Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-06-29
2001-04-17
Elliott, George C. (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C544S001000, C548S100000, C548S146000, C548S148000, C548S152000
Reexamination Certificate
active
06218359
ABSTRACT:
The current invention concerns the use of a receptor from the RZR/ROR receptor family or of a functional fragment thereof in a test of a compound for anti-autoimmune, anti-arthritic, anti-tumor, melatonin-like and/or melatonin-antagonistic activity and the production of a receptor ligand complex comprising said receptor or a functional fragment thereof and a ligand of said receptor. Described is also a method for testing compounds for said agonists or antagonists (screening for ligands) and the active compounds identified therewith.
INTRODUCTION
Small lipophilic substances like retinoic acid (RA), 1,25-dihydroxyvitaminD
3
(VD), thyroid hormone (T3) and steroid hormones regulate a number of developmental and physiological processes in vertebrates and in invertebrates by binding to specific receptors that function directly as transcription factors. These ligand-dependent transcription factors are members of the nuclear receptor superfamily.
The nuclear receptor superfamily also includes structurally related proteins for which no ligand has been identified yet and therefore are referred to as orphan receptors (O'Malley et al., Mol. Endocrinol. (1992), 6, 1259-1361). Examples of such orphan receptors are peroxisome-proliferator activated receptors (PPARS) and chicken ovalbumin upstream promoter transcription factor (COUP-TFs). Despite large diversity in function, two conserved zinc-finger motifs which are involved in binding to DNA appear in all members of this superfamily.
Recently, a novel orphan receptor family has been identified via a reverse transcription-polymerase chain reaction (RT-PCR) strategy (Becker-Andre et al., Biochem. Biophys. Res. Com. (1993), 194, 1371-1379; Becker-Andre et al., Keystone Symposium, Feb. 7-13, 1994, Taos, N.Mex. p.376). RZR/RORs are able to bind as monomers to their specific response elements, but they seem to interact with certain constellations of binding sites cooperatively as homodimers. RZR/RORs show constitutive transactivation and despite different approaches no ligands have been isolated so far for the RZR/ROR receptor family and therefore, it has been assumed, that RZR/RORs may provide constitutive rather than ligand-inducible transactivation.
Surprisingly, it has now been found, that melatonin is a natural ligand of the RZR/ROR receptor family. It was only known that melatonin is a ligand of a membrane receptor, which has been recently cloned from frog skin (Ebiswawa et al., Proc. Natl. Acad. Sci. USA (1994), 91, 6133-6137) and from mamalian tissues (Reppert et al., Neuron (1994), 13, 1177-1185). Melatonin is the major hormon of the pineal gland, but it is also produced in extrapinal tissues. It lightens skin color in amphibians by reversing the darkening effect of MSH (melanotropin). Melatonin is a transmitter of photoperiodic information and is a regulator of seasonal reproductive cycles in photoperiodic animals. It has been shown also that melatonin is involved in thermoregulation and neuroimmunoregulation (Fraschini and Reiter, Eds., Plenum Press N.Y., London 1991).
Melatonin has a short half life in animals and man and it is therefore surprising for melatonin to be a ligand of a nuclear receptor.
As a further surprise, synthetic chemical substances have also been identified as artificial ligands of the RZR/ROR receptor family. Said compounds are known and show anti-autoimmune, anti-arthritic and/or anti-tumor activity (EP-A-494047, EP-A-508955, EP-A-548017, EP-A-548018, CH-511877 and BE-753532). These properties can be demonstrated in vivo, for example in the adjuvant arthritis model in rats in accordance with Wiesenberg et al., Clin. Exp. Immunol. (1989), 78, 245 and the DMBA-tumor model in rats (Schmidt-Ruppin et al., Experentia (1973), 29, 823-825).
These compounds and the pharmaceutically acceptable salts thereof are known to have valuable pharmacological properties in the treatment of diseases of the rheumatoid type. Those diseases include, especially, rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, and other seronegative spondylarthritises, Colitis ulcerosa and Crohn's disease, and also reactive arthritises, collagen diseases, such as Lupus erythematosus and scleroderma, degenerative rheumatic diseases, extra-articular rheumatic and para-rheumatic diseases, for example gout, osteoarthritis and osteoporosis. Furthermore, compounds of this type have immuno-modulating and anti-tumor activities and, hence, can be administered generally in tumor therapy and in autoimmune based or related diseases such as multiple sclerosis, Hashimoto thyriditis, juvenile diabetes and psoriasis.
Nobody could expect or predict that these compounds function as ligands of the RZR/ROR receptor family, too. The anti-autoimmune, anti-arthritic and/or anti-tumor activity observed, implies that the binding of the ligand to the receptor enhances the affinity of the receptor to specific DNA regions (so called hormone response elements) in genes, which are involved in the regulation of cell proliferation and/or differentiation. The transcription of these response genes is either up or downregulated after binding of the ligand-receptor complex. Based on this novel observations it is now possible to use this receptor family for the screening of further compounds (ligands) having anti-autoimmune, anti-arthritic, anti-tumor, melatonin-like and/or melatonin-antagonistic activity.
DETAILED DESCRIPTION OF THE INVENTION
Thus the present invention preferably relates to the use of a receptor of the RZR/ROR receptor family or a functional fragment thereof in a test for identifying a compound with anti-autoimmune, anti-arthritic, anti-tumor, melatonin-like and/or melatonin-antagonistic activity.
In general, these compounds (ligands) can be tested for agonistic and antagonistic activity in respect to the RZR/ROR receptor family.
The RZR/ROR receptor family embraces nuclear receptors that consist of several domains, each domain having a specific function. Nuclear receptors generally act via suppression or activation of transcription after the binding of a ligand to the receptor. Members of the RZR/ROR receptor family show affinity to a certain kind of ligand. Individual members of the RZR/ROR family may, for example, be produced by alternative splicing of a common DNA coding for said receptors.
Functional fragments of these receptors are, e.g.,. constructs that have the same properties with respect to ligand selectivity, e.g., constructs comprising the ligand binding domain and the DNA binding domain of a receptor of the RZR/ROR receptor family but are devoid of other domains or wherein other protein fragments to address some special properties to the RZR/ROR have been inserted. Also included are fragments that are constructed by combination of the ligand binding domain with other fragments that allow the decision whether a ligand is bound or not as, e.g., another DNA binding domain; or constructs wherein the ligand binding domain is connected via a spacer group to a solid carrier for fishing ligands.
Fragments of RZR/RORs including the functional ligand binding domain can also be labeled using one or more groups that can be identified easily, as for example a fluorescent, chemiluminescent or a radioactive group or can be connected to avidin, biotin, a reporter enzyme or any group easily detectable by spectroscopic or immunogenic methods like NMR, IR, UV, NMR, MS and ELISA. Constructs of this type can be used in the screening of compound libraries as described for example in Walter et al., TIBTECH (1993), 11, 247-254.
Most preferred members of the RZR/ROR receptor family are RZR/ROR&agr;, RZR/ROR&bgr; or RZR/ROR&ggr; (Hirose et al., Biochem, Biophys Res. Comm. (1994), 205, 1976-1983). RZR/RORs can be used as monomers or dimers including homodimers and heterodimers. Also possible are the splicing variants like ROR&agr;1 (Giguere et al., Genes and Development (1994), 8, 538-553) and the like.
The synthetical ligands known so far, and referred to above, have valuable pharmacological properties in the treatment of diseases of the autoimmune, rheum
Missbach Martin
Wiesenberg Irmgard
Elliott George C.
Ferraro Gregory D.
Novartis Corporation
Sandals William
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