Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-06-18
2003-10-28
McKane, Joseph K. (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S247000, C548S249000
Reexamination Certificate
active
06639078
ABSTRACT:
BACKGROUND OF THE INVENTION
Farnesoid X Receptor (FXR) is an orphan nuclear receptor initially identified from a rat liver cDNA library (8) that is most closely related to the insect ecdysone receptor. FXR is a member of the nuclear receptor family of ligand-activated transcription factors that includes receptors for the steroid, retinoid, and thyroid hormones (6). Northern and in situ analysis show that FXR is most abundantly expressed in the liver, intestine, kidney, and adrenal (8:22). FXR binds to DNA as a heterodimer with the 9-cis retinoic acid receptor RXR. The FXR/RXR heterodimer preferentially binds to response elements composed of two nuclear receptor half sites of the consensus AG(G/T)TCA organized as an inverted repeat and separated by a single nucleotide (IR-1 motif (8). An early report showed that rat FXR is activated by micromolar concentrations of farnesoids such as farnesol and juvenile hormone (8). However, these compounds failed to activate the mouse and human FXR, leaving the nature of the endogenous FXR ligand in doubt. The present invention demonstrates that several naturally-occurring bile acids bind and activate FXR at physiological concentrations (see also recent publications 17; 19; 24). As discussed in the present invention, the bile acids that serve as FXR ligands include chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine and glycine conjugates of these bile acids.
Bile acids are cholesterol metabolites that are formed in the liver and secreted into the duodenum of the intestine, where they have important roles in the solubilization and absorption of dietary lipids and vitamins. Most bile acids (~95%) are subsequently reabsorbed in the ileum and returned to the liver via the enterohepatic circulatory system. The conversion of cholesterol to bile acids in the liver is under feedback regulation: Bile acids down-regulate the transcription of cytochrome P450 7a (CYP7a), which encodes the enzyme that catalyzes the rate limiting step in bile acid biosynthesis. There are data to suggest that FXR is involved in the repression of CYP7a expression by bile acids, although the precise mechanism remains unclear (21). In the ileum, bile acids induce the expression of the intestinal bile acid binding protein (IBABP), a cytoplasmic protein which binds bile acids with high affinity and may be involved in their cellular uptake and trafficking. Two groups have now demonstrated that bile acids mediate their effects on IBABP expression through activation of FXR, which binds to an IR-1 type response element that is conserved in the human, rat, and mouse IBABP gene promoters (14; 17). Thus FXR is involved in both the stimulation (IBABP) and the repression (CYP7a) of target genes involved in bile acid and cholesterol homeostasis.
The present invention has made the useful discovery of ligands for FXR as well as methods of determining genes regulated by FXR interaction and affecting those genes. Thus this discovery provides methods of regulating bile acid and cholesterol homeostasis, fatty acid absorption, and protein and carbohydrate digestion.
SUMMARY OF THE INVENTION
The present invention provides a method of identifying compounds for the treatment of diseases or disorders modulated by FXR, comprising the step of determining whether the compound interacts directly with FXR, wherein a compound that interacts directly with FXR is a compound for the treatment.
The present invention also provides a compound that binds Farnesoid X receptor wherein the compound is of the following formula (II):
wherein X
1
is CH or N; X
2
is O or NH; R and R
1
may independently be H, lower alkyl, halogen, or CF
3
; R
2
is lower alkyl; R
3
and R
4
may independently be H, lower alkyl, halogen, CF
3
, OH, O-alkyl, or O-polyhaloalkyl.
The present invention further provides a method of modulating a gene whose expression is regulated by FXR in a mammal comprising administering to the mammal a ligand of FXR.
REFERENCES:
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Knowles AM, Lawson A. Action of 5-isoxazolones on enamines. J Chem Soc, Perk Trans.1. 1972;9-10:1240-3. (abstract only).*
Maloney et al., “Identification of a chemical tool for the orphan nuclear receptor FXR,”Journal of Medicinal Chemistry43(16):2971-2974 (Aug. 2000).
Krey et al., “Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay,”Molecular Endocrinology11(6):779-791 (Jun. 1997).
Nichols et al., “Development of a scintillation proximity assay for peroxisome proliferator-activated receptor &ggr; ligand binding domain,”Analytical Biochemistry257, 2):112-119 (Mar. 1998).
Nolte et al., “Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-&ggr;,”Nature395:137-143 (Sep. 1998).
Zhou et al., “Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer,”Molecular Endocrinology12(10):1594-1604 (Oct. 1998).
Makishima, M. et al.,Identification of a Nuclear Receptor for Bile Acids, Science, 284:1362-1365 (1999).
Parks, D.J. et al.,Bile Acids: Natural Ligands for an Orphan Nuclear Receptor, Science, 284:1365-1368 (1999).
Yao, T-P et al.,The nuclear hormone receptor coactivator SRC-1 is a specific target of p300, Proc. Natl. Acad. Sci. USA, 93:10626-10631 (1996).
Comment of Saul J. Karpen, M.D., Ph.D. on Parks et al.,Hepatology, 30:4 1107-1109 (Oct. 1999).
Haffner Curt Dare
Maloney Patrick Reed
Wilson Timothy Mark
Bennett Virginia C.
McKane Joseph K.
Small Andrea D.
SmithKline Beecham Corporation
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