Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-11-06
2002-11-19
Ketter, James (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C536S024100
Reexamination Certificate
active
06482610
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a process for preparing agents for treating tumour diseases and for immunosuppression. The agents selected in this context are those which exhibit particularly effective binding to the Ah receptor.
The Ah receptor is a member of the bHLH-PAS protein family. The members of this protein family act as conditionally regulated transcription factors which are activated by various chemical compounds, for example by dioxins, aromatic hydrocarbons, (heterocyclic) food mutagens and the like. In that which follows, such chemical compounds which activate the Ah receptor are also termed Ah ligands.
Ah ligands transform the Ah receptor into an active state in which the Ah receptor dimerizes, for example, with the nuclear factor ARNT and, as a consequence, induces the expression of target genes. To date, it is known that target genes which correlate with the metabolism of foreign substances are expressed. These include the cytochrome P450I subfamily and enzymes of phase II metabolism (Schmidt, J. V. et al., Ann. Rev. Cell Dev. Biol. 12, 55 (1996); Hankinson, Annu. Rev. Pharmacol. Toxicol. 35, 307 (1995)). It is furthermore known that dioxins exert an influence on the proliferation and differentiation of cells, with phenomenological effects of this influence which may be mentioned by way of example being augmented differentiation of the skin, reduced body growth, thymus atrophy, inhibition of spermatogenesis and carcinogenesis (e.g. of the liver in rodents). The Ah receptor plays a role in these processes, as is demonstrated by the correlation between Ah receptor activation and the toxicity of dioxin-related ligands and also by genetic investigations carried out on mice which carry different alleles of the Ah receptor gene or targeted null mutations (Schmidt, loc. cit.; Hankinson, loc. cit.; Fernandez-Salguero, et al. Science 268, 722 (1995); Sewall, et al. Mutat. Res. 333, 111 (1995). It is unlikely that a dioxin-induced metabolism of foreign substances is responsible for the effects of dioxins on cell proliferation and differentiation. The mechanisms and genes by which the Ah receptor exhibits the abovementioned pronounced effects in this regard are so far unknown. While IL-1 and tissue plasminogen activator inhibitor 2 have been identified as being target genes for the Ah receptor (Sutter, et al. Science 254, 415 (1991)), these genes appear, like those of other cytokines, not to be regulated directly by the Ah receptor.
It is known that the cell cycle inhibitor gene p27-Kip1 is a factor for regulating cell proliferation (Polyak et al. Cell 78, 59 (1994); Loda et al. Nat. med. 3,231 (1997); Porter et al. Nat. med. 3, 222 (1997); Catzavelos et al. Nat. Med. 3, 227 (1997); Fredersdorf et al. Proc. Natl. Acad. Sci. USA 94, 6380 (1997). This follows from investigations which have shown that the expression of Kip1 is reduced in various forms of tumour. Kip1 is consequently a tumour suppresser gene. It would therefore be desirable to find substances which are able to increase the cellular level of the protein which is expressed by the Kip1 gene. To date, it is known to use transforming growth factor &bgr; (TGF&bgr;); however, this factor only stabilizes the protein expressed by the Kip1 gene and does not induce Kip1 messenger RNA.
SUMMARY OF THE INVENTION
In relation to the prior art, the invention is based on the technical problem of making available a process for preparing agents which are suitable for treating tumour diseases or for immunosuppression.
For solving this technical problem, the invention teaches that target cells are treated with chemical compounds which bind to the Ah receptor of the target cells, that the binding of the chemical compound(s) to the Ah receptor of the target cells is determined by measuring the expression, which is induced thereby, of a detector gene which is relevant for the effects of the chemical compound(s), and that the chemical compounds which have been found in this way are employed for preparing the agent.
The detector gene is preferably under the control of the regulatory elements of the Kip1 gene. For example, a gene which is the Kip1 gene product which is naturally controlled by the Kip1 promoter is described as being a detector gene. In this case, Kip1 expression can be detected, for example, using Northern blot or Western immunoblot methods. In this context, the expression detector gene refers to the fact that a protein which can be expressed thereby can be readily detected at least semiquantitatively using means which are technically customary.
Alternatively, synthetic genes which contain the essential regulatory elements of the Kip1 gene are also suitable. In this case, it is advantageous, for example, to use the luciferase gene under the control of the Kip1 promoter since this gene is particularly easy to measure. Luciferase is an enzyme which is present in luminous organs and which converts luciferin into an optically excited state in the presence of oxygen. Luciferase can therefore easily be measured using optical detection systems, which systems can be employed to achieve high sample throughputs and to screen low molecular weight substances efficiently within the context of the process according to the invention. However, it is also possible to conceive of using other detector systems, such as alkaline phosphatase, chloramphenicol acetyltransferase or &bgr;-alactosidase, for the process according to the invention.
The invention is accordingly based on the novel finding that the Ah receptor directly induces expression of the Kip1 gene, more specifically its transcription, and that use can be made of a detector gene which is under the control of the Kip1 gene.
Using the process according to the invention, it is possible, on the one hand, to determine agents for treating tumour diseases. On the other hand, it is possible to investigate which chemical compounds, by means of inducing expression of the Kip1 gene, are suitable for use as tumour suppresser substances (and are not merely protein-stabilizing as is TGF&bgr;). The chemical compounds which are found in this way are advantageous as compared with TGF&bgr; since they are comparitively simpler to produce (namely by means of organic synthesis instead of using recombinant methods) and employ as pharmaceutical active compounds. Their simpler employment is based on the fact that such chemical compounds have the potential to be effective following oral administration. It is furthermore advantageous that the chemical compounds that can be found in this way are inducers of Kip1 messenger RNA and not merely of protein stability. Thus, adequate expression of the messenger RNA is a primary requirement for enabling accumulation of the Kip1 protein in cells to be further increased by means of protein stabilization.
According to the above, therefore, the process according to the invention is suitable for screening both chemical compounds for treating tumour diseases and chemical compounds for immunosuppression. The tested agents which are selected in this context are those which exhibit a particularly effective induction of the Kip1 gene.
The chemical compounds which are found using the process according to the invention are also better tolerated than is TGF&bgr; since the latter also promotes inflammatory reactions, for example. While side-effects, such as immunosuppression and the induction of foreign substance-degrading enzymes, are also to be expected when using the found substances, these side-effects are comparatively acceptable. Moreover, substances which are found using the process according to the invention can also be employed for treating immune diseases such as lymphoproliferative syndromes or autoimmune diseases, or in association with transplantations, since the cellular (T-cell) immune system, in particular, can be suppressed by Ah ligands.
The present invention consequently also relates to chemical compounds which are suitable for preparing agents for treating tumour diseases or for immunosuppression. These agents are characterized in that
Göttlicher Martin
Kolluri Siva Kumar
Weiss Carsten
Forschungszentrum Karlsruhe GmbH
Ketter James
Kueffner Friedrich
Li Q Janice
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