Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
METHOD FOR THE PREVENTION AND/OR TREATMENT OF
ATHEROSCLEROSIS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to US provisional application SN
60/170,403, filed December 13, 1999, herein incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
Recent publications in Nature Genetics, August, 1999 (Young et al,
page 316; Bodzioch et al, page 347; Brooks-Wilson et al, page 335, and Rust et
al,
page 352 ) showed that humans with mutations in the gene ABC1 have low levels
of
high density lipoprotein (HDL). Low HDL levels are a risk factor for
atherosclerosis,
myocardial infarction and related conditions such as ischemic stroke.
Therefore,
increasing the expression of the ABC1 gene would be expected to increase HDL
levels and decrease the occurrence of atherosclerosis, myocardial infarction
and
related conditions such as ischemic stroke. It has been reported that
expression of the
ABC1 gene is increased by cholesterol loading of cells (Langmann et al,
Biochem.
Biophys. Res. ComnZ., 257, 29-33 (1999)). LXRa is a nuclear receptor that is
required
for the induction of cholesterol 7a-hydroxylase in mouse liver following
cholesterol
feeding (Peet et al, Cell, 93, 693-704 (1998)). LXRa and LXR(3 are activated
by 22-
(R)-hydroxycholesterol and other oxysterols (Janowski et al. Proc. Natl. Acad.
Sci
USA , 96, 266-271 (1999)). As part of the instant invention it was found that
LXRa
and/or LXR~3 cause the induction or regulation of ABCl expression. We conclude
that
small molecule ligands of LXR are useful as drugs to increase the expression
of
ABC1, increase levels of HDL and thereby decrease the risk of atherosclerosis,
myocardial infarction and related conditions such as peripheral vascular
disease and
ischemic stroke.
SUMMARY OF THE INVENTION
One object of the instant invention is to provide a method for raising
serum HDL cholesterol levels comprising administering a therapeutically
effective
amount of an LXR ligand to a patient in need of such treatment.
Another object is to provide a method for stimulating the expression of
the ABCI gene which comprises administering an effective amount of an LXR
ligand
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to a patient in need of such treatment whereby the patient's serum HDL level
is
increased.
As a further object, methods are provided for preventing or reducing
the risk of developing atherosclerosis, as well as for halting or slowing the
progression of atherosclerotic disease once it has become clinically evident,
comprising the administration of a prophylactically or therapeutically
effective
amount, as appropriate, of an LXR ligand to a patient who is at risk of
developing
atherosclerosis or who already has atherosclerotic disease. The method of this
invention also serves to remove cholesterol from tissue deposits such as
xanthomas
and atherosclerotic lesions by hastening the efflux of cholesterol from cells
in those
lesions. Additional objects will be evident from the following detailed
description.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows displacement of [3H2]Compound A from GST-
LXRa,with percent inhibition by Compound 1 at ~M concentrations. Compound 1
ICSp is 80 nM (calculated K; ~ 30 nM).
Figure 2 shows displacement of [3HZ]Compound A from GST-
LXR(3, with percent inhibition by Compound 1 at ~M concentrations. Compound 1
IC50 is 40 nM (calculated K; ~ 14 nM).
Figure 3 shows LXRa-GAL4 fusion protein transactivation in cultured
cells by various concentrations of Compound 1.
Figure 4 shows LXR(3-GAL4 fusion protein transactivation in cultured
cells by various concentrations of Compound 1.
DETAILED DESCRIPTION OF THE INVENTION
Any patient desiring to increase their HDL cholesterol level may use
this treatment. Particularly suitable patients in need of such treatment are
those whose
HDL level is below the clinically desirable level of HDL cholesterol, i.e,
about 40
mg/dl in men and about 50 mg/dl in women.
Atherosclerosis encompasses vascular diseases and conditions that are
recognized and understood by physicians practicing in the relevant fields of
medicine.
Atherosclerotic cardiovascular disease including restenosis following
revascularization procedures, coronary heart disease (also known as coronary
artery
disease or ischemic heart disease), cerebrovascular disease including mufti-
infarct
dementia, and peripheral vessel disease including erectile dysfunction are all
clinical
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manifestations of atherosclerosis and are therefore encompassed by the terms
"atherosclerosis" and "atherosclerotic disease."
An LXR ligand may be administered to prevent or reduce the risk of
occurrence, or recurrence where the potential exists, of a coronary heart
disease event,
a cerebrovascular event, and/or intermittent claudication. Coronary heart
disease
events are intended to include CHD death, myocardial infarction (i.e., a heart
attack),
and coronary revascularization procedures. Cerebrovascular events are intended
to
include ischemic or hemorrhagic stroke (also known as cerebrovascular
accidents) and
transient ischemic attacks. Intermittent claudication is a clinical
manifestation of
peripheral vessel disease. The term "atherosclerotic disease event" as used
herein is
intended to encompass coronary heart disease events, cerebrovascular events,
and
intermittent claudication. It is intended that persons who have previously
experienced
one or more non-fatal atherosclerotic disease events are those for whom the
potential
for recurrence of such an event exists.
Accordingly, the instant invention also provides a method for
preventing or reducing the risk of a first or subsequent occurrence of an
atherosclerotic disease event comprising the administration of a
prophylactically
effective amount of an LXR ligand to a patient at risk for such an event. The
patient
may already have atherosclerotic disease at the time of administration, or may
be at
risk for developing it.
The method of this invention also serves to remove cholesterol from
tissue deposits such as atherosclerotic plaques or xanthomas in a patient with
atherosclerotic disease manifest by clinical signs such as angina,
claudication, bruits,
one that has suffered a myocardial infection or transient ischemic attack, or
one
diagnosed by angiography, sonography or MRI.
The term LXR includes all subtypes of this receptor and corresponding
genes which encode such subtypes. Specifically LXR includes LXRa and LXR(3,
and
a ligand of LXR should be understood to include a ligand of LXRa or LXR~i.
LXRa
has been referred to under a variety of names and for purposes of this
application
LXRa should be understood to mean any gene referred to as LXRa, LXRa,
LXRalpha, RLD-1, NR1H3 or a gene with homology to accession number U22662 or
a protein with homology to a protein encoded by such a polynucleotide.
Similarly,
LXR~i should be understood to include any gene referred to as LXRb, LXR(3,
LXRbeta, NER, NER1, UR, OR-l, R1P15, NR1H2 or a gene with homology to
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accession number U07132 or a protein with homology to a protein encoded by
such a
polynucleotide.
The term ligand throughout this application should be understood to
include an agonist, partial agonist or antagonist of LXR. The ligand may be
selective
for LXRa or LXR(3, or it may have mixed binding affinity for both LXRa and
LXR(3.
Particularly, compounds within the scope of this invention include those which
have
greater selectivity as determined by binding affinity for LXRa and/or LXR(3
receptors
than they have for each of the PPARa, y and 8 receptors. More particularly,
the
compounds included within the scope of this invention have an IC50 less than
or
equal to 100nM for at least one of either the LXRa or LXR(3 receptors, and
have an
IC50 equal to or greater than 1 pM for each of the PPARa, PPAR~y and PPARB
receptors, and even more particularly they have an ICSp equal to or greater
than lOpM
for each of the PPARa, PPARy and PPARB receptors. For example, the selectivity
of
suitable LXR receptor ligands can be determined from IC50 results obtained
employing the LXR radioligand competition scintillation proximity assays
described
below in the Example section, and from PPAR competition binding assays
described
in Berger J, et al., Novel peroxisome proliferator-activated receptory
(PPAR~y) and
PPARB ligands produce distinct biological effects, J Biol Chem 274: 6718-6725
(1999), herein incorporated by reference in its entirety.
The term "patient" includes mammals, especially humans, who use the
instant active agents for the prevention or treatment of a medical condition.
Administering of the drug to the patient includes both self-administration and
administration to the patient by another person. The patient may be in need of
treatment for an existing disease or medical condition, or may desire
prophylactic
treatment to prevent or reduce the risk for diseases and medical conditions
affected by
HDL cholesterol.
The term "therapeutically effective amount" is intended to mean that
amount of a drug or pharmaceutical agent that will elicit the biological or
medical
response of a tissue, a system, animal or human that is being sought by a
researcher,
veterinarian, medical doctor or other clinician. The term "prophylactically
effective
amount" is intended to mean that amount of a pharmaceutical drug that will
prevent or
reduce the risk of occurrence of the biological or medical event that is
sought to be
prevented in a tissue, a system, animal or human by a researcher,
veterinarian, medical
doctor or other clinician. Particularly, the dosage amount of an LXR ligand
that a
patient receives can be selected so as to achieve the amount of HDL
cholesterol
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raising desired; the dosage a patient receives may also be titrated over time
in order to
reach a target HDL level.
An effective amount of an LXR ligand in the method of this invention
is about 0.01 mg/kg to about 140 mg/kg of body weight per day, or about 0.5 mg
to
about 7 g per patient per day. For example, adequate elevation of ILL can be
accomplished by the administration of about 0.5 mg to about 3.5 mg per patient
per
day.
It will be understood, however, that the specific dose level for any
particular patient will depend upon a variety of factors including the age,
body weight,
general health, sex, diet, time of administration, route of administration,
rate of
excretion, drug combination and the severity of the particular HDL deficiency.
A
consideration of these factors is well within the purview of the ordinarily
skilled
clinician for the purpose of determining the therapeutically effective or
prophylactically effective dosage amount needed to prevent, counter, or arrest
the
progress of the condition.
An example of an LXR ligand suitable for use in the method of this
invention is represented by Compound 1 having the following structural formula
O
O
0
O
O ~~ H
O\ /
~O
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Compound 1
In the method of treatment of this invention, the LXR receptor ligands
described above may be administered orally, topically, parenterally, by
inhalation
spray or rectally in dosage unit formulations containing conventional non-
toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The term
parenteral as
used herein includes subcutaneous injections, intravenous, intramuscular,
intrasternal
injection or infusion techniques.
The pharmaceutical compositions of this invention containing the
active ingredient may be in a form suitable for oral use, for example, as
tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders or
granules,
emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended
for oral
use may be prepared according to any method known to the art for the
manufacture of
pharmaceutical compositions and such compositions may contain one or more
agents
selected from the group consisting of sweetening agents, flavoring agents,
coloring
agents and preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in admixture
with non-
toxic pharmaceutically acceptable excipients, which are suitable for the
manufacture
of tablets. These excipients may be for example, inert diluents, such as
calcium
carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or alginic
acid;
binding agents, for example starch, gelatin or acacia, and lubricating agents,
for
example, magnesium stearate, stearic acid or talc. Oral immediate-release and
time-
controlled release dosage forms may be employed. Tablets may be uncoated or
they
may be coated by known techniques to delay disintegration and absorption in
the
gastrointestinal tract and thereby provide a sustained action over a longer
period. For
example, a time delay material such as glyceryl monostearate or glyceryl
distearate
may be employed. They may also be coated by the technique described in the
U.S.
Patent 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets
for
controlled release.
Formulations for oral use may also be presented as hard gelatin
capsules wherein the active ingredient is mixed with an inert solid diluent,
for
example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin
capsules
wherein the active ingredients is mixed with water or miscible solvents such
as
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propylene glycol, PEGs and ethanol, or an oil medium, for example peanut oil,
liquid
paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with
excipients suitable for the manufacture of aqueous suspensions. Such
excipients are
suspending agents, for example sodium carboxymethylcellulose, methylcellulose,
hydroxy-propylmethycellulose, sodium alginate, polyvinyl-pyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents may be a naturally-
occurnng
phosphatide, for example lecithin, or condensation products of an alkylene
oxide with
fatty acids, for example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with
partial
esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol
monooleate, or condensation products of ethylene oxide with partial esters
derived
from fatty acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives,
for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring
agents, one
or more flavouring agents, and one or more sweetening agents, such as sucrose,
saccharin or aspartame.
Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil
or coconut
oil, or in mineral oil such as liquid paraffin. The oily suspensions may
contain a
thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
Sweetening
agents such as those set forth above, and flavouring agents may be added to
provide a
palatable oral preparation. These compositions may be preserved by the
addition of
an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an
aqueous suspension by the addition of water provide the active ingredient in
admixture with a dispersing or wetting agent, suspending agent and one or more
preservatives. Suitable dispersing or wetting agents and suspending agents are
exemplified by those already mentioned above. Additional excipients, for
example
sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the
form of an oil-in-water emulsions. The oily phase may be a vegetable oil, for
example olive oil or arachis oil, or a mineral oil, for example liquid
paraffin or
mixtures of these. Suitable emulsifying agents may be naturally-occurnng
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phosphatides, for example soy bean, lecithin, and esters or partial esters
derived from
fatty acids and hexitol anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene oxide, for
example
polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening
and flavouring agents.
Syrups and elixirs may be formulated with sweetening agents, for
example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may
also
contain a demulcent, a preservative and flavouring and colouring agents. The
pharmaceutical compositions may be in the form of a sterile injectable aqueous
or
oleagenous suspension. This suspension may be formulated according to the
known
art using those suitable dispersing or wetting agents and suspending agents
which
have been mentioned above. The sterile injectable preparation may also be a
sterile
injectable solution or suspension in a non-toxic parenterally-acceptable
diluent or
solvent, for example as a solution in 1,3-butane diol. Among the acceptable
vehicles
and solvents that may be employed are water, Ringer's solution and isotonic
sodium
chloride solution. Cosolvents such as ethanol, propylene glycol or
polyethylene
glycols may also be used. In addition, sterile, fixed oils are conventionally
employed
as a solvent or suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In addition, fatty acids
such as
oleic acid find use in the preparation of injectables.
Compounds useful in the method of treatment of the invention may
also be administered in the form of a suppository for rectal administration of
the drug.
These compositions can be prepared by mixing the drug with a suitable non-
irritating
excipient which is solid at ordinary temperatures but liquid at the rectal
temperature
and will therefore melt in the rectum to release the drug. Such materials are
cocoa
butter and polyethylene glycols.
For topical use, creams, ointments, gels, solutions or suspensions, etc.,
containing the compound of are employed. For purposes of this application,
topical
application shall include mouth washes and gargles. Topical formulations may
generally be comprised of a pharmaceutical carrier, cosolvent, emulsifier,
penetration
enhancer, preservative system, and emollient.
The amount of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon the host
treated
and the particular mode of administration. For example, a formulation intended
for
the oral administration of humans may contain from 0.5 mg to 5 g of active
agent
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compounded with an appropriate and convenient amount of Garner material which
may vary from about 5 to about 95 percent of the total composition. Dosage
unit
forms will generally contain between from about 1 mg to about 500 mg of an
active
ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg,
600
mg, 800 mg, or 1000 mg.
One or more additional active agents may be used in combination with
the LXR ligands of this invention in a single dosage formulation, or may be
administered to the patient in a separate dosage formulation, which allows for
concurrent or sequential administration of the active agents. The additional
active
agent or agents can be lipid altering compounds such as HMG-CoA reductase
inhibitors, or agents having other pharmaceutical activities, or agents that
have both
lipid-altering effects and other pharmaceutical activities. Examples of HMG-
CoA
reductase inhibitors include statins in their lactonized or dihydroxy open
acid forms
and pharmaceutically acceptable salts and esters thereof, including but not
limited to
lovastatin (see US Patent No. 4,342,767); simvastatin (see US Patent No.
4,444,784),
pravastatin, particularly the sodium salt thereof (see US Patent No.
4,346,227);
fluvastatin particularly the sodium salt thereof (see US Patent No.
5,354,772);
atorvastatin, particularly the calcium salt thereof (see US Patent No.
5,273,995);
cerivastatin, particularly the sodium salt thereof (see US Patent No.
5,177,080), and
nisvastatin also referred to as NK-104 (see PCT international publication
number WO
97/23200). Additional active agents which may be employed in combination with
an
LXR ligand include but are not limited to HMG-CoA synthase inhibitors;
squalene
epoxidase inhibitors; squalene synthetase inhibitors (also known as squalene
synthase
inhibitors), acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors
including
selective inhibitors of ACAT-1 or ACAT-2 as well as dual inhibitors of ACAT1
and -
2; microsomal triglyceride transfer protein (MTP) inhibitors; probucol;
niacin;
cholesterol absorption inhibitors such as SCH-58235; bile acid sequestrants;
LDL
(low density lipoprotein) receptor inducers; platelet aggregation inhibitors,
for
example glycoprotein IIb/>IIa fibrinogen receptor antagonists and aspirin;
human
peroxisome proliferator activated receptor gamma (PPARy) agonists including
the
compounds commonly referred to as glitazones for example troglitazone,
pioglitazone
and rosiglitazone and, including those compounds included within the
structural class
known as thiazolidinediones as well as those PPAR~y agonists outside the
thiazolidinedione structural class; PPARa agonists such as clofibrate,
fenofibrate
including micronized fenofibrate, and gemfibrozil; PPAR dual a/~y agonists;
vitamin
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B( (also known as pyridoxine) and the pharmaceutically acceptable salts
thereof such
as the HCl salt; vitamin B 12 (also known as cyanocobalamin); folic acid or a
pharmaceutically acceptable salt or ester thereof such as the sodium salt and
the
methylglucamine salt; anti-oxidant vitamins such as vitamin C and E and beta
carotene; beta-Mockers; angiotensin II antagonists such as losartan;
angiotensin
converting enzyme inhibitors such as enalapril and captopril; calcium channel
Mockers such as nifedipine and diltiazam; endothelian antagonists; agents
other than
LXR ligands that enhance ABC1 gene expression; bisphosphonate compounds such
as alendronate sodium; and cyclooxygenase-2 inhibitors such as rofecoxib and
celecoxib.
Compound A is used in the following assays and has the following
structural formula:
CF3
HO
\~
N
O / S~O~ / O
CI
Compound A
Compound A and related compounds are disclosed along with methods for making
them in W097/28137 herein incorporated by reference in its entirety (US Serial
No.
08/791211, filed January 31, 1997.
EXAMPLE 1
Radioligand Competition Binding Scintillation Proximity Assays:
Preparation of Recombinant Human LXRa and LXR(3:
Human LXRa and LXR(3 were expressed as GST-fusion proteins in E.
coli.. The ligand binding domain cDNAs for human LXRa (amino acids 164-447)
and
human LXR(3 (amino acids 149-455) were subcloned into the pGEX-KT expression
vector (Pharmacia). E. coli containing the respective plasmids were
propagated,
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induced, and harvested by centrifugation. The resuspended pellet was broken in
a
French press and debris was removed by centrifugation. Recombinant human LXR
receptors were purified by affinity chromatography on glutathione sepharose
and
receptor was eluted with glutathione. Glycerol was added to a final
concentration of
50% to stabilize the receptor and aliquots were stored at -80 °C.
Binding to LXRa:
For each assay, an aliquot of human GST-LXRa receptor was
incubated in a final volume of 100 p1 SPA buffer (10 mM Tris, pH 7.2, 1 mM
EDTA,
10°lo glycerol, 10 mM Na molybdate, 1 mM dithiothreitol, and 2 ~g/ml
benzamidine)
containing 1.25 mg/ml yttrium silicate protein A coated SPA beads (Amersham
Pharmacia Biotech, Inc.), 8.3 ~g/ml anti-GST antibody (Amersham Pharmacia
Biotech, Inc.) 0.1°lo non-fat dry milk and 25 nM [3H2]Compound A
(13.4
Ci/mmole), ~ test compound. After incubation for ~16 h at 15°C with
shaking, the
assay plates were counted in a Packard Topcount. In this assay the Kd for
Compound
A for LXRa is ~ 15 nM.
Binding to LXR(3:
For each assay, an aliquot of human GST-LXR(3 ligand binding
domain receptor was incubated in a final volume of 100 p,1 SPA buffer (10 mM
Tris,
pH 7.2, 1 mM EDTA, 10°Io glycerol, 10 mM Na molybdate, 1 mM
dithiothreitol, and
2 ~g/ml benzamidine) containing 1.25 mg/ml yttrium silicate protein A coated
SPA
beads (Amersham Pharmacia Biotech, Inc.), 8.3 pg/ml anti-GST antibody
(Amersham
Pharmacia Biotech, Inc.) 0.1% non-fat dry milk and 25 nM [3H2]Compound A (13.4
Ci/mmole), ~ test compound. After incubation for ~16 h at 15°C with
shaking, the
assay plates were counted in a Packard Topcount. In this assay the Kd for
Compound
A for LXR~3 is ~ 10 nM.
Results
Compound 1 is a ligand for human LXRa and human LXR(3, having an IC50 = 80nM
for the LXRa receptor, and an IC50 = 40nM for the LXR~3 receptor. as shown in
Figures 1 and 2. Compound 1 has an IC50 greater than 10 ~,M in binding assays
for
human PPAR~y, PPARB and PPARa.
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EXAMPLE 2
Transactivation Assay
Plasmids
Expression constructs were prepared by inserting the ligand binding
domain (LBD) of human LXRa and LXR(3 cDNAs adjacent to the yeast GAL4
transcription factor DNA binding domain (DBD) in the mammalian expression
vector
pcDNA3 to create pcDNA3-LXRoc/GAI~ and pcDNA3-LXR(3/GAL4, respectively.
The GAL4-responsive reporter construct, pUAS(SX)-tk-luc , contained 5 copies
of the
GAL4 response element placed adjacent to the thymidine kinase minimal promoter
and the luciferase reporter gene. The transfection control vector, pEGFP-Nl,
contained the Green Fluorescence Protein (GFP) gene under the regulation of
the
cytomegalovirus promoter.
Assay
HEK-293 cells were seeded at 40,000 cells/well in 96 well plates in Dulbecco's
modified Eagle medium (high glucose) containing 10% charcoal stripped fetal
calf
serum, 100 units/ml Penicillin G and 100 ~.g/ml Streptomycin sulfate at
37°C in a
humidified atmosphere of 5% C02. After 24 h, transfections were performed with
Lipofectamine (Gibco-BRL, Gaithersburg, MD) according to the instructions of
the
manufacturer. In general, transfection mixes contained 0.002 ~g of LXRaJGAL4
or
LXR(3/GAL4 chimeric expression vectors, 0.02 ~g of reporter vector pUAS(SX)-tk-
luc and 0.034 ~.g of pEGFP-Nl vector as an internal control of transfection
efficiency.
Compounds were characterized by incubation with transfected cells for 48h
across a
range of concentrations. Cell lysates were prepared from washed cells using
Cell
Lysis Buffer (Promega) according to the manufacturer's directions. Luciferase
activity
in cell extracts was determined using Luciferase Assay Buffer (Promega) in a
ML3000 luminometer (Dynatech Laboratories). GFP expression was determined
using the Tecan Spectrofluor Plus at excitation wavelength of 485nm and
emission at
535nm. Luciferase activity was normalized to GFP expression to account for any
variation in efficiency of transfection.
Results with Compound 1 for LXRa transactivation are shown in
Figure 3, and results for LXR(3 transactivation are shown in Figure 4.
EXAMPLE 3
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Induction of ABC1 mRNA levels
Cultured human THP-1 cells were stimulated to differentiate into
macrophages by incubation with 100 nM tetradecanoyl phorbol acetate for three
days.
All cell culture incubations were performed at 37°C under 95°70
air/5% carbon dioxide
using culture conditions as recommended by ATCC. After differentiation, the
THP-1
macrophages were incubated with the test LXR agonist. After 6 hours at
37°C, the
cells were harvested and total RNA prepared using the phenol/guanidine
isothiocyanate method as supplied and described by Molecular Research Center,
Inc.
(TRI REAGENT~ Cat. No. TR 118). ABC 1 mRNA levels in the total RNA were
measured using the TaqMan~ mRNA quantitation system, following protocols
published by the manufacturer (Perkin-Elmer). The oligonucleotide PCR primers
used to detect ABC1 were:
GAGGCTCCCGGAGTTGTTGandGTATAAAAGAAGCCTCCGAGCATC
The oligonucleotide probe used was:
6FAM-AAACTTTAACAAATCCATTGTGGCTCGCCTGT-TAMRA
ABC1 mRNA levels in each sample were normalized to the mRNA levels for the 23
kDa highly basic protein. The oligonucleotide PCR primers used to detect the
23 kDa
highly basic protein were:
GCTGGAAGTACCAGGCAGTGAandACCGGTAGTGGATCTTGGCTTT
The oligonucleotide probe used was:
VIC- TCTTTCCTCTTCTCCTCCAGGGTGGCT-TAMRA
The results from this experiment for Compound 1 and 22-(R)-
hydroxycholesterol are as follows:
Fold Induction of
ABC1
Compound mRNA (Mean + SEM) P Value vs DMSO Control
15 ~M 22-(R)- 6.7 + 1.2 0.008
hydroxycholesterol
6.9 + 0.6 0.0009
0.10 ~,M Compound
1
EXAMPLE 4
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Preparation of Compound 1
Podocarpic acid (550 mg) was dissolved in 2 ml of acetic anhydride in
a 10 ml flask and heated to reflux (150 °C) for 30 minutes and cooled.
The reaction
was analyzed by HPLC. The major product was the mixed anhydride and about 1%
of
the reaction mixture was the acetate dimer Compound 1. The solvent was blown
off
under nitrogen and the resultant oil was charged to a 200 cc Sephadex LH20
column
in MeOH (methanol). Compound 1 eluted in cuts 75-80 (2 ml each, 0.8 cv). The
mixed anhydride eluted in cuts 80-100. Cuts 75-80 were dried down and
dissolved in
300 u1 CH3CN and loaded on a semi-preparative Zorbax RX-C8 column. The
column was eluted with a 40-min gradient of 50 to 90°70 aqueous CH~CN
at 4 mL per
min. One-minute fractions were collected. Compound 1 eluted at 35 min. The
pooled fractions gave 0.7 mg of Compound 1. Mass spectral analysis of this
compound gave a molecular weight of 614 amu and molecular formula of C38H460~.
Mass spectral data: Found: 632.3620; Calculated: 632.3587; Formula: C38HSON0~;
Assignment: [M+NH4].
'H NMR data: (8, 500 MHz, CDC13): 8 1.14(1H, dt, 13.5,4.0 Hz), 1.20(3H,s),
1.39(3H,s), 1.44(lH,dt, 13.5, 4.0 Hz), 1.63(1H, d, 12.5), 1.67(1H, m), 2.0(1H,
d, 14.0
H ), 2.05(lH,m), 2.2(1H, dd,13.5, 6.0 Hz), 2.25(lH,d, 12 Hz), 2.28(3H,s),
2.30(lH,d,
13.5 Hz ), 2.80(1H, ddd, 13.0, 12.5,6.5 Hz), 2.95(1H, dd, 16.5,5.0 Hz),
6.83(1H, dd,
8.0,2.0 Hz), 6.96(lH,d, 2.OHz), 7.05(lH,d, 8.0 Hz).
Equipment: Mass spectra were recorder on an LCQ (LC-MS-ESI, Liquid
chromatography-Electrospray ionization) and exact mass measurements were
recorded on a Finnigan NewStar FTMS mass spectrometer. 'H spectra were
recorded
in either CDC13 or CD30D on a Varian Unity 500 NMR Spectrometer operating at
500 MHz for'H. Chemical shifts are given in ppm relative to tetramethylsilane
(TMS) at zero ppm using the respective solvent peaks as an internal standard.
While the invention has been described and illustrated with reference
to certain particular embodiments thereof, those skilled in the art will
appreciate that
various changes, modifications and substitutions can be made therein without
departing from the spirit and scope of the invention. For example, effective
dosages
other than the particular dosages as set forth herein above may be applicable
as a
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CA 02392568 2002-05-24
WO 01/41704 PCT/US00/33098
consequence of variations in the responsiveness of the mammal being treated
for any
of the indications for the active agents used in the instant invention as
indicated
above. Likewise, the specific pharmacological responses observed may vary
according to and depending upon the particular active compound selected or
whether
there are present pharmaceutical carriers, as well as the type of formulation
employed,
and such expected variations or differences in the results are contemplated in
accordance with the objects and practices of the present invention. It is
intended,
therefore, that the invention be defined by the scope of the claims which
follow and
that such claims be interpreted as broadly as is reasonable.
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