in vivo because of its resistance to the serum effects .
  • Jurkat-Tat cells were infected with HXBc2/ ⁇ tat virus as described in the experimental example ⁇ 1-1>, and then 2/3 volume of culture medium containing cells was harvested at an interval of 3 days. Fresh medium and the test compounds were replaced for further culturing. The harvested culture medium was vortexed vigorously for 1 minute, followed by centrifugation for 10 seconds at 15,000 rpm to remove cell debris.
  • Reverse transcriptase activity was measured by the method reported previously with a slight modification. Specifically, the supernatant was transferred into a new test tube, to which PEG/NaCl solution equivalent to 1/2 volume of supernatant (30% PEG in 0.4 M NaCl) was added. Mixed thoroughly, the sample was left overnight at 4 ° C . The solution was centrifuged for 45 minutes at 15,000 rpm. After removal of supernatant, 10 ⁇ of lysis buffer(0.25% Triton X-100, 20% glycerol, 50 mM Tris-HCl pH 7.5, 0.1% DTT, 250 mM KC1) was added to the virus sediments for dissolution.
  • lysis buffer 0.25% Triton X-100, 20% glycerol, 50 mM Tris-HCl pH 7.5, 0.1% DTT, 250 mM KC1
  • Reverse transcriptase reaction solution (1 unit/ ⁇ ⁇ of RNasin, 1 ⁇ Ci of 3 H-TTP, 0.025 unit Poly (A) - (dT) , 50 ⁇ M Tris-HCl pH 7.5, 5 mM DTT, 0.1% Triton X- 100) was added into each test tube (50 /z ⁇ /test tube), and incubated for 1 hour at 37 ° C. After reaction, the solution was absorbed into DE51 filter paper (Whatman) .
  • the filter paper was dried, washed with 2X SSC solution
  • the reverse transcriptase activity was rapidly increased from day 6 till day 12 after infection. After day 12, the enzyme activity was decreased and maintained at a certain level. Meanwhile, when the cells were cultured in medium containing 0.5 ⁇ M oligonucleotides of the SEQ. ID. NO: 2 and 22 of the present invention, the reverse transcriptase activity was not increased at all until 18 days after infection (Fig. 3) .
  • Anti-HIV-1 activities of oligonucleotides of the SEQ. ID. NO: 2, 17, 20 and 22 in the serum-supplemented medium were similar to those shown in serum-free medium when measured by reverse transcriptase activity (Fig. 4) .
  • the reverse transcriptase activity was not inhibited, rather increased at earlier days after infection than that of control, which indicated that viral replication was accelerated by the treatment of oligonucleotide of the SEQ. ID. NO: 4 as shown in serum-free medium.
  • the present inventors have further investigated whether the oligonucleotides of the present invention showed anti-viral activity against MB and SHIV 89 .6 (obtained from AIDS Research and Reference Reagent Program, NIH, USA) in addition to HXBc2/ ⁇ tat (tat- defective HIV-1 strain) .
  • C8166 cells obtained from AIDS Research and Reference Reagent Program, NIH, USA were infected with SHIV 89 . 6 as described above experimental example ⁇ 1-1>.
  • the number of syncytium formed in the presence of oligonucleotides of the present invention was determined at the following days after infection and summarized in Table 6.
  • syncytium formation was not observed when infected cells were treated with the oligonucleotides of the SEQ. ID. NO: 20 and 22 even at the final concentration of 0.2 ⁇ M. While the oligonucleotides of the SEQ. ID. NO: 2 and 17, showed sterile inhibition of syncytium formation during the experiment when the infected cells were treated at 0.5 ⁇ M, they were not so effective when treated at 0.2 ⁇ M, and the number of syncytium increased as time went on after first detection on 4 days after infection. On the other hand, syncytium formation began 2 days after infection in untreated control or in culture treated with the oligonucleotide of the SEQ. ID. NO: 4.
  • the oligonucleotides of the SEQ. ID. NO: 2, 17, 20, 22 and 29 showed effective anti-viral activity against virulent strains such as HIV-1 MB and HIV-1 89 . 6P (0.05 ⁇ M ⁇ EC 50 ⁇ 0.12 ⁇ M) as well as against laboratory strain such as HXBc2/ ⁇ tat (0.05 ⁇ M ⁇ EC 50 ⁇ 0.12 ⁇ M) .
  • the oligonucleotide of the SEQ. ID. NO: 22 was the most potent for its antiviral activity against a broad spectrum of HIV-1. It is particularly noted that the anti-viral activity of the oligonucleotides of the present invention (NO: 2, 17, 20, 22 and 29) was much more effective than those of other AIDS therapeutic drugs such as ddC or ddl .
  • the anti-viral activity of AZT (Sigma, USA) , widely used for AIDS therapeutic drug, against HXBc2/ ⁇ tat and MB was about 10-times as strong as those of oligonucleotides of the present invention for a short period of time (for 4 days) after infection, but the oligonucleotides of the present invention were much more effective than that of AZT for a long term antiviral activity (for 9-12 days) after a single treatment (Table 2 and Table 3). Meanwhile, AZT did not show anti-viral activity at all against SHIV-1 89 . 6P .
  • the oligonucleotides of the present invention are to be effective for the treatment of the AIDS patients who have drug-resistant mutant against the commercialized RT (reverse transcriptase) inhibitors such as AZT, ddC, ddl etc, since the antiviral mechanism of the oligonucleotides of the present invention is quite different from that of RT inhibitors .
  • RT reverse transcriptase
  • C8166, 174xCEM and MT-4 cells (AIDS Research and Reference Reagent Program, NIH, USA) , HeLa (ATCC CCL 2), Jurkat E6 (ATCC TIB 152), Vero (ATCC CCL 81) and U-937 (ATCC CRL 1593) cells were used. Each cell lines were treated with oligonucleotides for 4 days at varying concentrations, and then the viable cells were measured using modified MTT assay.
  • MT-4 cells (1x10 s cells/well) in RPMI1640 supplemented with 10% FBS was loaded into 96-well microtiter plate (200 ⁇ /well ) , and different concentrations (0 ⁇ M - 100 ⁇ M) of oligonucleotides were added thereto, followed by culturing for 4 days.
  • 10 ⁇ of MTT solution (7.5 mg/ml, Sigma Chem. Co., dissolved in PBS, stored at 4 ° C) was added to each well and the cultures were incubated for 4 hours at 37 ° C. Removing supernatant carefully, 100 ⁇ of HC1 solution containing isopropanol (0.4 M) was added.
  • cytotoxic concentration 50 was determined by the concentration of the compound required for 50% reduction of the cell viability. When the CC 50 was above 100 ⁇ M, cytotoxicity of the sample was not re-determined at the concentration more than this. Cytotoxicity of each oligonucleotide of the present invention was summarized in Table 8.
  • the number in parentheses is the number of six- membered azasugars contained in each oligonucleotide.
  • CC 5 o values of oligonucleotides of the present invention were above 50 ⁇ M in most cells except U937. It means that the Tl
  • Tl of the oligonucleotides of the SEQ. ID. NO: 22 and 29 was over 250 even in U937 cells.
  • the Tl value in medication represents its safety and effectiveness. The greater Tl is the safer and more effective the drug is. Considering all these together, it was expected that the oligonucleotides of the present invention might not cause any harmful effect when used as a therapeutic drug in vivo.
  • oligonuclotides were added (final cone. 13 ⁇ M) into culture supernatant (Fig. 5) or fresh medium (RPMI1640 supplemented with 10% FBS) (Fig. 6), and incubated at 37 ° C, C0 2 incubator.
  • each reaction 10 ⁇ l was withdrawn at 0, 1, 2 hours and 1 st , 2 nd , 4 th , 6 th day for culture supernatant, and at 0, 1 st , 2 nd , 3 rd , 6 th , 9 th , 12 th , 15 th day for fresh medium, respectably, and these aliquots were run on 20% SDS- PAGE electrophoresis .
  • the stability of each oligonucleotide of the present invention was determined by the band integrity after staining the gel with ethidium bromide solution.
  • S backbone represented by the SEQ. ID. NO: 2 or 4
  • the oligonucleotides of the present invention are expected to be effective enough to overcome the limitation of the instability of the general antisense oligonucleotides in vivo.
  • it was noteworthy that even a nucleotide having P 0 bonds was resistant against nuclease, if it has more than 3 six-membered azasugar nucleotides, which seemed to be an important information for designing the antisense oligonucleotides .
  • oligonucleotides (the SEQ. ID. NO: 2 and 22) of the present invention keep their stability for over 15 days in fresh medium containing 10% FBS.
  • MAGI cells HeLa cell line containing LTR- ⁇ -galactosidase gene, designed to express ⁇ -galactosidase under the control of LTR promoter and HIV-1 Tat protein
  • pSV2-Tat plasmid Teat-expressing vector
  • LTR-CAT plasmid AIDS Research and Reference Reagent Program
  • oligonucleotides of the present invention were added directly into medium
  • the number of blue- colored cells was markedly decreased by transfection of the oligonucleotide of the SEQ. ID. NO: 4, which has no six-membered azasugar nucleotide.
  • transfection of the oligonucleotides of the SEQ. ID. NO: 2 and 22 having 6-membered azasugar nucleotides did not suppress the number of the blue-colored cells (Fig. 7B) .
  • oligonucleotides of the present invention seems to be similar to the antisense mechanism as reported previously in the point nonspecific reactions. However, anti-viral activity, safety and stability of oligonucleotides of the present invention have greatly improved by introducing the modified nucleotides containing 6-membered azasugar, which is the novelty of the present invention.
  • oligonucleotides of the present invention were due to the inhibition of HIV-1 infection by acting outside of the cells, rather than their sequence-specific inhibition by binding to the TAR RNA sequence.
  • Jurkat-tat cells (2xl0 6 ) were transfected with 5 ⁇ g of LTR-CAT plasmid, followed by addition of 2 ⁇ g of oligonucleotides into medium (Fig. 9) .
  • Jurkat-tat cells were transfected with 5 ⁇ g of LTR/CAT and 2 ⁇ g of antisense oligonucleotides simultaneously (Fig. 10) .
  • CAT chloramphenicol acetyl transferase activity was measured, which is proportional to the LTR/Tat activity.
  • GenePorterTM transfectant kit (Gene Therapy System Inc. San Diego, CA, USA) was used for the transfection and experiment was performed according to the supplier' s manual .
  • Forty-eight hours after transfection cells were harvested and washed twice with PBS(4°C) . After adding 1 m of TNB solution (40 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl) , cell number was counted with hemocytometer .
  • CAT activity was not affected by oligonucleotides when treated in the medium, which means oligonucleotides of the present invention could not inhibit LTR/Tat activity within the cells (Fig. 9) .
  • oligonucleotides were introduced into the cells by transfection, CAT activity was remarkably decreased due to the suppression of LTR/Tat activity by the oligonucleotides having no six-membered azasugar nucelotide (the SEQ. ID. NO: 4 and 13) or weakly inhibited by the oligonucleotide (the SEQ. ID. NO: 9) having six-membered azasugar nucleotides at both ends .
  • oligonucleotides containing six- membered azasugars on HIV-1 replication was not mediated by the sequence specific antisense mechanism against HIV gene, but rather mediated by inhibiting virus attachment on the cell surface.
  • oligonucleotides of the present invention were to have anti-viral activity against broad spectrum of HIV-1 strains including drug resistance mutant strains.
  • oligonucleotides of the present invention (the SEQ. ID. NO: 20 and 22) showed potent anti-viral activity not only against laboratory strain and wild-type strain (MB), but also against SHIV89.6, an AZT-resistant and lethal recombinant virus.
  • oligonucleotides of the present invention block the virus infection by working on the outside of the cells.
  • anti-viral activity of the oligonucleotides of the present invention against HIV-1 strains, resistant to reverse transcriptase inhibitor (Y181C, K103N, Y188H, L1001 and V106A) was determined.
  • the oligonucleotides of the present invention showed strong anti-viral activity against these resistant strains.
  • oligonucleotides of the present invention are expected to have anti-viral activity against wide variety of HIV-1 strains, thus they can be developed as therapeutic anti-AIDS agents.
  • Jurkat-Tat cells were infected with HXBc2/ ⁇ tat as described above experimental example ⁇ 1-1>, and cultured in medium containing 10% FBS.
  • the oligonucleotides of the present invention (0.5 ⁇ M) were added to the medium 5 days after infection, when many syncytia were formed. After 36 hours of treatment, the disappearance of syncytium formation was measured (Fig. 11) .
  • oligonucleotides containing 6-membered azasugars can suppress virus infection in the initial stage as well as virus propagation, which has shown by the inhibitory effect on syncytium formation mediated by cell-to-cell fusion through gpl20-CD4 binding. Therefore, if oligonucleotides of the present invention were given to AIDS patients, they would block the virus infection to the normal cells and suppress syncytium progression, resulting in recovering of the AIDS patients.
  • the anti-viral activity of oligonucleotides containing six-membered azasugars of the " present invention is HIV-1 specific. While SIV has a similar gene structure with HIV-1, the sequence homology between SIV and HIV-1 genes is about 50%, and host specificity is also different, therefore human beings are not infected with SIV. Considering that the host- specificity is different between SIV and HIV-1, it is explained that the oligonucleotides of the present invention exert anti-viral activity through the mechanism of blocking HIV-1 infection specifically by binding to receptor or envelope proteins that are essential for the virus attachment.
  • HeLa cells cultured by mono-layer on 30 mm culture dish were infected with poliovirus (Sabin type I, obtained from Dr. E. Wimmer, State University of New York, USA) for 1 hour (0.1 MOI) followed by washing twice with PBS to remove uninfected virus.
  • DMEM medium supplemented with 10% FBS, GIBCO/BRL
  • TCID 50 assay Fig. 13
  • oligonucleotides of the present invention affect on poliovirus replication regardless of sequence specificity. This result suggests that the oligonucleotides could not exert anti-viral activity within the cells since they could not get into the cells without special delivery system such as liposome, and also had no effect on poliovirus receptor highly expressed on the cell surfaces.
  • oligonucleotides of the present invention inhibit the HIV-1 replication not by intracellular antisense mechanism but by blocking the cell surface receptor as reported previously (P. Hawley, et al . , Antisense Nucleic Acid Drug Dev. , 9, 61-69, 1999; P. Rockwell, et al., Proc . Na tl . Acad . Sci . USA, 94, 6523-8, 1997), or by blocking V3 region of HIV-1 envelope proteins (J. Suzuki, et al . , Nucleic Acids Symp . Ser. , 42, 227-228, 1999; J. R. Wyatt, et al . , Proc . Na tl .
  • the oligonucleotides of the present invention had no influence on the replication of other viruses than HIV-1, suggesting that the treatment of the oligonucleotides of the present invention did not affect on the function of other cell surface molecules .
  • Oligonucleotides of the present invention should have an anti-viral activity in the presence of high concentration of serum when they are administered to human beings.
  • anti-viral activities of oligonucleotides in serum-free medium Opti-MEM, GIBCO/BRL
  • RPMI-1640 medium GIBCO/BRL
  • Jurkat- tat cells were infected with HIV-l/ ⁇ tat as described in the experimental example ⁇ 1-1>, followed by adding 0.1 ⁇ M of oligonucleotides. And then, the date was checked when the syncytium formation was first observed and the results were summarized in Table 5.
  • oligonucleotides represented by the SEQ. ID. NO: 2, 11, 17, 22 and 29
  • Table 5 shows that oligonucleotides (represented by the SEQ. ID. NO: 2, 11, 17, 22 and 29) of the present invention showed high anti-viral activity except the oligonucleotide of the SEQ. ID. NO: 4, where the syncytium formation was first observed 8 days after infection.
  • serum was added, anti-viral activity was rather decreased, resulting in that the syncytium formation was observed 2-3 days earlier than the serum-free condition.
  • the oligonucleotide of the SEQ. ID. NO: 2 was most affected by serum, and the anti- viral activity of the oligonucleotide of the SEQ. ID.
  • oligonucleotides of the SEQ. ID. NO: 22 and 29 keep their anti-viral activity without being influenced by the presence of serum, type or concentration of serum.
  • mice 5-week old SPF ICR line mice were used for acute toxicity test.
  • Each oligonucleotide of the present invention was dissolved in PBS and injected intravenously at the dosage of 100 Eg/kg to 3 mice per group. Death, clinical symptoms, and weight change in mice were observed, hematological and biochemical tests in blood were performed, and any abnormal signs in chest and abdomen were checked by naked eyes after autopsy, in which no specific clinical symptoms, weight change, death, nor changes in hematological and biochemical tests were resulted. These results indicate that the oligonucleotides of the present invention are considered to be safe by intravenous administration into mice up to lOOmg/kg.

Abstract

La présente invention concerne l'utilisation d'oligonucléotides antisens utilisés comme agent thérapeutique contre le SIDA. Plus précisément, la présente invention concerne une nouvelle utilisation d'oligonucléotides antisens comme agent thérapeutique contre le SIDA, constitués par des dérivés nucléotidiques dans lesquels un cycle ribose à cinq éléments, un sucre nucléotidique naturel, est substitué par un azasucre hexagonal, sur un squelette P=S. Les oligonucléotides de la présente invention présentent une faible toxicité par rapport aux cellules et agissent en dehors des cellules pour inhiber la fixation virale du VIH. Ces oligonucléotides présentent une activité antivirale à large spectre contre des variants du VIH. Ainsi, les oligonucléotides de la présente invention peuvent être utilisés comme agent thérapeutique efficace contre le SIDA.
PCT/KR2002/000325 2001-02-27 2002-02-27 Oligonucleotides a phosphorothioate (p=s) renfermant des nucleotides modifies par des azasucres hexagonaux et leur utilisation dans une therapie contre le sida WO2002068582A2 (fr)

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