Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-07
2001-09-18
LeGuyader, John L. (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06291668
ABSTRACT:
The present invention relates to an oligonucleotide derivative directed against human Bcl-2 mRNA. The present invention further relates to a pharmaceutical composition comprising said oligonucleotide derivative, uses thereof and methods of treatment and diagnosis utilizing said oligonucleotide derivative.
Human Bcl-2 is a protein which is closely associated with the process of programmed cell death (apoptosis). Lack of programmed cell death plays an important role in cancer and other hyperproliferative diseases like restenosis, fibrosis or psoriasis, in particular in tumor progression and, importantly, might contribute to the clinical problem of resistance to anti-neoplastic regimens (standard chemotherapeutic drugs/gamma-irradiation).
Oligonucleotides or, in particular, oligonucleotide derivatives directed against human Bcl-2 mRNA may be used in an antisense technology strategy interferring with expression of Bcl-2. For example, it has been reported that such an approach results in decreased cell survival (L. Campos et al., Blood 84 (1994), pp. 595-600; F. E. Cotter et al, Oncogene 9 (1994), pp. 3049-3055; M. R. Smith et al., Cancer Gene Ther. 2 (1995), pp. 207-212), the induction of apoptotic death (F. J. Keith et al., Leukemia 9 (1995), pp. 131-138), and increased drug-sensitivity (S. Kitada et al., Antisense Res. Dev. 4 (1994), pp. 71-79; J. C. Reed et al., see above) in lymphoid tumor cells.
However, there is an ongoing need for further oligonucleotides or oligonucleotide derivatives showing high efficacy, preferably improved efficacy, in modulating the biosynthesis or expression of human Bcl-2, in particular for the treatment of hyperproliferative diseases, e.g. those as mentioned above.
Surprisingly, it has been found that the compounds according to the present invention, mentioned below, show the capability to modulate human Bcl-2 biosynthesis in a cell. They are therefore appropriate for the therapeutic treatment of diseases that respond to this modulation, especially inhibition, of Bcl-2 biosynthesis, such an inibition having as one effect an induction of apoptosis, resulting in inhibition of cell (hyper)proliferation.
In the context of the present invention it has been identified that a region encompassing nucleotide nos. 1880 to 1899, or encompassing at least a part thereof, of the coding region of a RNA deriving from the gene encoding human Bcl-2 protein, and the region comprising the translation termination codon, which codon encompasses nucleotides having nos. 2176 to 2178 of said RNA, are particularly accessible, for hybridization with an antisense oiigonucleotide or oligonucleotide derivative, in particular under physiological conditions for example as encountered in a cellular environment, resulting in modulation of the biosynthesis of the human Bcl-2 protein in a cell. Preferably, said region around the translation termination codon comprises nucleotide having nos. 2175 to 2194, or comprises at least a part thereof, of said RNA. In one aspect, the present invention relates to such an oligonucleotide derivative.
Accordingly, it is an object of the present application to provide an oligonucleotide derivative which is specifically hybridizable to a region of a RNA deriving from the gene encoding human Bcl-2 protein, said region being selected from the group consisting of a region comprising a sequence corresponding to a sequence ranging from base position no. 1880 (5′) to no. 1899 (3′), or comprising at least a part thereof, of the human Bcl-2 cDNA, and a region comprising the translation termination codon of said RNA.
Within the context of the present invention, a RNA, deriving from the gene encoding human Bcl-2 protein, is pre-mRNA or, preferred, mRNA.
Within the context of the present invention, an oligonucleotide derivative is preferred, which is capable of modulating the biosynthesis of the human Bcl-2 protein.
A preferred embodiment thereof is directed to an oligonucleotide derivative as mentioned above, wherein said region is selected from the group consisting of a region comprising a sequence corresponding to a sequence ranging from base position no. 1880 (5′) to 1899 (3′) of the human Bcl-2 cDNA, and a region comprising the translation termination codon of said RNA, the former being preferred.
A likewise preferred embodiment thereof relates to an oligonucleotide derivative as mentioned above, wherein said region comprising the translation termination codon of said RNA comprises a sequence corresponding to a sequence ranging from base position no. 2175 (5′) to 2194 (3′), or comprises at least a part thereof, of the human Bcl-2 cDNA. Preferably, said region comprising the translation termination codon of said mRNA comprises a sequence corresponding to a sequence ranging from base position no. 2175 (5′) to 2194 (3′) of the human Bcl-2 cDNA.
In an even more preferred embodiment thereof, said respective region consists of the respective sequence as specified, i.e. the sequence ranging from base position no. 1880 (5′) to 1899 (3′) of the human Bcl-2 cDNA, or the sequence ranging from base position no. 2175 (5′) to 2194 (3′) of the human Bcl-2 cDNA, the former being preferred.
Within the context of this invention, the term “oligonucleotide derivative” therefore preferably denotes an oligonucleotide which is structurally modified, as compared with a corresponding natural oligonucleotide, at at least one position of at least one building block (this can e.g. relate to the sugar or the base of a nucleoside building block, or to an internucleosidic bridging group). An oligonucleotide derivative can also comprise, in place of at least one nucleoside building block, at least one nucleoside analogue which encompasses a non-sugar backbone to which a nucleic acid base is linked.
Derivatized oligonucleotides, nucleosides, internucleosidic bridging groups and analogues have been described (cf., for example, De Mesmaeker, A. et al., Curr. Op. Struct. Biol. 5 (1995), pp. 343-355; Sanghvi, Y. S. et al., (Ed.), “Carbohydrate Modifications in Antisense Research”, ACS Symposium Series 580 (1994); S. T. Crooke, “Therapeutic Applications of Oligonucleotides”, R. G. Landes Company Publisher (1995)).
Within the context of the present invention, “modulation” of the biosynthesis or expression of the human Bcl-2 protein denotes an interference with the biosynthesis or expression, in particular a partial or complete inhibition thereof, in particular in connection with the translation or transcription process. Such an inhibition, in particular due to partial or complete degradation of the target nucleic acid, due to the process for translating the target nucleic acid being completely or partially inhibited, or due to the transcription process being completely or partially inhibited, can be determined by means of known methods, for example by means of the Northern blot technique at the level of the target nucleic acid, or by means of the Western blot technique at the protein level (cf., for example, Sambrook,J., Fritsch, E. F. and Maniatis, T.: “Molecular Cloning: A Laboratory Manual”, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989). For further means, reference is made to the Examples as mentioned below. In this connection, the term “hybridization” in particular denotes binding by way of hydrogen bonds, known as “Watson-Crick base-pairing”, between complementary bases of an oligonucleotide derivative according to the invention, on the one hand, and of a target nucleic acid, on the other hand. “Specific hybridization” denotes that a sufficient degree of complementarity exists between the oligonucleotide derivative according to the invention and the target nucleic acid to enable specific binding between the oligonucleotide derivative and the nucleic acid to be achieved. In this context, it is not absolutely necessary, for achieving specific hybridization, for 100% or full complementarity to exist between the oligonucleotide derivative according to the invention and the target nucleic acid. An oligonucieotid
Altmann Karl-Heinz
Fabbro Doriano
Zangemeister-Wittke Uwe
Ziegler Annemarie
Epps Janet
LeGuyader John L.
Novartis AG
Pfeiffer Hesna J.
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