U.S. patent application number 09/352816 was filed with the patent office on 2002-09-26 for use of rar antagonists as modulators of hormone mediated processes.
Invention is credited to EVANS, RONALD M., NAGY, LASZLO, TONTONOZ, PETER J..
Application Number | 20020137794 09/352816 |
Document ID | / |
Family ID | 23386621 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137794 |
Kind Code |
A1 |
EVANS, RONALD M. ; et
al. |
September 26, 2002 |
USE OF RAR ANTAGONISTS AS MODULATORS OF HORMONE MEDIATED
PROCESSES
Abstract
In accordance with the present invention, it has been discovered
that retinoic acid receptor (RAR) antagonists are capable of
modulating processes mediated by other members of the
steroid/thyroid hormone receptor superfamily, including permissive
receptors such as PPARs (e.g., PPAR.alpha., PPAR.delta. and
PPAR.gamma.). Indeed, it has been discovered that RAR antagonists,
in combination with agonists for members of the steroid/thyroid
hormone receptor superfamily, are capable of inducing and/or
enhancing processes mediated by such members.
Inventors: |
EVANS, RONALD M.; (LA JOLLA,
CA) ; TONTONOZ, PETER J.; (LOS ANGELES, CA) ;
NAGY, LASZLO; (SAN DIEGO, CA) |
Correspondence
Address: |
STEPHEN E. REITER
FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Family ID: |
23386621 |
Appl. No.: |
09/352816 |
Filed: |
July 13, 1999 |
Current U.S.
Class: |
514/549 |
Current CPC
Class: |
A61K 2800/70 20130101;
A61K 45/06 20130101 |
Class at
Publication: |
514/549 |
International
Class: |
A61K 031/47 |
Claims
That which is claimed is:
1. A composition for modulating hormone mediated process(es)
comprising: at least one agonist for a member of the
steroid/thyroid hormone receptor superfamily, at least one RAR
antagonist, and optionally, at least one agonist for a heterodimer
partner for said member.
2. A composition according to claim 1, wherein said member of the
steroid/thyroid hormone receptor superfamily is a permissive
receptor.
3. A composition according to claim 2, wherein said permissive
receptor is PPAR, NGFI-B, NURR1, FAR or LXR.
4. A composition according to claim 3, wherein said agonist for
said permissive receptor is a PPAR agonist.
5. A composition according to claim 4, wherein said PPAR agonist is
a hypolipidemic drug, a polyunsaturated fatty acid, an eicosanoid,
a thiazolidine, a benzene compound, an anti-inflammatory compound
(NSAID) , or mixtures thereof.
6. A composition according to claim 5, wherein said PPAR agonist is
troglitazone, WY14,643, GW0072, rosiglitazone (BRL 49653),
L-764406, 15-deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) and
oxidized linoleic acid (9- and 13-HODE), (2S)-((2-benzoylphenyl)
amino)-3-4-[2-(5-methyl-2-- phenyloxazol-4-yl) ethoxy]
phenylpropanoic acid, 2(S)-((2-benzoylphenyl)am-
ino)-3,4-[2-(5-methyl-2-pyridin-4-yloxazol-4-yl) ethoxy]
phenylpropionic acid, 2(S)-((2-benzoylphenyl)amino)-3-(4-2-
[5-methyl-2-(4-methylpiperazi- n-1-yl)thiazol-4-yl]ethoxyphenyl)
propionic acid, (2S)-3-(4-(benzyloxy)phe-
nyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)amino) propionic acid,
(2S)-((2-benzoylphenyl)amino)-3-4- [2-(methylpyridin-2-ylamino)
ethoxy ]phenylpropionic acid, 3-4-[2-(benzoxazol-2-ylmethylamino)
ethoxy] phenyl-(2S)-((2- benzoylphenyl)amino)propanoic acid, or
mixtures thereof.
7. A method according to claim 4 wherein said process is mediated
by PPAR-.alpha..
8. A method according to claim 4 wherein said process is mediated
by PPAR-.delta..
9. A method according to claim 4 wherein said process is mediated
by PPAR-.gamma..
10. A composition according to claim 1, wherein said heterodimer
partner is RXR or ultraspiracle.
11. A composition according to claim 10, wherein said agonist for
said heterodimer partner is an RXR agonist.
12. A composition according to claim 11, wherein said RXR agonist
is a 3-substituted (tetramethyltetrahydronaphthyl)carbonylbenzoic
acid, an
(E,E,E)-7-(1,2,3,4-tetrahydroquinolin-6-yl)-7-alkyl-6-fluoro-3-methylhept-
a-2, 4, 6-trienoic acid derivative, a diaryl sulfide retinoid
analog, phytanic acid, a tricyclic or trienic compound, or mixtures
thereof.
13. A composition according to claim 1, wherein said RAR antagonist
is a dicarba-closo-dodecaborane, a hydroanthracenyl, a
benzochromenyl and/or a benzothiochromenyl retinoid, a
diarylacetylene, a benzoic acid derivative; naphthalenyl analog, an
aryl-substituted, aryl and/or (3-oxo-1-propenly)-substituted
benzopyran, a benzothiopyran, a 1,2-dihydroquinoline, a
5,6-dihydronaphthalene derivatives, an adamantyl-substituted
biaromatic compound, a 1-phenyl-adamantane derivative, a
polyaromatic heterocyclic compound, a dihydronaphthalene
derivative, a 4-phenyl (benozopyranoyl or naphthoyl) amidobenzoic
acid derivative, a diazepinylbenzoic acid derivative, a
tetrahydronaphthalene derivative, an aryl- or
heteroarylcyclohexenyl substituted alkene, a dibenzofuran compound,
a N-aryl substituted tetrahydroquinolines, a
benzo[1,2-g]-chrom-3-ene or benzo[1,2-g]-thiochrom-3-ene
derivative, an aryl dihydronaphthalenyl derivative of acetylene, or
mixtures thereof.
14. A composition according to claim 1, wherein said RAR antagonist
is LE135, LE511, LE540, LE550, Ro41-5253, SR11330, SR11334, SR
11335, BMS453, BMS411, CD2366, CD2665, ER27191, AGN193109,
4-[4,5,7,8,9,10-hexahydro-7,7,10,-10-tetramethyl-1-(3
-pyridylmethyl)anthra[1,2-b]pyrrol-3-yl]benzoic acid,
4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)-5-thi-
aanthral[1,2-b]pyrrol-3-yl]benzoic acid,
4-[4,5,7,8,9,10-hexahydro-7,7,10,-
10-tetramethyl-1-(3pyridylmethyl)anthra[2,1 -d]pyrazol-3-yl]benzoic
acid, AGN193109, 4-[[5,6-dihydro-
-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalen- yl]ethynyl] benzoic
acid, or mixtures thereof.
15. A method for modulating hormone mediated process(es) in a
biological system, said method comprising introducing an effective
amount of at least one retinoic acid receptor (RAR) antagonist into
said system in combination with an effective amount of at least one
agonist for a member of the steroid/thyroid hormone superfamily of
receptors, wherein said agonist is not an agonist for retinoic acid
receptor.
16. A method according to claim 15, wherein said method relieves
the inhibition of retinoic acid receptor on process(es) mediated by
members of the steroid/thyroid hormone superfamily of
receptors.
17. A method according to claim 15, wherein said method enhances or
induces process(es) mediated by members of the steroid/thyroid
hormone superfamily of receptors.
18. A method according to claim 15 wherein said RAR antagonist is a
dicarba-closo-dodecaborane, a hydroanthracenyl, a benzochromenyl
and/or a benzothiochromenyl retinoid, a diarylacetylene, a benzoic
acid derivative; naphthalenyl analog, an aryl-substituted, aryl
and/or (3-oxo-1-propenly)-substituted benzopyran, a benzothiopyran,
a 1,2-dihydroquinoline, a 5,6-dihydronaphthalene derivatives, an
adamantyl-substituted biaromatic compound, a 1-phenyl-adamantane
derivative, a polyaromatic heterocyclic compound, a
dihydronaphthalene derivative, a 4-phenyl (benozopyranoyl or
naphthoyl) amidobenzoic acid derivative, a diazepinylbenzoic acid
derivative, a tetrahydronaphthalene derivative, an aryl- or
heteroarylcyclohexenyl substituted alkene, a dibenzofuran compound,
a N-aryl substituted tetrahydroquinolines, a
benzo[1,2-g]-chrom-3-ene or benzo[1,2-g]-thiochrom-3-ene
derivative, an aryl dihydronaphthalenyl derivative of acetylene, or
mixtures thereof.
19. A method according to claim 18 wherein said RAR antagonist is
an aryl dihydronaphthalenyl derivative of acetylene.
20. A method according to claim 15 wherein said RAR antagonist is
LE135, LE511, LE540, LE550, Ro41-5253, SR11330, SR1334, SR11335,
BMS453, BMS411, CD2366, CD2665, ER27191, AGN193109,
4-[4,5,7,8,9,10-hexahydro-7,7,10,-10--
tetramethyl-1-(3-pyridylmethyl)anthra[1,2-b]pyrrol-3-yl]benzoic
acid,
4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)-5-thi-
aanthral[1,2-b]pyrrol-3-yl]benzoic acid,
4-[4,5,7,8,9,10-hexahydro-7,7,10,-
10-tetramethyl-1-(3-pyridylmethyl)anthra[2,1-d]pyrazol-3-yl]benzoic
acid, AGN193109,
4-[[5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl-
]ethynyl] benzoic acid, or mixtures thereof.
21. A method according to claim 15, wherein said agonist is an
agonist for a permissive receptor.
22. A method according to claim 21, wherein said agonist is a PPAR
agonist.
23. A method according to claim 22, wherein said PPAR agonist is a
hypolipidemic drug, a polyunsaturated fatty acid, an eicosanoid, a
thiazolidine, a benzene compound, an anti-inflammatory compound
(NSAID), or mixtures thereof.
24. A method according to claim 15 wherein said process is mediated
by PPAR-.alpha..
25. A method according to claim 15 wherein said process is mediated
by PPAR-.delta..
26. A method according to claim 15 wherein said process is mediated
by PPAR-.gamma..
27. A method according to claim 15, wherein said method further
comprises introducing an effective amount of at least one agonist
for a heterodimer partner for said member.
28. A method according to claim 27, wherein said agonist for said
heterodimer partner is an RXR agonist.
29. A method according to claim 28, wherein said RXR agonist is a
3-substituted (tetramethyltetrahydronaphthyl)carbonylbenzoic acid,
a (E,E,E)-7-(1,2,3
,4-tetrahydroquinolin-6-yl)-7-alkyl-6-fluoro-3-methylhep- ta-2, 4,
6-trienoic acid derivative, a diaryl sulfide retinoid analog,
phytanic acid, a tricyclic or trienic compound, or mixtures
thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the modulation
of nuclear receptor mediated processes. In a particular aspect, the
present invention relates to methods for modulating the activity of
members of the steroid/thyroid hormone receptor superfamily by
relieving the inhibition of hormone mediated processes caused by
retinoic acid receptor, or agonists thereof. In another aspect, the
present invention relates to methods for inducing hormone mediated
processes.
BACKGROUND OF THE INVENTION
[0002] A central problem in eukaryotic molecular biology continues
to be the elucidation of molecules and mechanisms that mediate
specific gene regulation. As part of the scientific attack on this
problem, a great deal of work has been done in efforts to identify
ligands (i.e., exogenous inducers) which are capable of mediating
specific gene regulation. Additional work has been done in efforts
to identify other molecules involved in specific gene
regulation.
[0003] Although much remains to be learned about the specifics of
gene regulation, it is known that ligands modulate gene
transcription by acting in concert with intracellular components,
including intracellular receptors and discrete DNA sequences known
as hormone response elements (HREs). The identification of
compounds which directly or indirectly interact with intracellular
receptors, and thereby affect transcription of hormone-responsive
genes, would be of significant value, e.g., for therapeutic
applications.
[0004] The actions of steroids, retinoids and thyroid hormones are
mediated by intracellular nuclear receptors whose coordinate
activity defines the physiological response (Mangelsdorf and Evans,
Cell 83:841-850 (1995)). These receptors are all structurally
related and constitute a superfamily of nuclear regulatory proteins
that modulate gene expression in a ligand-dependent fashion.
Previous studies have demonstrated that the 9-cis retinoic acid
receptor (RXR) serves as a common heterodimeric partner for thyroid
hormone receptor (TR), retinoic acid receptor (RAR), vitamin D
receptor (VDR), prostanoids (PPAR), as well as numerous orphan
receptors (Kliewer et al. (1992) Nature 355:446-449).
[0005] Nuclear hormone receptor heterodimers can be classified into
two distinct groups based upon their transcriptional responses to
synthetic RXR ligands. So called "permissive" heterodimers such as
PPAR:RXR, respond to either RXR and/or PPAR ligands and the two
together have, at least, an additive effect (see, e.g., Mukherjee
et al., Nature 386:407-10 (1997)). In contrast, so called
"non-permissive" heterodimers, such as RAR:RXR, do not respond to
RXR ligands unless ligands for RAR are already present, in which
case they yield an additive or synergistic response (Apfel et al.,
J Biol Chem. 270(51):30765-72.(1995); Chen et al. PNAS 93:7567-7571
(1996)). Other non-permissive heterodimers include TR:RXR and
VDR:RXR heterodimers, which also do not appear to be activated by
RXR ligands. Indeed, the RXR ligand, LG100268, appears to partially
antagonize the action of thyroid hormone.
[0006] This difference between permissive and non-permissive
heterodimers is likely to be important for regulating the activity
of naturally occurring RXR ligands (Heyman et al., Cell (1992)
68:397-406; Mascrez et al., Development (1998) 125(23):4691-707;
Solomin et al., Nature (1998) 395(6700):398-402; Fujita and
Mitsuhashi, Biochem Biophys Res Commun (1999) 255(3):625-30)] as
well as being crucial to understanding the behavior of synthetic
compounds currently under development as both anti-cancer and
anti-diabetic agents (see, e.g., Anzano et al., Cancer Research
(1994) 54:4614-4617, Gottardis et al., Cancer Research (1996)
56:5566-5570, Mukherjee et al., supra). It has been also suggested
that RXR can function as a homodimer (Mangelsdorf et al., Cell
66(3):555-61 (1991)). By competing for dimerization with RXR on
response elements, the relative abundance of RAR and PPAR
determines whether the RXR signaling pathway will be
functional.
[0007] PPAR.alpha. is a permissive member of the nuclear receptor
superfamily, which includes receptors for the steroid, thyroid and
retinoid hormones (see Mangelsdorf & Evans in Cell 83:841-50
(1995)). Two other PPAR.alpha.-related genes (PPAR.gamma. and
PPAR.delta.) have been identified in mammals. PPAR.gamma. is highly
enriched in adipocytes, while the .delta. isoform is ubiquitously
expressed (see Schoonjans et al., in Biochim Biophys Acta
1302:93-109 (1996)). Like other members of this receptor
superfamily, all of the PPAR isoforms contain a central DNA binding
domain that recognizes response elements in the promoters of their
target genes (see, for example, Latruffe et al. in Biochimie
79:81-94 (1997)). PPAR response elements (PPRE) are composed of a
directly repeating core-site separated by 1 nucleotide (see Kliewer
et al., in Nature 358:771-4 (1992)). In order to recognize a PPRE,
PPARs must heterodimerize with the 9-cis retinoic acid receptor
(RXR).
[0008] The peroxisome proliferator activated receptors (PPARs)
preferentially bind to DNA, i.e., response elements, as
heterodimers with a common partner, the retinoid X (or 9-cis
retinoic acid) receptor (RXR; see, for example, Kliewer et al., in
Nature 355:446-449 (1992); Leid et al, in Cell 68:377-395 (1992);
Marks et al., in EMBO J 11:1419-1435 (1992); Zhang et al., in
Nature 355:441-446 (1992); and Issemann et al., in Biochimie.
75:251-256 (1993). Once bound to a response element, PPARs activate
transcription following binding of ligand to the C-terminal ligand
binding domain thereof. Due to the key role of ligands for the
activation of transcription, an intense search for the
identification of ligands for members of the PPAR family has been
undertaken by a number of research groups.
[0009] PPAR.alpha. has been identified as a vertebrate nuclear
hormone receptor which regulates genes involved in fatty acid (FA)
degradation (.delta. and .omega.-oxidation; see Schoonjans et al.,
in Biochim Biophys Acta 1302:93-109 (1996)). PPAR.alpha. is highly
expressed in the liver and was originally identified by Green and
colleagues as a molecule that mediates the transcriptional effects
of drugs that induce peroxisome proliferation in rodents (see
Issemann & Green in Nature 347:645-50 (1990)). Mice lacking
functional PPAR.alpha. are incapable of responding to these agents
and fail to induce expression of a variety of genes required for
the metabolism of FAs in peroxisomes, mitochondria and other
cellular compartments (see Lee et al., in Mol Cell Biol
15:3012-3022 (1995)). As a result, PPAR.alpha.-deficient mice
inappropriately accumulate lipid in response to pharmacologic
stimuli.
[0010] PPAR.alpha. appears to regulate FA oxidation, suggesting
that PPAR.alpha. ligands may represent endogenous signals for FA
degradation (see Schoonjans et al., supra). Fatty acids (FAs) are
ubiquitous biological molecules that are utilized as metabolic
fuels, as covalent regulators of signaling molecules and as
essential components of cellular membranes. It is thus logical that
FA levels should be closely regulated. Indeed, some of the most
common medical disorders in industrialized societies (e.g.,
cardiovascular disease, hyperlipidemia, obesity and insulin
resistance) are characterized by altered levels of FAs or their
metabolites (see, for example, Durrington, in Postgrad Med J 69
Suppl 1, S18-25; discussion S25-9 (1993) and Reaven, in J Intern
Med Suppl 736:13-22 (1994)).
[0011] PPAR.gamma. is preferentially expressed in adipose tissue.
PPAR.gamma.-activation leads to adipocyte differentiation and
improved insulin signaling of mature adipocytes.
15-deoxy-.DELTA..sup.12,14-prosta- glandin J.sub.2 (15d-J.sub.2)
has been identified as a ligand for PPAR.gamma. (see, for example,
Forman et al., in Cell 83:803-12 (1995) and Kliewer et al., in Cell
83:813-9 (1995)). Activation of PPAR.gamma. by 15d-J.sub.2 or its
synthetic analogs (e.g., thiazolidinediones; see Forman et al.,
supra) promotes differentiation of pre-adipocytes into mature,
triglyceride-containing fat cells. Similarly, thiazolidinediones
have been shown to increase body weight in animals (see, e.g.,
Zhang et al. (1996) J Biol Chem 271:9455-9459), suggesting that
15d-J.sub.2 may be utilized as an in vivo signal to store fatty
acids (FAs) in the form of triglycerides.
[0012] Accordingly, there is a need in the art for new agents and
compositions which allow the modulation of hormone mediated
processes. This and other needs in the art are addressed by the
present invention.
BRIEF DESCRIPTION OF THE INVENTION
[0013] In accordance with the present invention, it has been
discovered that retinoic acid receptor (RAR) antagonists are
capable of modulating processes mediated by other members of the
steroid/thyroid hormone receptor superfamily, including permissive
receptors such as PPARs (e.g., PPAR.alpha., PPAR.delta. and
PPAR.gamma.). Indeed, it has been discovered that RAR antagonists,
in combination with agonists for members of the steroid/thyroid
hormone receptor superfamily, are capable of inducing and/or
enhancing processes mediated by such members.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 illustrates the effects of AGN193109 on PPAR.gamma.
response (induction of CD14 expression in myeloid cell lines), by
comparing HL-60 (intact PPAR.gamma. response) and HL-60-CDM-1 cells
(impaired PPAR.gamma. response, wherein no PPAR.gamma. is
expressed).
[0015] FIG. 2 illustrates the effects of the combination of the
PPAR-.gamma. agonist, PG-J2, the RXR agonist, LG 268, and the RAR
antagonist, AGN 193109, on HL-60 cells.
[0016] FIG. 3 provides several graphs summarizing the flow
cytometry data for experiments depicted in FIG. 2.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In accordance with the present invention, there are provided
compositions for modulating hormone mediated process(es)
comprising:
[0018] at least one agonist for a member of the steroid/thyroid
hormone receptor superfamily, and
[0019] optionally, at least one agonist for a heterodimer partner
for said member; and
[0020] at least one antagonist for a second member of the
steroid/thyroid hormone receptor superfamily, preferably for a
non-permissive member such as RAR.
[0021] As employed herein, the phrase "members of the nuclear
receptor superfamily" (also known as "members of the
steroid/thyroid hormone superfamily of receptors" or "intracellular
receptors") refers to hormone binding proteins that operate as
ligand-dependent transcription factors, including identified
members of the steroid/thyroid hormone superfamily of receptors for
which specific ligands have not yet been identified (referred to
hereinafter as "orphan receptors"). These hormone binding proteins
have the intrinsic ability to bind to specific DNA sequences.
Following binding, the transcriptional activity of target gene
(i.e., a gene associated with the specific DNA sequence) is
modulated as a function of the ligand bound to the receptor.
[0022] A member of the superfamily can be identified as a protein
which contains the above-mentioned invariant amino acid residues,
which are part of the DNA-binding domain of such known steroid
receptors as the human glucocorticoid receptor (amino acids
421-486), the estrogen receptor (amino acids 185-250), the
mineralocorticoid receptor (amino acids 603-668), the human
retinoic acid receptor (amino acids 88-153), and the like. The
highly conserved amino acids of the DNA-binding domain of members
of the superfamily are as follows:
1 Cys-X-X-Cys-X-X-Asp*-X-Ala*-X-Gly*-X-Tyr*-X-
X-X-X-Cys-X-X-Cys-Lys*-X-Phe-Phe-X-Arg*-X-X
-X-X-X-X-X-X-X-(X-X-)Cys-X-X-X-X-X-(X-X-
X-)Cys-X-X-X-Lys-X-X-Arg-X-X-Cys-X-X-Cys-
Arg*-X-X-Lys*-Cys-X-X-X-Gly*-Met (SEQ ID NO:1);
[0023] wherein X designates non-conserved amino acids within the
DNA-binding domain; the amino acid residues denoted with an
asterisk are residues that are almost universally conserved, but
for which variations have been found in some identified hormone
receptors; and the residues enclosed in parenthesis are optional
residues (thus, the DNA-binding domain is a minimum of 66 amino
acids in length, but can contain several additional residues).
[0024] Members of the steroid/thyroid hormone superfamily of
receptors (including the various isoforms thereof) include steroid
receptors such as glucocorticoid receptor (GR), mineralocorticoid
receptor (MR), estrogen receptor (ER), progesterone receptor (PR),
androgen receptor (AR), vitamin D.sub.3 receptor (VDR), and the
like; plus retinoid receptors, such as the various isoforms of
retinoic acid receptor (e.g., RAR.alpha., RAR.beta. or RAR.gamma.),
the various isoforms of retinoid X (or 9-cis retinoic acid)
receptor (e.g., RXR.alpha., RXR.beta., or RXR.gamma.), various
isoforms of peroxisome proliferator-activated receptors (e.g.,
PPAR.alpha., PPAR.gamma., PPAR.delta.) and the like (see, e.g.,
U.S. Pat. Nos. 4,981,784; 5,171,671; and 5,071,773); thyroid
hormone receptor (T.sub.3R), such as TR.alpha., TR.beta., and the
like; steroid and xenobiotic receptor (SXR, see for example,
Blumberg et al., Genes Dev (1998) 12(20):3195-205), RXR-interacting
proteins (RIPs; see, e.g., Seol et al., Mol Endocrinol (1995)
9(1):72-85; Zavacki et al., Proc Natl Acad Sci USA (1997)
94(15):7909-14) including farnesoid X receptor (FXR; see for
example, Forman et al., Cell (1995) 81(5):687-93), BXR (Blumberg et
al., Genes Dev (1998) 12(9):1269-77), Hanley et al., J Clin Invest
(1997) 100(3):705-12, O'Brien et al., Carcinogenesis (1996)
17(2):185-90), pregnenolone X receptor (PXR; see for example,
Schuetz et al., Mol Pharmacol (1998) 54(6):1113-7), liver X
receptor (LXR, see, e.g., Peet et al., Curr Opin Genet Dev (1998)
8(5):571-5), insect derived receptors such as the ecdysone receptor
(EcR), the ultraspiracle receptor (see, for example, Oro et al., in
Nature 347:298-301 (1990)), and the like; as well as other gene
products which, by their structure and properties, are considered
to be members of the superfamily, as defined hereinabove, including
the various isoforms thereof (see, e.g., Laudet, V., J Mol
Endocrinol (1997) 19(3):207-26).
[0025] In accordance with the present invention, the compositions
for modulating hormone mediated process are capable of modulating
the activity of complexes comprising homodimeric or heterodimeric
member(s) of the steroid/thyroid hormone superfamily of receptors.
It is readily recognized that a number of receptors preferentially
bind to DNA as homodimers or heterodimers. Homodimeric members of
the steroid/thyroid hormone receptor superfamily include GR, TR,
RAR, RXR, and the like (see, e.g., Beato et al., Steroids
61(4):240-51 (1996)). Alternatively, RAR, VDR, TR, PPAR, SXR, ORI,
SHP, LXR, BXR, and the like, preferentially form heterodimers with
a common partner, e.g., RXR (see, for example, Dong et al.,
Biochemistry (1998) 27(30):10691-700; Yu et al., in Cell
67:1251-1266 (1991); Bugge et al., in EMBO J. 11:1409-18 (1992);
Kliewer et al., in Nature 355:446-449 (1992); Leid et al, in Cell
68:377-395 (1992); Marks et al., in EMBO J 11:1419-1435 (1992);
Zhang et al., in Nature 355:441-446 (1992); and Issemann et al., in
Biochimie. 75:251-256 (1993). Similarly, other receptors, e.g.,
EcR, will form heterodimers with the RXR homolog, ultraspiracle. In
a preferred embodiment of the present invention, the invention
compositions will modulate the activity of permissive heterodimers.
Permissive heterodimeric members of the steroid/thyroid hormone
receptor superfamily are well known to those skilled in the art and
include PPAR:RXR, LXR:RXR, NGFI-B:RXR, NURR1:RXR, FXR:RXR, BXR:RXR,
SXR:RXR, and the like.
[0026] As employed herein, the term "agonist (or agonist precursor)
for a member of the steroid/thyroid hormone superfamily of
receptors" (i.e., intracellular receptor) refers to a substance or
compound which, in its unmodified form (or after conversion to its
"active" form), inside a cell, binds to receptor protein, thereby
creating an agonist/receptor complex, which in turn can activate an
appropriate hormone response element. An agonist therefore is a
compound which acts to modulate gene transcription for a gene
maintained under the control of a hormone response element, and
includes compounds such as hormones, growth substances, non-hormone
compounds that modulate growth, and the like. Agonists include
steroid or steroid-like hormone, retinoids, thyroid hormones,
pharmaceutically active compounds, and the like. Individual
agonists may have the ability to bind to multiple receptors.
Preferably, the agonist is for a member which forms a heterodimer
(or homodimer) with additional or other members. In a more
preferred embodiment, the agonist for a member of the
steroid/thyroid hormone superfamily of receptors is an agonist for
a permissive receptor, such as PPAR, LXR, RXR interacting proteins
(RIPs including, for example, FXR), and the like. In the most
preferred embodiment, the agonist is for a member other than
retinoic acid receptor.
[0027] Agonists for individual members of the steroid/thyroid
hormone superfamily of receptor are well known in the art. For
example, agonists for retinoids are described in The Retinoids :
Biology, Chemistry, and Medicine (Sporn, Roberts & Goodman,
eds. (Raven Press, 1993), the entire contents of which is hereby
incorporated by reference herein).
[0028] Peroxisome proliferator-activated receptor (PPAR) agonist(s)
contemplated for use herein are well known in the art. See, for
example, Peroxisome Proliferators: Unique Inducers of
Drug-Metabolizing Enzymes (Pharmacology and Toxicology) David E.
Moody (Editor) (July 1994), the entire contents of which are hereby
incorporated by reference herein. As readily recognized by those of
skill in the art, a variety of PPAR agonists, both synthetic and
naturally occurring, can be used in accordance with the present
invention. Exemplary PPAR agonists include hypolipidemic drugs,
polyunsaturated fatty acids, eicosanoids, thiazolidines (Komers et
al., Physiol Res (1998) 47(4):215-25), benzene compounds,
anti-inflammatory compounds (NSAIDs), and the like (see, e.g.,
Tajima et al., W09915520, Collins et al., J Med Chem 41(25):5037-54
(1998), Forman et al., Proc Natl Acad Sci U S A (1997)
94(9):4312-7, Willson et al., Curr Opin Chem Biol (1997)
1(2):235-41, Sorensen et al., Vitam Horm (1998) 54:121-66, Berger
et al., J Biol Chem (1999) 274(10):6718-25, Wilson et al., Ann N Y
Acad Sci (1996) 804:276-83). Preferred PPAR agonists include
troglitazone, WY14,643, GW0072, rosiglitazone (BRL 49653),
L-764406, 15-deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) and
oxidized linoleic acid (9-and 13-HODE), (2S)-((2-benzoylphenyl)
amino)-3-4-[2-(5-methyl-2-phenyloxazol-4-yl) ethoxy]
phenylpropanoic acid, 2(S)-((2-benzoylphenyl)amino)-3,4-
[2-(5-methyl-2-pyridin-4-yloxazol-4-yl) ethoxy] phenylpropionic
acid, 2(S)-((2-benzoylphenyl)amino)-3-(4-2-
[5-methyl-2-(4-methylpiperazin-1-yl- )thiazol-4-yl]ethoxyphenyl)
propionic acid, (2S)-3-(4-(benzyloxy)phenyl)-2-
-((1-methyl-3-oxo-3-phenylpropenyl)amino) propionic acid,
(2S)-((2-benzoylphenyl)amino)-3-4- [2-(methylpyridin-2-ylamino)
ethoxy ]phenylpropionic acid, 3-4-[2-(benzoxazol-2-ylmethylamino)
ethoxy] phenyl-(2S)-((2- benzoylphenyl)amino)propanoic acid, and
the like.
[0029] Additional agonists contemplated for use in the practice of
the present invention depend on the target receptor and are known
to those skilled in the art, including benzoate metabolites for BXR
(e.g., Blumberg et al., Genes Dev. (1998) 12(9):1269-77),
farnesoids and bile acids for FXR (e.g., Parks et al., Science
(1999) 284(5418): 1365-8), oxysterols for LXR (e.g., Janowski et
al., Proc Natl Acad Sci U S A (1999) 96(1):266-71), and the
like.
[0030] The invention composition further optionally comprises at
least one agonist for a second member of the steroid/thyroid
hormone superfamily of receptors, i.e., a heterodimer partner.
Those of skill in the art readily recognize those members which can
form heterodimer partners, including RXR, ultraspiracle NGFI-B,
NURR1, and the like. RXR agonist(s) contemplated for use herein are
well known in the art. See, for example, The Retinoids, supra. As
readily recognized by those of skill in the art, a variety of RXR
agonists, both synthetic and naturally occuring, can be used in
accordance with the present invention. Examplary RXR agonists
include 3-substituted
(tetramethyltetrahydronaphthyl)carbonylbenzoic acids (Canan Koch et
al., J Med Chem (1999) 42(4):742-50),
(E,E,E)-7-(1,2,3,4-tetrahydroquinolin-6-yl)-7-alkyl-6-fluoro-3-methylhept-
a-2, 4, 6-trienoic acid derivatives (Hibi et al., J Med Chem (1998)
41(17):3245-52), diaryl sulfide retinoid analogs (Beard et al., J
Med Chem (1996) 39(18):3556-63), phytanic acid (Lemotte et al., Eur
J Biochem (1996) 236(1):328-33), tricyclic compounds (U.S. Pat.
Nos. 5,770,383, 5,770,382 and 5,770,378), trienic compounds (U.S.
Pat. No. 5,721,103), and the like (see also, Jiang et al., Biochem
Pharmacol (1995) 50(5):669-76), Heyman WO9710819). Preferred RXR
agonists include LGD1069 (Bischoff et al., Cancer Res (1998)
58(3):479-84), LG100153,
E(-2-[2-(5,6,7,8-Tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)
propen-1-yl]-4-thiophenecarboxylic acid (AGN 191701),
2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-2-(4-carboxylphenyl-
)-1,3-dioxolane (SR11237), Standeven et al., Biochem Pharmacol
(1997) 54(4):517-24), HX600 or HX630, and the like.
[0031] In accordance with the present invention, the invention
compositions will comprise at least one antagonist for members of
the steroid/thyroid hormone superfamily of receptors, including
antagonists for other non-permissive receptors, such as antagonists
for RAR, TR, VDR, and the like. Preferably, the antagonist will be
an RAR antagonist. Those of skill in the art will readily recognize
antagonists which can be employed in the practice of the present
invention. As readily recognized by those of skill in the art, a
variety of retinoic acid receptor (RAR) antagonists, both synthetic
and naturally occuring, can be used in accordance with the present
invention. Examplary RAR antagonists include
dicarba-closo-dodecaboranes (Iijima et al., Chem Pharm Bull (Tokyo)
(1999) 47(3):398-404), hydroanthracenyl, benzochromenyl and
benzothiochromenyl retinoids (Vuligonda et al., Bioorg Med Chem
Lett (1999) 9(5):743-8), diarylacetylenes, benzoic acid derivatives
(see, e.g., Kagechika, H. (1994) Yakugaku Zasshi 114(11):847-862;
Eckhardt et al. (1994) Toxicol Lett 70(3):299-308; Yoshimura et al.
(1995) J Med Chem 38(16):3163-3173; Chen et al. (1995) EMBO
14(6):1187-1197; Teng et al. (1997) J Med Chem 40(16):2445-2451);
naphthalenyl analogs (see, e.g., Johnson et al. (1995) J Med Chem
38(24):4764-4767; Agarwal et al. J Biol Chem 271(21):12209-12212:
Umemiya et al. (1996) Yakugaku Zasshi 116(12):928-941);
aryl-substituted and aryl and (3-oxo-1-propenly)-substi- tuted
benzopyran, benzothiopyran, 1,2-dihydroquinoline, and
5,6-dihydronaphthalene derivatives (Klein et al. U.S. Pat. Nos.
5,877,207 and 5,776,699), adamantyl-substituted biaromatic
compounds (Bernardon and Charpentier, U.S. Pat. No. 5,877,342),
1-phenyl-adamantane derivatives (Bernardon and Bernardon EP
776885), polyaromatic heterocyclic compounds (Charpentier et al.
U.S. Pat. No. 5,849,798), dihydronaphthalene derivatives (Beard et
al., U.S. Pat. No. 5,808,124 and Johnson et al. U.S. Pat. No.
5,773,594), 4-phenyl (benozopyranoyl or naphthoyl) amidobenzoic
acid derivatives (Chandraratna et al. WO 98/US/13065),
diazepinylbenzoic acid derivatives (Umemiya et al., J Med Chem
(1997) 40(26):4222-34), tetrahydronaphthalene derivatives
(Vuligonda et al. U.S. Pat. No. 5,763,635, 5,741,896 and
5,723,666), aryl- and heteroarylcyclohexenyl substituted alkenes
(Beard et al. U.S. Pat. No. 5,760,276), dibenzofuran compounds,
including aromatic dibenzofuran compounds (Charpentier et al. U.S.
Pat. No. 5,702,710, Charpentier and Bernard U.S. Pat. No.
5,747,530), N-aryl substituted tetrahydroquinolines (Beard et al.
U.S. Pat. No. 5,739,338), benzo[1,2-g]-chrom-3-ene and
benzo[1,2-g]-thiochrom-3-ene derivatives (Vuligonda et al. U.S.
Pat. No. 5,728,846), and the like (see also, Chandraratna, R A,
Cutis (1998) 61(2 Suppl):40-5).
[0032] Examples of specific RAR antagonists contemplated for use
herein include LE135 (Umemiya et al. (1996) Yakugaku Zasshi
116(12):928-941), LE511, LE540, LE550 (Li et al., J Biol Chem
(1999) 274(22):15360-6; Umemiya et al. (1996) Yakugaku Zasshi
116(12):928-941), Ro4l-5253 (Keidel et al. (1994) Mol Cell Biol
14(1):287-298), SR11330, SR11334, SR11335 (Lee et al. (1996) J Biol
Chem 271(20):11897-11903), BMS453, BMS411 (Chen et al. (1995) EMBO
14(6):1187-1197), CD2366 and CD2665 (Meister et al., Anticancer
Res. (1998) 18(3A):1777-1786), ER27191 (Uemo et al., Leuk. Res.
(1998) 22(6):517-525), AGN 193109 (Johnson et al., Bioorg Med Chem
Lett (1999) 9(4):573-6), 4-[4,5,7,8,9,10-hexahydro-7,7,10,-i
0-tetramethyl- 1 -(3-pyridylmethyl)anthra[1,2-b]pyrrol-3
-yl]benzoic acid,
4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)-
-5-thiaanthral[1,2-b]pyrrol-3-yl]benzoic acid,
4-[4,5,7,8,9,10-hexahydro-7-
,7,10,10-tetramethyl-1-(3-pyridylmethyl)anthra[2,1-d]pyrazol-3-yl]benzoic
acid (Yoshimura et al. (1995) J Med Chem 38(16):3163-3173),
AGN193109 (Agarwal et al. J Biol Chem 271(21):12209-12212), and the
like. A presently preferred class of RAR antagonists contemplated
for use according to the invention are aryl dihydronaphthalenyl
derivatives of acetylene. An especially preferred RAR antagonist
contemplated for use herein is
4-[[5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]-
ethynyl] benzoic acid.
[0033] In accordance with another aspect of the present invention,
there are provided methods for modulating hormone mediated
processes, the methods comprising introducing an effective amount
of at least one retinoic acid receptor (RAR) antagonist in
combination with at least one agonist for the member into the
system. Preferably, the agonist is an agonist for a member of the
steroid/thyroid hormone superfamily of receptors which form
heterodimers or homodimers with other or additional members. More
preferably, the agonist is for a permissive receptor. In the
presently most prefered embodiment, the agonist is not for retinoic
acid receptor.
[0034] In yet another preferred embodiment of the present
invention, there are provided methods for relieving, in a
biological system, the inhibition of hormone mediated process(es),
such as process(es) mediated by peroxisome proliferator activated
receptor(s) (PPARs). In a presently preferred aspect, the invention
method relieves inhibition of hormone mediated processes caused by
retinoic acid receptor (RAR) and agonist(s) thereof. Alternatively,
or in addition, there are provided methods for inducing hormone
mediated process(es) in a biological system by peroxisome
proliferator activated receptor(s) (PPARs). Invention methods
comprise introducing an effective amount of at least one retinoic
acid receptor (RAR) antagonist, alone, or in combination with PPAR
agonists, into said system.
[0035] In accordance with yet another embodiment of the present
invention, the invention method further comprises administering or
co-administering an effective amount of at least one agonist for a
heterodimer partner for the member, e.g., RXR, NURR1,
ultraspiracle, and the like.
[0036] As employed herein, the term "modulate" refers to the
ability of compound(s) to either directly (by binding to the
receptor as a ligand) or indirectly (by relieving the inhibition of
process(es) mediated by members of the steroid/thyroid hormone
receptor superfamily, or as a precursor and/or facilitator for a
ligand or an inducer which promotes production of ligand from a
precursor, or combinations thereof) induce and/or enhance
expression of gene(s) maintained under hormone expression control,
or to repress expression of gene(s) maintained under such
control.
[0037] As employed herein, the phrase "relieving . . . the
inhibition of process(es) . . . " refers to the ability of a
suitable compound, e.g., RAR antagonist, to either directly (by
binding to the receptor as a ligand) or indirectly (as a precursor
for a ligand or an inducer which promotes production of ligand from
a precursor) counteract or divert the ability of members of the
steroid/thyroid hormone receptor superfamily, and agonists thereof,
to inhibit or interfere with the expression of gene(s) maintained
under hormone expression control. Preferably, members of the
steroid/thyroid hormone receptor superfamily contemplated for use
in the practice of the present invention include non-permissive
receptors such as retinoic acid receptor.
[0038] As employed herein, the term "inducing" refers to the
ability of a modulator for a member of the steroid/thyroid hormone
receptor superfamily to either directly (by binding to the receptor
as a ligand) or indirectly (as a precursor for a ligand or an
inducer which promotes production of ligand from a precursor)
promote expression of gene(s) maintained under hormone expression
control.
[0039] As employed herein, the phrase "biological system" refers to
an intact organism or a cell-based system containing the various
components required for response to the ligands described herein,
e.g., a member of the steroid/thyroid hormone receptor superfamily,
a heterodimer partner for the member (e.g., RXR), and a reporter
responsive to the heterodimer (which typically comprises a response
element (RE) in operative communication with a reporter gene;
suitable reporters include luciferase, chloramphenicol transferase,
.beta.-galactosidase, and the like).
[0040] As employed herein, the phrase "hormone mediated processes"
refers to biological, physiological, endocrinological, and other
bodily processes which are mediated by receptor or receptor
combinations which are responsive to the ligands described herein.
Modulation of such processes can be accomplished in vitro or in
vivo. In vivo modulation can be carried out in a wide range of
subjects, such as, for example, humans, rodents, sheep, pigs, cows,
and the like. Exemplary processes contemplated to be modulated
include neoplastic diseases, inflammatory or infectious diseases,
and the like.
[0041] As employed herein, the phrase "process(es) mediated by
peroxisome proliferator activated receptor(s) (PPARs)" refers to
processes which are manifested by expression or repression of
expression of PPAR-responsive genes. Thus, for example,
"PPAR.alpha.-responsive genes" refers to genes whose expression
products are involved in the biological, physiological,
endocrinological, and other bodily processes which are mediated by
receptor or receptor combinations which are responsive to the
PPAR.alpha. ligands described herein (e.g., genes involved in fatty
acid metabolism in peroxisomes, mitochondria and other cellular
compartments (including FA degradation (by .beta.- and
.omega.-oxidation), and the like). Modulation of such processes can
be accomplished in vitro or in vivo. In vivo modulation can be
carried out in a wide range of subjects, such as, for example,
humans, rodents, sheep, pigs, cows, and the like.
[0042] As employed herein, the phrase "PPAR.delta.-responsive
genes" refers to genes whose expression products are involved in
the biological, physiological, endocrinological, and other bodily
processes which are mediated by receptor or receptor combinations
which are responsive to PPAR.delta. ligands. Modulation of such
processes can be accomplished in vitro or in vivo. In vivo
modulation can be carried out in a wide range of subjects, such as,
for example, humans, rodents, sheep, pigs, cows, and the like.
[0043] As employed herein, the phrase "PPAR.gamma.-responsive
genes" refers to genes whose expression products are involved in
the biological, physiological, endocrinological, and other bodily
processes which are mediated by receptor or receptor combinations
which are responsive to PPAR-.gamma. ligands (e.g., cell
differentiation to produce lipid-accumulating cells, regulation of
insulin-sensitivity and blood glucose levels, especially as related
to hypoglycemia/hyperinsulinism (resulting, for example, from
abnormal pancreatic beta-cell function, insulin-secreting tumors
and/or autoimmune hypoglycemia due to autoantibodies to insulin,
the insulin receptor or autoantibodies that are stimulatory to
pancreatic beta-cells), the formation of macrophages which lead to
the development of atherosclerotic plaques, and the like).
Modulation of such processes can be accomplished in vitro or in
vivo. In vivo modulation can be carried out in a wide range of
subjects, such as, for example, humans, rodents, sheep, pigs, cows,
and the like.
[0044] As employed herein, the phrase "effective amount" refers to
levels of compound sufficient to provide circulating concentrations
high enough to modulate the expression of an isoform of PPAR. Such
a concentration typically falls in the range of about 10 nM up to 2
mM; with concentrations in the range of about 100 nM up to 500 nM
being preferred. Since the activity of different compounds
described herein may vary considerably, and since individual
subjects may present a wide variation in severity of symptoms, it
is up to the practitioner to determine a subject's response to
treatment and vary the dosages accordingly.
[0045] The above-described biologically active compounds can be
administered in a variety of forms (e.g., in combination with a
pharmaceutically acceptable carrier therefor) and by a variety of
modes of delivery. Exemplary pharmaceutically acceptable carriers
include carriers suitable for oral, intravenous, subcutaneous,
intramuscular, intracutaneous, and the like administration.
Administration in the form of creams, lotions, tablets, dispersible
powders, granules, syrups, elixirs, sterile aqueous or non-aqueous
solutions, suspensions or emulsions, and the like, is
contemplated.
[0046] For the preparation of oral liquids, suitable carriers
include emulsions, solutions, suspensions, syrups, and the like,
optionally containing additives such as wetting agents, emulsifying
and suspending agents, sweetening, flavoring and perfuming agents,
and the like.
[0047] For the preparation of fluids for parenteral administration,
suitable carriers include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized, for example, by filtration through
a bacteria-retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured in the form
of sterile water, or some other sterile injectable medium
immediately before use.
[0048] As used herein, co-administration of two pharmacologically
active compounds refers to the delivery of two separate chemical
entities, whether in vitro or in vivo. Co-administration refers to
the simultaneous delivery of separate agents; to the simultaneous
delivery of a mixture of agents; as well as to the delivery of one
agent followed by delivery of the second agent. In all cases,
agents that are co-administered are intended to work in conjunction
with each other.
[0049] Each of the references and U.S. and foreign patents cited
herein are hereby incorporated by reference in its entirety. The
invention will now be described in greater detail by reference to
the following non-limiting examples.
Example 1
Cell Culture and Transfection
[0050] CV-1 cells were grown and transfected as described by Forman
et al., in Cell 83:803-12 (1995). The reporter construct, PPREx3
TK-LUC, contained 3 copies of the acyl CoA oxidase PPRE upstream of
the Herpes virus thymidine kinase promoter (see Kliewer et al., in
Nature 358:771-4 (1992)). Expression vectors contained the
cytomegalovirus IE promoter/enhancer (pCMX) upstream of wild-type
mouse PPAR.alpha., mouse PPAR.gamma.1-DN (Met.sup.105-Tyr.sup.475),
mouse PPAR.alpha.*-DN (Leu.sup.69-Tyr.sup.440), mouse PPAR.alpha.-G
(Glu.sup.282->Gly) (see Hsu et al., in Mol Pharmacol 48:559-67
(1995)) or E. coli.beta.-galactosidase as an internal control.
Cells were exposed to the compounds for 24 hours then harvested and
assayed for luciferase and .beta.-galactosidase activity. All
points were performed in triplicate and varied by less than 10%.
Normalized luciferase activity was determined and plotted as
fold-activation relative to untreated cells. Each experiment was
repeated three or more times with similar results.
Example 2
Electrophoretic Mobility Shift Assays
[0051] In vitro translated mouse PPAR.alpha.(0.2 ml) and human
RXR.alpha. (0.1 ml) were incubated for 30 minutes at room
temperature with 100,000 cpm of Klenow-labeled acyl CoA oxidase
PPRE as described by Forman et al., in Cell 81:687-93 (1995), but
with 150 mM KCl.
[0052] Example 3
Activation of PPAR.alpha. by Diarylacetylenes
[0053] In order to evaluate the ability of RAR antagonists to
relieve the inhibition of processes mediated by PPARs, CV-1 cells
are transiently transfected with a PPAR responsive reporter, PPAR
expression vectors and then treated with
4-[[5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-napht-
halenyl]ethynyl] benzoic acid. Wy 14,643 and rosiglitazone (BRL
49653) are included as positive controls since these compounds
selectively activate PPAR .alpha. and .gamma., respectively (see
Forman et al., in Cell 83:803-12 (1995), Kliewer et al., in Cell
83:813-9 (1995) and Kliewer et al., in Proc Natl Acad Sci USA
91:7355-9 (1994)).
[0054]
4-[[5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethy-
nyl] benzoic acid is found to activate PPAR.alpha. maximally at
about 300 mM.
Example 4
Activation of PPAR.alpha. by Benzoic Acid Derivatives
[0055] In order to evaluate the ability of RAR antagonists to
relieve the inhibition of processes mediated by PPARs, CV-1 cells
are transiently transfected with a PPAR responsive reporter, PPAR
expression vectors and then treated with 4-[4-(2,2,2,-trifluoro- 1
-methoxyethyl)5,6,7,8-tetrahy-
dro-5,5,8,8-tetramethyl-2-anthracenyl]benzoic acid (SR11335; Lee et
al. J Biol Chem 271:11897-11903). Wy 14,643 and BRL 49653 are
included as positive controls since these compounds selectively
activate PPAR .alpha. and .gamma., respectively (see Forman et al.,
in Cell 83:803-12 (1995), Kliewer et al., in Cell 83:813-9 (1995)
and Kliewer et al., in Proc Natl Acad Sci USA 91:7355-9
(1994)).
[0056] 4-[4-(2,2,2,-trifluoro- 1
-methoxyethyl)5,6,7,8-tetrahydro-5,5,8,8--
tetramethyl-2-anthracenyl]benzoic acid is found to activate
PPAR.alpha. maximally at about 300 mM.
Example 5
Retinoid Antagonists Relieve the Retinoic Acid
Induced Inhibition of PPAR Mediated Processes
[0057] To confirm the effect of an RAR antagonist on
PPAR.alpha./RXR.alpha., studies are done with the homologous malic
enzyme gene promoter (pMECAT -490/+31; see Castelein et al. (1994)
J Biol Chem 269:26754-26758). The -490/+31 malic enzyme promoter
sequence is inserted in a pOCAT2 reporter and used in this
transfection experiment with receptor plasmids in COS cells.
Increasing amounts of an RAR agonist (e.g., retinoic acid) is
presented with a constant amount of PPAR.alpha./RAR.alpha.
expressing vectors. The cells are treated with either DMSO,
ciprofibrate (100 mM) or with the combination of ciprofibrate and
an RAR antagonist.
[0058] The activity of the pOCAT2 reporter is reduced alone in the
presence of an RAR agonist. PPAR.alpha./RXR.alpha. stimulate CAT
activity of the pMECAT reporter to almost the same extent,
regardless of the absence or presence of their respective ligands.
RAR agonists suppress CAT activation by the non-liganded
PPAR.alpha./RAR.alpha. in a dose dependent manner, and, to a lesser
extent, also suppress CAT activation by the ciprofibrate activated
receptors. However, RAR agonist is completely unable to counteract
PPAR.alpha./RAR.alpha. activated by ciprofibrate and an RAR
antagonist. These results show that RAR antagonists may relieve the
repression of PPAR.alpha. transactivation caused by RAR
agonist.
Example 6
RAR Antagonists Enhances PPAR.gamma. Mediated Biological
Processes
[0059] A myeloid cell line HL-60 was treated with the PPAR gamma
activator, PG-J2 (5 .mu.M), the RXR activator, LG 268 (100 nM), the
RAR/RXR activator, 9-cis retinoic acid (100 nM), and the RAR
antagonist AGN 193109 (100 nM), individually or in different
combinations. The expression levels of monocytic cell surface
marker CD14 is measured over two days. As a comparison, HL-60-CDM-1
cells which do not express PPAR.gamma. and thus have impaired
PPAR.gamma. response, were also treated with the above ligands,
individually or in various combinations (FIG. 1).
[0060] FIG. 1 and 2 illustrate the dramatic effect of the
combination of PPAR-.gamma. ligand, PG-J2, the RXR agonist, LG268,
and the RAR antagonist, AGN193109, on HL-60 cells.
[0061] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
1 1 71 PRT Artificial Sequence general sequence for DNA binding
domain for members of the steroid/thyroid hormone receptor
superfamily 1 Cys Xaa Xaa Cys Xaa Xaa Asp Xaa Ala Xaa Gly Xaa Tyr
Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Cys Lys Xaa Phe Phe Xaa Arg
Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 Xaa Xaa Xaa Lys Xaa Xaa Arg
Xaa Xaa Cys Xaa Xaa Cys Arg Xaa Xaa 50 55 60 Lys Cys Xaa Xaa Xaa
Gly Met 65 70
* * * * *