Note: Descriptions are shown in the official language in which they were submitted.
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COIIiPOSITIONS r~ND t~I~THODS OP' TREATING, RIJDUCING, AND
PIZCYTN'I'ING CAIt.DI01'tISCUL.~.R bISCfIS~S AND D1SORDI~,RS 1~'IT~I
P O L'k' I\ I;CThI O ~1'~LA'~' O N ~ S
I. CROSS I~.CI!CCtI~NCC'I~0 RLL;~TCD APPLICtITIOIJS
This application is a Conlinuatiott-fn-Part oCco-pending United States Palenl
Alylication Serial Number 09/167,631, filed olt October G, 1998 entillc:d
"Compositions and
Mietltods of lnltibitittg l~Jeopla,stic Diseases 1','itlt Comrounds Related to
Limocilrin and S-
Desrnelltyl Sinenselilt", wlterein Ilte entire disclosure is herein
incorporated by
,A, Field of llte Irtveltlion reference
Z. LiACI~:GROUND OT T I-IC IN~'~NTION
Tloc present invention relates to cornposiliolts and ntetltods fur Ilte
prevenlioti,
reduction, andlor treatrttent of cardiovascular diseases wvllt synthetic and
naturally occurring
pofyotetltovyflavorte cuntpounds derived, some of wlticlt are derived front
IintociU'io and
quercelin. '
These corttpounds include, but are riot limited to, tire follov~ing evantples
of liotocitrin,
and quercetilt derivativesv
limocitrin-3,7,~I'-trintellt~~lether (5-It~~drox~~-J,7,S,]',4'-
penlamelltoxyflavone)
(imocilrin-3,5,,7,~I'-lelrantelltylcllter (3,5,7,8,3'4'-Itexamelhoxy(lavone)
Ilntocllrilt-3,5,7,1'-ietraetltylether (8,3'-dipetltox~~-3,5,7,4'-
letraelltox~~flavone)
lintocitrilt ],7,~I'-triotethvletltcr-S-acetate
querceliri letraotetlt~~letlter (S-Itydroxy-3,7,]',~l'-letrantetlto;yflavotte)
quercelilt 3,S-dirttetltylellter-7,3'~I'-tribenzyl elllcr
quercetin henlantetltyletlter (3,5,7,3',1'-pentantelhoxytlavone)
quercetin-5,7,]',d'-lelrantellt~~lelher-]-acetate
quercetilt-5,7,,]',~I'-lelrametltylellter (J-It,y~dro,y~-5,7,]',~I'-
IelrarttetltoYyflavone)
~.vamples" but not lilttitzd to. of mturall~~ occurring pol~~ntetltoxy(lavones
for the
CA 02403548 2002-09-17
2
purposes of the present invention include:
3,5,6,7,8, 3',4'-heptamethoxyflavone,
rtobiletin (5,6,7,8.3',4'-hexamethoxyflavone)
tangeretin (5,6,7,8,x'-pentamethoxyfSavone)
5-desntethylnobiletin (5-hydraw-G,7.8.3'4'-pentantethoxyflavone)
tetra-O-methylisoscutellarein (5.7,8,4'-tetramethoxyflayone) .
5-deswetltylsinensetin (S-hydrosy-G,7,3',4'-tetramethoxyflavone)
sinensetin (S,G,7,3',4'-pentamethoxyl7avone),.
B. Description of the Related Art
Lintocitrin derivatives are a group of citrus-derived flavonoids that are
naturally
occurring in the plant or ire chemicaSiy synthesized. 5-desmethylsinesetin is
chemically
5ynt1te51Zed fault Of sinensetin (Tatunt. .l.f-i, et al.., Pltytocltemistry
II, ZZ8,3-2288, 197Z).
5inensetin occurs in trace levels in ntaydaritt orange leaves (5ugiyama, S. et
al., Cheto.
Pharnt. Bull., Volume 41, 7i4-719. 4993), and in orange and mandarin peel.
F'lavonaids are
polyphenolic compounds that occur ubiquitously, in foods of plant origin. The
major dietary
sources of flavonaids are vegetables. fruits, and beverages suc.lt as tea and
red seine (I-Iertog,
I\~t.G.L. et al., J. Agric. Food Chen~.. Valuri~e ~41, IZ~Z-SZ~6, 1993).
Flavonotds have been
demonstrated to be the most potent dietary antioxidants and in fight of the
large dietary
consumption, Ilavonoids make a major contribution to the antioxidant potential
of the human
diet. The main Cood sources of ilavonols and flavones are black tea, onions,
apples, herbs,
arid spices such as cloves and black pepper (I-Iertog. M.G.L., et al,,J.
Agric. Food Chem.,
Volume ~10, Z379-2383, 1992).
The association between quercetin and cardiovascular disease,has been studied
in
prospective cohort studies arid cross-cultural epidemiological studies.
Flavonol and fla,vone
intake was inversely associated with mortality from coronary heart disease and
to a lesser
extent with incidence of first ntyocardial infarction. These effects were
independent of
known risk factors far coronary heart disease such as serum cltoiesterol, body
mass index,
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3
blood pressure, smoking and intake of antioxidant .vitamins, alcohol, and.
fat. Flavonol and
flavon,e intake (mainly quercetin) was also inversely associated with stroke
risk (I-Iertog et al.,
Lancet, Volume 324, 1007-101 1, 1993; Ke)i et al., Arch. Inter. Ivied., Volume
154, 637-642,
19,96). However, four thousand different types of flavonoids have been
described and it is
crucial that flue active components be identified not only to, make a positive
impact on
agriculture but also to more specifcal(y use, these nutraceuticals as
anticholesterol agents
and/or antitluombotic, anticoronary heart disease, antitnyocardial infarction
andlor antistrqke
agents.
In the United States, the complications of atherosclerosis account for about
one half of
all deaths and for about one tl5ird of deaths in persons between 35 and 65
years of age.
Atherosclerosis, or the developments of atlaeromatous plaques in large and
medium-sized
arteries, is the most common form of arteriosclerosis. /,-tarry factors a re
associated with the
acceleration of atherosclerosis, regardless, of the underlying primary
pathogenic change, for
example, age, elevated plasma cholesterol level, high arterial blood
pressure,,cigarette
sntoki,ng, reduced high-density lipoprotein (I-iDL) cholesterol level, or
family history of
premature coronary artery disease.
The risk, of death, from coronary artery disease has a continuous and graded
relation to
total serum cholesterol levels greater than 180 mg/dl (Stamler et~al,, JAMA,
Volume 256,
2823, 1986). Approximately one third of adults in the United States have
levels that exceed
24U mg/dl and, therefore, have a risk of coronar~~ artery disease that is
tGVice that of people
with cholesterol levels lower than I 80mg/dl, Acceleration of atherosclerosis
is principally
correlated with elevation of LDL, or beta fraction, v~hich is rich in
cholesterol but poor in
triglycerides, Elevation o,f I-IDL or alpha Fraction, leas a negative
carrelation with
atherosclerosis (Castelli et al,, JAMIA, Volume,256, 2835, 1986). /-/DL exerts
a protective
effect and the ratio of total cholesterol to I-IDL cholesterol is a better
predictoi~ of coronary
artery disease than the level of either alone. Total cholesterol levels are
classified as being
desiral.~le (<200mg1d1), borderline (200-239 mgld.l), or high (>240 mg/d()
(Report of the
National Education Expert Panel on Detection, Evaluation, and Treatment of I-
ligit C3lood
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4
Clylesteral in .Adults, flrclo Ilrtern. lv~ied.. ~'olun~e 1~5, 3G, 19SS).
/Advances in the study oFcliolesterol metabolism and coronarw disease have
initiated
an era of increased emphasis on preventive therapy. New guidelines far the
detectibn and
treatment of high blood cholesterol in adults recommend that patients with
High cholesterol
levels or with borderline-high levels and, two or mare additional risk factors
should have a
measurement of LDL. LDL cholesterol levels are then classilled as borderline-
high risk
(130-i 59 nlg/di) or high risk (% 160 mgldl). Dietary treatment is recommended
for those
patients with high-risk levels who liave two or more additional risk factors.
Drug treatment
is recommended for all patients with LDL levels greater than 1 S9 rng/dl and
for those patierots
with LDL cholesterol levels between 159 and 1 S9 mg/df who have rlvo or more
additional
risk factors. ,4mong the many drugs that have been used to.reduce serum
cholesterol levels
are cholestyranline, colestipol, clot brace, eernftbrozil, and lovastatin.
Platelet-blood vessel interactions are implicated in the development of
thrombosis.
flavonoids inhibit platelet aggregation and adhesion (Fraukel et al., Lancet,
Volume 341,
l 103-1 104. 1993). Flavonoids antagonize tltrot~~boxane formation and
increase platelet
cyclic AI\~fP levels. This is it»portant because Flavonoids
additionallwscaver~ge free radicals
and their antioxidant actions participate in their antitlaron~botic action
(Gryelevvski et al.,
8iochem. Pharmacol., Volume 36,.317-322. 1987). Drug treatment is recommended
for
patients «''Ith tlll'al11bo51s alld lschel»1C heart disease. The medical
therapy comprrses
pl~larmaceutical drugs including, but, is not limited to, aspirin (anti-
platelet aggregating agents)
and the combined use of beta-adrenergic blocking agents (e.g.propranonol,
nadolol, timoloi,
ate.), nitrates (e.g., nitrogfs~cerin) and calcium channel blockers (e.g.,
vrerapamil, nifedipine,
diltiazem, etc.): ,
There remains a need iti the art for methods and compositions for at feast
reducing the
development of and/or treating vascular diseases. The present invention
provides new
compositions and methods directed to this need. The use of limocitt~in
derivatives, quercetirt
derivatives andlor, n,aturallv-occurring polvmethoxyf7avones attd r»ixtures
thereof alone or in
combination with a cholesterol-lo,werine drub has not been report,~d for at
least reducing the
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development of and:'or treating vascular diseases and disorders.
3. SUk~II~~I,~.RY OF T~-I~ INVENTION
It is therefore an object of the present invention to provide compositions and
methods
for the redu,ctian, prevention, and/or treatment of cardiovascular diseases
and disorders 1
wherein an effective amount of a C0111p05ttlorl leaving at feast one
limocitrin andlar quercetin
derivative is administered to reduce, prevent or treat a mammal at high risk
for or suffering
from a cardiovascular disease.
Another object of the present invention is to provide compositions and methods
for
the reduction, prevention, and/or treatment of cardiovascular diseases ar
disorders wherein an
effective amount of a composition having at least one flavonoid is
administered to reduce,
prevent or treat a mammal at high risk for or suffering from a cardiovascular
disease
A further object of the present invention is to provide compositions and
n7et110d5 fox
the reduction, preverition, and/or treatment of cardiovascular diseases or
disorders wherein an
effecti>>e amount of a composition having at least one limocitrin. quercetin
derivative,
tocotrienol, and mivures thereof is administered to reduce, prevent or. treat
a mammal at high
risk for or suffering from a cardiovascular disease.
A still further object of the present invention is to provide compositions and
tt~ethods
for the reduction, prevention, and/or treatment of cardiovascular diseases or
disorders wherein
an effective amount of a composition having at least one naturally occurring
polyme,thovyaflavone is administered to reduce. prevent, or treat a, mantntal
at high risk for or
suffering from a cardiovascular disease.
Another abject of the present invention is to provide compositions and yethod5
for.
the reduction, prevention, and/or treatment, af.cardiovascular diseases or
disorders wherein an
effective amount of a composition having at feast one naturally occurring
polymethoxyflavone, toeotriet~ols, and mixtures thereof, is administered to
reduce, prevent or
treat a mammal at liigh risk far or suffering from a cardiovascular disease.
A further object of tl~e present invention is to provide compositions and
methods for
the,reduction, pret~eoUion, andior treatment of cardiovascular diseases or
disorders wherein an
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6
effective amount of a conipas,ition having at least otte a tocotrieno~l,
flavonoid, aitd mixtures
thereof, is administered tb reduce, prevent, or treat a mammal at high risk
for or suffering
from a cardiovascular disease.
A still further object of the presenf invention is to provide compositions and
methods
for the reduction, preventian, and/or treatment of cardiavascular.diseases or
disorders wherein
an effective amount of a composition having at least one lintacitrin
derivative, quercetin
derivative, naturally occurring po(yntethoxyailavone, tocotrienol, and
mixtures thereof, is
administered to a mamrttal to loner serum chalesteral, apo-$, and/or LDL,
cholesterol.
Another object of the preset invention is to provide compositions and methods
for
the reduction, prevention, and/or treatttient of card'tavascular diseases or
disorders wherein an
effective amount of a composition having at least one limocitrin derivative,
quercetin
derivative, naturally occurring pals~methox5~aflavone, tocotrienol, arid
mixtures thereof, in
combination with a cholesterol-lowering drug, is administered to a mammal to
lower serum
cholesterol, apa-$, and/or LDL cholesterol
Another,object of the present invention is to provide compositions and methods
for
the reduction, prevention, andlor treatment of cardiovascular diseases or
disorders wherein att
effective amount of a composition having at least one tin tocitrin derivative,
quercetin
derivative, naturall~~ occurring polvmethoxyaflavone, tocotrienol, and
mixtures thereof, in
combination with a phamtaccutical drug including anti-platelets agents, ,beta-
adrenergic
blocking agents, nitrates or calcium channel blockers.
Further objects and advantages of the present invention will become apparent
from the
fallpWirtg description.
4. DETt~,ILED DESCRIPTION OF THE INVENTION
The,present invention relates to the use of at least one of limocitrin
derivative,
quercetin derivative. polymethoyflavone, tocotrienol and mixtures thereof
alone or in
combination with at least one cholesterol-lowering drub for the treatment of
cardiovascular
diseases or disorders. Liotocitrin occurs in the peel al' terror as limocitrin-
3-O-glucoside,
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7
arid can be produced from the 3-glycoside by enzymatic and acid hydrolysis
(Harowitz et al.,
J. Org. Chem., Volume Z5, Z 1885-2,1887, 1960) or by a chemical synthesrs
procedure such as
reported by Dryer et al., Tetrahedcort, Volume 2D, 2977-2983, 1964. Two
limociirin
analogues, limocitrin 3,7.4'-trin~etlwle,ther and limocitrin-3,,5,6-4'-
tetramethylether, also occur
in orange peel (Tatum et al., Phytochemistry, volume II, 2283-2285; 1972).
Several
polymethoxyflavones were tested and found to be active as i,nlyibitors of
apolipoprotein B
(apoB) production and had negligible cytotoxicity in the human liver carcinoma
cell line
IIepG2. It has been shown that humans with coronary heart disease (CAD) have
higher
levels of apoB in their blood. ApoB concentrations also reflect the number of
LDL, and
VLDL (very low density lipoprotein) particles in arteries. Administering
polymethoxylatedflavone of the invention.to a mammal results in a reduction in
the amount
of substances in the blood which contribute to CAD, such as for example apoB,
LDL,
cholesterol, etc; preferably reduction of the seruin, plasma, or whole blood
concentration or its
r.ivo amounts of these substances. Preferably the concentration or ire vivo
atoount of these
substances is, reduced to.t~orr~tal levels typically found in such a mammal.
Also, preferably,
the pol~nnethoxylatedflauone of the present invention are administered in
amounts which
prad~tce little ~r nu cytotosicity, nlore prefera,b(y where no cytotoxicity is
produced.,
I3~~ v~ay ofdeCnition, a polymethoxylatedllavone is a flavone substituted with
methoxy groups, preferably at least ?, more preferably at least 3, more
preferably at least 4,
snore preferably 4-S, and most prefecably,4-7 toethaxy groups and optionally
substituted by
one or more hydroxy groups, preferably 1-3, and store preferably 1-2 hydroxy
groups.
Cour COt11p0u11d5 Of the present invention v~ere synthesized from the lemon
flavonoid
(irnocitrin (3',8-dimethoxv.3,5,7,4'-tetrahydroxyflavone) for use in the
present invention:
limocitrin-3,7,4'-tritneth~~lether (5-hydroxy-3,7,8,3',4'-
pentamethoxyflavone); limocitrin-
3,5,7,4'-tetramethylether (3,5,?,8,3'4'-ltexamethoxyflavone); and limocitrin-
3,7,4'-
trimethylether-5-acetate.
. A nur~iber of methovylated flavo~~es, most of which occur naturally in
citrus, have
been found to be useful in the present invention. Also included are
substituted derivatives of
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8
quercetin. Tlte compounds its these groups include 5-desmetl~~~lnob'sletin (5-
hydrovy-
G,7,8,3',4'-pentametltoxyflavone); tetra-O-trtetli5~lisosculellarein (5,7,8,4'-
tetraroetl~oxyflavolte); .,5,6,7,8,J',4'-lteptametliox~~flavooe; nobiletin
(5,6,7,8,J',4'-
hexametllovyflavone); tartgeretin (5,6,7,8,4'-penlametltoxyflavone);
sinensetin (5,G,7,3',4'-
pentanietltoy~flavone); 5-desntel,ltylsiltelseli~n (5-hydroy~-6,7,J',4'-
,lelramelho;c~rflavrjnc);
quercetin lelramethyletl~er (5-hydroxy-3,7,3',4'-tetrametltox)~Ilavone);
quercetiri 3,5-
dimelhyletlner-7,3',4.'-tribenz,ylether; quercetin penlamethyl ell,ter
(3,5,7,3',4'-
pentamethoayflavone); quercelin-5,7,3',4'-tetramelfiylether-J-acetate;
quercelin-5,7,3',4'-
telramethylether (3-h~~droxy-5,7,J',4'-tetramethotyflavone).
'fhe basic StflJCtUrCS for limicitrin derIVOtIVeS arid S-
de5rr10lltyI5111en5etin 8re
represented
below:
OCI-I~ OCI-13
OCI-fo
OCH
OCH~
Cf 130 Cl-1~0' ~ ~O~'
OCI-l3 .
Ac
(t11~;7Crtr-trl-3,7,4'-trinrthylether linx~citriv-3,7,4'-trimelhylellter-5-
aoetale
OCI-1~ OCI-fi
OCH~ . OCi-1
O C l-I~
CH30 / O \ CI t 0
w
OCI-fi
C I-15.0 '
l-I3
lit~-rocitr'tw-3 , 5.7 .~~'- tetrame lhyletlver 5-desn5eth~~l5'rve ttse tin
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9
Examples of tocokrienol compounds useful in the present invention include, but
are
not lirT~ited to. are alpha-tocotrienol, gamma-tocotrienol, delta-tocotrienol,
and mixtures
thereof.
Examples of cholesterol-lowering dt'UES for the treatment of cardiovascular
diseases
or disorders useful in the present invention include, but are not limited to,
are cholestyramine,
colestipol, cloFbrate, gemftbrozil or lovastatin,
The methods of the present invention rnay be administered to any mammal. Most
preferably, the polyrnetl~oxylatedflavone useful in the methods of the present
invention are
administered to humans.
In another aspect of tile present invention, the polymethoxylatedflavone tray
be
formulated into a pharmaceutical preparation by a conventional method usually
employed in
the art, .
Dosages for the compositions of the present invention may be formulated into
pharn~aceutical,preparations for administration to mammals for reduction,
prevention, and
tt'eacoelat of cardiowscular diseases. Examples. not limited thereto, of
cardiovascular
disease treatable by~ tile COtI'1p051ttUr15 Of the present in~~ention include
hypercholesteroletnta,
hyperlipidemia. atl~erosclerosis, tllron2bosls, nmocardial infarction, etc
1'~lany of the limocitrin derivatives. quercetin derivatives. naturally-
occurring
polymethoxyflavones, tocotrienal compounds and mixtures thereof may 6e
provided as
compounds,with pharmaceutically compatible counterions , a fOCnl In WhLCh they
may be
salable, Counterio,ns for the purposes of this invention include, for example,
hydrophilic and
h7'dr017110biC aotnts.
The polymethoxylatedflavone can be administered by a variety of routes,
including
oral, transdern~al, rectal, intrartieular, intravenous, and intramuscular
introduction. However,
it should,be,understood that the amount of the polymethoxylatedflavone
actually
administered ought to be determined in light of various relavent,factors
includipg the ,
condition to be treated, the chosen route of administration, tIe age and
weight of the
individual patient. end tl~e severity of the patient's condition, and
therefore, the doses given
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Herein should not be construed to limit tl~e scope of the itwer~tion in any
wa~~. The
polyniethoxylatedflavone useful in the present invention may be administered
in a
pharmaceutically or: physiologically acceptable carrier. The pharmaceutically
or
physiologically acceptable carrier is any solvent with which the
polymethoxylatedflavone is
compatible and ~~.'111C11 15 n011-toxlC to individuals treated at the amounts
administered. A
variety. of delivery 5f 5te1715 for pharmacological coti~positions may be
employed including,
but not limited~to,, liposomes and emulsions., The pharmaceutical compositions
also may
comprise suitable solid or ge) phase carriers or excipients. Examples of
excipients include,
but are not limited to, calcium carbonate, calcium phosphate, various sugars,
starches,
cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Formulations suitable for oral administration include liquid solutions of the
active
compound or compounds dissolved in a diluent such as, for example, saline,
water, P~'G 400;
solid preparations such as capsules or tablets, each containing a
predetermined amount of tl~e
active agent as solids, granules, gelatins, suspensions, and/or emulsions.
FoCtllUlat1o11S SLlltable For parenteral administration include aqueous and
non-aqueous
isotonic sterile solutions which contain buffers. antioxidants, and
preservatives. The
fore aulations may be in unit dose or mufti-dose containers.
Dosages administered are any effective amount of a p,olytivethoxylatedflavone
which
will, when given for the treatment, prophylactically or therapeutically,
reduce or prevent
cardiovascular diseases by reducing levels of substances wHicH contribute to
cardiovascular
diseases to normal or near normal levels in the, blood or in vivo. By way of
deFtnition
substances which contribute to.cardioyscular diseases, include but are not
limited to
apohrotein B, low density lipoproteins, very law density lipoproteins.
cholesterol; etc.
Fatient dosages for oral administration of f(avonoids range from about 1-1000
mg/day, commonly 1-S00 mglday, and typically 1-100 mgiday. Stated in terms of
patient
with a 70 kg body weight, usual dosages range from about 0.01-15 mg/kglday,
commonly'
from about O.b,l-7.0 mg/kg/day, and typically from,about 0.01-2.0 mg/kg/day.
C'atient dosages for oral adnoni,stration oi-synthetic flavonoid analogues
range from
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11
about 2000-5000 n~g/day. con~noonly front about 1000-2000 mg/dav, and
typically front
about 500-1500 mglday.
Patient dosages for oral administration of limocitrin deri~~atives, quercetin
derivatives,
naturally-occurring polymethoxyflavones, and tocotrienols range from about I-
1000 mg/day,
COrllmOtlly about l-500 mglda_,~, and typically from about 1-100 n~g/day .
Patient dosages for oral, administratian of s~~nthetic limocitrin derivatives
range from
about 200-500 ntglday, commonly about 1000-2000 mg/day, and typically from
about 500-
1500 mg/day,
Patient dosages Cor oral administration of naturally-occurring
polymeihoxyflavones
range fram about 1-1000 mglday, commonly fTOm about 1-500 mglday, and
ri~pically front
about 1-100 mglday. Stated in terms of patient body weight, for about 70 kg
bodyyveight,
usual dosages range from about 0,01-15 mglkglda5~, commonly from about 0,01-
7.0
mglkg/day, and typically from about 0.01-2,0 mgJhglday.
Dosage amount and interval tray be adjusted individually to provide plasma
levels of
1
the active moiet~~ v.vltich are sufficient to maintain the anti-proliferative
and antioxidative
effects of the disease being treated;
For local administration. the camposition can be administered by injection.
directly
into a tissue, often in a depot or sUStalIled release farrrtulation.
The following examples illustrate the use of the inventian for lowering
substances which contribute to cardiovascular diseases and disorders. They are
intended to
further illustrate the invention and are not intended to limit the scope of
th,e invention as
defined by the claints.
EXAI'~LPLE 1
Limocitrin derivatives, quercetin derivatives, naturally occurring
polymethoxyflavones, and tocotri,enoIs were tested for cholesterol lowering
activity in HepG2
cells. Canfluent 1-iepG2 cells were preincubated for 24 hours in a lipoprotein-
free medium
(M~inimuni Essential l~~Sediuni (1~~1C1vI), Life Technola~ies, Burlington,
Canada) in which the
feral bovine serum (Li'fe Tecl~nolo~.:ies, Burlington, Canada) was replaced by
bovine serum
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12
albun3in (BS:4, Life Technologies, Burlington. Canada to inhibit cell
proliferation and to
stimulate synthesis of cholesterol-containing lipoproteins. Cells were
subsequently incubated
for another 24 hours in tl~e same med,iunt in the presence,ar absence of
limocitrin derivatives,
quercetin derivatives, naturally occurring polv~nethowtlavooes, or
tocotrienols (See Table 1)
at the highest concentrations sustaining about 100°ro X10°o cell
viability, as determined by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diplzetsyltetrazolium bromide (IvSTT)
viability assay, The.
test compounds were added to the cell culture n~ediunoaf~ter solubilization in
dimethyl
sulfoxide (DI\~ISO) and f Iter sterilized. The final concentration of DIufSO
in culture media
did not exceed U.3% by volume, to prevent anv effects of DIvI50 on apo B
metabolism. ror
each experiment, equal cancentration$ of solvent were added to all treatment
media and to a
control medium (without tested compounds). ,fit the end of the incubation,
culture tiledia
were collected and the concentration of the LDL structural protein, apo-B, was
measured by
ELIS~.. The competitive Elisa assay kit Far determination of human apa B
(Ortha Diagostics,
La.lalla, CA) treasures medium apo B concentrations within the range 0:4-0.031
~tg/mL. In
this assay, microtiter 96-well plaies are coated withmonoclotlal antibody
against human apo 1
B, which binds apo B epitope fror~i hun~ar~ LDL (standard curve, control sera
with high and
lour content of human apo B) or from LDL-like lipaprat2itt: secreted by HepG?
cells (tested
media, usually 2 x diluted with CaZi- and l'~Ig-'--free plospb.ate buffered
saline (PBS)). After,,
the exposure, a fraction of antibody is blocked and the remaining amount is
captured by a
subsequent incubation with conjugate cantainino a secondazy antibody (against
anti-human
apo B) coupled with horseradish peraxidase. Tl~e falfowin~ incubation with
liorseradisft
peroxidase substrate, o-phenyler~edi,atnioe, produces a yellow color change
proportional to the
amount of apo L present, and tlye optical density of samples is treasured at
490-492 nm
against tl~e blank PB5/canjugate wells. Ta determine whether evaluated
compounds cross-
react with Elisa assay, in separate study. various doses of compounds were
tested in.presence .
of cell-free culture media.
Amount of lipoprotein-associated apo-B in tl~e media ~ nzt apo-B secretion)
was determined
CA 02403548 2002-09-17
WO 01/70029 PCT/USO1/08395
13
as descriL,ed above, and calculated per nag cell protein. Net apo B secretion
was also
expressed as percent of control (amount of apo B in medium from cells
incubated without
tested compounds).
Table 1: IC5° concentrations were 2.~ ~rg/mL for tange,retin, 4.9
~tg/mL for nobiletin, 7.8
i.rg/mL for ltehtamethosyflavone and 17,8 ~Cg/mL for sinensetin.
Compound ConcentrationApo B ' apo B % apo
' B .
pg/mL mediumug/mL medium in medium reduction
5, 6, 7, 8, 4'-
pentamethoxyfl.avone0 0.252 ~ 0.20 100 ~ 4 -
3.12 0.080 ~ 0.02 32 ~ 8 GS
G.25 0.055 ~ 0.01 22 ~ 2 78
12.5 0.048 1 0.01 19 1 2 81
25.0 0.035 ~ 0.01 14 ~ 1 8G
5, 6, 7. 8, 3'. 4'- 0 ~ 0.55 t 0.21 100 t 15 -
heaamcthoayflavone 6.25 0.37 X0.09 G7 ~ 1G 33
12.5 0.1750.03 321G G8
25.0 0.10 ~ 0.01 18 1 1 82
50.0 0.095 ~ 0.01 17 t 1 83
1-leptamethovyflavone0 0.250 t 0.02u100 ~ 8 -
6.25 0.138y0.02 555 45
12.5 0.080 ~ 0.02 32 f 9 G8
25.0 0.0480.01 19~ , 81
50.0 0.045 :~ 0.0118 t 2 82
Sinensetin 0 0.93 ~ 0.07 100 f 7 -
G.25 0.70 ~ 0,13 75 ~ 14 25
~
12.5 0.54 X0.04 584 ~ 42
25.0 0.35 ~ 0,04 38 t 5 62
50.0 0.26 ~ 0.02 28 ~ 5 ' 72
'Tetra-O-methyl- 0 . 0.430 i 0.01 100 ~ 10 -
5cutellarein G.25 0.305 :~ 0.0371 ~ 7 29 .
12.5 0.255 ~ O.OG 59 ~ 13 41
5-de5methylsinensetin0 1.15 ~ 0.29 100 ~ 25 -
25.0 0.84 ~ 0.21 73 ~ 18 27
~
50.0 0.41 t 0.20 3G ~ 1 G4
~
