Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-06-26
2002-12-17
Wang, Andrew (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S375000
Reexamination Certificate
active
06495675
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a pharmaceutical composition for treating or preventing influenza, and a novel capped oligonucleotide.
2. Description of the Related Art
Influenza is extremely prevalent, repeatedly on a worldwide scale. Most people are infected by airborne germs. The influenza virus is highly infectious, and therefore, when a new subtype of the virus is generated, the prevailing scale thereof is enlarged beyond comparison with the infection of those suffering from the acquired immunodeficiency syndrome (AIDS).
The influenza virus belongs to orthomyxoviridae, and has a minus strand, i.e. a single strand RNA virus. The gene of the influenza virus is composed of eight segments. Among proteins encoded by the eight segmentation genes are hemagglutinin (HA) and neuraminidase (NA), which are two spikes on a surface of a virus particle and project from an envelope. There is also a segmentation gene encoding M2, one of the membrane proteins. On the surface of the virus particle exist two glycoproteins in the form of spikes embedded in a lipid bilayer (envelope) stemming from a host. Furthermore, there is another membrane protein, M1. The segmentation gene encoding M2 is also contained in the virus gene, A ribonucleoprotein (RNP) complex is located at the center of the virus, and composed of the gene RNA, three RNA polymerase subunits (PB1, PB2, and PA), and a nucleoprotein (NP). The segments encoding the proteins, PB1, PB2, PA, and NP, are also contained in the virus gene. A non-structural protein is synthesized from the 8th segmentation gene.
The influenza viruses are classified into three types, A, B, and C, on the basis of the differences in serotypes of the nucleoprotein (NP) and the membrane protein M2. The above three proteins PB1, PB2, and PA in the influenza virus are subunits which constitute an RNA polymerase. Amino acid sequences of the RNA polymerase are conserved in the influenza A, B, C or a mutant thereof. The RNA polymerase catalyzes a synthesizing reaction of RNA, an addition reaction of poly A, a restriction reaction of a cap, or the like. In particular, PB1 participates in the synthesizing reaction of RNA, and PB2 recognizes an mRNA cap structure of a host cell, and cleaves mRNA. PA has a role in transcription and elongation reactions. NP is a polynucleotide-binding protein non-specific to a base sequence. An RNA in an NP-RNA complex forms a double strand or a helix, and is important for a transcriptional reaction of a virus. Further, PB2, PB1, PA, NP, and NS are synthesized in an initial stage of the infection, respectively.
Of the influenza viruses, the influenza A virus undergoes a substantial change in antigenecity, and prevails above all others. In view of an acute infectivity thereof, the influenza A virus is most malignant. As an antiviral agent for the influenza A virus, amantadine or rimantadine are known, but these cannot cope with mutants and have strong side effects. Thus, a medicament exhibiting a satisfactorily antiviral activity has not been developed. Further, a treatment with an inactivated vaccine has been attempted, but there is a difference between the inactivated vaccine and an antibody generated by a natural infection, and thus there are fewer people to whom the vaccine is administered. Further, the vaccine does not have a sufficient effect in a sustainability of producing antibodies, and thus cannot completely prevent the spread of infection. The effect of the inactivated vaccine can not be sustained for a long period, and thus the development of an attenuated vaccine is still desired. Therefore, there exists no vaccine that can be put to practical use. A difficult problem is the substantial changes in viral antigenecity, and this is one of causes of the delay in the development of the vaccine.
Recently, the development of a gene-analyzing technique has made it possible to easily determine a mutant gene sequence. As an antiviral agent for the influenza viruses encountering considerable mutations, it would be suitable to use a method wherein a gene is a target, e.g., an antisense oligonucleotide method, such as an antisense DNA method.
In the antisense oligonucleotide method, an oligonucleotide having a base sequence complementary to that of a target gene is used to inhibit a transcription, splicing, or translation of the target gene, at an mRNA level. This technique can specifically prevent the expression of viral proteins, in a mechanism different from a medicament [S. T. Crooke, Therapeutic Applications of Oligonucleotides, Springer-Verlag, (1995)].
However, it would be difficult to completely prevent the expression of the viral proteins by the antisense oligonucleotide method, because a main target in the antisense oligonucleotide method is a viral mRNA, which is an intermediate product. More particularly, a synthesizing rate of the viral proteins from the viral mRNAs is very rapid in a cell infected with an influenza virus, and thus a part of the viral proteins may be produced before the antisense nucleotide functions to the targent mRNA. This would lead to an investigation of the potentiality of an antiviral agent utilizing a decoy RNA against a final product, i.e., a viral protein, such as a viral RNA polymerase, which plays an important role in the proliferation of viruses.
For example, the PB2 protein, one of the influenza virus RNA polymerases, utilizes a part of the 5′ end of a host cell mRNA, when an mRNA is transcribed from an influenza viral RNA (vRNA) in an initial stage of the infection with the influenza virus. More particularly, the PB2 protein recognizes and binds to an oligoribonucleotide moiety of a first 10 to 13 base sequence containing a cap structure located at the 5′ end of the mRNA of the infected host cell. Thereafter, the rest of the mRNA is cleaved out, and a complementary chain is elongated after the bound first 5′ terminal oligoribonucleotide moiety, using the vRNA having genentic information of the influenza virus as a template, to generate an mRNA having the genentic information of the influenza virus.
The present inventors conducted in vivo experiments using similar oligoribonucleotides (synthesized mRNAs), and surprisingly, found that oligoribonucleotides (synthesized mRNAs) having 10 to 12 bases (nucleotides) effectively inhibit the RNA elongation reaction. The present inventors also found that the in vitro results obtained by D. Y. Thomas, et al cannot be applied to an in vivo experiment. Further, the present inventors found that oligoribonucleotides having 8 to 9 bases effectively inhibit the RNA elongation reaction in vivo.
The present invention is based on the above findings.
SUMMARY OF THE INVENTION
Accordingly, the object of the present invention is to provide a method or a pharmaceutical composition for treating or preventing an influenza virus.
Another object of the present invention is to provide a novel oligoribonucleotides.
Other objects and advantages of the present invention will be apparent from the following description.
In accordance with the present invention, there is provided a pharmaceutical composition comprising a capped oligonucleotide capable of binding to a PB2 protein of an influenza virus RNA polymerase, and a pharmaceutically acceptable carrier or dilute, the capped oligonucleotide having a structure of the formula (I):
m
7
GpppXpY (I)
wherein ppp is a triphosphate bridge, p is a monophosphate bridge, m
7
G is 7-methylguanosine group binding at the 5′ end thereof to the ppp, X is 2′-O-methyl guanosine group or guanosine group, the 2′-O-methyl guanosine group or guanosine group binds at the 5′ end thereof to the ppp, and at the 3′ end thereof to the p, and Y is an oligoribonucleotide moiety binding at the 5′ end thereof to the p, and having 5 to 11 bases.
In accordance with the present invention, there is also provided a method for treating or preventing influenza, comprising administering to a subject in need thereof a capped oligonucleo
Hatta Toshifumi
Ishikawa Masahide
Miura Kin-ichiro
Takai Kazuyuki
Takaku Hiroshi
Chiba Institute of Technology
Wang Andrew
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