Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • 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 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • 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 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

• This invention generally relates to a method for inhibiting expression or activity of an adhesion molecule associated with an endothelial cell by contacting the adhesion molecule or endothelial cell with one or more isoflavone compounds or derivatives thereof.
• the invention also generally relates to a method of preventing or reducing the risk of restenosis after angioplasty, and to a method for the treatment or prophylaxis of atherosclerosis, coronary artery diseases, other cardiovascular diseases and inflammatory diseases mediated by adhesion molecules.
• the invention further generally relates to pharmaceutical compositions useful in these methods, and to methods for the manufacture of such medicaments. Further aspects of the invention will become apparent from the description, which follows.
• Atherosclerosis is a chronic inflammatory disease of the arterial intima characterised by the focal accumulation of leukocytes, smooth muscle cells, lipids and extra cellular matrix. Deposits of lipids and other blood derivatives in blood vessel walls, especially of the large arteries, results in the formation of plaques. As the adherence of lipids and leukocytes to the blood vessel wall continues, there is a concomitant thickening of the vessel wall. The size increase of the plaque gains a stenosing character which becomes responsible for vascular occlusions by atheroma, thrombosis or embolism. Serious vascular problems can result including infarction, cardiac insufficiency, stroke and sudden death.
• Atheromatous plaque is composed primarily of cells containing cholesterol; consequently a high blood cholesterol level is associated with increased risk of atherosclerosis. While a high absolute cholesterol level is an important risk factor, the risk is more specifically associated with the type of lipoprotein present in the blood.
• LDL low density
• VLDL very low density
• HDL high density lipoprotein
• Atherosclerosis progressed by the oxidation of LDL cholesterol in the arterial wall, leading to an inflammatory lesion. If left unchecked the inflammatory process proceeds until the lesion causes vascular construction, obstruction and infarct.
• Antioxidants are molecules or compounds that can inhibit oxidation (i.e. damage) by chemically inactivating the free radicals produced during normal physiological function. If the body is lacking in a supply of antioxidants, some free radicals remain active and attack healthy cells or convert safe compounds into damaging ones. Antioxidants play many important roles in cardiovascular disease. They have attracted particular interest as potential inhibitors of atherosclerosis via inhibition of the oxidative modification of the low density lipoprotein (LDL). Lipoproteins are the major carriers of cholesterol in the body. The majority of the cholesterol associates with LDL to be distributed throughout the body from the liver. The high density lipoprotein (HDL) "mops up" excess cholesterol present in the blood and returns it to the liver.
• LDL low density lipoprotein
• Lipoproteins are very susceptible to oxidation, and once oxidised can accumulate in healthy arterial and vascular walls without being metabolised. Such gathering of lipoproteins greatly increases the risk of atherosclerosis.
• the oxidation of LDL in the sub-endothelial space represent an early and causative step in atherogenesis (Steinberg et al, 1989).
• Oxidized LDL' formation by antioxidants is generally thought to slow down the progression of the disease.
• cardioprotective agents can have utility, and in which more and better agents are sought.
• cell adhesion molecules are involved in the adhesion of leukocytes and monocytes to tissues including the vascular endothelium.
• Known cell adhesion molecules include intercellular adhesion molecules (ICAM 1, 2 and 3), vascular cell-adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1).
• ICM 1, 2 and 3 intercellular adhesion molecules
• VCAM-1 vascular cell-adhesion molecule-1
• PECAM-1 platelet endothelial cell adhesion molecule-1
• cell adhesion molecules are upregulated by several coronary heart disease risk factors and antibodies to cell adhesion molecules are believed to prevent reperfusion injury in animal models. Accordingly, modulation of the expression or activity of adhesion molecules associated with endothelial cells may be useful in the treatment or prevention of cardiovascular pathology by limiting the development of atherosclerotic plaque.
• E-selectin is a cell surface protein inducibly expressed in cytokine-activated endothelial cells in response to inflammatory factors such as occurs with tissue injury.
• the expression of E-selectin by endothelial cells can also be induced by inflammatory factors including interleukin-1 (IL-1), tumour necrosis factor- ⁇ (TNF- ⁇ ) and various endotoxins.
• IL-1 interleukin-1
• TNF- ⁇ tumour necrosis factor- ⁇
• E-selectin is associated with the binding of endothelial cells and platelets with leukocytes and lipids.
• leukocytes The binding of leukocytes to endothelial cells is observed at an early stage after tissue injury and is associated with various acute and chronic inflammation. Suppression or inhibition of the expression or activity of adhesion molecules associated with endothelial cells will limit the focal accumulation and adhesion of leukocytes to vessel walls, particularly at areas of injury, damage or infection.
• angioplasty In the treatment of atherosclerosis, angioplasty has been developed to permit non-surgical intervention of the atherosclerotic plaque.
• dilation of the blood vessel beyond its ability to recoil completely with a balloon catheter increases the vessel lumen, thereby allowing for increased blood flow.
• This procedure however causes mechanical injury of the arterial wall, following which restenosis commonly occurs.
• restenosis is a major problem following traumatic injury rendered to vessels during vascular surgery and treatment.
• Such therapies include the administration of compounds which arrest cell division and hyperproliferative disorders, necrose vascular smooth-muscle cells and block the expression of endothelial cell adhesion molecules.
• Further compounds useful in addressing restenosis include lipid lowering agents, anti- platelet agents, anti-thrombotic agents, calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors and ⁇ -blockers.
• ACE angiotensin converting enzyme
• ⁇ -blockers Generally though, as therapeutic agents are not selective, there are often side effects which need to be monitored or countered.
• drugs currently in use such as Ticlid (ticlopidine), Plavix (clopidrogel) and Cardiprin (aspirin).
• gastrointestinal disturbances and skin rashes are commonly reported side effects with their use.
• Other agents such as heparin reportedly inhibits smooth muscle cell proliferation in vitro but when used in vivo has the adverse side effect of inhibiting coagulation.
• vascular disease is currently a leading cause of death in today's society, there is a strong need to identify new, improved, better and alternative methods and pharmaceutical agents for its treatment and prevention.
• isoflavone compounds, metabolites and derivatives thereof are particularly useful for inhibiting or down-regulating the expression or activity of adhesion molecules in endothelial cells. It has also been found that isoflavones and derivatives thereof are particularly useful for inhibiting endothelial cell surface adhesion molecules, and in particular E-selectin and VCAM-1.
• isoflavones and derivatives thereof find use in preventing or reducing the risks of restenosis associated with vascular intervention including angioplasty treatment of atherosclerosis, and the resultant mechanical injury at the angioplasty site during treatment of an atherosclerotic lesion.
• Isoflavone compounds, metabolites, derivatives and analogues thereof useful in the methods of the present invention are depicted by the general formula I as set out below.
• a method for inhibiting expression or activity of an adhesion molecule associated with an endothelial cell comprises the step of contacting the adhesion molecule or endothelial cell with one or more compounds of formula I in an amount sufficient to inhibit said expression or activity.
• the adhesion molecule is E-selectin or vascular cell surface adhesion molecule (VCAM-1).
• a method for inhibiting the expression or activity of adhesion molecules associated with endothelial cells in a subject comprises the step of administering to the subject a therapeutically effective amount of one or more compounds of formula I.
• a method of treating a disease mediated by expression or activity of adhesion molecules associated with endothelial cells in a subject comprises the step of administering to the subject one or more compounds of formula I in an amount sufficient to inhibit said expression or activity of the adhesion molecules associated with the endothelial cells.
• the disease is a vascular disease including restenosis, inflammatory disease, coronary artery disease, angina or small vessel disease, more preferably post-angioplasty restenosis.
• a method for the treatment, amelioration, prophylaxis or reduction in the risk of restenosis in a subject comprises the step of administering to the subject a therapeutically effective amount of one or more compounds of formula I.
• the restenosis is associated with vascular intervention such as coronary intervention.
• vascular intervention such as coronary intervention.
• the vascular coronary intervention is percutaneous transluminal coronary angioplasty, direction coronary atherectomy or stent, more preferably angioplasty.
• a method for the treatment of procedural vascular trauma in a subject comprises the step of administering to the subject a therapeutically effective amount of one or more compounds of formula I.
• the procedural vascular trauma is angioplasty, vascular surgery, graft or transplant procedure.
• a method for the treatment or prophylaxis of vascular disease in a subject comprises the step of administering to the subject a therapeutically effective amount of one or more compounds of formula I.
• the vascular disease is restenosis, inflammatory disease, coronary artery disease, angina or small vessel disease, more preferably post-angioplasty restenosis.
• a pharmaceutical composition in a dosage form suitable for use in the treatment of a disease mediated by expression or activity of adhesion molecules associated with endothelial cells in a subject which composition comprises one or more compounds of formula I in association with a pharmaceutical acceptable carrier.
• compositions in a dosage form suitable for use in preventing or reducing the risk of vascular disease in a subject which composition comprises one or more compounds of formula I in association with a pharmaceutical acceptable carrier.
• a ninth aspect of the invention there is provided the use of one or more compounds of Formula I in the manufacture of a medicament for the treatment of a disease mediated by expression or activity of adhesion molecules associated with endothelial cells.
• stenosis is taken in its broadest sense to mean a narrowing or constriction of the diameter of a bodily passage or orifice, such as in particular a blood vessel or artery, and generally leads to reduced blood flow and the concomitant problems of vessel occlusion. Typically stenosis occurs as a result of the focal accumulation and deposit of lipids and other blood derivatives.
• restenosis or re-stenosis or secondary stenosis is taken in its broadest sense to mean a recurrence of stenosis typically after vascular intervention, injury or surgery, including balloon catheter treatment. Stenosis and restenosis can occur in blood vessels throughout the body, and of particular medical importance is the life threatening and often fatal effects of stenosis in the coronary arteries.
• isoflavones and derivatives thereof block the induced expression of the endothelial cell surface adhesion molecules, in particular E- selectin and VCAM-1, in response to many signals known to be active in atherosclerosis, restenosis, inflammatory response and other diseases mediated by cell adhesion molecule expression.
• endothelial cell surface adhesion molecules in particular E- selectin and VCAM-1
• isoflavone compounds and derivatives thereof are useful in the treatment or prophylaxis of restenosis, coronary artery diseases, angina and other vascular and cardiovascular diseases, inflammatory diseases mediated by adhesion molecules E-selectin and VCAM-1.
• the specific molecular mechanisms by which the isoflavones and derivatives function in inhibiting cell adhesion molecule expression are not fully understood.
• isoflavones and derivatives thereof inhibit cellular proliferation of human vascular smooth muscle cells, and also inhibit PDGF-induced Erk activation in human vascular smooth muscle cells. This activity shows the potential for isoflavones and derivatives thereof to prevent the development and progression of atherosclerotic lesions, providing a potential benefit in vascular protection
• Isoflavones and derivatives thereof have also been shown to inhibit endothelial cell proliferation and to inhibit endothelial cell migration, and thus have potential for use as cardioprotective therapeutics including the treatment, amelioration or prophylaxis of restenosis after vascular intervention.
• Isoflavones and derivatives thereof have also been shown to have potent vascular regulatory capacity.
• isoflavones and derivatives thereof are capable of antagonising contractile activity, antagonising direct vasodilatation, and protecting against endothelium damage by oxidised low density lipoprotein. While comparable to the ovarian steroid 17 ⁇ - estradiol, their activities also appear to be unique in their mechanism of action.
• the isoflavones and derivatives again show potential for use as cardioprotective therapeutics including the treatment, amelioration or prophylaxis of restenosis after vascular intervention
• the isoflavone compounds and derivatives can also be administered to treat small vessel disease that is not treatable by surgery or angioplasty, or other vessel disease in which surgery is not an option, and to stabilise patients prior to and after revascularisation therapy.
• the active compounds can also be administered in the period immediately prior to and following vascular intervention such as coronary or vascular angioplasty as a means to reduce or eliminate the abnormal proliferative and inflammatory response that currently leads to clinically significant restenosis.
• isoflavones can also be used in the treatment of cardiac transplant rejection and vascular graft and transplant procedures.
• the methods of this invention represent a significant advance in treating vascular conditions and disease, in that they go beyond well known therapies designed simply to inhibit the progression of disease.
• the experimental data provided herein has unexpectedly shown that restenosis after vascular intervention or angioplasty is inhibited or at least markedly reduced in various mammalian animal models.
• the present methods demonstrate the potential of isoflavone compounds and derivatives thereof to medically address restenosis, and possibly to cure atherosclerosis by preventing new lesions from developing and causing established lesions to stabilise or regress.
• the isoflavones encompass a class of phytoestrogens found predominantly in leguminous plants with estrogenic activity related to their structural similarity with steroidal estrogen. It is only until recently that the importance of isoflavones has been recognised by its apparent association with a reduction in risk of developing osteoporosis, some forms of cancer and its ability to lower cholesterol levels and blood pressure in mammals. It has also been suggested that many of the biological effects of isoflavones can been accounted for by their conversion in vivo to various other active metabolites. Given the importance of isoflavone metabolites, synthetic derivatives of isoflavones and metabolites are also seen as important therapeutic agents. Further isoflavones have been ingested for years as components of staple foods being very well tolerated with little or no known side effects, unlike many known symptomatic remedies of vascular diseases and related surgery.
• Ri, R 2 and Z are independently hydrogen, hydroxy, OR 9 , OC(O)R ⁇ 0 , OS(O)R 10 , CHO, C(O)R ⁇ o, COOH, CO 2 R ⁇ o, CONR 3 R 4 , alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or R is as previously defined, and Ri and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from
• Ri is as previously defined, and R 2 and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from
• W is Ri
• A is hydrogen, hydroxy, NR 3 R or thio
• B is selected from
• W is Ri, and A and B taken together with the carbon atoms to which they are attached form a six-membered ring selected from
• R 3 is hydrogen, alkyl, arylalkyl, alkenyl, aryl, an amino acid, C(O)Ru where Rn is hydrogen, alkyl, aryl, arylalkyl or an amino acid, or CO 2 R ⁇ 2 where R 12 is hydrogen, alkyl, haloalkyl, aryl or arylalkyl,
• R 4 is hydrogen, alkyl or aryl, or R 3 and 4 taken together with the nitrogen to which they are attached comprise pyrrolidinyl or piperidinyl, R 5 is hydrogen, C(O)Rn where Rn is as previously defined, or CO2R 1 2 where R i2 is as previously defined,
• R 6 is hydrogen, hydroxy, alkyl, aryl, amino, thio, NR 3 R , COR ⁇ where Rn is as previously defined, CO2R 12 where R ⁇ 2 is as previously defined or CONR 3 R , R 7 is hydrogen, C(O)Rn where Rn is as previously defined, alkyl, haloalkyl, alkenyl, aryl, arylalkyl or Si(R ⁇ 3 ) 3 where each R ⁇ 3 is independently hydrogen, alkyl or aryl, Rs is hydrogen, hydroxy, alkoxy or alkyl,
• R 9 is alkyl, haloalkyl, aryl, arylalkyl, C(O)Rn where Rn is as previously defined, or Si(R 13 ) 3 where R 13 is as previously defined, Rio is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an amino acid, alkylamino or dialkylamino, the drawing "TM" represents either a single bond or a double bond, T is independently hydrogen, alkyl or aryl, X is O, NR 4 or S, and Y is
• R 14 , R 15 and R 16 are independently hydrogen, hydroxy, OR 9 , OC(O)R 10 , OS(O)R 10 , CHO, C(O)R ⁇ 0 , COOH, CO 2 R ⁇ o, CONR 3 R 4 , alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, including pharmaceutically acceptable salts thereof.
• the invention in particular relates to the use of compounds of the general formulae II - VIII:
• Ri, R 2 , R 5 , R 6 , R ⁇ 4 , Ri 5 , W and Z are as defined above,
• Ri, R 2 , R ⁇ , Ri 5 , W and Z are independently hydrogen, hydroxy, OR 9 , OC(O)Rio,
• R 5 is hydrogen, C(O)Rn where Rn is hydrogen, alkyl, aryl, or an amino acid, or CO 2 R 12 where R 12 is hydrogen, alkyl or aryl, R 6 is hydrogen, hydroxy, alkyl, aryl, CORn where Rn is as previously defined, or
• R 9 is alkyl, haloalkyl, arylalkyl, or C(O)Rn where Rn is as previously defined, and Rio is hydrogen, alkyl, amino, aryl, an amino acid, alkylamino or dialkylamino,
• Ri and R ⁇ are independently hydroxy, OR 9 , OC(O)Rio or halo
• R 2 , R ⁇ 5 , W and Z are independently hydrogen, hydroxy, OR 9 , OC(O)R ⁇ 0 , C(O)Rio, COOH, CO 2 R 10 , alkyl, haloalkyl, or halo
• R 5 is hydrogen, C(O)Rn where Rn is hydrogen or alkyl, or CO2R12 where R 12 is hydrogen or alkyl
• R 6 is hydrogen or hydroxy
• R 9 is alkyl, arylalkyl or C(O)Rn where Rn is as previously defined, and Rio is hydrogen or alkyl,
• Ri and R ⁇ 4 are independently hydroxy, methoxy, benzyloxy, acetyloxy or chloro
• R 2 , R 15 , W and Z are independently hydrogen, hydroxy, methoxy, benzyloxy, acetyloxy, methyl, trifluoromethyl or chloro
• R 5 is hydrogen or CO2R12 where R 1 2 is hydrogen or methyl
• R 6 is hydrogen.
• Particularly preferred compounds of the present invention are selected from:
• the preferred compounds of the present invention also include all derivatives with physiologically cleavable leaving groups that can be cleaved in vivo from the isoflavone or derivative molecule to which it is attached.
• the leaving groups include acyl, phosphate, sulfate, sulfonate, and preferably are mono-, di- and per-acyl oxy-substituted compounds, where one or more of the pendant hydroxy groups are protected by an acyl group, preferably an acetyl group.
• acyloxy substituted isoflavones and derivatives thereof are readily cleavable to the corresponding hydroxy substituted compounds.
• the protection of functional groups on the isoflavone compounds and derivatives of the present invention can be carried out by well established methods in the art, for example as described in T. W. Greene (1981).
• isoflavone compounds contemplated for use in accordance with the invention include formononetin, biochanin, genistein, daidzein and equol, and functional derivatives, equivalents or analogues thereof.
• isoflavone metabolites including dihydrodaidzein, cis- and trans-tetrahydrodaidzein and dehydroequol, and derivatives and prodrugs thereof.
• Chemical and functional equivalents of a particular isoflavone should be understood as molecules exhibiting any one of more of the functional activities of the isoflavone and may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening.
• alkyl is taken to include straight chain, branched chain and cyclic (in the case of 5 carbons or greater) saturated alkyl groups of 1 to 10 carbon atoms, preferably from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl, cyclopentyl, and the like.
• the alkyl group is more preferably methyl, ethyl, propyl or isopropyl.
• the alkyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C ⁇ -C -alkoxycarbonyl, C ⁇ -C -alkylamino- carbonyl, di-(C ⁇ -C -alkyl)-amino-carbonyl, hydroxyl, C ⁇ -C -alkoxy, formyloxy, C ⁇ -C - alkyl-carbonyloxy, C ⁇ -C 4 -alkylthio, C 3 -C 6 -cycloalkyl or phenyl.
• alkenyl is taken to include straight chain, branched chain and cyclic (in the case of 5 carbons or greater) hydrocarbons of 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, with at lease one double bond such as ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 2-methyl-l-peopenyl, 2-methyl-2-propenyl, and the like.
• the alkenyl group is more preferably ethenyl, 1-propenyl or 2-propenyl.
• the alkenyl groups may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C ⁇ -C - alkoxycarbonyl, C ⁇ -C 4 -alkylamino-carbonyl, di-(C ⁇ -C 4 -alkyl)-amino-carbonyl, hydroxyl, C ⁇ -C -alkoxy, formyloxy, C ⁇ -C 4 -alkyl-carbonyloxy, C ⁇ -C 4 -alkylthio, C 3 -C 6 -cycloalkyl or phenyl.
• alkynyl is taken to include both straight chain and branched chain hydrocarbons of 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, with at least one triple bond such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.
• the alkynyl group is more preferably ethynyl, 1-propynyl or 2-propynyl.
• the alkynyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C ⁇ -C -alkoxycarbonyl, C ⁇ -C -alkylamino-carbonyl, di-(C ⁇ -C -alkyl)-amino- carbonyl, hydroxyl, C ⁇ -C -alkoxy, formyloxy, C ⁇ -C -alkyl-carbonyloxy, C ⁇ -C 4 -alkylthio, C 3 -C 6 -cycloalkyl or phenyl.
• aryl is taken to include phenyl, biphenyl and naphthyl and may be optionally substituted by one or more C ⁇ -C -alkyl, hydroxy, C ⁇ -C 4 -alkoxy, carbonyl, C ⁇ -C 4 - alkoxycarbonyl, C ⁇ -C 4 -alkylcarbonyloxy or halo.
• heteroaryl is taken to include five-membered and six-membered rings which include at least one oxygen, sulfur or nitrogen in the ring, which rings may be optionally fused to other aryl or heteroaryl rings including but not limited to furyl, pyridyl, pyrimidyl, thienyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isopuinolyl, purinyl, morpholinyl, oxazolyl, thiazolyl, pyrrolyl, xanthinyl, purine, thymine, cytosine, uracil, and isoxazolyl.
• the heteroaromatic group can be optionally substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C ⁇ -C -alkoxycarbonyl, C ⁇ -C 4 - alkylamino-carbonyl, di-(C ⁇ -C -alkyl)-amino-carbonyl, hydroxyl, C ⁇ -C -alkoxy, formyloxy, C ⁇ -C 4 -alkyl-carbonyloxy, C ⁇ -C -alkylthio, C 3 -C 6 -cycloalkyl or phenyl.
• the heteroaromatic can be partially or totally hydrogenated as desired.
• halo is taken to include fluoro, chloro, bromo and iodo, preferably fluoro and chloro, more preferably fluoro.
• Reference to for example "haloalkyl” will include monohalogenated, dihalogenated and up to perhalogenated alkyl groups. Preferred haloalkyl groups are trifluoromethyl and pentafluoroethyl.
• pharmaceutically acceptable salt refers to an organic or inorganic moiety that carries a charge and that can be administered in association with a pharmaceutical agent, for example, as a counter-cation or counter-anion in a salt.
• Pharmaceutically acceptable cations are known to those of skilled in the art, and include but are not limited to sodium, potassium, calcium, zinc and quaternary amine.
• Pharmaceutically acceptable anions are known to those of skill in the art, and include but are not limited to chloride, acetate, citrate, bicarbonate and carbonate.
• pharmaceutically acceptable derivative refers to a derivative of the active compound that upon administration to the recipient, is capable of providing directly or indirectly, the parent compound or metabolite, or that exhibits activity itself.
• treatment includes amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
• the amount of one or more compounds of formula I which is required in a therapeutic treatment according to the invention will depend upon a number of factors, which include the specific application, the nature of the particular compound used, the condition being treated, the mode of administration and the condition of the patient.
• Compounds of formula I may be administered in a manner and amount as is conventionally practised. See, for example, Goodman and Gilman, et al. (1995).
• the specific dosage utilised will depend upon the condition being treated, the state of the subject, the route of administration and other well known factors as indicated above.
• a daily dose per patient may be in the range of 0.1 mg to 5 g; typically from 0.5 mg to 1 g; preferably from 50 mg to 200 mg.
• the length of dosing may range from a single dose given once every day or two, to twice or thrice daily doses given over the course of from a week to many months to many years as required, depending on the severity of the condition to be treated or alleviated. It will be further understood that for any particular subject, specific dosage regimens should be adjust over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
• Relatively short term treatments with the active compounds can be used to cause stabilisation or shrinkage of coronary artery disease lesions that cannot be treated either by angioplasty or surgery. Longer term treatments can be employed to prevent the development of advanced lesions in high-risk patients.
• compositions for the treatment of the therapeutic indications herein described are typically prepared by admixture of the compounds of the invention (for convenience hereafter referred to as the "active compounds") with one or more pharmaceutically or veterinary acceptable carriers and/or excipients as are well known in the art.
• the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject.
• the carrier or excipient may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose, for example, a tablet, which may contain up to 100% by weight of the active compound, preferably from 0.5% to 59% by weight of the active compound.
• One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients.
• the preferred concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art.
• the formulations of the invention include those suitable for oral, rectal, optical, buccal (for example, sublingual), parenteral (for example, subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.
• Formulation suitable for oral administration may be presented in discrete units, such as capsules, sachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
• Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).
• the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture such as to form a unit dosage.
• a tablet may be prepared by compressing or moulding a powder or granules containing the active compound, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing, in a suitable machine, the compound of the free-flowing, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s).
• Moulded tablets may be made by moulding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
• Formulations suitable for buccal (sublingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
• compositions of the present invention suitable for parenteral administration conveniently comprise sterile aqueous preparations of the active compounds, which preparations are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the compound with water or a glycine buffer and rendering the resulting solution sterile and isotonic with the blood.
• Injectable formulations according to the invention generally contain from 0.1% to 60% w/v of active compound and are administered at a rate of 0.1 ml/minute/kg.
• Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
• Formulations or compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
• Carriers which may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combination of two or more thereof.
• the active compound is generally present at a concentration of from 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples of such compositions include cosmetic skin creams.
• Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
• patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the said active compound. See for example Brown, L., et al. (1998).
• Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Panchagnula R, et al, 2000) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2 M active ingredient. Formulations suitable for inhalation may be delivered as a spray composition in the form of a solution, suspension or emulsion. The inhalation spray composition may further comprise a pharmaceutically acceptable propellant such as carbon dioxide or nitrous oxide.
• a pharmaceutically acceptable propellant such as carbon dioxide or nitrous oxide.
• the active compounds may be provided in the form of food stuffs, such as being added to, admixed into, coated, combined or otherwise added to a food stuff.
• food stuff is used in its widest possible sense and includes liquid formulations such as drinks including dairy products and other foods, such as health bars, desserts, etc.
• Food formulations containing compounds of the invention can be readily prepared according to standard practices.
• Therapeutic methods, uses and compositions may be for administration to humans or animals, including mammals such as companion and domestic animals (such as dogs and cats) and livestock animals (such as cattle, sheep, pigs and goats), birds (such as chickens, turkeys, ducks) and the like.
• mammals such as companion and domestic animals (such as dogs and cats) and livestock animals (such as cattle, sheep, pigs and goats), birds (such as chickens, turkeys, ducks) and the like.
• the active compound or pharmaceutically acceptable derivatives prodrugs or salts thereof can also be co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, or antiviral compounds.
• the active agent can comprise two or more isoflavones or derivatives thereof in combination or synergistic mixture.
• the active compounds can also be administered with lipid lowering agents such as probucol and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents such as coumadin; calcium channel blockers such as verapamil, diltiazem, and nifedipine; angiotensin converting enzyme (ACE) inhibitors such as captopril and enalapril, and ⁇ - blockers such as propanolol, terbutalol, and labetalol.
• lipid lowering agents such as probucol and nicotinic acid
• platelet aggregation inhibitors such as aspirin
• antithrombotic agents such as coumadin
• calcium channel blockers such as verapamil, diltiazem, and nifedipine
• angiotensin converting enzyme (ACE) inhibitors such as captopril and enalapril
• ⁇ - blockers such as propano
• the compounds can also be administered in combination with nonsteriodal antiinflammatories such as ibuprofen, indomethacin, aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac.
• nonsteriodal antiinflammatories such as ibuprofen, indomethacin, aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac.
• the compounds can also be administered with corticosteroids.
• the co-administration may be simultaneous or sequential. Simultaneous administration may be effected by the compounds being in the same unit dose, or in individual and discrete unit doses administered at the same or similar time. Sequential administration may be in any order as required and typically will require an ongoing physiological effect of the first or initial active agent to be current when the second or later active agent is administered, especially where a cumulative or synergistic effect is desired.
• the isoflavones for use in the present invention may be derived from any number of sources readily identifiable to a person skilled in the art. Preferably, they are obtained in the form of concentrates or extracts from plant sources. Again, those skilled in the art will readily be able to identify suitable plant species, however, for example, plants of particular use in the invention include leguminous plants. More preferably, the isoflavone extract is obtained from chickpea, lentils, beans, red clover or subterranean clover species and the like.
• Isoflavone extracts may be prepared by any number of techniques known in the art.
• suitable isoflavone extracts may be prepared by water/organic solvent extraction from the plant source. It will be appreciated that an isoflavone extract may be prepared from any single tissue of a single species of plant or a combination of two or more different tissues thereof. Similarly, an extract may be prepared from a starting material which contains a heterogeneous mixture of tissues from two or more different species of plant.
• the material may be comminuted or chopped into smaller pieces, partially comminuted or chopped into smaller pieces and contacted with water and an organic solvent, such as a water miscible organic solvent.
• an organic solvent such as a water miscible organic solvent.
• the plant material is contacted with water and an organic solvent without any pre-treatment.
• the ratio of water to organic solvent may be generally in the range of 1:10 to 10:1 and may, for example, comprise equal proportions of water and solvent, or from 1% to 30% (v/v) organic solvent. Any organic solvent or a mixture of such solvents may be used.
• the organic solvent may preferably be a C2-10, more preferably a Cl-4 organic solvent (such as methanol, chloroform, ethanol, propanol, propylene glycol, erythrite, butanol, butanediol, acetonitrile, ethylene glycol, ethyl acetate, glycidol, glycerol dihydroxyacetone or acetone).
• the water/organic solvent mixture may include an enzyme which cleaves isoflavone glycosides to the aglycone form.
• the mixture may be vigorously agitated so as to form an emulsion.
• the temperature of the mix may range, for example, from an ambient temperature to boiling temperature. Exposure time may be between one hour to several weeks.
• the extract may be separated from undissolved plant material and the organic solvent removed, such as by distillation, rotary evaporation, or other standard procedures for solvent removal.
• the resultant extract containing water soluble and non-water soluble components may be dried to give an isoflavone-containing extract, which may be formulated with one or more pharmaceutically acceptable carriers, excipients and/or auxiliaries according to the invention.
• An extract made according to the description provided in the previous paragraphs may contain small amounts of oil which include isoflavones in their aglycone form (referred to herein as isoflavones).
• This isoflavone enriched oil may be subject to HPLC to adjust the isoflavone ratios, or, if it is at the desired isoflavone ratio, may be dried, for example in the presence of silica, and be formulated with one or more carriers, excipients and/or auxiliaries to give an isoflavone containing extract.
• the isoflavones contained in said small amounts of oil may be further concentrated by addition to the oil of a non-water soluble organic solvent such as hexane, heptane, octane acetone or a mixture of one or more of such solvents.
• a non-water soluble organic solvent such as hexane, heptane, octane acetone or a mixture of one or more of such solvents.
• a non-water soluble organic solvent such as hexane, heptane, octane acetone or a mixture of one or more of such solvents.
• a non-water soluble organic solvent such as hexane, heptane, octane acetone or a mixture of one or more of such solvents.
• 80% hexane, 20% acetone w/w having high solubility for oils but low solubility for isoflavones.
• the oil readily partition
• the present invention also contemplates the production of suitable isoflavones, functional derivatives, equivalents or analogues thereof, by established synthetic techniques well known in the art. See, for example, Chang et al. (1994) which discloses methods appropriate for the synthesis of various isoflavones.
• Each ring was mounted on two parallel stainless steel hooks in a single water-jacketed 3 ml organ bath containing modified Krebs solution maintained at 37 C and bubbled with 95% O 2 plus 5% CO 2 .
• the lower hook was attached to a movable support leg and the upper hook to an FTO3 force transducer (Grass Scientific Instruments, Mitutoyo, Japan) for isometric recordings. Changes in force were amplified (Quadbridge amplifier, Scientific Concepts Inc., Victoria, Australia) and recorded on a MacLab 8E data acquisition system (Adinstruments Pty Ltd., NSW, Australia) linked to an Apple computer (Apple Computer Inc, Cupertina, CA). Rings were set at an initial tension of 2 g and allowed to equilibrate for 30 minutes after which time they were re-set to 2 g tension before the commencement of each experimental protocol.
• Protocol 1 The effect of isoflavone metabolites on contractile curves to noradrenaline Full concentration-contractile curves were obtained to noradrenaline (0.1 nM - 10 mM) in the absence and presence of ⁇ -estradiol (1 ⁇ g/ml); Cpd. 5 (0.1 and 1 ⁇ g/ml); Cpd. 7 (0.1 and 1 ⁇ g/ml); Cpd. 8 (0.1 and 1 ⁇ g/ml) and Cpd. 12 (1 ⁇ g/ml) and the vehicle equi-volume DMSO. Only one compound at any one concentration was tested on any one ring from any one animal.
• Protocol 2a The vasodilatory capacity of isoflavone metabolite derivatives A full concentration response to noradrenaline was obtained. From this the concentration producing a submaximal contraction of approximately 80% was selected (0.03 - 0.3 ⁇ M). The ring was then constricted with this submaximal concentration and allowed to plateau before full concentration-relaxation curves were obtained to ⁇ -estradiol and Cpds. 5, 7, 8 and 12 and the vehicle DMSO (at equi-volume). Only one compound was studied with any one ring from any one animal.
• Protocol 2b The vasodilatory capacity of isoflavone metabolite derivatives: mechanism of action
• KC1 40 mM to inhibit endothelium derived hyperpolarising factor
• the cyclo-oxygenase inhibitor indomethacin (10 ⁇ M)
• the soluble guanylate cyclase inhibitor 1H- [l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ; 10 ⁇ M). Only one compound with only one intervention was tested on any one ring from any one animal.
• Protocol 3 Protective effect of isoflavone metabolite derivatives against endothelium damage induced by oxidised low-density lipoprotein
• Human plasma was obtained from the blood bank. Individual lipoprotein fractions were obtained using discontinuous density gradient ultracentrifugation on a Beckman vertical rotor (70 Ti) with a Beckman centrifuge. In short, 0.01 8g of KBr was added to each ml of plasma, this was transferred to quick seal tubes and spun at 65,000 rpm, 4°C overnight. The top (VLDL) fraction was then removed and 0.064g of KBr added to each ml of the bottom section. This was transferred to quick-seal tubes and spun at 65000 rpm, 4°C overnight. The top (LDL) fraction was transferred to quick seal tubes and topped up with dl .063 solution and spun.
• the top LDL fraction was collected following each spin and dialysed against 0.05M H ⁇ HCOs pH 8.0 over 4 days with changes of solution each day.
• the protein content of the final LDL fraction obtained was determined by the simplified protein assay method of Lowry. LDL was oxidised by a 2 hr incubation with CuSO 5 ⁇ M. Data presentation and statistical analysis
• Contractile responses are expressed in g tension (mean + standard error of the mean). Dilatory responses are expressed as a percentage of the contractile force generated by the pre-constricting agent used. Individual concentration-response curves for noradrenaline and acetylcholine ⁇ the interventions used were fitted to a logistic equation of the form
• E is the response
• M is the maximum response
• K is the concentration eliciting 50% of the maximum response (ie neg log EC 50 ).
• Results were analysed by 2 way repeated measure analysis of variance where appropriate followed by post-hoc t-tests with the appropriate corrections using Sigmastat Statistical software (Jandel Scientific, San Rafael, CA) which inherently tests data for normality prior to performing parametric analysis.
• 1,3,5 [10]-estratriene-3,17 ⁇ -diol (17 ⁇ -estradiol, SIGMA), Cpd. 5, Cpd. 7, Cpd. 8 and Cpd. 12 were dissolved in DMSO and diluted to the required concentrations in Krebs.
• N ⁇ -nitro-L-arginine (SIGMA), indomethacin (SIGMA), norepinephrine bitartrate salt (SIGMA), acetylcholine chloride (SIGMA) and 1H- [l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (SIGMA) were dissolved to the manufacturers specifications.
• Protocol l The effect of isoflavone metabolites on contractile curves to noradrenaline Table 1 lists the maximal force obtained to noradrenaline in the absence and presence of ⁇ - estradiol, the synthetic isoflavone derivatives (Cpds. 5, 7, 8 and 12) and the used.
• Fig 1 shows in graphical form the effects on responses to noradrenaline for various compounds when used at 1 ⁇ g/ml.
• Fig 2 shows full concentration-contractile responses to noradrenaline in the absence (pre-compound) and presence following a 30 min incubation (post-compound).
• Protocol 2a The vasodilatory capacity of isoflavone metabolites: mechanisms of action Fig 3 depicts the concentration-dilatory effects of the compounds studied. Concentration- dilatation curves obtained for 17 ⁇ -estradiol, and Cpds. 5, 7, 8 and 12 with rat isolated aortic rings pre-constricted with a submaximal concentration of noradrenaline. All values are mean ⁇ standard error of the mean. All four compounds were at least as potent as ⁇ - estradiol in their vasodilatory capacity. The mechanism by which these compounds exerted the dilatory effect was further examined using specific antagonists.
• Fig 3a Vasodilatory responses to ⁇ -estradiol were inhibited by removal of the endothelium (Fig 3a), incubation with nitro-L-arginine (Fig 4b), high KCl levels (Fig 4c) and ODQ (Fig 4d). Indomethacin did not influence the dilatory effects of this steroid (Fig 4e) and no time dependent changes were observed (Fig 4f).
• Fig 5a Vasodilatory responses to Cpd. 5 were inhibited by removal of the endothelium (Fig 5a), incubation with nitro-L-arginine (Fig 5b), high KCl levels (Fig 5c) and ODQ (Fig 5d). Indomethacin did not influence the dilatory effects of this steroid (Fig 5e) and no time dependent changes were observed (Fig 5f).
• Fig 6a Vasodilatory responses to Cpd. 7 were inhibited by removal of the endothelium (Fig 6a), high KCl levels (Fig 6c) and ODQ (Fig 6c). There was a trend towards inhibition of these responses by NOLA but this was not statistically significant. Indomethacin did not influence the dilatory effects of this steroid (Fig 6d) and no time dependent changes were observed (Fig 6e).
• Fig 7a Vasodilatory responses to Cpd. 8 were inhibited by removal of the endothelium (Fig 7a), high KCl levels (Fig 7b) but not by ODQ (Fig 7c). Indomethacin potentiated the inhibitory effects of Cpd. 8 (Fig 7d). No time dependent changes were observed (Fig 7e).
• NOLA 10 ⁇ M nitric oxide synthase inhibitor N ⁇ -nitro-L-argin
• Protocol 3 Protective effect of isoflavone metabolites against endothelium damage induced by oxidised low-density lipoprotein
• Fig 9 depicts full concentration-dilatation curves to acetylcholine obtained with rat isolated aortic rings pre-constricted with a submaximal concentration of phenylephrine in the absence and presence following a 30 min incubation with (a) oxidised low density lipoprotein (ox-LDL 0.3 mg protein/ml); (b) Ox-LDL + Cpd. 8 (3 ⁇ g/ml).
• ox-LDL oxidised low density lipoprotein
• Ox-LDL + Cpd. 8 3 ⁇ g/ml
• Fig 10 represents histograms depicting maximal dilatation obtained to acetylcholine with rat isolated aortic rings pre-constricted with a submaximal concentration of phenylephrine in the absence and presence of sole incubation with ox-LDL and co-incubation of oxLDL+17 ⁇ -estradiol or Cpds. 5, 7, 8 or 12.
• n number of experiments.
• Cpds. 5, 7, 8 and 12 were all able to antagonize the contractile effects of noradrenaline but to differing degrees. While Cpd. 12 was most effective and comparable to 17 ⁇ -estradiol, Cpd. 5 and Cpd. 7 appeared equipotent but less potent than Cpd. 12. Cpd. 8 was the least potent. The antagonising action of Cpds. 5, 7 and 8 were dose dependent but dose dependency of Cpd. 12 and ⁇ -estradiol were not examined. At equi-volume, the vehicle (DMSO) was ineffective at inhibiting responses to noradrenaline.
• DMSO vehicle
• vasodilatory effects of the isoflavone metabolite synthetic derivatives were at least as potent as, if not more potent than, 17 ⁇ -estradiol, the mechanism of action differed from that of 17 ⁇ -estradiol in that the dilatory action of all four compounds diminished upon removal of the endothelium.
• Cpd. 8 in this context is at least 10 times more potent than 17 ⁇ -estradiol. Since Cpd. 8 is not the most potent compound in the other protocols, ie. in either antagonising noradrenaline nor in its direct vasodilatory capacity, the mechanism of the cardioprotective action of this compound is likely to be different to the others tested and may lie in its anti-oxidant capacity.
• Cpds. 5, 7, 8 12 have potent vascular regulatory capacity.
• Vascular smooth muscle cell proliferation is an important step in the atherosclerotic process and is inhibited by estrogens.
• experiments were performed to examine the effects of the selected synthetic derivatives of isoflavone metabolites, Cpds. 5, 7, 8 and 12, on simple markers of DNA synthesis and cell proliferation, namely [3H]-thymidine inco ⁇ oration and cell numbers.
• the signalling pathways mediating these activities were examined by determining the effect of Cpd. 7 on the mitogen-activated protein (MAP) kinases (Erk, JUNK, p38).
• MAP mitogen-activated protein
• vascular smooth muscle cells VSMC derived from female internal mammary artery
• IMA IMA were cultured in 10% FBS-DMEM. Cells were grown to confluence in LG (glucose 5 mM)-medium then trypsinized, diluted with 10% FBS-LG medium to about 10,000 cells/ml and 1ml of suspension was aliquoted into four 24-well plates. Cells were then allowed to grow for 2 days to visual confluence. LG serum-free media was changed for 48 h to synchronize most cells at Go/Gi.
• FBS + isoflavone treatment at O.OlmM (2) FBS + isoflavone treatment at O.OlmM (5) FBS + isoflavone treatment at lOmM (3) FBS + isoflavone treatment at O.lmM (6) FBS + isoflavone treatment at lOOmM
• PDGF platelet derived growth factor
• Cpd. 7 inhibited platelet-derived growth factor (PDGF) BB-induced DNA synthesis, assessed by [3H] -thymidine inco ⁇ oration, in a dose-dependent manner with 10% inhibition at 1 nmol/L and 17% inhibition at 10 and 100 nmol/L. The inhibition was statistically significant but less compared to the effect of 17 ⁇ -estradiol (27% at 1 or 10 nmol/L and 33% at 100 nmol/L).
• ICI 182780 a non-selective estrogen receptor (ER) antagonist (100 nmol/L) completely abolished the inhibitory effect of Cpd. 7 at 1-10 nmol/L) and partly abolished that of Cpd.
• isoflavones and derivatives thereof inhibits cellular proliferation of human vascular smooth muscle cells, and also inhibits PDGF-induced Erk activation in human vascular smooth muscle cells.
• This activity show the potential for isoflavones and derivatives thereof to prevent the development and progression of atherosclerotic lesions, and in particular accelerated stenosis, providing a potential benefit in vascular protection.
• EXAMPLE 3 Antioxidant Activity Oxidation is a process which plays a role in many major disease processes.
• one of the major contributors to development of atherosclerosis is the oxidation of LDL cholesterol in the arterial wall, leading to an inflammatory lesion. If left unchecked the inflammatory process proceeds until the lesion causes vascular obstruction and infarct, and presents a particularly important problems in the accelerated intimal thickening of restenosis.
• selected isoflavone compounds of the invention were subjected to assays to determine their antioxidant activity.
• the LDL antioxidation test measures the ability of a compound to directly scavenge free radicals or to chelate transition metals.
• the isoflavones and their derivatives were tested for their ability to act as free radical scavengers.
• Fresh whole blood was obtained by venipuncture from healthy human volunteers under 25 years of age and supplemented immediately with EDTA (91 mg/ml) and BHT (4.4 mg/ml).
• LDL was prepared by step-wise ultracentrifugation within a density gradient of 1.02-1.05 g/cm 3 .
• EDTA and BHT were present throughout all the steps of the isolation.
• the EDTA BHT containing stock solution (15-30 mg LDL/ml) was stored in the dark in a nitrogen atmosphere until use, but never longer than two weeks.
• the LDL stock solution was dialyzed in the 100-fold volume of 0.01 M phosphate buffer pH 7.4, 0.16 M NaCl, 0.1 mg/ml chloroamphenicol, which was made oxygen-free by vacuum degassing following by purging with nitrogen. The buffer was changed four times.
• This EDTA- and BHT-free LDL stock solution was used for all oxidation studies. The stock solution was stored not longer than 24 hr at 4°C.
• the EDTA- and BHT-free LDL stock solution was diluted with oxygen-saturated 0.01 M phosphate buffer pH 7.4, 0.16 M NaCl and the oxidation was initiated by the addition of a freshly prepared aqueous CuCl solution. The final conditions were in all experiments: room temperature, 0.25 mg LDL/ml and 1.66 mM CuCl 2 (see Esterbauer et. al, 1989)
• the antioxidant activities of the following compounds were tested: daidzein (lOmM), genistein (10 mM), glycitein (10 mM), biochanin (10 mM), formononetin (10 mM), PromensilTM (Novogen) (2.5 mg/ml, 5 mg/ml), Cpd. 5 (10 mM), Cpd. 7 (10 mM), Cpd. 8 (10 mM), Cpd. 12 (10 mM), Cpd. 4 (10 mM) and Cpd. 6 (10 mM).
• controls were used to compare the relative effectiveness of the Novogen isoflavone derivatives to common and well-known antioxidants such as ascorbate (2.5mM; Vitamin C).
• Antioxidant activity of the isoflavone parent compounds expressed as the lag time, relative to the effect produced by ascorbate. Note: the longer the lag time, the more active the compound as an antioxidant.
• Cpd. 12 (10 mM) and Cpd. 8 (10 mM) were very active scavengers, increasing the lag time by over 600%, when compared to the positive control (see Table 3).
• Cpd. 7 was found to be significantly less active than Cpd. 8 and Cpd. 12 but still almost as active as ascorbate.
• antioxidant-mediated Per oxidation Studies were carried out to determine whether the isoflavone derivatives can prevent LDL oxidation in the presence of Vitamin E. This test is physiologically important, since Vitamin E (a-tocopherol) is present with LDL in the blood stream, and LDL oxidation is believed to be one of the major factors of the development of atherosclerosis.
• the anti-TMP test has been described in detail by Bowry et al. (1995).
• the test indirectly assesses the ability of a compound to synergise with a-tocopherol in human LDL undergoing mild and chemically controlled oxidation. Oxidation is measured by the accumulation of cholesterol ester hydroperoxides at a time point corresponding to 20% consumption of endogenous a-tocopherol.
• ascorbate and ubiquinol-10-free LDL (1 mM in apoB) was supplemented with an aliquot of a stock solution of the compound to be tested.
• the mixture was incubated at 37°C for 10 min and subsequently oxidized at 37°C with a low flux of water-soluble ROO generated from AAPH (4 mM).
• the time-dependent consumption of LDL a-tocopherol and accumulation of cholesteryllinolate hydroperoxide (Chl8:2-OOH) were monitored by HPLC with electrochemical and post-column chemiluminescence detection, respectively.
• redox index The effectiveness of an antioxidant, assigned as its anti-TMP index (redox index), was defined as the relative amounts of Chl8:2-OOH formed with versus without the added antioxidant measured after 20% consumption of the endogenous a-tocopherol in the control sample (ie. in the absence in the antioxidant). Active compounds give rise to low redox index. A high redox activity suggests that the compounds are capable of interacting with the a-tocopherol in LDL, perhaps by reducing the a-tocopheroxyl radical. Butylated hydroxytoluene (BHT, 10 mM) was used as a positive control.
• BHT butylated hydroxytoluene
• Cpd. 12 (10 mM) has strong LDL protective action (low redox index), preventing LDL oxidation in the presence of Vitamin E. Furthermore, Cpd. 7 and Cpd. 8 showed low levels of activity. Results are summarised in Table 4.
• the aim of this study was to assesses the ability of the isoflavone derivatives to attenuate a-tocopheroxyl radicals in cetyltrimethyl ammonium chloride and sodium dodecyl sulphate micelles.
• a-tocopheroxyl radical attenuating ability (TRAA) assay has been described in detail by Witting et al. (1997). Briefly, one hundred mM stock solutions of cetyltrimethyl ammonium chloride (HTAC) and sodium dodecyl sulphate (SDS) were prepared in phosphate buffer. Micellular dispersions of a-tocopherol were prepared by diluting an ethanolic solution of a-tocopherol (0.2 M) into micelles at a final vitamin concentration of 500 mM. This resulting solution was sonicated for 15 sec at which time it was completely homogenous.
• HTAC cetyltrimethyl ammonium chloride
• SDS sodium dodecyl sulphate
• LDL was isolated by ultracentrifugation of freshly heparinized plasma obtained from non-fasted healthy male donor (27 years of age). LDL was obtained by direct aspiration and stored at 4°C for 16 hr before use. Immediately prior to use, excess KBr and remaining low molecular weight water-soluble antioxidants were removed from the LDL by gel filtration chromatography, using a PD-10 column (Pharmacia, Uppsala, Sweden) and the concentration of LDL was determined as described by Lowry et al (1951).
• the flat cell arrangement containing a-TO was removed from the ESR cavity and the solution was gently coaxed into the neck of the flat cell under positive pressure.
• the compound of choice was then added to give a final concentration of 10 mM and the treated sample was replaced in the cavity and allowed to equilibrate to standard conditions, and sampling was resumed.
• the time-dependent decay of ESR signal intensity for a-TO was measured both in the presence and absence of the added co-antioxidant (10 mM) using a sweep time of 20.5 sec, averaging the output from three successive sweeps at each time point, and averaging the results of three separate experiments.
• Results are expressed as the relative rate constant of decay of a-tocopheroxyl radicals in the presence of the test sample divided by the relative rate constant of decay of a- tocopheroxyl radicals in the absence of the test sample.
• TRAA approaching unity is considered to have poor synergistic activity, whereas active compounds show large values because they eliminate the a-tocopheroxyl radicals immediately upon mixing.
• Ascorbate was used as a positive control. It was found that Cpd. 12 induced high rate of decay and was active at eliminating a-tocopheroxyl radicals. Results are summarized in Table 6.
• Peroxyl radicals are natural side-products of a number of enzymes, including lipoxygenases and cyclo-oxygenase. Peroxyl radicals were generated by the thermo-labile azo-initiator AAPH. AAPH-induced oxidation of linoleate was performed as described previously (Pryor et al, 1993), except that a lower linoleate concentration was used and SDS was omitted. An aliquot of a linoleate stock solution was added to PBS at 37°C to give a final concentration of 500 mM. Oxidation was initiated by the addition of AAPH (87.5 mM final concentration) in the absence and presence of either 10 mM of Cpd.
• Dubecco's PBS was purchased from Sigma, St. Louis, MO.
• the water-soluble azo peroxyl radical generator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Polysciences, Warrington, PA.
• Ascorbate (sodium salt) was purchased from Merck.
• AAPH (350 mM) stock solutions were prepared by serial dilution with Dubecco's PBS.
• Stock solutions of the test compounds (10 mM) were prepared as ethanolic solutions while stock solution of linoleate (300 mM) was made by dissolving in isopropanol.
• the aim of this study was to test whether the isoflavone derivatives can affect heme-catalyzed oxidation reactions. Such oxidation reactions are thought to be relevant in vivo and can take place using lipid, protein and/or DNA as substrate(s).
• the test is similar to the peroxyl scavenging described in section 4.5 above, except that horseradish peroxidase (HRP) plus hydrogen peroxide (H 2 O 2 ), were used instead of the peroxyl radical generator.
• HRP horseradish peroxidase
• H 2 O 2 hydrogen peroxide
• Increased level of heme is considered as a potential risk factor for atherosclerosis, and heme-containing proteins may be involved in the oxidative modification of LDL.
• H 2 O 2 heme proteins give rise to a powerful oxidant (referred to as compound I) that can oxidatively modify lipids, including those in LDL.
• compound I a powerful oxidant
• HRP/ H 2 O 2 -induced oxidation of linoleate was as described principally by Witting et al (1997) by replacing LDL with linoleate. Linoleate stock was added to PBS at 37°C to give a final concentration of 300 mM and oxidized by HRP (40 U/mL)/ H 2 O 2 (2mM) in the absence and presence of either 10 mM of Cpd. 7, Cpd. 8 or Cpd. 12, or 25 mM BHT (positive control). Formation of linoleate hydroperoxide was monitored by UV 234 nm using the Perkin Elmer's UV/Vis Lambda 40 spectrophotometer. The test was performed twice as single analysis. An appropriate concentration of alcohol (ethanol and isopropanol; 0.13% - 0.2% v/v) was included in the respective control samples.
• Dubecco's PBS, butylated hydroxytoluene (BHT) and acid free linoleate were purchased from Sigma, St. Louis, MO.
• Horse radish peroxidase (HRP; Grad II; 100,000 U/ 502.5 mg lyop. powder) was purchased from Boehringer Mannheim, Germany.
• Hydrogen peroxide (H 2 O 2 ; 30% w/v) was purchased from Merck.
• HRP (6000 U/mL) and H 2 O 2 (1M) stock solutions were prepared by serial dilution with Dubecco's PBS.
• Stock solutions of BHT (25 mM) and the test compounds (10 mM) were prepared as ethanolic solutions while stock solution of linoleate (300 mM) was made by dissolving in isopropanol.
• Lipoxygenase is a potentially pro-inflammatory enzyme that that requires oxidative activation and releases peroxyl radicals upon catalytic action.
• the isoflavone derivatives were tested for their ability to affect lipoxygenase activity in vitro, using both soybean and rabbit reticulocyte 15 -lipoxygenase and linoleic acid as substrate.
• 15-Lipoxygenase has been suggested to be involved in the initial stages of in vivo LDL oxidation and has been suggested to contribute to atherogenesis (Kuhn et al, 1994; Folcik et al, 1995).
• Soybean 15-lipoxygenase (SLO) is a suitable model of 15 -lipoxygenase and was therefore used to assess the ability of Cpd. 7, Cpd. 8 and Cpd. 12 to inhibit this enzyme.
• SLO-induced oxidation of linoleate was performed as described previously (Upston et al, 1996).
• linoleate (100 mM) in PBS was oxidized by SLO (60 U/mL) in the absence and presence of 10 mM Cpd. 7, Cpd. 8 or Cpd. 12, or 100 mM eicosatetraynoic acid (ETYA, positive control). Formation of linoleate hydroperoxide was monitored by UV 234 nm using the Perkin Elmer's UV/Vis Lambda 40 spectrophotometer. The test was performed twice as single analysis. An appropriate concentration of alcohol (ethanol and isopropanol; 0.13% - 0.2% v/v) was included in the respective control samples.
• Dubecco's PBS and acid free linoleate, soybean 15-lipoxygenase (SLO; 630,000 U/mg protein) were purchased from Sigma, St. Louis, MO.
• Ascorbate (sodium salt) and eicosatetraynoic (ETYA) were respectively purchased from Aldrich and Cayman
• Cpd. 12 is an effective inhibitor of lipoxygenase type-1, as indicated by the strongly attenuated increase in 234 nm absorbance. In fact, even at 10 mM, Cpd. 12 showed comparable inhibitory action than 100 mM ETYA, a well-established inhibitor of lipoxygenase. As for peroxyl radicals, neither Cpd. 7 nor Cpd. 8 were able to inhibit SLO.
• Cpd. 1 (10 mM and 50 mM), was incubated with whole plasma for 0 h, 4 h and 8 h. The different subfractions LDL/VLDL, HDL and protein (lipoprotein free) were then analysed for their Cpd. 1 concentration. Cpd. 1 was used because it had the most similar polarity to the isoflavone synthetic derivatives. Results.
• Cpd. 1 (daidzein) associated with the lipoproteins.
• 2% of Cpd. 1 associated with the LDL, 6% was found to associate with the HDL and 92% associated with the protein fraction.
• the amount associated with the lipoproteins increased with time, but not with concentration. The results are demonstrated in Fig 13 (Cpd. 1, 10 mM) and Fig 14 (Cpd. 1, 50 mM).
• a high risk factor for cardiovascular disease is an elevated LDL cholesterol level. Excess cholesterol results in a greater chance of oxidative damage, and therefore atherosclerosis.
• Estrogens have a number of potentially beneficial effects on the development of atherosclerosis and the outcome of cardiovascular events. 17 ⁇ -estradiol has been shown to increase LDL receptor activity. There is also evidence that plant-derived estrogens (i.e., phytoestrogens such as isoflavones) may have a protective activity against the incidence of cardiovascular disease, although the underlying mechanism for this remains unknown. Given the structural similarity of isoflavones and their derivatives with certain moieties of estrogen, it is of interest to test whether isoflavones and related compounds may have similar molecular actions.
• Confluent human hepatoma (HepG2) cells were preincubated in the absence or presence of the test compound (Cpd. 7, Cpd. 8 and Cpd. 12 at final concentration of 50 nM, 500 nM and lOmM) for 48 hours, in complete DMEM medium containing 10% fetal bovine serum (FBS). Serum deprivation (i.e. 0.5% FBS) was used as a positive control to induce LDL receptor expression.
• FBS fetal bovine serum
• HepG2 cells were incubated in 12-well plates with increasing concentrations of [ I]-LDL in the presence of excess (i.e., 500 mg) unlabelled LDL. Saturation was reached at around 50mg [ 125 I]-LDL. Using this concentration, the time required to reach saturation binding at 4°C was determined to be approximately 4 hours. These established optimal conditions were used for all subsequent binding assays.
• Cpd. 12 was without consistent effect, while Cpd. 7 and Cpd. 8 increased LDL binding.
• Cpd. 7 (but not Cpd. 8) the expression of LDL receptor increased concentration-dependently although the extent of this increase was small when compared to that obtained with serum deprivation.
• Cpd. 8 had no clear concentration-dependent effect.
• Angiogenesis occurs through a number of distinct steps that can be defined as endothelial cell activation, migration, proliferation, and reorganisation to form the mature vessel characterised by a lumen.
• the effect of isoflavone derivatives on angiogenesis and in particular endothelial cell proliferation and endothelial cell migration uses assessed by in vitro assays.
• Human umbilical vein endothelial cells between passage 1 and 3 were used. Cells were grown in gelatin coated flasks in medium 199 with Earle's Salts supplemented with 20% foetal calf serum, 25 mg/ml endothelial cell growth supplement and 25 mg/ml heparin. Cells from two different donors were used.
• Results were compared to a standard curve performed in each experiment. Two different endothelial cell isolates were used and the assays were performed on two different days.
• Figures 15 and 16 show the effect of Cpd. 5 , Cpd. 7, Cpd. 8 and Cpd. 12 on endothelial cell proliferation.
• Both Cpd. 8 (1-lOmg/ml) and Cpd. 12 (0.01-10 mg/ml) inhibited cellular proliferation and did not cause toxicity.
• there was considerable line variation in the responsiveness of the cells to these compounds ( Figure 15 a and Figure 16a).
• Cpd. 5 (0.01 - 10 mg/ml) was found to have no apparent effect on endothelial cell proliferation, in either of the two cell isolates studied ( Figure 15b and Figure 16b).
• Cpd. 7 (0.01-10 mg/ml) displayed no appreciable antiproliferative action on the two cell isolates studied ( Figure 15c and Figure 16c).
• Migration Assay 5 x 105 cells per well in growth medium were plated onto fibronectin coated 6 well trays and grown to confluence. A wound was produced along the monolayer with the use of a cell scraper (Costar). The cells were then washed once and fresh medium with the test compounds was added. Defined points along the wound were marked using an ink condenser (Olympus microscopes) allowing the movement of cells to be visualized at constant points over the next 48 hours. Photographs were taken at relevant times and the degree of cell movement from the wound front assessed.
• Figure 17 shows the effect of Cpd. 12 on endothelial cells migration.
• the photos show the wound at baseline (time 0 hr) ( Figure 17a) and the amount of cell movement 30 hours after wounding.
• DMSO solvent control
• Figure 17b Photos were taken at different points along the wound but the points were constant over the course of the experiment. The initial wound front is marked.
• Figure 18 provides comparative data on the ability of a number of isoflavone compounds to inhibit the expression of E-selectin conducted in accordance with the method of Litwin et al.
• isoflavone Cpd. 8 showed very good inhibition of E- selectin expression, especially at 10 ⁇ g/ml.
• Isoflavone Cpd. 12 showed excellent inhibition of E-selectin expression, especially at concentrations of 1 and 10 ⁇ g/ml.
• the example establishes that the claimed isoflavones and derivatives specifically block the ability of E-selectin to be expressed by vascular endothelial cells in response to many signals known to be active in restenosis, inflammatory response and other diseases mediated by cell adhesion molecule expression.
• vascular smooth muscle cell vascular smooth muscle cell
• the animal model of atherosclerosis used in this experiment aimed to induce VSMC migration and neointimal proliferation by mechanical damage to the endothelium.
• a probe was introduced into the femoral artery of the mouse and was passed through the iliac artery to the bifurcation of the aorta. The probe was then withdrawn and the femoral artery ligated to prevent haemorrhage. Colateral circulation prevents any limb-associated ischaemia. Neointimal proliferation occurred in the iliac artery over the following 4 weeks.
• Neointimal proliferation is expressed as the ratio of the intimal area to the intrmal plus media area.
• Figure 19 shows a typical profile of proliferation in normal mice. Minimal proliferation is seen at 3 weeks, with a substantial rise in proliferation at 4 weeks, followed by a further rise in week 5. Minimal neointimal proliferation occurs in the control non-operated vessel ("non-angio”) from the opposite side.
• non-angio control non-operated vessel
• isoflavone derivatives to reduce and/or prevent the "atherosclerotic" response in a mouse model of neointimal hyperplasia was investigated.
• the compounds tested were Cpd. 5 and Cpd. 12 .
• Atherosclerosis was induced in the femoral artery of each mouse by mechanically removing the endothelium using a probe. The ability of isoflavone compounds to inhibit the atherosclerotic response was assessed by comparing treatment and placebo groups.
• C57/B16 mice (8-10 week old male) were anaesthetized, and the right femoral artery was de-endothilialized using a dissection microscope. These mice were also fed with a 2% cholesterol diet to enhance the progression of the disease. The mice were sacrificed at week 4. Their treated (right) and control (left) arteries were both collected and fixed in formalin for histopathological evaluation.
• mice were fed a diet of 2% cholesterol mixed with normal mouse chow for a week prior to surgery.
• the treatment groups were fed the chow with Cpd. 5 or Cpd. 12 or Cpds. 5 + 12.
• Mice were anaesthetised with Avertin, and angioplasty of the femoral artery was performed. After surgery, the mice were moved onto a warm mat in a quiet, darkened area and their recovery closely monitored. They were housed in sterile cages with sterile bedding to avoid infection.
• the mice continued a diet of cholesterol mixed with normal mouse chow for the next 4 weeks.
• the mice were euthanased at 4 weeks postsurgery. Their femoral arteries were collected for pathological examination.
• Sections were evaluated by image analysis to determine the area of the various sections of the arterial wall, and the numbers of cells in each area.
• the primary parameter being evaluated was the increase in the intimal area, expressed as the ratio of the area of the intima versus the area of the intima plus media.
• the interval sections (every 50 ⁇ m, or every ten sections) were mounted and stained with H & E.
• the cross-sectional areas of the endothelium, intima and media were evaluated, using image analysis.
• Figure 20 shows a transverse section through the iliac artery 4 weeks after surgery.
• Figure 20a shows the absence of neointimal proliferation in the iliac artery from the non-operated side, where the intima is about one cell thick.
• Figure 20b shows substantial neointimal proliferation in the iliac artery in response to surgery, showing a thickness of approximately 50% of the vessel wall.
• Figures 20c and 20d are post-surgical iliac arteries from mice treated with Cpd. 12 and Cpd. 5, respectively. Neointimal proliferation can be seen in each case, but the proliferation was significantly reduced in Cpd. 5.
• Figure 21 quantitates the effects of the test compounds on neointimal proliferation, both individually and together.
• the untreated vessels (no surgery) show 5 + 1% intimal thickness, while the surgical-treated only vessels show 50 + 5% intimal thickness.
• Cpd. 5, both alone and in combination with Cpd. 12 reduced neointimal proliferation to 30 + 6% and 32 +/- 10%, respectively, while Cpd. 12 had no detectable effect of the extent of neointimal proliferation (50 ⁇ 29%).
• Cpd. 5 significantly reducing neointimal proliferation, but Cpd. 12 having no appreciable effect.
• the area of the neointimal proliferation corresponds well with the numbers of cells within the intima determined using image analysis.
• Beyond cholesterol Modifications of low-density lipoprotein that increase its atherogenicity.
• Folcik VA Nivar-Aristy RA, Krajewski LP, Cathcart MK "Lipoxygenase contributes to the oxidation of lipids in human atherosclerotic plaques".
• Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women.

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