MXPA00003200A - Inhibtion of lipoprotein oxidation - Google Patents

Abstract

Hydroxylated derivatives of cholesterol lowering agents inhibit the oxidation of lipoproteins, and are thus useful for preventing the progression of atherogenesis and resultant vascular diseases, including heart attacks.

Description

INHIBITION OF LIPOPROTEIN OXIDATION FIELD OF THE INVENTION This invention is related to a method to inhibit the oxidation of lipoproteins, thereby decreasing or stopping atherogenesis, the method is related to the use of hydroxylated derivatives of known cholesterol lowering agents .

BACKGROUND OF THE INVENTION Atherosclerotic cardiovascular diseases and related conditions and cases of diseases associated with hyperlipidemia are major causes of disability and death. It is now well recognized that decreasing certain forms of cholesterol, both in healthy mammals as well as in individuals already experiencing hyperlipidemic states, can dramatically reduce heart attacks, vascular disease, and other diseases associated with atherosclerotic conditions. Hyperlipidemia is a condition characterized by an abnormal increase in blood lipids, such as cholesterol, triglycerides and phospholipids. These lipids do not circulate freely in solution in the plasma, but they are bound to proteins and transported as macromolecular complexes called lipoproteins. There are five classifications of lipoproteins based on their degree of density: cycle microns, very low density lipoproteins (VLDL), low density lipoproteins (LDL), intermediate density lipoproteins (IDL), and high density lipoproteins (HDL). One form of hyperlipidemia is hypercholesterolemia, characterized by the existence of elevated LDL cholesterol levels. The initial treatment for hypercholesterolemia is often to modify the diet to a low in fat and cholesterol, along with appropriate physical exercise, followed by drug therapy when the goals of lowering LDL can not be met simply by diet and exercise . LDL is commonly known as "bad" cholesterol, while HDL is "good" cholesterol. Although it is desirable to lower high levels of LDL cholesterol, it is also desirable to increase HDL cholesterol levels. Generally, it has been found that increased levels of HDL are associated with a lower risk for coronary heart disease (CHD). While LDL cholesterol is recognized as bad, most cholesterol lowering agents operate by lowering the plasma concentration of the LDL form, there is another key process in the early stages of atherogenesis, which is the oxidation of LDL. Oxidation of VLDL and HDL also occurs, which also contributes to atherogenesis. Oxidation leads to an increase in intracellular calcium calcium, a decrease in energy production, cytokine activation, membrane damage, all resulting in apoptosis, necrosis, and finally cell death. Oxidation typically begins when a radioactive radical extracts a hydrogen atom from a polyunsaturated fatty acid in the LDL particle. Peroxyl lipids and alkoxy radicals are formed, which in turn can initiate oxidation in the surrounding fatty acids, resulting in a propagation of lipid peroxidation. You are oxidized forms of lipoproteins are absorbed by macrophages faster than native lipoprotein, and this results in a cholesterol accumulation of macrophages, and a subsequent foam cell formation and inhibition of the mobility of tissue and cell macrophages endothelial This cascade of events results in vascular dysfunction and formation and activation of atherosclerotic lesions. It has now been discovered that certain hydroxysubstituted derivatives of commonly used cholesterol lowering agents are effective antioxidants for lipoprotein. In addition, these compounds are useful for the elimination of free radicals and the chelation of metal ions, which are also mechanisms by which lipoproteins are oxidized.

BRIEF DESCRIPTION OF THE INVENTION This invention provides a method for inhibiting the oxidation of lipoproteins in mammals comprising the administration of an effective amount of antioxidant of an agent that lowers the hydroxylated cholesterol. The invention also provides a method for removing free radicals in mammals which comprises administering an amount to remove free radicals from an agent that lowers the hydroxylated cholesterol. The invention also provides a method for inhibiting the chelation of metal ions by lipoproteins which comprises administering an effective amount of an agent that lowers the hydroxylated cholesterol. In a preferred embodiment, the methods are practiced using a hydroxylated form of a statin, especially atorvastatin, whose compounds are described in the patent.

North American No. 5,385,929, which is incorporated herein by reference. In another preferred embodiment, the methods are practiced using a hydroxylated gemfibrozil, for example, a compound of the formula In another embodiment, a hydroxylated fluvastatin is used, for example, compounds of the formula In another embodiment, a hydroxylated cerivastatin is used, especially a compound of the formula In another preferred embodiment, hydroxylated derivatives of lovastatin are used, for example, compounds of the formulas BRIEF DESCRIPTION OF THE FIGURES Figure 1. Structural formulas of atorvastatin in its hydroxylated metabolites. A- Atorvastatin, B-Metabolite Para-Hydroxy, C- Metabyte Orto-Hydroxy. Figure 2. They are structural formulas of gemfibrozil and its hydroxylated metabolite. A- Genfibrozil, B- Metabolite I Figure 3. The effect of atorvastatin and its hydroxylated metabolites on LDL oxidation in the copper ion (A) oxidative system, the AAPH oxidative system (B), and the macrophage oxidative system J- 774A.1 (C). LDL (100 μg protein / mL) was incubated in the three oxidative systems with increased concentrations of drug or its metabolites for four hours at 37 ° C in systems A and B, and for 20 hours with cells (C). At the end of the incubation, LDL oxidation was measured by the assay TBARS. LDL oxidation mediated by macrophages was calculated by subtracting the values obtained in the absence of cells from those obtained in the presence of cells. The results are given as the average standard deviation + (SD) (n = 3). A- CuSO4, A'- Atorvastatin, A "- Para-hydroxy, A" 'Ortho-hydroxy; B- AAPH, B'- Atorvastatin, B "- Para-hydroxy, B" '- Ortho-hydroxy; C- Macrophages, C- Atorvastatin, C "- Para-hydroxy, C "'- Ortho-hydroxy; D- Drug Concentration (μM); LDL E-Oxidation (LDL protein nmol MDA / mg) Figure 4. The effect of atorvastatin and its hydroxylated metabolites on the oxidation of VLDL in the oxidative system of copper ions (A), and the AAPH oxidation system (B) VLDL (100 μg protein / mL) was incubated with 10 μM of atorvastatin or its metabolites for 4 hours at 37 ° C. At the end of incubation , the oxidation of VLDL was measured by the TBARS assay, the results are given as mean ± SD (n = 3), A- CuS04, B-AAPH, C- Atorvastatin Control Ortho-hydroxy, Para-hydroxy, D-Oxidation of VLDL (VLDL protein nmol MDA / mg), Figure 5. Free radical elimination activity (A), and chelation capacity of copper ions of atorvastatin and its hydroxylated metabolites (B) A. Atorvastatin or its hydroxylated metabolites ( 20 μM) were incubated with a mM DPPPH and kinetic determination of the absorption at 517 nm was performed. representative of 3 different studies with a similar pattern. Vitamin E (20 μM) was used as a positive control for the free radical scavenger. B. LDL (100 μg protein / mL was incubated with atorvastatin or its metabolites (10 μM) and with increased CuSO concentrations for 4 hours at 37 ° C, before analysis of lipoprotein oxidation by the TBARS assay the results were give as average ± SD (n = 3) .A- A'- Atorvastatin, A "- Para-hydroxy, A" 'Ortho-hydroxy; A "" Vitamin E, A1 Time (sec), A2 Elimination of Free Radicals, Absorption (517 nm); B- B'- Control, B "- Para-hydroxy, B" '- Ortho-hydroxy, B1- Concentration of CuSO4 (μm), B2- Oxidation of LDL (protein LDL nmol / MDA / mg) Figure 6. Effect of gemfibrozil and concentration of gemfibrozil metabolite in LDL oxidation in the copper ion (A) oxidative system, the AAPH oxidative system (B), and the macrophage oxidative system J-774A.1 (C). LDL (100 μg protein / mL) was incubated in the three oxidative systems with increased concentrations of drug or its metabolite for four hours at 37 ° C in systems A and B, and for 20 hours with cells (C). At the end of the incubation, LDL oxidation was measured by the TBARS assay. The oxidation of the LDL mediated by macrophages was calculated by subtracting the values obtained in the absence of cells from those obtained in the presence of cells. The results are given as the mean ± SD (n = 3). A- CuSO4, A 'Gemfibrozil, A "Metabolite I; B- AAPH, B' Gemfibrozil, B" Metabolite I; C. Macrophages, C- Gemfibrozil, C "-Metabolite I, D- Drug Concentration (μM), E- Oxidation LDL (LDL protein nmol MDA / mg) Figure 7 The effect of gemfibrozil and its metabolite on the oxidation of LDL in the oxidative copper ion system (A), and in the AAPH oxidation system (B), incubated LDL (100 μg protein / mL) with 4 μM of gemfibrozil or its metabolites for 4 hours at 37 ° C. At the end of the incubation, the VLDL oxidation was measured by the TBARS assay.The results are given as mean ± (SD) (n = 3) .A- CuSO4; B- AAPH; C Control, C "- Gemfibrozil, C" Metabolite I; D Oxidation VLDL (nmol MDA / mg) Figure 8. VLDL lipoprotein electrophoresis after lipoprotein oxidation induced by copper ions (10 μM CuSO) in the absence or presence of atorvastatin, gemfibrozil, or its metabolites. A. Metabolite I, Gemfibrozil, Control, B- Para-hydroxy, Ortho-hydroxy, Atorvastatin, Control Figure 9. Radical elimination activity free (A), and chelating capacity of copper ions of gemfibrozil and its metabolite (B). A. Gemfibrozil or its metabolite (20 μM) were incubated with 1 mM DPPH and the kinetic determination of the absorbance was performed 517 mm. A representative experiment of 3 different studies with a similar pattern is shown. Vitamin E (Vit E) was used at a similar concentration as a free radical scavenger. B. LDL (100 μg protein / mL) was incubated with gemfibrozil or its metabolite (3 μM) and with increased CuS04 concentrations for 4 hours at 37 ° C before analysis of lipoprotein oxidation by the TBARS assay. The results are given as an average ± SD (n = 3). A- A'- Gemfibrozil, A "- Metabolite I, A1- Time (sec), A2- Elimination of Free Radicals, Absorption (517 nm), B- B'- Control, B" - Metabolite I, B1 Concentration CuS04 ( μm), B2- Oxidation LDL (LDL protein nmol MDA / mg) Figure 10 Combined effect of metabolite I of gemfibrozil and the ortho-hydroxy metabolite of atorvastatin in the oxidation of LDL. LDL (100 μg protein / mL) was incubated with 10 μM CuS04 for 4 hours at 37 ° C on its own (Control) or in the presence of metabolite I of gemfibrozil (3 μM), or the ortho-hydroxy metabolite of atorvastatin ( 4 μM) alone, or in combination.

The oxidation of lipoprotein was then measured by the TBARS assay. * p < 0.01 (vs. Control) and p < 0.01 (vs. Metabolite I), #P < 0.01 (vs. Ortho-Hydroxy). The results are given as the average ± SD (n = 3). A- Oxidation LDL (LDL protein nmol MDA / mg), B- Control, B 'Gemfibrozil, Metabilito I, B "- Atorvastatin, Ortho-hydroxy, B"' - Metabolite I + Ortho-Hydroxy Figure 11. Dependent antioxidant effects of the metabolite dose for atorvastatin hydroxy in membrane preparations enriched with polyunsaturated fatty acids. A- Effect of Metabolite of Atorvastatin in the Peroxidation of Lipids in Membrane; B- Drug concentration (μm), C-% inhibition of lipid peroxidation; D- * p < 0.01, ** p < 0.001; the values are average ± SD, Control LOOH = 1300 μM; 48 h of incubation at 37 ° C.

Figure 12. The comparative antioxidant potency of the para-hydroxy metabolite of atorvastatin, Vitamin E, and probucol. A- Inhibition of Lipid Peroxidation by Atorvastatin Metabolites in Atherosclerotic Membranes; B-Metabolite of Atorvastatin, B'- Vitamin E, B "- Probucol, C-% inhibition of lipid peroxidation, D- * p <0.01, The values are average ± SD, Cholesterol: mol ratio of phospholipids of 0.9: 1, 1.0 μm drug concentration Figure 13. The antioxidant potency of atorvastatin hydroxy metabolite and Vitamin E under high-membrane atherosclerotic type conditions A- Effect of Cholesterol Enrichment on Antioxidant Activities of Atorvastatin Metabolites; B-Atorvastatin; C- Vitamin E; D- Atherosclerotic; E- Normal; F-% inhibition of lipid peroxidation; G- * p < 0.001, values are average + SD (N = 3); drug = 1.0 μM.

DETAILED DESCRIPTION OF THE INVENTION The term "agent lowering hydroxylated cholesterol" means any chemical compound that is effective for lowering LDL cholesterol in mammals, which has at least one hydroxy group substituted in the original structure, and has antioxidant activity. Examples include hydroxylated statins. Statins are a known class of HMG.CoA reductase inhibitors, such as atorvastatin, fluvastatin, and cerivastatin. Hydroxylated statins are the original statin compound that has at least one substituted hydroxy group, examples are atorvastatin ortho-hydroxy and para-hydroxy atorvastatin as shown in Figure 1. Other hydroxylated cholesterol lowering agents are idroxysubstituted fibers. , as hydroxylated gemfibrozil as shown in Figure 2 (metabolite I). The hydroxy compound to be used in the method of this invention is preferably a compound having a hydroxy group linked to a phenyl ring. The increased risk of atherosclerosis in hyperlipypedic patients results from the improved oxidizability of their plasma lipoproteins. While hypercholesterolemic drug therapy, including the reductase inhibitors 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) such as atorvastatin, and the drug besafibra ipotriglyceridemic, reduce the enhanced susceptibility to the oxidation of lipoprotein low density (LDL) isolated from hyperlipidemic patients, this antioxidant effect may not be obtained in vitro with these drugs. The following experiments establish the effect of atorvastatin and gemfibrozil, as well as specific hydroxylated metabolites, on the LDL susceptibility of VLDL, and HDL on oxidation (for example, lipid peroxidation). Lipid peroxidation, induced either by copper ions (10 μM CuSO4), by the 2'2'-azobis2-amidino propane hydrochloride free radical generating system (5 mM AAPH), or by the cell line type J-774A.1 macrophages, were not inhibited by the original forms of atorvastatin or gemfibrozil, but were substantially inhibited (by 57% -97%), in a concentration-dependent manner, by pharmacological concentrations of the ortho-hydroxy and para-hydroxy metabolites of atorvastatin as well as by the para-hydroxy metabolite (metabolite I) of gemfibrozil. When using the ortho-hydroxy metabolite of atorvastatin and the metabolite I of gemfibrozil in combination, an additive inhibitory effect on the oxidation of LDL was found. Similar inhibitory effects (37% -96%) of the above metabolites were obtained for the susceptibility of VLDL and HDL on oxidation in the aforementioned oxidation systems. The effects of inhibition of these metabolites on the oxidation of LDL, VLDL and HDL may be related to their activity of elimination of free radicals, as well as (mainly for metabolite I of gemfibrozil) to their metal ion chelation capacities. . In addition, the inhibition of HDL oxidation was associated with the preservation of paraoxonase activity associated with HDL. The data establish that the hydroxy metabolites of atorvastatin, and the metabolite I of gemfibrozil, possess a great antioxidant potential, and as a result protect from oxidation to LDL, VLDL and HDL. The cholesterol lowering agents hydroxylated in this way are useful for reducing the atherogenic potential of lipoproteins through their antioxidant properties.

Oxidation of LDL is a key process in early atherogenesis and thus, oxidation of LDL is antiatherogenic. Oxidation of LDL and HDL also occurs during oscillatory stress and also contributes to atherogenesis. Antioxidants are derived environmentally as well as genetically. For example, diet antioxidants, such as vitamin E, carotenes, or polyphenolic flavonoids, associated with lipoproteins, protect them from oxidation. In addition, genetic factors such as paraoxonase associated with HDL, also protects this lipoprotein from the damage of oxidative stress. The improved susceptibility of LDL to the oscillation derived from hypercholesterolemic patients is significantly reduced by hypocholesterolemic therapy. In this way, the hipolipedimica therapy can be considered as beneficial not only due to its effects on plasma levels of VLDL, LDL and HDL, but also because it can reduce the formation of atherogenic oxidized lipoproteins. Ex vivo inhibition of LDL oxidation has been demonstrated after administration of the HMG-CoA reductase inhibitors lovastatin, simvastatin, pravastatin, or fluvastatin in hypercholesterolemic patients. It is suggested that the effects of inhibition of these drugs on the oxidation of LDL results from the improved removal of the "aged" in plasma which is more prone to oxidation than the recently synthesized LDL. This effect would be secondary in the stimulation induced by the statin of the activity of the LDL receptor in liver cells and in the inhibition of the production of the LDL and VLDL hepatic. The metabolites of the original statins, which are produced in the liver during drug therapy, can also be mechanistically involved. The activity of the system to metabolize the hepatic P450 drug participates by altering the structure of the original statin, usually by hydroxylation. In fact, all previous statins, with the exception of fluvastatin, did not demonstrate direct antioxidant effects on LDL oxidation in vitro when tested at concentrations comparable to the blood drug levels observed in treated hypercholestorlemic subjects. Atorvastatin, a new inhibitor of HMG-CoA, is the most effective statin to reduce both the level of LDL and total blood cholesterol. This compound also has significant hypotriglyceridemic properties towards all lipoprotein fractions. Therapy with atorvastatin increases the LDL receptor activity and inhibits direct production of apolipoprotein B-100 containing lipoprotein. Both the original drug and its metabolites have relatively long average circulation lives of 14 to 36 hours. Fiber drugs can also affect the susceptibility of lipoproteins to oxidation; for example, bezafibra has this capacity. The fibric acid derivatives are drugs that regulate the lipids that promote the catabolism of triglyceride-rich lipoproteins, secondary to the activation of lipoprotein lipase, and the reduction of apoC-III synthesis. Another fiber, gemfibrozil, has been shown not only to reduce plasma triglycerides, but also to increase plasma HDL concentration in humans and reduce plasma lipoprotein levels (a) in male monoscinomolgus. In humans, gemfibrozil is metabolized into gemfibrozil acylglucoronides, and these metabolites are found in the blood and urine of volunteers after treatment. The level of the hydroxy metabolite of gemfibrozil (metabolite I) found in plasma from rodents treated with gemfibrozil is much higher than that of treated humans and possibly reflects differences in doses and metabolisms. The effects of atorvastatin and gemfibrozil have been shown, as have hydroxylated metabolites (alone or in combination) on LDL susceptibility, VLDL and HDL to oxidation. The results clearly demonstrate the inhibitory effects of drug metabolites (but not of the original drugs) on the plasma lipoprotein oxidation individually, and an additional effect, when combined. The data establish that the hydroxylated derivatives are useful to prevent the oxidation of lipoproteins and thereby reduce their atherogenic potential. The following detailed examples demonstrate the antioxidant activity of various hydroxylated lowering agents.

EXAMPLE 1 Materials- Atorvastin and its ortho-hydroxy and para-hydroxy metabolites (Fig 1) were synthesized, as well as gemfibrozil and its metabolite I (Figure 2) by prior art methods. 2,2-Azobis 2-amidinopropane hydrochloride (AAPH) was purchased from Wako Chemical Industries, Ltd. (Osaka, Japan). 1,1-Diphenyl-2-pyridylhydraz (DPPH) was purchased from Sigma (St. Louis, MO). Lipoproteins were isolated from VLDL, LDL, and HDL in serum from fasting normolipidemic volunteers. The lipoproteins were prepared by an ultra-centrifugation of discontinuous density gradient. The lipoproteins were washed at their appropriate densities (1,006 g / mL, 1063 g / mL, and 1210 g / mL, respectively), and dialyzed against 150 mM NaCl, (pH 7.4) at 4 ° C. The lipoproteins were then sterilized by filtration (0.45 μM), kept under nitrogen in the dark at 4 ° C, and used in 2 weeks. Prior to the oxidation studies, the lipoproteins were dialyzed against a free solution of PBS, EDTA, pH 7.4 under nitrogen at 4 ° C. The lipoproteins were found to be free of lipopolysaccharide (LPS) contamination when tested by the Limulus Ameboccyte Lysate assay (Associated of Cape Cod, Inc., Woods Hole, MA, USA). The protein and lipoprotein content was determined by standard methods.

Oxidation of lipoproteins- Lipoproteins (100 μg protein / mL) were incubated with 10 μM CuSO4 or with 5 mM AAPH for 4 hours at 37 ° C. AAPH is a water-soluble azo compound that thermally decomposes and generates water-soluble peroxyl radicals at a constant rate. Oxidation was determined by the addition of 10 μM of butylated hydroxytoluene (BHT) and cooling at 4 ° C. The extent of lipoprotein oxidation was measured by the assay of thiobarbituric acid reagent substances (TBARS), using malodialdehyde (MDA) for the standard curve. In addition, the oxidation of lipoproteins was also determined by the lipid peroxidation test that analyzes the lipid peroxides by its ability to convert iodide to iodine which can be measured photometrically at 365 nm. The kinetics of LDL oxidation were continually built up by measuring the formation of conjugated dienes as the increase in absorption at 234 nm. Oxidation of LDL by Macrophages - Murine J-774A.1 macrophage cell lines were purchased from the American Type Culture Collection (Rockville, MD). The macrophages were cultured in the Dulbecco's Moodified Eagles Medium (DMEM) supplemented with 5% hot inactivated calf fetal calf serum (FCS). For lipoprotein oxidation studies, cells (1 x 106/35 mm disc) were incubated with LDL (100 μM protein / mL) in RPMI medium (without rojofenol) in the presence of CuSO for 20 hours at 37 ° C in the incubator. The LDL control is also incubated in a cell-free system under the same conditions. At the end of the incubation period, the extent of LDL oxidation was measured in the medium (after centrifugation at 100 x g for 10 minutes) by the TBARS assay. The oxidation mediated by LDL cells was calculated by subtracting the values obtained in the cell-free system from those obtained with cells. Lipoprotein electrophoresis- Lipoproteins (100 μg protein / mL) were incubated without or with drugs after oxidation in the presence of 10 μM CuSO. Then, the electrophoresis of the lipoproteins was performed in 1% agarose using a Hydragel-Lipo kit (Sebia France). Free radical scavenging capacity- The free radical scavenging capabilities of the drugs were analyzed by the 1, 1-diphenyl-2-picrylhydraz (DPPH) assay. Each drug (20 μM) was mixed with 3mL of 0.1 nmol DPPH / 1 (in ethanol). The time course of change in optical density at 517 nm was then verified kinetically. Paraoxonase activity measurements- The rate of paraoxon hydrolysis was evaluated by measuring the formation of p-nitrophenol at 412 nm at 25 ° C. The basal assay mixture included 1.0 nM paraoxon and 1.0 mM CaCl2 in 50 mM glycine / NaOH pH 10.5. One unit of paraoxonase activity produces 1 nmole of p-nitrophenol per minute.

Statistical analysis- The t-Student test used to compare two averages was used, while the variant analysis (ANOVA) was used when more than two groups were compared. The data are presented as averages ± standard deviation is (SD).

RESULTS The effect of atorvastatin and its hydroxy metabolites, as well as that for gemfibrozil and its metabolites, on the susceptibility of lipoproteins to oxidation were studied in several oxidation systems including those containing metal ions (10 μM CUSO4), those that have the capacity to generate free radicals (5 mM AAPH), and those that mimic biological oxidation (macrophage cell line J-774A.1). Oxidation of lipoproteins and atorvastatin- Oxidation of LDL was inhibited by the metabolites of atorvastatin ortho-hydroxy and para-hydroxy, but not by atorvastatin in all studies of oxidative systems. These inhibition effects depend on the concentration (Fig 3) at 10 μM, both ortho-hydroxy and para-hydroxy metabolites inhibited the oxidation of LDL measured by the TBARS assay in the CuSO system, by 73% and 60%, respectively (Figure 3A); in the AAPH system, by 44% and 34%, respectively (Figure 3B); and in the macrophage system by 50% and 46%, respectively (Figure 3C). In all the concentrations studied and in all the oxidation systems, the ortho-hydroxy metabolite was a better inhibitor of LDL oxidation than the para-hydroxy metabolite (Fig 3). A more potent inhibitory effect of both atorvastatin metabolites was obtained in the metal ion oxidation system (Fig 3A), compared to that by the free radical generation system (Fig 3B). Similar results were obtained in the other oxidation systems when LDL oxidation was determined by analysis of lipoprotein associated peroxides. The ortho-hydroxy and para-hydroxy metabolites of atorvastatin reduced the peroxide content associated with the LDL from 710 ± 51 in the LDL control, to 192 ± 15 and 284 ± 13 nmol / mg of LDL protein in the CuSO4 system respectively, and from 990 ± 89 in the LDL control, to 554 ± 32 and 624 ± 38 nmol / mg of LDL protein in the AAPH system, respectively. In addition, the kinetic analysis of the formation of conjugated dienes at 234 nm during the oxidation of LDL induced by copper ions (10 μM CuSO), revealed that the delay time required for the initiation of LDL oxidation was 50 ± 7 minutes (n = 3) both for the control and for the LDL treated with atorvastatin, while the formation of conjugated LDL diene started only after 180 + 25 minutes (n = 3) for both metabolites of atorvastatin. The effect of atorvastatin and its metabolites on the oxidation of LDL is shown in Figure 4. In the copper ion oxidation system, the ortho-hydroxy and para-hydroxy metabolites (10 μM) inhibited the oxidation of lipoproteins by a % and 37%, respectively (Figure 4A) while atorvastatin pos alone did not have any effect. In the AAPH oxidation system, the inhibitory effects of these metabolites were only 43% and 16%, respectively (Figure 4B), and once again atorvastatin alone had no effect. Similar results were found when oxidation of LDL was analyzed by peroxide formation. The ortho-hydroxy and para-hydroxy metabolites of atorvastatin reduced the VLDL-associated peroxide content of 1818 ± 33 in the VLDL control, to 242 ± 22 and 1088 ± 10 nmol / mg of LDL protein in the CuSO4 system, respectively, and of 2169 + 329 in the control of VLDL, at 1228 ± 210 and 1819 ± 228 nmol VLDL in the AAPH system, respectively. Similarly, oxidation of HDL in the presence of CuSO4 under similar incubation conditions revealed that the ortho-hydroxy metabolite completely inhibited the oxidation of HDL, while the metabolite for para-hydroxy inhibited HDL oxidation, whereas the metabolite para-hydroxy inhibited the oxidation of lipoproteins by approximately 50% (Table 1). The effects of inhibition of these metabolites in the oxidation of HDL were associated with the protection of paraoxonase by 54% and 27%, respectively. Elevated activities of paraoxonase associated with HDL were noted, compared to the activity of paraoxonase in HDL that was oxidized in the absence of the original drug added (Table 1).

TABLE 1 The Effect of Atorvastatin and its Metabolites on HDL Oxidation and HDL-Associated Paraoxonase Activity Oxidation of HDL-induced CuSO4 Specific Activity of paraoxonase (nmoL / mg HDL Protein) (nMoL / mg HDL protein / min) MDA Peroxides Control 9.1 ± 0.1 122 + 14 26 + 2 Atorvastin 9.6 + 0.5 122 + 15 29 ± 4 Metabolite Ortho-hydroxy 0.2 + 0.1 * 9 + 1 * 40 + 4 * Metabolite Para-Hydroxy 4.5 + 0.3 65 ± 9 * 33 ± 3 * * p < 0.01 (vs. Control) The effects of inhibition of atorvastatin metabolites on lipoprotein oxidation are also related to a free radical scavenging activity and to a capacity for chelation of metal ions. In the DPPH assay, a time-dependent reduction in absorption at 517 nm by both metabolites of atorvastatin (20 μM), but were not observed by atorvastatin (Figure 5A). After 300 seconds of incubation, the ortho-hydroxy and para-hydroxy metabolites reduced the absorption at 517 nm by 37% and 28%, respectively. In comparison, a 95% reduction in absorption was obtained by 20 μM of the free radical scavenging antioxidant, Vitamin E (Figure 5A). These results establish that the metabolites of atorvastatin possess substantial free radical scavenging abilities. The ability of atorvastatin metabolites to act as inhibitors of LDL oxidation by chelating copper ions was tested by incubating LDL with increased concentrations of CuSO for 2 hours at 37 ° C in order to determine whether excessive concentrations of copper ions they can solve the effect of inhibition of these metabolites in the oxidation of LDL (Figure 5B). The addition of increased concentrations of copper ions to the incubation system caused only a minor increase in the oxidation of LDL when the metabolites were present, in comparison with the control LDL (Figure 5B), indicating only minimal capacities of these metabolites to inhibit the oxidation of LDL by chelation of metal ions.

EXAMPLE 2 Oxidation of lipoprotein and gemfibrozil The previous experiments were conducted to determine the effects of gemfibrozil and one of its metabolites (metabolite I) in the oxidation of LDL, and is similar to that shown for atorvastatin (Figure 3-5). Oxidation of LDL was inhibited by metabolite I, but not by gemfibrozil by itself, in all the oxidation systems studied. This inhibition effect of metabolite I was concentration dependent (Figure 6). At a concentration as low as 4 μM, gemfibrozil metabolite I inhibited the oxidation of LDL, measured by the TBARS assay, by 96% in the CuS04 oxidation system (Figure 6A), by 26% in the oxidation system AAPH (Figure 6B), and by 99% in the system of oxidation mediated by macrophages J-774 A.1 (Figure 6C). Similar results were found when the oxidation of LDL was analyzed by the amount of peroxides formed. The metabolite I of gemfibrozil reduced the peroxides associated with LDL from 710 ± 57 to 28 + 7 nmol / mg of LDL protein in the CuSO4 system, and from 917 + 78 to 703 + 38 nmol / mg of protein in LDL in the AAPH system. In addition, the time required for the initiation of LDL oxidation (measured by the kinetic analysis of the conjugate formation), revealed a delay time of 60 ± 9 minutes for LDL alone or for LDL in the presence of gemfibrozil. In comparison, even after 240 minutes of incubation with metabolite I of gemfibrozil, no formation of conjugated dienes of LDL was observed. The analysis of the effect of gemfibrozil and its metabolites on the oxidation of LDL once again showed a very potent inhibition effect of metabolite I (4 μM), but not of gemfibrozil, with 96% inhibition of VLDL oxidation in the system of oxidation of CuSO (Figure 7A) and 91% of inhibition in the AAPH oxidation system (Figure 7B).

VLDL lipoprotein electrophoresisAfter oxidation with atorvastatin and its metabolites, with the presence of gemfibrozil and its metabolite, clearly he demonstrated the potency of the ortho-hydroxy metabolite of atorvastatin and metabolite I of gemfibrozil to reduce the electrophoretic migration of lipoproteins (Figure 8). Similar results were obtained for LDL and HDL. Upon the oxidation of HDL in the presence of 10 uM CuSO, metabolite I of gemfibrozil substantially inhibited lipoprotein oxidation (Table 2), with a concomitant protection activity oxonasa, preserving the initial level of activity for oxonasa associated with HDL (Table 2). The gemfibrozil by itself had no effect. The oxidation of lipoprotein was carried out for 4 hours at 37 ° C with 10 μM CuSO, in the absence (Control) or presence of 10 μM of the drugs. The activity of paraoxonase HDL before its incubation with copper ions was 50 + 3 nmol // mg HDL protein / min. The results are given as the average + SD (n = 3).

TABLE 2 Effect of Gemfibrozil and its Metabolites on HDL Oxidation and HDL-Associated Paraoxonase Activity Oxidation of CuSO-Induced HDL Specific Activity of paraoxonase (nmoL / mg HDL Protein) (nmoL / mg HDL protein / min) MDA Peroxides Control 9.1 ± 0.1 122 + 14 26 ± 3 Gemfibrozil 8.2 ± 0.4 134 ± 13 27 + 5 Metabolite 0.8 ± 0.1 * 18 ± 4 * 50 ± 7 * * p < 0.01 (vs Control) In analyzing the mechanism responsible for the inhibition of lipoprotein oxidation by means of gemfibrozil metabolite I, they demonstrated both the ability to eliminate free radicals (Figure 9A), and the ability to chelate copper ions from its metabolite (Figure 9B). When using the DPPH assay, only metabolite I, but not gemfibrozil per se (20 μM), demonstrated a time-dependent reduction in absorption at 517 nm, with up to 86% reduction in optical density after 300 seconds of incubation (Figure 9A). Incubation of LDL with increased concentrations of CuSO4? during 2 hours at 37 ° C in the presence of metabolite I of gemfibrozil revealed that when using 20 μM of CuSO, the inhibition effect of metabolite I was completely prevented (Figure 9B) indicating that in this LDL oxidation system, the chelation of copper ions by means of metabolite I plays a role in the inhibition of lipoprotein oxidation.

EXAMPLE 3 Interaction between atorvastatin and gemfibrozil The general procedures described above were repeated to determine whether the in vitro addition of the combined potent metabolites (metabolite I of gemfibrozil and the ortho-hydroxy metabolite of atorvastatin) produces a greater inhibitory effect on the oxidation of the LDL than any of the agents by itself. When using low concentrations of metabolite I of gemfibrozil (3 μM) or ortho-hydroxy metabolite of atorvastatin (4 μM), only 40% to 43% of inhibition effect of each of these drugs was observed in the LDL oxidation induced by copper ions, respectively, compared to the control LDL (Figure 10). However, when using a combination of these metabolites at the above concentrations, a significant additional inhibition effect of 88% was observed for the oxidation of LDL (Figure 10).

EXAMPLE 4 The metabolite for para-hydroxy of atorvastatin, and the known antioxidants Vitamin E and probucol, were valued in membrane vesicles enriched with polyunsaturated fatty acids. For the lipid peroxidation experiments, 500 μL of membrane vesicles were enriched with phosphatidylcholine of d i I i noleoi I (DLPC) at a concentration of 1.0 mg DLPC / mL. The enriched vesicles were freshly prepared in a HEPES buffer (N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid) (0.5 mM HEPES, 154.0 mM NaCl, pH 7.3) The buffer solution was prepared without antioxidants additional (as control), and with (1) various concentrations of atorvastatin para-hydroxy metabolite, (2) Vitamin E, and (3) probucol, which is 4,4 '- [(1-methylethylmilidene) bid (thio) )] [2,6-bis (1,1-dimethylethyl) -phenol.) The membrane vesicle solutions were immediately placed in a shaking water bath at 37 ° C. During the incubation period (0-72 hours) ), 100 μL aliquots were removed and the peroxidation reaction was terminated by adding 25 μL of 5.0 mM ethylenediaminetetraacetic acid (EDTA) and 20 μL of 35.0 mM butylated hydroxytoluene, The extent of lipid peroxidation in each sample was determined by an aspectrophotometric assay for peroxide lipids in serum lipoproteins using a decolorizing reagent known as CHOD-iodide (Merck, Darmstadt, FRG, Merck Cat. No. 14106). The coloring reagent has the following composition: Potassium phosphate, pH 6.2 0.2M Potassium iodide 0.12 M Sodium azide 0.15 μM Polyethylene glycol mono [p- (1,1 ', 3,3'-tetramethyl-butyl-phenyljether 2 g / L Alkylbenzyl dimethyl ammonium chloride 0.1 g / L 10 μM ammonium molybdate The concentration formed of triidiode was measured spectrophotometrically according to the formula (L = lipoprotein) LOOH + 2H + + 21"? LOH + H2O + l2 l2 + I"? L3"To each of the aliquots extracted from the membrane vesicles were added 1.0 mL of CHOD coloring reagent, and the mixture was incubated in the absence of light for 4 hours. The absorption of the solution was measured at 365 nm (e = 2.4 x 104M "1 cm" 1). The formation of peroxide in lipids was measured in triplicate and the values were expressed as mean ± SD. The significance of the differences between the results of the different experimental conditions was tested using the two-tailed Student t-test. The antioxidant activity of atorvastatin para-hydroxy metabotryl is shown in Figure 11 for various dose concentrations. The results establish that the para-hydroxy compound has a dose-dependent antioxidant activity, and at 10.0 μM it causes 80% inhibition of lipid peroxidation. Even at concentrations as low as 10.0 μM, the para-hydroxy compound inhibited high levels (>; 102 μM) of lipid peroxidation. The results shown in Figure 12 establish that the para-hydroxy metabolite of atorvastatin is significantly more active than other known antioxidants, specifically Vitamin E and probucol. The antioxidant activity of atorvastatin para-hydroxy metabolite increased under atherosclerotic high-cholesterol type conditions, and this is shown in Figure 13. The above experiments establish that the metabolites of HMG-CoA reductase inhibitors, such as atorvastatin for example , and of the fibric acid derivatives, for example gemfibrozil, significantly inhibited the oxidation of lipoproteins in various oxidation systems. Oxidation in LDL is a key case in atherogenesis, as it contributes to the accumulation of macrophage cholesterol and the formation of foam cells, as does cytotixity, thrombosis, and inflammation. In this way, the inhibition of LDL oxidation contributes to the attenuation of the atherosclerotic process. Although not studied as extensively, the oxidation of VLDL and HDL also occurs under oxylative stress, and also facilitates the development of atherosclerosis. In VLDL, lipid peroxidation mainly involves the oxidation of central triglyceride polyunsaturated fatty acids, whereas in HDL, the surface phospholipid fatty acids are the main substrates susceptible to oxidation. In hypercholesterolemic and hypertiglycerimic patients, the high concentration of triglycerides and blood cholesterol are risk factors for atherosclerosis. The increased risk is due to the improved susceptibility of lipoproteins to oxidation. Several hypolipidemic drugs have been shown to reduce the improved propensity of LDL to oxidation in hypercholesterolemic patients. This effect of inhibition on the oxidation of LDL can result from an improved removal (by means of an increased LDL receptor activity induced by drugs, mainly in iron) of "aged LDL" which is more prone to oxidative modifications. In addition, this protective effect against oxidation can result from drug metabolites formed in vivo that possess antioxidant properties. However, with the exception of fluvastatin, none of the original forms of the hypolipimed drugs studied demonstrated a direct inhibition effect on LDL oxidation when tested in vitro at pharmacological concentrations. The above data demonstrate that the original drugs, atorvastatin and gemfibrozil, do not affect the oxidation of LDL, VLDL, or HDL in vitro, even when used at high concentrations. However, low pharmacological concentrations of specific hydroxylated metabolites induce very potent inhibitory effects in the oxidation of LDL, VLDL, and HDL, both in independent systems and dependent on metal ions. The effect of inhibition of drug metabolites on lipoprotein oxidation was found to be more pronounced in the CuSO4 system compared to the AAPH system, and this phenomenon may be related to the effects of the metabolites on both the elimination of free radicals as in the copper ion bond. Both metabolite I of gemfibrozil and the hydroxy metabolites of atorvastatin proved to be potent scavengers of free radicals. The ortho-hydroxy metabolite of atorvastatin compared to metabolite I of gemfibrozil, acted on the CuSO4 oxidation system as a better metal ion chelator. The increased copper ion concentrations completely extinguished the inhibition effect of metabolite I of gemfibrozil, but not that of the metabolites of atorvastatin, on the oxidation of LDL. The molecular structure of atorvastatin hydroxy metabolites, where the idroxyl group binds to the carboxamide portion of the molecule, allows these metabolites to act as electron donors, and in this way, as potent antioxidants (Figure 1). The ortho-hydroxy metabolite is a more potent antioxidant than the para-hydroxy metabolite of atorvastatin, since the hydroxyl group in the ortho position in the amine group (but not the hydroxyl group in the para position), can form a transition state relatively stable peroxide radical, and in this way, act as a potent antioxidant. Similarly, in metabolite I of gemfibrozil (but not in gemfibrozil), the hydroxyl group of the aromatic ring can substantially contribute to the antioxidant properties of this compound (Figure 2). Under oxidative stress, the oxidation of lipoproteins involves the action of reactive oxygen species, and since the transition metal ions are known to be present in areas of atherosclerotic lesions, the oxidation models used in the previous experiments are representative of the situation in vivo. The inhibitory effects of both the metabolites of atorvastatin and gemfibrozil, in the oxidation of LDL, were also demonstrated for VLDL and HDL. The inhibition pattern was similar in all the oxidation systems studied. These results establish that metabolisms exert their inhibitory effect on the oxidation of lipoproteins by means of common mechanisms, that is, elimination of free radicals and chelation of metal ions. In one study, in patients with familial combined hyperlipidemia, gemfibrozil therapy did not significantly affect the oxidation of LDL. This observation, however, could have resulted from too low a concentration of drug metabolite to exert an antioxidant effect on the oxidation of LDL, or at the time of sample collection. In addition, the metabolites of the drug may be associated with non-lipoprotein components of the plasma (e.g. albumin) or isolated within the cells or interstitial compartments. Thus, ex vivo examination of the oxidation potential of lipoproteins isolated from treated humans or from experimental animals may not necessarily reflect the lipoprotein environment in vivo. The data presented above establish that the agents that lower the hydroxylated cholesterol inhibit the oxidation of lipoproteins by eliminating free radicals and reducing the chelation of metal ions of lipoproteins. Accordingly, the invention provides a method for inhibiting the oxidation of lipoproteins, as well as a method for inhibiting the chelation of metal ions of lipoproteins, and a method for eliminating free radicals. The amounts of agents that lower the hydroxylated cholesterol required to inhibit the metal ion chelation of lipoproteins, and to eliminate free radicals, are all referred to herein as "antioxidant amount". The hydroxylated cholesterol lowering agents will be administered in an antioxidant amount, mainly an amount that is effective to cause an inhibition of lipoprotein oxidation. Such effective antioxidant amounts will be from about 1 to 100 mg / kg. Such amounts of active agent will be administered one to four times a day in order to inhibit the oxidation of lipoproteins.

Hydroxylated compounds will be formulated for convenient oral or parenteral administration, and will be combined with common excipients and vehicles such as calcium carbonate, candelilla wax, hydroxypropyl cellulose, lactose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, talc, and dioxide. of titanium. For oral administration, the formulations can be compressed to form tablets, or they can be encapsulated in gelatin capsules. Typical tablets will contain from about 10 mg of active ingredient to about 80 mg. The compounds can be further formulated as slow release dosage forms, for example using osmotic pump technology, as well as transdermal skin patches. For parenteral doses, the compounds typically dissolve isotonic saline for convenient intravenous administration, or by injection.

Claims (7)

1. A method for inhibiting the oxidation of lipoproteins in mammals characterized in that it comprises administering an antioxidant effective amount of an agent that lowers the hydroxylated cholesterol.

2. The method according to claim 1, characterized in that it employs hydroxylated gemfibrozil, hydroxylated atorvastatin, or hydroxylated fluvastatin.

3. The method according to claim 2, characterized in that it employs atorvastatin ortho- or para-hydroxy side.

4. The method for eliminating free radicals in mammals comprising administering an amount of free radical removal of an agent that lowers the hydroxylated cholesterol.

5, The method according to claim 4, characterized in that the agent lowering the hydroxylated cholesterol is ortho- or para-hydroxylated atorvastatin, hydroxylated gemfibrozil, or hydroxylated fluvastatin.

6. A method for inhibiting chelation of metal ions of lipoproteins in mammals, characterized in that it comprises administering an amount of inhibition of metal ion chelations of an agent that lowers the hydroxylated cholesterol.

7. The method according to claim 6, characterized in that the agent lowering the hydroxylated cholesterol is ortho- or para-hydroxylated atorvastatin, hydroxylated gemfibrozil or hydroxylated fluvastatin.