Inhibition of cytokine production

Details Inhibition of cytokine production Inhibition of cytokine production

2000-07-14

2002-11-05

Ketter, James (Department: 1632)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S069100, C435S069500, C424S450000

Reexamination Certificate

active

06476214

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to inhibiting the production of cytokines in cells of the body, and thus to the treatment or prevention of diseases associated with excessive production of cytokines. Of especial interest is the production of interleukin-5, a cytokine which is implicated in inflammation of the pulmonary airways, especially in asthma; and TNF-&agr;, a cytokine shown to play a very important role in rheumatoid arthritis.
2. Description of the Related Art
There is now evidence that interleukin-5 (IL-5) plays an important role in the pathogenesis of asthma (Corrigan, Clin. Exp. Allergy 25, 485-487 (1995)). IL-5 is one of the commonest cytokines which are produced by T-helper cells. It is particularly implicated in control of eosinophil functions, including selective chemotaxis, growth differentiation, proliferation, release of cytotoxic proteins and the survival of these cells (Takatsu et al., Adv. in Immunol. 57, 145-190 (1995)). IL-5 also enhances T-cell adhesion to vascular endothelial cells. IL-5 has been detected at the site of the allergic inflammation and in the peripheral blood of patients, its level correlating to the extent of eosinophilia, the severity, and the response of the treatment of asthma (H. Okudaira et al., Int. Arch. Allergy Immunol. 107, 255-258 (1995); Corrigan, Clin. Exp. Allergy 25, 485-487 (1995)). Animal models from murine to monkey have demonstrated the involvement of IL-5 in asthma and the beneficial effects due to the inhibition of the cytokine.
A prophylactic and therapeutic effect of an anti-IL-5 monoclonal, TRFK-5, in a mouse model of allergic pulmonary inflammation has recently been demonstrated (T. T. Kung et al., American Journal of Respiratory Cell and Molecular Biology 13, 360-365 (1995)). The number of eosinophils in the bronchoaveolar lavage (BAL) fluid and lung tissue was reduced and the decrease in bone marrow eosinophils was prevented. Both these effects were being seen to occur in a dose-dependent fashion. After the neutralisation of IL-5, the ovalbumin-allergic mice showed no evidence of increased epithelial damage, oedema, or the presence of mucus that could have resulted from eosinophil apoptosis and release of toxic proteins. In IL-5“knock-out” mice, the airway pathology was abolished (P. S. Foster et al., J. Exp. Med. 183, 195-201 (1996)).
Other groups have worked on guinea pig models and have reported that in studies using anti-IL-5 antibodies, a total inhibition of the development of bronchohyperreactivity to histamine and arecoline after ovalbumin challenge (A. J. M. Van Oosterhout et al., American Review of Respiratory Disease 147, 548-552 (1993)). The number of eosinophils was markedly reduced and the number of neutrophils was not affected, verifying the specificity of IL-5 on eosinophils in asthma. However, antibody therapy in humans has many well-known problems.
An alternative idea which could be contemplated is to inhibit production of IL-5 prior to RNA transcription using a modified synthesised double stranded (msds) DNA sequence to which is similar or identical to a particular part of the promoter region of IL-5, for example the part which binds to a transcription factor.
As a general principle, a DNA “decoy” would be provided which would compete with the native gene for a limited supply of transcription factor. This would be a double-stranded (ds) DNA oligomer modified to prevent it from being destroyed by endogenous DNases. For example, the phosphate linkages could be replaced to a large extent by phosphorothioate linkages. See Bieliaska et al., Science 250, 997-1000 (1990), in relation to interleukin-2.
NOMENCLATURE NOTE
All 5′-non-coding region nucleotide nomenclature used in this patent specification is based on the first nucleotide of the start of the coding region (A of the ATG) being +1 and the nucleotide preceding it −1. All nomenclature used in references has been converted to this basis.
V. Gruart-Gouilleux et al., Eur. J. Immunol 25, 1431-1435 (1995) studied the transcriptional activity of the human IL-5 gene promoter region in mouse EL4 cells, using a series of deletion constructs linked to a luciferase reporter gene. They found that deletions of nucleotides between −358 and −274 or −124 to −79 inhibited the induction of IL-5 promoter activity. Investigation of the −358 to −274 region revealed that the octamer-binding motif 5′-atgcaaat-3′ at −290 to −283 was required for full transcriptional activity. See nomenclature note above. Very minor errors of numbering in this paper have also been corrected, taking the sequence of oligo probes as correct.
At about the same time D. Z. Staynov et al., Proc. Natl. Acad. Sci. USA 92, 3606-3610 (1995) and Int. Arch. Allergy Immunol. 107, 217-219 (1995) described a regulatory element in the promoter of the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene that has related sequences in the gene promoters of human interleukins (hIL-2, hIL-4, hIL-5, hIL-13). The authors identified a palindrome-containing sequence as involved in a DNA-protein interaction, by using nuclear extract from Jurkat cells as the source of protein. A 40 bp sequence of hGMCSF promoter DNA containing cttgg . . . (22N) . . . ccaag “outer” palindromic sequences near each end and a 12 bp-long “inner”, AT-rich, palindrome in the centre was found to bind to the nuclear extract by a gel retardation assay (in which the protein-DNA complex migrates more slowly than the DNA alone on a gel). Some of the binding was ascribed exclusively to the outer palindrome and some to the inner. However, the use of these crude nuclear extracts calls into question which proteins were bound. Noting that the outer palindrome motif or a shorter version, ttgg . . . ccaa, is present in the promoters of certain interleukins, the authors speculated that some of the interacting proteins may be gene-specific. As regards IL-5, the conterpart palindrome-containing sequence occurs at nucleotides −514 to −504. However, V. Gruart-Gouilleux et al., above, reported that a deletion of nucleotides −552 to −449 (thus encompassing the whole of Staynov et al.'s palindrome-containing sequence) had little effect on induction of IL-5 promoter activity.
SUMMARY OF THE INVENTION
It has now surprisingly been found that a double-stranded oligomeric deoxyribonucleotide (oligo-DNA), modified to make it resistant to degradation, containing a similar palindrome in hIL-5, inhibits production of hIL-5 in T-cells, apparently by competing with hIL-5 for a transcription factor. It has further been found that there are palindrome-containing sequences comprising the ttgg . . . ccaa motif in the gene promoter region of other cytokines besides those mentioned by Staynov et al., such as TNF-&agr;. These are also inhibitors of transcription of the specific cytokines. The inhibition referred to herein comprises partial or complete prevention of the transcription.
By delivering these oligonucleotides to cells, it will be possible to treat diseases characterised by over-expression of a target gene, leading to unwanted effects at the cellular level, at the level of tissue or organ function and/or throughout the body. These include but are not limited to allergy, asthma, chronic obstructive airways disease or conditions characterised as being inflammation, including autoimmune diseases, or cancer.
In one aspect, the present invention provides a ds-DNA oligomer which has from 25 to 150 base pairs, preferably 25 to 60 and more preferably 50 base pairs, and comprises (i.e. consists of or includes) the palindrome-containing sequence (one strand only shown) of formula 1:
5′B
1
. . . x
N . . . B
2
3′  (formula 1)
wherein B
1
is ttgg or ccaa and B
2
is correspondingly ccaa or ttgg and xN represents a sequence of from 1 to 30 intervening nucleotides, preferably 3 to 30 nucleotides, which is substantially native to the promoter region of the gene coding for a cytokine, said inter

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