WO1999059561A2

WO1999059561A2 - Resveratrol inhibition of myeloperoxidase

Info

Publication number: WO1999059561A2
Application number: PCT/US1999/010969
Authority: WO
Inventors: Medical Research Foundation Oklahoma
Original assignee: Hensley, Kenneth, L.; Floyd, Robert, A.
Priority date: 1998-05-18
Filing date: 1999-05-18
Publication date: 1999-11-25
Also published as: AU4084599A; WO1999059561A3

Abstract

Resveratrol and its derivatives are potent inhibitors of myeloperoxides, an enzyme released by neutrophils which causes tissue damage in diseases involving chronic inflammation. Such diseases include but are not limited to atherosclerosis, arthritis, Alzheimer's disease, sepsis (endotoxic shock), inflammatory skin disease (psoriasis), inflammatory gum disease (gingivitis), and inflammatory bowel disease.

Description

RESVERATROL INHIBITION OF MYELOPEROXIDASE

TECHNICAL FIELD OF INVENTION

This invention relates to relates to a bio-affecting and body treating composition and to a biological diagnostic agent.

BACKGROUND OF THE INVENTION

Inflammatory diseases such as atherosclerosis, arthritis, and Alzheimer's disease are a major health concern. Tissue damage, the operation of joints, and pain are only a few of the deleterious effects of such diseases. One factor in inflammatory disease is myeloperoxidase (MPO), an enzyme released by neutrophils, which causes tissue damage in a variety of disease states that involve a chronic inflammatory component.

The release of MPO by neutrophils at sites of inflammation gives rise to MPO-catalyzed formation of HOCl and aryl radicals (Ar^«) using hydrogen peroxide and Cl- or ambient aromatic compounds (e.g. tyrosine) as substrates. HOCl, Ar», and other oxidizing agents damage ambient lipids and proteins (Hazan,

S.L. and Heinecke, J.W. 1997. "3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density Hpoprotein isolated from human atherosclerotic intima," JClin Invest 99:2075- 2081) and may stimulate proinflammatory protein kinase cascades. Neutrophils and/or MPO-derived products (e.g., dityrosine and chlorotyrosine) have been detected in atherosclerotic plaques (Hazan S.L. and Heinecke J.W. 1997. "3- Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density Hpoprotein isolated from human atherosclerotic intima," JClin Invest 99:2075-2081), arthritic synovial fluid (Nurcombe, et al. 1991. "Activation of the neutrophil myeloperoxidase-H₂0₂ system by synovial fluid isolated from patients with rheumatoid arthritis," Ann Rheum Dis 50:237-242; and De Clerck, et al. 1995. "Expression of neutrophil activation markers and neutrophil adhesion to chondrocytes in rheumatoid arthritis patients: Relationship with disease activity, " Res Immunol 146:81-87) and Alzheimer's diseased brain

(Hensley, et al. 1997, unpublished manuscript).

The correlation between MPO and inflammatory disease has led scientists to attempt to find MPO inhibitors. MPO inhibitors have been reported in the literature. Certain non-steroidal anti-inflammatory compounds which inhibit MPO do so reversibly and/or require high concentrations to inhibit MPO. (Kettle, A. and

Winterbourn, C. C. 1991. "Mechanism of inhibition of myeloperoxidase by anti- inflammatory drugs," Biochem Pharmacol 41:1485-1492; Davies, B. and Edwards, S.W. 1989. "Inhibition of myeloperoxidase by salicylhydroxamic acid," Biochem J 258:801-806). Additionally, many known MPO inhibitors are very toxic, can be metabolized to toxins, or are easily oxidized with loss of function (e.g., CN" and aminobenzoic acid hydrazide) making them difficult to formulate and deliver to sites of action.

Resveratrol, or trans-3,4',5-trihydroxystilbene, is synthesized by a narrow range of plant species including grape vines, peanuts and pines, and is thought to serve an antifungicidal role in damaged plant tissue. (Soleas, et al. 1997.

"Resveratrol: A molecule whose time has come? And Gone?," Clin Biochem 30:91-113). Resveratrol has been reported to inhibit platelet aggregation and to exhibit vasorelaxing activity (Bertelli, et al. 1995. "Antiplatelet activity of synthetic and natural resveratrol in red wine, " IntJ Tissue React 17:1-3; and Bertelli, et al. 1996. "Kinetics of trans and cis-resveratrol (2,4',5- trihydroxystilbene) after red wine oral administration in rats," Int J Clin Pharmacol Res 16:77-81). It is also reported to exhibit anticarcinogenic activity (Jang, et al. 1997. "Cancer chemopreventive activity of resveratrol, a natural product derived from grapes," Science 275:218-220). The mechanism for the beneficent action of resveratrol has been postulated to be the inhibition of cyclooxygenase and lipooxygenase enzymes in platelets and neutrophils. (Kimura, et al. 1985. "Effects of stilbenes on arachidonate metabolism in leukocytes, " Biochim BiophysActa 834:275-278; Pace-Asciak, et al. 1995. "The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: Implications for protection against coronary heart disease, " Clin Chem Ada 235:207-219; and Jang, et al. 1997. "Cancer chemopreventive activity of resveratrol, a natural product derived from grapes," Science 275:218-220). Resveratrol has also been previously reported to act as a nonsteroidal anti-inflammatory agent by virtue of cyclooxygenase inhibition.

(Jang, et al. 1997. Science 275:218-220). Acute cellular inflammation is mediated by the products of arachidonic acid. Physical or chemical perturbations of platelets, endothelial cells, or neutrophils leads to immediate release of free arachidonate, or arachidonic acid, from phospholipids. The enzymatic oxygenation of the arachidonic acid by cyclooxygenase forms endoperoxides which are subsequently converted to biologically active compounds such as prostaglandins and thromboxanes. In leukocytes and mast cells, 5-lipooxygenase also converts free arachidonic acid to pro-inflammatory compounds as well as leukotrienes which affect chemotaxis of neutrophils and eosinophils. Thus, it is believed that resveratrol mediates inflammation by inhibiting the cyclooxygenase pathway to prostaglandin and thromboxane production and the lipooxygenase pathway to leukotriene production.

Resveratrol has also been shown to act as an efficient chain-breaking antioxidant which inhibits free radical-mediated lipid damage. (Fauconneau, et al. 1997. "Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests," Life Sci 61:2103-2110; Sun, at al. 1997. "Ethanol-induced cell death by lipid peroxidation in PC12 cells," Neurochem Res 22: 1187-1192). By virtue of structural similarity to tyrosine kinase substrates and known tyrosine kinase inhibitors (e.g. tyrphostins and genisteins), resveratrol subserves an anti-inflammatory function via inhibition of cytokine-activated, receptor-linked tyrosine kinase cascades (Jayatilake, et al. 1993. "Kinase inhibitors from Polygonum cuspidatum, " J Nat Prod 56:1805-1810). Therefore, resveratrol has been reported to be a multifunctional compound with specific applicability to the treatment of inflammatory disease states involving neutrophil infiltration and resultant oxidative stress.

It has now been found that resveratrol has the ability to inhibit myeloperoxidase (MPO), providing a new therapeutic utility for treatment of inflammatory diseases of interest. Further, it has been found that resveratrol inhibits MPO at lower concentrations than required for other MPO inhibitors.

SUMMARY OF THE INVENTION

In one aspect of the invention, resveratrol or its derivatives or both are used to effect inhibition of myeloperoxidase.

In another aspect, resveratrol or its derivatives or both are used to manufacture a pharmaceutical composition for treatment of a disease state wherein neutrophil myeloperoxidase is released.

In yet another aspect, resveratrol or its derivatives or both are used in an assay for screening a patient's body fluid or tissue to determine resveratrol- inhibitable peroxidase activity. In yet another aspect, resveratrol or its derivatives or both are used in a method of treating a patient to cause a reduction in myeloperoxidase.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph depicting resveratrol inhibition of myeloperoxidase activity, using guaiacol/hydrogen peroxide as substrate reagents. The reaction mixtures contained 0.5 mM hydrogen peroxide, 5 mM guaiacol, 10 mM sodium acetate, pH 6.5, and the indicated concentrations of resveratrol. Reactions were monitored optically at 450 run and 37°C. DETAILED DESCRIPTION

Resveratrol is a potent MPO inhibitor with additional antioxidant and anti- inflammatory action and as such can be used to treat any disease state associated with neutrophil recruitment and MPO release (degranulation), including but not limited to atherosclerosis, arthritis, Alzheimer's disease, sepsis (endotoxic shock), inflammatory skin and gum disease (e.g. psoriasis, gingivitis), and inflammatory bowel disease.

Resveratrol differs from previously reported MPO inhibitors in several respects. Resveratrol is a potent MPO inhibitor (IC₅₀ = 2.5 μM using guaiacol as a peroxidase substrate, Fig. 1). Kinetics data indicate resveratrol inhibition is mechanism-based and irreversible. Resveratrol's structure is unique among reported MPO inhibitors, and this imparts multiple synergistic anti-inflammatory and antioxidant functions to the molecule.

Resveratrol is available commercially from Sigma Chemical Co. (St. Louis, MO). Resveratrol and other stilbene derivatives may be isolated from plant sources such as grapes and peanuts. Alternatively, suspension cultures of plant cells may be used to biosynthesize these materials. (Schopner, A. and Kindl, H. 1984. "Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut," JBiol Chem 259:6806-6811; Orsini, et al. 1997. "Isolation, synthesis, and antiplatelet aggregation activity of resveratrol 3-O-beta-

D-glucopyranoside and related compounds," JNat Prod 60: 1082-1087). Chemical synthesis of resveratrol and related hydroxy stilbenes by Wittig reaction chemistry has also been described. (Orsini, et al. 1997. "Synthesis of biologically active polyphenolic glycosides (combrestastatin and resveratrol series)," Carbohydr Res 301:95-109; Reimann, E. 1969. "Synthesis ofpolyhydroxystilbene ethers by the

Wittig reaction," Chem Ber 102:2281-2288).

Resveratrol can be absorbed orally by mammals and can be orally administered. (Bertelli, et al. 1996. Kinetics of trans- and cis-resveratrol (2,4',5- trihydroxy stilbene) after red wine oral administration in rats," Int J Clin Pharmacol Res 16: 77-81). Resveratrol can also be administered topically to inflamed skin or gum/mouth tissue as a cream or a gel, or inhaled as an aerosol. For inflammation of the gums or mouth, resveratrol can be applied to inflamed tissue via toothpaste containing resveratrol. The relative stability and lipophilicity of resveratrol make this compound amenable to delivery in numerous possible formulations. It can be administered via ingestion of a food substance containing resveratrol in an amount sufficient to achieve therapeutic levels. Alternatively, it can be enclosed in capsules, compressed into tablets, microencapsulated, entrapped in liposomes, in solution or suspension, alone or in combination with a substrate immobilizing material such as starch or poorly absorbable salts. Pharmaceutically compatible binding agents and/or adjuvant materials can be used as part of a composition.

Tablets or capsules can contain any of the following ingredients, or compounds of similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; an integrating agent such as alginic acid; corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; and additional sweetening and flavoring agents. When a capsule form is used the liquid carrier such as a fatty oil may be used. Capsules and tablets can be coated with sugar, shellac and other enteric agents as is known. Resveratrol can also be administered in a controlled-release formulation. In summary, resveratrol can be administered orally, topically, parenterally, intraperitoneally, or sublingually.

An effective therapeutic amount of resveratrol to inhibit MPO is from about 0.1 to about 10 milligrams per kilogram body weight.

Derivatives of resveratrol which retain the structure of phenolic or polyphenolic rings linked by a rigid connecting moiety (e.g.. an alkene bond in the case of resveratrol or other limiting factors such as a third ring as in hydroxy flavone and hydroxy flavanone derivatives) can also be useful as myeloperoxidase- inhibiting anti-inflammatory agents. Derivatives useful in the present invention can also be characterized by the presence of multiple -OH groups oriented against a rigid aromatic molecular backbone and a relative lipophilicity of the presence of conjugated phenolic moieties in the molecule. For example, esters or glucosides of resveratrol or related stilbenes and flavanoids are representative of chemically modified agents which, upon bioconversion in the stomach or within inflamed tissue, can be used as MPO inhibitors of the present invention. Esters or glucosides of resveratrol useful in the present invention have the following chemical structure:

where R,, R₂, and R₃ can be hydrogen, saturated or unsaturated, straight chained, or branched alkyls of 1 to 5 carbons, or glucose, wherein R,, R₂, and R₃ can be the same or different.

Other derivatives of the present invention include flavones or flavanoids having the following chemical structure:

where R,, R₂, R₃, R₄, R₅, and Rή can be hydrogen, saturated or unsaturated, straight chained, or branched alkyls of 1 to 5 carbons, or glucose, wherein R R₂, R₃, R₄, R₅, and Rg can be the same or different.

Piceatarmol (trans-3,3',4,5'-tetrahydroxystilbene) shown below is a resveratrol derivative which has been assayed for MPO inhibition. Results

showed an IC₅₀ = 0.8 μM by the guaiacol assay. Compared to resveratrol at IC₅₀ = 2.5 μM, piceatarmol is approximately three times more effective in inhibiting MPO than resveratrol. Resveratrol can also be used in as assay for the determination of resveratrol-inhibitable peroxidase activity. As an example, a 1 ml reaction mixture is made containing 0.5 mM hydrogen peroxide and 5 mM guaiacol in 10 mM sodium acetate pH 6.5. A second reaction mixture is made containing the same reagents and also 100 μM resveratrol. To each reaction mixture is added 0.1 ml of body fluid or tissue suspected of containing myeloperoxidase activity. After 30 minutes at 37 °C, the optical density of both solutions is measured at 450 nm. Resveratrol-inhibitable peroxidase activity is expressed as the difference in optical densities normalized to total protein concentration in the original fluid or tissue, wherein peroxidase activity is inhibited in the resveratrol-containing reaction mixture.

In summary, resveratrol has been found to be a potent inhibitor of human neutrophil myeloperoxidase. Resveratrol protects tissue in any disease state involving neutrophil infiltration. Isomers and derivatives of resveratrol are also considered as myeloperoxidase inhibitors.

Claims

WE CLAIM:

1. A method of inhibiting neutrophil myeloperoxidase comprising contacting cells with an effective amount of resveratrol or resveratrol derivative to effect inhibition of neutrophil myeloperoxidase.

2. The method of Claim 1, wherein said resveratrol derivative retains the structure of phenolic or polyphenolic rings linked by a rigid connecting moiety.

3. The method of Claim 1, wherein said resveratrol derivative is represented by the following chemical structure:

wherein R,, R₂, and R₃ is hydrogen; saturated or unsaturated, straight chained, or branched alkyls of 1 to 5 carbons; or glucose, and wherein R,, R₂, and R₃ can be the same or different.

4. The method of Claim 1, wherein said resveratrol derivative is represented by the following chemical structure:

where R_l5 R₂, R₃, R₄, R₅, and Rg can be hydrogen, saturated or unsaturated, straight chained, or branched alkyls of 1 to 5 carbons, or glucose, wherein RΓÇ₧ R₂, R₃, R₄, R₅, and Rg can be the same or different.

5. The method of Claim 1, wherein said resveratrol derivative is trans- 3 ,3 ',4,5'-tetrahydroxystilbene.

6. The method of Claim 1, wherein said effective amount is from about 0.1 to about 10 milligrams per kilogram body weight.

7. The method of Claim 2, wherein said effective amount is from about 0.1 to about 10 milligrams per kilogram body weight.

8. The method of Claim 3, wherein said effective amount is from about 0.1 to about 10 milligrams per kilogram body weight.

9. The method of Claim 4, wherein said effective amount is from about 0.1 to about 10 milligrams per kilogram body weight.

10. The method of Claim 5, wherein said effective amount is from about 0.1 to about 10 milligrams per kilogram body weight.

11. A use of resveratrol or resveratrol derivative to manufacture a pharmaceutical composition for treatment of a disease state comprising neutrophil infiltration and myeloperoxidase release.

12. The use of Claim 11, wherein said resveratrol derivative retains the structure of phenolic or polyphenolic rings linked by a rigid connecting moiety.

13. The use of Claim 11, wherein said resveratrol derivative has the chemical structure of Claim 3.

14. The use of Claim 11, wherein said resveratrol derivative has the chemical structure of Claim 4.

15. The use of Claim 11 , wherein said resveratrol derivative is trans- 3 ,3',4,5'-tetrahydroxystilbene.

16. An assay for screening a patient's body fluid or tissue to determine resveratrol-inhibitable peroxidase activity comprising the step of a. making a first reaction mixture comprising 0.5 mM hydrogen peroxide and 5 mM guaiacol in 10 mM sodium acetate at pH 6.5; b. making a second reaction mixture comprising 0.5 mM hydrogen peroxide, 5 mM guaiacol, and 100 ╬╝M resveratrol in 10 mM sodium acetate at pH 6.5; c. adding to said first reaction mixture a volume of body fluid or tissue to form a first test sample, wherein said volume is about 0.1 of the volume of the first reaction mixture; d. incubating said first test sample for thirty minutes at about 37┬░C; e. measuring the optical density of said first test sample at 450 run; f. adding to said second reaction mixture a volume of body fluid or tissue to form a second test sample, wherein said volume is about 0.1 of the volume of the second reaction mixture; g. incubating said second test sample for thirty minutes at room temperature; h. measuring the optical density of said second test sample at 450 nm; and i. comparing the optical density of said first test sample to the optical density of said second test sample, wherein the optical density of said second test sample being less than the optical density of said first test sample indicates the presence of resveratrol-inhibitable peroxidase activity.

17. A method for treating a patient with resveratrol or resveratrol derivative, comprising the steps of administering to said patient an effective amount of resveratrol or resveratrol derivative to cause a reduction in myeloperoxidase activity.

18. The method of claim 17, wherein said amount of resveratrol or resveratrol derivative is administered orally.

19. The method of claim 17, wherein said amount of resveratrol or resveratrol derivative is administered topically.

20. The method of claim 17, wherein said effective amount of resveratrol or resveratrol derivative is from about 0.1 to about 10 milligrams per kilogram body weight.