CA2403548A1 - Compositions and methods of treating, reducing, and preventing cardiovascular diseases and disorders with polymethoxyflavones - Google Patents
Compositions and methods of treating, reducing, and preventing cardiovascular diseases and disorders with polymethoxyflavones Download PDFInfo
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- quercetin
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
Compositions and methods for the treatment, reduction and/or prevention of cardiovascular diseases and disorders are described. Individuals at high ris k for developing or having cardiovascular disease or disorder may be treated with an effective dose of a polymethoxyflavone including limocitrin derivatives, quercetin derivatives, naturally occurring polymethoxyflavones, tocotrienols, and mixtures of these compounds.
Description
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 purposes of the present invention include:
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 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, 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 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 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 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, 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 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 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 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 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 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 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 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 ~
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, 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 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 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 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, 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 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 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 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 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 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 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 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 ~
Claims (12)
quercetin tetramethylether (5-hydroxy-3,7,3',4'-tetramethoxyflavone);
quercetin-3,5-dimethylether-7,3',4'-tribenzyl ether; quercetin pentamethyl ether (3,5,7,3',4'-pentamethoxyflavone); quercetin-5,7,3',4'-tetramethylether-3-acetate; and quercetin-5,7,3',4'-letramethylether (3-hydrovy-5;7,3',4'-tetramethoxyflavone); and the naturally occurring polymethoxyflavones: 3,5,6,7,8,3',4'-heptamethoxytlavone; nobiletin (5,6,7,8,3'.4'-.
hexamethoxyflavone); tangeretin (5,6.7,5,4'pentamethoxyflavone); 5-desmethylnobiletin (5-hydroxy-6,7,8,3',4'-pentametliovyllavone); tetra-O-methylisoscutellarein(5,7,5,4',-tetramethoxyflavone); 5-desmethylsinensetin (5-hydroxy-6,7,3',4'-tetramethoxyflavone); and sinensetin (5,6.7,3'.4'-pentamethoaytlavone), The present invention is not to be limited in scope by embodiments disclosed in the examples that are intended illustration purposed and any methods which are functionally equivalent are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to one of ordinary skill in, the from the foregoing description, Such modifications are intended to fall within the scope of the appended claims. The various publications cited herein are herein incorporated by, reference in their entirety.
We claim:
1. A method for reducing the level of substances in a mammal which contribute to cardiovascular diseases or disorders comprising administering to a mammal in need of such treatment an effective amount of a polymethoxyflavone to reduce the production of substances in a mammal which contribute to cardiovascular diseases and/or disorders.
2. The method of claim 1 wherein the substances are selected from the group consisting of apolipoprotein B, cholesterol, low density lipoprotein, very low density lipoprotein and mixtures thereof.
3. The method of claim 2 wherein the substance is apolipoprotein,B.
4. The method of claim 1 wherein said mammal is a human.
5. The method of claim 1 wherein in said polymethoxyflavone is selected from the group consisting of limocitrin-3,7,4'-trimethylether (5-hydroxy-3,7,8,3',4'-pentamethoayflavone), limocitrin-3,5,7,4'-tetratnethylether (3,5.7,8,3',4'-hexamethoxyflavone), limocitrin- 3,5,7,4'-tetraethylether (8.3-dimethoxy-3,5,7,4'-tetramethoxyflavone), limocitrin-3,7,4'-trimethylether-5-acetate, and mixture thereof.
6. The method of claim 1 wherein said polymethoxyflavone is selected from the group consisting of 5-desmethylnobiletin (5-hydroxy-6,7,8,3',4'-pentamethoxyflavone); tetra-O-methylisoscutellarein (5,7,8.4'-tetramethoxyflavone), sinensetin (5,6,'7,3,'4'-pentamethoxyflavone), 5-desmethylsinensetin (5-hydroxy-6,7,3',4'-tetramethoxyflavone), quercetin tetramethylether (5-hydroxy-3,7,3',4'-tetramethoxyflavone), quercetin-3.5-dimethylether-7,3',4'-tribenzyl ether, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), quercetin pentamethyl ether (3,5,7,3',4'-pentametltoyflavone), tangeretin (5,6,7,8,4'-pentamethoxyflavone), quercetin-5,7,3',4'-tetramethyl ether-3-acetate, 3,5,6,7,8,3',4'-heptamethoxyflavone, 5,7,3',4'-tetramethylether (3-hydroxy-5,7,3',4'-tetramethoxyflavone), and mixtures thereof.
7. The method of claims 5 or 6 further comprising administering at least one tocotrienol wherein said tocotrienol is selected from the group consisting of alpha-tocotrienol, gamma-tocotrienol, delta-tocotrienol, and mixtures thereof.
8. The method of claims 5 or 6 further comprising administering at least one pharmaceutical drug used in inhibiting cardiovascular diseases.
9. A pharmaceutical, composition suitable for administering to a mammal at risk or suffering from a cardiovascular disease or disorder comprising an effective amount of at least one polymethoxyflavone to reduce the production of substances in a mammal which contribute to cardiovascular diseases or disorders selected from the group consisting of limocitrin-3,7,4'-trimethylether (5-hydroxy-3,7,8,3',4'-pentamethoxyflavone), limocitrin-3.5,7.4'-tetramethylether (3,5.7.8,3',4'-hexamethoxyflavone), limocitrin-.
3,5,7,4'-tetraethylether (8.3-dimethoxy-3.5,7,4'-tetraethoxyflavone), limocitrin-3.7,4'-trimethylether-5-acetate, and mixtures thereof.
3,5,7,4'-tetraethylether (8.3-dimethoxy-3.5,7,4'-tetraethoxyflavone), limocitrin-3.7,4'-trimethylether-5-acetate, and mixtures thereof.
10. A pharmaceutical composition suitable for administering to a mammal at risk or sufferine from a cardiovascular disease or disorder, said contposition'comprising an effective amount of at least one polymethowflavone to reduce the production of substances in a mammal which contribute to cardiovascular diseases or disorders selected from the group consisting of 5-desmethylnobiletin (5-hydroxy-6,7,8,3',4'-pentametltovyflavone); tetra-O-methylisoseutellarein (5,7,8.4'-tetramethaxyflavone), sinensetin (5,6,7,3,'4'-pentamethoxyflavone), 5-desmethyl,sinensetin (5-hydroxy-6,7.3',4'-tetramethoxyflavone), quercetin tetramethyether (5-hydroxy-3,7,3',4'-tetramethoxyflavone), quercetin-3,5-ditttethylether-7,3',4'-tribenzyl ether, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), quercetin pentamethyl ether (3,5,7.3',4'-pentamethoxyflavone), tangeretin (5,6,7,8,4'-pentamethoayflavone), quercetin-5,7,3',4'-tetramethyl ether-3-acetate, 3,5,6,7,8,3',4'-heptamethoayflavone, quercetin-5,7,3',4'-tetramethyl ether (3-hydroxy-5,7,3',4'-tetramethoxyflavone), and mixtures thereof.
11. The composition of claims 9 or 10 further comprising at least one tocotrienol wherein said tocotrienol is selected from the group consisting of alpha-tocotrienol, gamma-tocotrienol, delta-tocotrienol, and mixtures thereof.
12. The composition of claims 9 or 10 further comprising administering at least one pharmaceutical drug used in inhibiting cardiovascular diseases.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/528,488 US6987125B1 (en) | 1998-10-06 | 2000-03-17 | Compositions and methods of treating, reducing and preventing cardiovascular diseases and disorders with polymethoxyflavones |
| US09/528,488 | 2000-03-17 | ||
| PCT/US2001/008395 WO2001070029A1 (en) | 2000-03-17 | 2001-03-16 | Compositions and methods of treating, reducing, and preventing cardiovascular diseases and disorders with polymethoxyflavones |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2403548A1 true CA2403548A1 (en) | 2001-09-27 |
Family
ID=24105873
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002403548A Abandoned CA2403548A1 (en) | 2000-03-17 | 2001-03-16 | Compositions and methods of treating, reducing, and preventing cardiovascular diseases and disorders with polymethoxyflavones |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1278417A1 (en) |
| AU (1) | AU2001247471A1 (en) |
| CA (1) | CA2403548A1 (en) |
| WO (1) | WO2001070029A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005115378A1 (en) * | 2004-05-26 | 2005-12-08 | Kgk Synergize Inc | Compositions comprising flavonoids and tocotrienols and methods thereof |
| WO2005115377A1 (en) * | 2004-05-26 | 2005-12-08 | Kgk Synergize Inc | Functional foods comprising flavonoids and tocotrienols and methods thereof |
| US7683095B2 (en) | 1998-10-06 | 2010-03-23 | The United States Of America As Represented By The Secretary Of Agriculture | Compositions and methods of treating, reducing and preventing cardiovascular diseases and disorders with polymethoxyflavones |
| US9132117B2 (en) | 2013-06-17 | 2015-09-15 | Kgk Synergize, Inc | Compositions and methods for glycemic control of subjects with impaired fasting glucose |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7201928B1 (en) | 1999-09-21 | 2007-04-10 | Rutgers, The State University Of New Jersey | Extracts of orange peel for prevention and treatment of cancer |
| WO2002087567A2 (en) * | 2001-05-02 | 2002-11-07 | Kgk Synergize Inc. | Polymethoxylated flavones for treating insulin resistance |
| JP2008513349A (en) * | 2005-05-24 | 2008-05-01 | ケージーケー シナジャイズ インコーポレイテッド | Functional food containing flavonoid and tocotrienol and method thereof |
| US7887852B2 (en) | 2005-06-03 | 2011-02-15 | Soft Gel Technologies, Inc. | Soft gel capsules containing polymethoxylated flavones and palm oil tocotrienols |
| WO2007109071A2 (en) * | 2006-03-15 | 2007-09-27 | Rutgers, The State University Of New Jersey | Hydroxylated polymethoxyflavone compositions |
| KR101340675B1 (en) | 2011-12-05 | 2013-12-12 | 대구한의대학교산학협력단 | Pharmaceutical composition comprising tangeretin for preventing or treating cardiovascular diseases |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3867541A (en) * | 1972-05-03 | 1975-02-18 | Ralph C Robbins | Compositions and methods for disaggregating blood cells |
-
2001
- 2001-03-16 WO PCT/US2001/008395 patent/WO2001070029A1/en not_active Application Discontinuation
- 2001-03-16 AU AU2001247471A patent/AU2001247471A1/en not_active Abandoned
- 2001-03-16 EP EP01920415A patent/EP1278417A1/en not_active Withdrawn
- 2001-03-16 CA CA002403548A patent/CA2403548A1/en not_active Abandoned
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7683095B2 (en) | 1998-10-06 | 2010-03-23 | The United States Of America As Represented By The Secretary Of Agriculture | Compositions and methods of treating, reducing and preventing cardiovascular diseases and disorders with polymethoxyflavones |
| WO2005115378A1 (en) * | 2004-05-26 | 2005-12-08 | Kgk Synergize Inc | Compositions comprising flavonoids and tocotrienols and methods thereof |
| WO2005115377A1 (en) * | 2004-05-26 | 2005-12-08 | Kgk Synergize Inc | Functional foods comprising flavonoids and tocotrienols and methods thereof |
| US9132117B2 (en) | 2013-06-17 | 2015-09-15 | Kgk Synergize, Inc | Compositions and methods for glycemic control of subjects with impaired fasting glucose |
| US9610276B2 (en) | 2013-06-17 | 2017-04-04 | Kgk Synergize, Inc. | Compositions and methods for glycemic control of subjects with impaired fasting glucose |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2001247471A1 (en) | 2001-10-03 |
| WO2001070029A1 (en) | 2001-09-27 |
| EP1278417A1 (en) | 2003-01-29 |
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