RU2235541C2 - Treatment of atherosclerosis and other cardio-vascular diseases - Google Patents

Abstract

FIELD: medicine, therapy.

SUBSTANCE: the present innovation deals with treating cardio-vascular and inflammatory VCAM-1-mediated (by the factor of vascular cellular adhesion 1) diseases or due to expression of redox-sensitive gene. For this purpose, one should introduce a substance to either prevent or minimize oxidation of polyunsaturated fatty acid. Also, this innovation deals with testing the compound for its ability to treat VCAM-1-mediated disease and evaluate sensitization of vascular endothelial cells to polyunsaturated fatty acids or their oxidized derivatives. The innovation provides identification of selective methods of therapy in case of above-mentioned diseases and adequate matching for necessary therapeutic means.

EFFECT: higher efficiency of therapy.

37 cl, 21 dwg, 17 ex

Description

 BACKGROUND OF THE INVENTION

The present application relates to methods and compositions for the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

Leukocyte adhesion to endothelium is the cause of various inflammatory diseases, including atherosclerosis, autoimmune diseases, and bacterial and viral infections. Endothelial leukocyte recruitment begins when adhesion-induced receptor molecules on the surface of endothelial cells interact with a counterreceptor on immune cells. Vascular endothelial cells determine which type of white blood cells (monocytes, lymphocytes, or neutrophils) is updated by the expression of specific adhesion factors, such as vascular cell adhesion factor 1 (VCAM-1), intracellular adhesion factor 1 (ICAM-1), and E-selectin. At the earliest stage of atherosclerotic damage, localized endothelial expression of VCAM-1 and selective recruitment of mononuclear leukocytes that express the integrin VLA-4 counterceptor occurs. Due to the selective expression of VLA-4 on monocytes and lymphocytes, but not on neutrophils, VCAM-1 plays an important role in mediating selective adhesion of mononuclear leukocytes. The subsequent conversion of leukocytes into foamy macrophages leads to the synthesis of a wide variety of inflammatory cytokines, growth factors and chemoattractants, which contribute to an increase in leukocytes and recruitment, proliferation of smooth muscle cells, activation of endothelial cells and the synthesis of a characteristic intercellular matrix of atherosclerotic plaques.

VCAM-1 is expressed in cultured human vascular endothelial cells after activation by lipopolysaccharide (LPS) and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF-α).

These factors are not selective in the activation of the expression factor of cell adhesion. Figure 1 illustrates the cytokine activation process of activation of gene expression of VCAM-1 in vascular endothelial cells.

Figure 1 regulatory patterns of cytokine activation of vascular cell adhesion-1 factor gene expression mediated by reduction-oxidation sensitive factors such as NF-kB in vascular endothelial cells (IkB is an inhibitory subunit; NF-kB is a nuclear factor of kV; NH ₃ refers to the amino terminus of a protein and RNA Pol II is RNA polymerase P).

1. The cytokine binds to its receptor.

2. An activated receptor generates an intracellular signal.

3. Activation of transcriptional regulatory protein.

4. Broadcast to the core.

5. Binding to the enhancer element of specific DNA on the VCAM-1 promoter.

6. Activation of RNA transcription.

7. RNA processing.

8. Translation into protein.

9. Post-translational processing.

10. Insertion of VCAM-1 into the membrane.

Molecular analysis of the regulatory elements of the human VCAM-1 gene that control its expression confirms the important role of nuclear factor-kB (NF-kB), transcription regulation factor, or similar NF-kB binding protein in the oxidation-reduction-sensitive regulation of VCAM-gene expression 1. Transcription factors are proteins that activate (or suppress) gene expression in the cell nucleus by binding to a specific DNA sequence called "enhancer elements", which are usually located next to the region of the gene called the "promoter" from which RNA synthesis begins. Nuclear factor kV is a ubiquitously expressed multisubunit transcription factor activated by certain cell types using a large and diverse group of inflammatory agents, such as TNF-α, IL-1β, bacterial endotoxin and RNA viruses. It plays a key role in mediating inflammation and other stress signals between the regulatory apparatus of the nucleus. Although the direct biochemical signals that activate NF-kB are unknown, this transcription factor may be included in the general molecular pathway of many risk factors and "causal" signals of atherosclerosis, such as hyperlipidimia, smoking, hypertension and diabetes mellitus.

Importantly, activation of NF-kB in vascular endothelial cells by various signals can be selectively inhibited by antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate (see USS No. 07/969934, now authorized). This leads to the hypothesis that oxygen radicals play an important role in the activation of NF-kB through an unidentified redox mechanism. Since the NF-kB-like enhancer element also regulates the transcription of the VCAM-1 promoter in an oxidation-reduction-sensitive manner, oxidative stress in atherosclerotic lesions can play a role in the regulation of the VCAM-1 expression gene through this oxidation-reduction-sensitive regulatory transcription protein.

It has been hypothesized that the modification of low density lipoprotein (LDL-LDL) into oxidized modified LDL (ox-LDL) using active oxygen is fundamental in the initiation and spread of atherosclerosis. Steinberg, et al., N. Engl. J. Med., 1989; 320-915-924. Oxidized LDL is a complex structure consisting of at least several different chemical oxidized materials, each of which, alone or in combination, can modulate cytokine-activated gene expression of the adhesion molecule. Fatty acid hydroperoxides, such as linoleyl hydroperoxide (13-HRODE), are derived from free fatty acids by lipoxygenases, and they are an important component of oxidized LDL.

It has been proposed that the generation of oxidized lipids is effected by the action of the cell lipoxygenase system and that oxidized lipids are sequentially transferred to LDL. This represents, therefore, the LDL propagation reaction in the medium, catalyzed by transition metals and / or sulfhydryl compounds. Previous studies have shown that fatty acid modification of cultured endothelial cells can alter their susceptibility to oxidizing agents. The addition of saturated or monounsaturated fatty acids to cultured endothelial cells lowers their susceptibility to oxidizing agents, while the addition of polyunsaturated fatty acids (PUFA) increases it.

Using reverse phase HPLC analysis of natural and saponified lipid extracts, it was shown that 13-HPODE is the dominant oxidized fatty acid in lipids oxidized by activated human monocytes. Prolonged exposure to oxidized LDL leads to an oxidative signal to the vascular endothelial cell, possibly through specific hydroperoxide of a fatty acid that selectively enhances cytokine-induced expression of the VCAM-1 gene.

The area of the vascular wall predisposed to atherosclerosis preferably captures circulating LDL through a mechanism that is not well defined. Then, through a poorly understood pathway, the endothelium, smooth muscle, and / or inflammatory cells convert LDL to oxidized LDL. Unlike LDL, which are captured by the LDL receptor, monocytes greedily capture oxidized LDL by the scavenger receptor, whose expression, unlike LDL receptor, is not inhibited by an increase in the content of intracellular lipid. Thus, monocytes continue to capture oxidized LDL and become crowded with lipids, foamy macrophage cells that form the fatty layer.

Since cardiovascular disease is currently the leading cause of death in the United States and ninety percent of cardiovascular disease is atherosclerosis, there is a need to develop new methods and pharmaceutical agents for treating it. To achieve this, it is important to identify and control specific oxidized biological compounds that act as selective regulators of the expression of inflammatory mediators, in particular VCAM-1. A more general goal is to identify selective methods to suppress the expression of oxidation-reduction-sensitive genes or to activate suppressed oxidation-reduction-sensitive genes.

Thus, an object of the present invention describes the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

Another subject of the present invention describes a method for selectively inhibiting VCAM-1.

Another object of the present invention still describes a method for treating a human disease or disorder that is mediated by expression or suppression of genes that are sensitive to redox potential.

Another subject of the present invention describes pharmaceutical compositions for the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

SUMMARY OF THE INVENTION

It was found that polyunsaturated fatty acids (PUFAS) and their hydroperoxides (ox-PUFAS), which are important components of oxidatively modified low density lipoproteins (LDL), induce the expression of VCAM-1 rather than intracellular adhesion factor-1 (ICAM-1) or E-selectin in the endothelial cells of a human aorta, through a mechanism that is not mediated by cytokines or other monocytokine signals. This is the fundamental discovery of an important, previously unknown biological pathway, in the VCAM-1 mediated immune response.

As examples, linoleic acid, linolenic acid, arachidonic acid, linoleyl hydroperoxide (13-HPODE) and arachidonic hydroperoxide (15-HPETE) cause expression of the VCAM-1 gene, but not ICAM-1 or E-selectin, on the cell surface. Unsaturated fatty acids (such as stearic acid) and unsaturated fatty acids (such as oleic acid) do not cause expression of VCAM-1, ICAM-1 or E-selectin.

Induction of VCAM-1 by the action of PUFAS and their hydroperoxides of fatty acids is inhibited by the antioxidant pyrrolidine dithiocarbamate (PDTC). This indicates that induction is mediated by an oxidized signal molecule and that induction is prevented when the oxidation of the molecule is blocked (i.e., no oxidation is observed), reversed (i.e., the signal molecule is reduced) or when the modified redox signal is somehow blocked for interactions with its regulatory target.

Cells that are aged for a long time with elevated levels of polyunsaturated fatty acids or their oxidized derivatives can trigger an immune response that is not normal, and which, outside the threatened part, lead to illness. For example, supersensitivity of vascular endothelial cells to PUFAS and ox-PUFAS can accelerate the formation of atherosclerotic plaques.

Based on these findings, a method is described for the treatment of atherosclerosis, postangioplastic restenosis, coronary artery disease, angina pectoris, and other cardiovascular diseases, as well as non-cardiovascular inflammatory diseases that are mediated by VCAM-1, including the removal, reduction of concentration or prevention the formation of oxidized polyunsaturated fatty acids, including, but not limited to, oxidized linoleic (C ₁₈ , Δ ^{9, 12} ), linolenic (C ₁₈ , Δ ^{6, 9, 12} ), arachidonic (C ₂₀ , Δ ^{5, 8, 11, 14} ) and eicosotrienoev Yu (C ₂₀ , Δ ^{8, 11, 14} ) acids.

Non-limiting examples of non-cardiovascular inflammatory diseases that are mediated by VCAM-1 include rheumatoid and osteoarthritis, asthma, dermatitis, and complex sclerosis.

This method has a clear advantage in the treatment of cardiovascular disease by preventing the development of new lesions and regression of existing lesions and, if used correctly, in addition to current therapy intended only to inhibit progression of the disease, provides the possibility of medical "treatment" of atherosclerosis.

In an alternative embodiment, a method is described for suppressing the expression of an oxidation-reduction-sensitive gene or activating a gene that is suppressed by an oxidation-reduction-sensitive pathway that comprises administering an effective amount of a substance that prevents oxidation of the oxidizable signal, typically oxidation of a polyunsaturated fatty acid. Typical oxidation-reduction-sensitive genes that are involved in the manifestation of an immune response include, but are not limited to, those expressed cytokines involved in triggering an immune response (e.g., 1L-1β), chemoattractants that stimulate the migration of inflammatory cells to the point of damage ( e.g., MCP-1), growth factors (e.g., IL-6 and a thrombin receptor) and adhesion factors (e.g., VCAM-1 and E-selectin).

The picture of diseases mediated by VCAM-1 or a gene sensitive to oxidation-reduction also includes the inclusion of surrogate markers of the disease. In one embodiment, the level of oxidized polyunsaturated fatty acids or other suitable markers in the host tissue or blood is determined, for example, as the average of the host's “oxidative environment” score and host susceptibility to VCAM-1 or disease-mediating oxidation-reduction gene.

In another embodiment, the level of VCAM-1 circulating either on the surface of the cell or other suitable marker is calculated and the effect of the administration of a suitable antioxidant on this level.

Another analysis evaluates the sensitivity of the endothelial cells of the host vessel to polyunsaturated fatty acids or their oxidized derivatives. This can be accomplished, for example, by inducing the host PUFA or ox-PUFA and comparing the final concentration of circulating VCAM-1 either on the surface of the cell or other surrogate markers with a normal population.

In another embodiment, in an in vivo model of atherosclerosis or other cardiac or inflammatory diseases that are mediated by VCAM-1, an excess of PUFA or oxidized polyunsaturated fatty acids may be administered to the host animal to induce the disease. These animals can be used in clinical studies to further analyze these diseases.

In another embodiment of the invention, the compounds can be evaluated further by their ability to treat VCAM-1 mediated diseases, based on their ability to inhibit the oxidation of polyunsaturated fatty acids, or by the interaction of PUFA or ox-PUFA with a protein target.

This can be done by stimulating the host, for example a human or animal, such as a mouse, with a high level of PUFA or ox-PUFA, and then determining the therapeutic efficacy of the test compound based on its ability to reduce the concentration of circulating VCAM-1 or on the cell surface. Alternatively, in vitro selection can be used, which is based on the ability of the test compound to prevent the oxidation of PUFA or the interaction of PUFA or ox-PUFA with a protein target in the presence of an oxidizing compound such as copper or an enzyme such as peroxidase, lipoxygenase, cyclooxygenase or P450 cytochrome.

In another embodiment, the vascular endothelial cells can withstand the appropriate time with TNF-α or other VCAM-1 inducing material, then destroyed using, for example, sound or freeze-thaw methods. Cytosolic and membrane fractions are isolated. To determine their amount, radiolabeled PUFA is added. The ability of a liquid to convert PUFA to ox-PUFA in the presence or absence of a test compound is investigated. Instead of destroyed cellular systems, intact cells can be used.

Pyrrolidine dithiocarbamate (PDTC), when administered orally at a dose of 25-50 mg / kg / day, inhibits the formation of an atherogenic fat strip, monocyte macrophage arterial inflammation, expression of VCAM-1 by endothelium and significantly normalizes the function of endothelium-dependent relaxation in hypercholesterolemic rabbits, causing cholesterol above 1000 mg / dl. At the same doses, other natural therapeutic agents, such as probucol antioxidants and vitamin E, did not exert such an effect on the formation of lesions in this model.

In experimental atherosclerosis, endothelium-dependent arterial relaxation is restored with the introduction of PDTS. With a diet, the induced hypercholesterolemic model of rabbits by oral administration of PDTS (25-50 mg / kg / day) restores the vascular reactivity dependent on the endothelium. This was determined by studying the contraction of the aortic ring excised in control and test animals. In patients with atherosclerosis, this manifests itself in the form of normalization of peripheral vascular reactivity in response to hyperemia, which was determined by non-invasive studies using the Doppler effect in the stream. This is a standardized, widely applicable and easily conducted test that can be used to titrate the functional level of the drug when administered orally. PDTC acts with anti-ischemic therapy by quickly normalizing disorders in pathological endothelium-dependent arterial contraction of blood vessels in case of cardiovascular disease and atherosclerosis. This improvement in blood flow in the vessel manifests itself in the form of improved symptomatology and the implementation of functions limited by ischemia and represents a non-invasive assessment of the protection of the vessel. Other clinical indications for abnormalities in endothelium-dependent vascular contraction include impotence.

The molecular regulatory factor that inhibits the transcription of the VCAM-1 gene represents a new complex regulatory transcription factor consisting of the p65 and p50 subunits of the NF-kB / Rel family cross-linked to the c-fos and c-jun subunits of the AP-1 family. Using structural and functional studies, it was found that these AP-1 factors play a role in the regulation of the VCAM-1 promoter, which seems to be central to the therapeutic regulation of VCAM-1 gene expression. This is the first evidence of the functional role of this cross-linked transcription complex in the regulation of the endogenous gene.

Brief Description of the Drawings

Figure 2 is a graph of expression of VCAM-1 on the cell surface (O.P. 450 nm) as a function of the number of hours in human aortic endothelial cells exposed to TNF-α cytokine (open circles); linoleic acid (filled triangles); linoleyl hydroperoxide (13-HPODE, filled squares); in the absence of exposure to these substances (control, open squares).

Figure 3 is a graph of the expression of VCAM-1 on the cell surface (O.P. 450 nm) in human aortic endothelial cells when interacting with linoleic acid (filled triangles) and linoleyl hydroperoxide (13-HPODE, filled squares) as a function of fatty acid concentration (μM).

Figure 4 is a graphical expression diagram (O.P. 450 nm) on the cell surface of VCAM-1, ICAM-1 and E-selectin by human aortic endothelial cells under the influence of cytokine TNF-α, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid.

Figure 5 is a graphical diagram of the expression of VCAM-1 (O.P. 450 nm) on the surface of human aortic endothelial cells under the action of linoleic acid, 13-HPODE, arachidonic acid and arachidonic acid hydroperoxide (15-HPETE), c (blacked out) with or without (dotted line) antioxidant pyrrolidine dithiocarbamate.

6 illustrates autoradiograms showing the abrupt induction of VCAM-1 mRNA under the action of linoleic acid and 13-HRODE. NAES (human aortic endothelial cells) was maintained with or without oleic acid (7.5 μM), 13-HOPODE (7.5 μM) or TNF-α (100 units / ml). Total RNA was isolated and 20 μg was size fractionated by denaturing electrophoresis on a 1.0% agarose formaldehyde gel, transferred to nitrocellulose and hybridized with either ³² P-labeled human: A) VCAM-1, or B) β-actin-specific cDNA. After washing, the filters were irradiated with an X-ray film at -70 ° C with one intensifying screen for 24 hours. Line Identification:

1) control;

2) linoleic acid (sharp response, 8 hours of exposure);

3) linoleic acid (sharp response, 48 hours of exposure);

4) 13-НРОDE (sharp response, 8 hours of exposure); and

5) TNF-α (100 units / ml), 4 hours exposure).

7 is an illustration of an autoradiogram that shows that the induction of VCAM-1 mRNA by polyunsaturated fatty acids is independent of cell protein synthesis. NAEC was incubated with linoleic (7.5 μM) or arachidonic (7.5 μM) acid in the presence or absence of cycloheximide (10 μg / ml) for a 4-hour period, and then processed as described for FIG. 5.

Fig. 8 is an illustration of an autoradiogram that shows that linoleic acid induces transcriptional activation of the VCAM-1 promoter using an oxidation-reduction sensitive NF-kB-like factor. HAECs were transfected with either 30 μg p288 VCAMCAT, p85 VCAMCAT, or pSV ₂ CAT plasmid by calcium phosphate coprecipitation using a conventional technique. After a 24-hour recovery period, HAEC was pretreated, or not, with 50 μM PDTC, and after 30 minutes with linoleic acid (7.5 μM) or TNF-α (100 u / ml), it was directly applied to the plates. After 18 hours, cell extracts were prepared by rapid freezing-thawing in 0.25 M Tris, pH 8.0. To determine the activity of chloramphenicol acetyl transferase (CAT), as previously described [Ausubel, 1989], the protein of each cell extract was studied (Ac-acetylated; N-non-acetylated chloramphenicol).

Fig. 9 illustrates a strip of acrylamide gel showing that polyunsaturated fatty acids activate NF-kB-like DNA binding activities that are inhibited by the antioxidant PDTC. Confluent HAECs in a medium containing 4% FBS (as described in FIG. 1) were pretreated with or without PDTC (50 μM) for thirty minutes and then incubated for three hours with linoleic acid (7.5 μM), oleic acid (7.5 μM) or TNF-α (100 u / ml), respectively. Five micrograms of core extract was incubated with a ³² -strand ³² P-labeled wt VCAM, fractionated by size in a 4% pure polyacrylamide gel and kept with an autoradiographic film at -70 ° C for 18 hours. Two bands A and C, representing NF-kB-like binding activity, were isolated. Weak band B was observed in control (untreated) cells.

10A and 10B are graphical bar graphs of the comparative reactivity of thiobarbituric acid (O.P. 532 nm) and arachidonic acid and 15-HPETE compounds in the presence or absence of PDTC. Thiobarbituric acid reactivity study (TBARS) determines the oxidative ability of a material in a cell-free, environment-free environment.

11 is an illustration of an mRNA autoradiogram obtained as described below hybridized with ³² P-labeled specific VCAM-1 cDNA (Panel A), specific E-selectin specific cDNA (ELAM-1) (Panel B), or specific ICAM-1 cDNA (Panel C). After treatment with 50 μM sodium pyrrolidine dithicarbamate (PDTC) for 30 minutes, HUVE cells (human allantoic vein) were kept with IL-1b (10 units / ml) with the constant presence of 50 μM PDTC. Identically conducted parallel control in the absence of PDTC. At a certain time, total RNA was isolated and 20 μg of material was fractionated by denaturing electrophoresis in 1.0% agarose formaldehyde gel, transferred onto nitrocellulose, hybridized as described above, and visualized by autoradiography. Line 1 - 0 hours; Lines 2, 4, 6, 8 - only OL - 1 for 2, 4, 8 and 24 hours, respectively; Lines 3, 5, 7, 9 - 1L-1 with PDTS for 2, 4, 8 and 24 hours, respectively.

12 is an autoradiogram of mRNA obtained as described below hybridized with ³² P-labeled specific human VCAM-1 (Panel A), specific cDNA-E-selectin (ELAM-1) (Panel B) or specific ICAM-1 cDNA (Panel C). HUVE cells were pretreated with specific concentrations of PDTC, then incubated with 1L-1b in the presence of PDTC for four hours, and mRNA accumulation was determined for VCAM-1 Northern analysis by filter hybridization. Line 1 - control, line 2 - IL-1 (10 units / ml), line 3 - IL-1b + PDTC (0.05 μM), line 4 - IL-1 LB + PDTC (0.5 μM), line 5 - IL-1b + PDTC (5.0 μM), line 6 - IL-1b + PDTC (50.0 μM), line 7 - IL-1b + PDTC (100 μM).

13 is an illustration of an mRNA autoradiogram obtained as described below hybridized with ³² P-labeled specific human VCAM-1 cDNA (Panel A), specific E-selectin specific cDNA (ELAM-1) (Panel B), or specific cDNA (ICAM -1) (Panel C). HUVE cells were pretreated as described in FIG. 10 with 50 μM PDTC, incubated for four hours with the agents noted below, and examined for accumulation of VCAM-1 (Panel A) and ICAM-1 (Panel B) mRNA. Line 1 - TNF-α (100 units / ml), line 2 - TNF-α + PDTC, line 3 - lipopolysaccharide (LPS) (100 ng / ml), line 4 - LPS + PDTC, line 5 - poly (I: C) (100 mg / ml), line 6 - poly (I: C) + PDTC.

Fig. 14 is a graph of the expression of VCAM-1 and ICAM-1 on the cell surface in the presence (dark columns) or absence (white columns) of PDTC and in the presence of many types of inducing stimuli. Confluent HUVEC cells were pretreated or not treated (CTL only) for 30 minutes with 50 μM PDTC, then the indicated time was maintained with the indicated agent in the presence or absence (CTL only) of PDTC. Cell surface expression was determined by primary binding to specific monoclonal antibodies to VCAM-1 (4B9) and ICAM-1 (84H10) and subsequent secondary binding to horseradish peroxidase bound to the goat anti-mouse (IgG). Quantitative calculation was carried out by determining the calorimetric conversion of TMB at 450 nm. Fig. 14 shows that multiple regulatory signals induce VCAM-1, but not ICAM-1, via the common, dithiocarbamate-sensitive pathway and endothelial cells of a human vessel.

Fig. 15 is a graph of the relative expression of VCAM-1 on the cell surface (O.P. 595 nm) of human endothelial vein of the allantoic vein activated by TNF-α in relation to the concentrations of various antioxidants. PDTS is sodium N-pyrrolidine dithiocarbamate; DETC is sodium N, N-diethyl-N-carbodithiolate, also referred to as sodium diethyl dithiocarbamate; NAC is N-acetylcysteine and DF is desferroximin).

Fig. 16 is a graph of the relative expression of VCAM-1 on the cell surface (O.P. 595 nm) in endothelial cells of a human allantoic vein activated with TNF-α in the presence of a specific amount of antioxidant. (PDTC represents sodium N-pyrrolidine dithiocarbamate; DIDTC represents sodium N, N-diethyl-N-carbodithioate; SarDTC represents sodium N-methyl-N-carboxymethyl-N-carbodithioate; IDADC represents trisodium N, N-di (carboxymethyl) -N β-carbodithioate; MGDTC represents sodium N-methyl-D-glucamine-N-carbodithioate; MeOBGDTS represents sodium N- (4-methoxybenzyl) -D-glucamine-N-carbodithioate; DEDTC represents sodium N, N-diethyl-N-carbothioate; DiPDTC is sodium N, N-diisopropyl-N-carbodithioate; NAC is N-acetylcysteine).

Fig.17 is a graph of the percentage of binding of Mo1t-4 cells to HUVE cells stimulated with TNF-α (100 units / ml) or not stimulated, for six hours in the presence or absence of PDTC.

Fig. 18 shows the chemical structures of the following active dithiocarbamates: sodium pyrrolidine-N-dithiocarbamate, sodium N-methyl-N-carboxymethyl-N-carbodithioate, trisodium N, N-di (carboxymethyl) -N-carbodithioate, sodium N-methyl-D -glucamine-N-carbodithioate, sodium N, N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate) and sodium N, N-diisopropyl-N-carbodithioate.

Fig. 19 is a bar graph diagram of the effect of PDTC on the formation of fluorescent addition products of BSA and 13-HOPODE, measured in units of fluorescence, with respect to the concentration of PDTC. One micromole of 13-HRODE was incubated with 200 micrograms of BSA in the presence of PDTC for six days. Fluorescence was measured at 430-460 nm, excitation at 330-360 nm.

FIG. 20 is a graphical representation of the effect of PDTC concentration on the formation of fluorescent BSA and ox-PuFA addition products as a function of wavelength (nm) and PDTC concentration. As the concentration of PDTC increases, the amount of fluorescent adducts decreases.

Fig.21 is a graphical representation of the effect of PDTC on LDL oxidation under the influence of horseradish peroxidase (HRP), measured by an increase in O.P. (234 nm), relative to time (minutes) at various concentrations of PDTC. It was found that after the incubation period, PDTC inhibits LDL oxidation under the influence of HRP in a concentration-dependent manner.

Fig. 22 is a diagram of the effect of PDTC on the formation of ox-PUFA induced by a cytokine in human aortic endothelial cells. As noted, both TNF-α and IL-1b cause oxidation of linoleic acid to oxidized linoleic acid. Oxidation is substantially prevented by PDTC.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

The term polyunsaturated fatty acid (also referred to as “PUFA”) as used herein refers to a fatty acid (usually C ₈ -C ₂₄ ) that has at least two alkenyl bonds and includes, but is not limited to, linoleic (C ₁₈ Δ ^{9 , 12} ), linolinic (C ₁₈ Δ ^{6, 9, 12} ), arachidonic (C ₂₀ Δ ^{5, 8, 11, 14} ) and ecosatrienic (C ₂₀ Δ ^{8, 11, 14} ) acids.

The term oxidized polyunsaturated fatty acid refers to an unsaturated fatty acid in which at least one of the alkenyl bonds is converted to hydroperoxide. Non-limiting examples are

The term alkyl, as used here, except in special cases, refers to a saturated straight, branched or cyclic (in the case of C ₅ or more) hydrocarbon C _1-10 (or lower alkyl, i.e. C ₁ -C ₅ ), which, in particular includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl. An alkyl group may not necessarily be substituted on any of the carbons with one or more radicals selected from the group consisting of hydroxyl, amino or mono- or disubstituted amino, wherein the substituent group is, independent, alkyl, aryl, alkaryl or aralkyl; aryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate or phosphonate, either unprotected or protected, as necessary, as is known to a person skilled in the art, for example, as described by Greene et at .; "Protective Groups in Organic Sunthesis", John Wiley and Sons. Second edition, 1991.

The term alkenyl, as used here and unless otherwise indicated, refers to a straight, branched or cyclic C ₂ -C ₁₀ hydrocarbon with at least one double bond.

The term alkynyl, as used here and unless otherwise indicated, refers to a C ₂ -C ₁₀ straight or branched hydrocarbon with at least one triple bond.

The term aralkyl refers to an aryl group with at least one alkyl substituent.

The term alkaryl refers to an alkyl group that has at least one aryl substituent.

The term halogen (alkyl, alkenyl or alkynyl) refers to an alkyl, alkenyl or alkynyl group in which at least one of the hydrogens in the group is substituted by a halogen atom.

The term aryl, as used here, and unless otherwise indicated, refers to phenyl, biphenyl or naphthyl, preferably phenyl. The aryl group may optionally be substituted with one or more radicals selected from the group consisting of alkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate or phosphonate, CO ₂ H or its pharmaceutically acceptable salt, CO ₂ (alkyl, aryl, alkaryl or aralkyl), or glucamine, either unprotected or protected, as necessary, as is known to a person skilled in the art, for example, as described by Jreene et al. "Protective Jroups in Organic Synthesis", John Wiley and Sons. Second edition. 1991.

The term alkoxy, as used herein and unless otherwise indicated, refers to a radical of the structure —O-alkyl.

The term acyl, as used here, refers to a group of the formula C (O) R ′, where R ′ is an alkyl, aryl, alkaryl or aralkyl group.

The term heteroaryl or heteroaromatic, as used here, refers to an aromatic radical that includes at least one sulfur, oxygen or nitrogen atom in the aromatic ring. Non-limiting examples are phenazine, phenothiazine, furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, indolyl, benzolylindol, indolyl, indolyl, benzolylindolyl, indolylphenolindinolind, , thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isoxoxazolyl, pyrrolyl, pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl, 5-azolithiyl, 5-azolithiyl, imidazolopiridinil, pyrrolopyrimidine, pyrazolopyrimidine, adenine, N ⁶ -alkilpuriny, N -benzilpurin ^6, N ⁶ -galopurin, N -vinilpurin ^6, N ⁶ -atsetilenovy purine, N ⁶ -atsilpurin, N -gidroksialkilpurin ^6, N ⁶ -tioalkilpurin, thymine , cytosine, 6-azapyrimidines, 2-mercaptopyrimidine, uracil, N ⁵ -alkilpirimidiny, -benzilpirimidiny N ^5, N ⁵ -galogenpirimidiny, -vinilpirimidin N ^5, N ⁵ -atsetilenovy pyrimidine, N -atsilpirimidin ^5, N ⁵ and N -gidroksialkilpurin ^6- thioalkylpurine and isoxazolyl. The heteroaromatic group may not necessarily be substituted as described above for aryl. The heteroaromatic group may be partially or fully hydrogenated, as desired. As a non-limiting example, dihydropyridine can be used instead of pyridine. Functional oxygen and nitrogen groups on the heterocyclic base can be protected, if necessary, during the reaction. Suitable protecting groups well known to those skilled in the art are 3-methylsilyl, dimethylhexylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl, tritylmethyl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyl and p-toluylsulfonyl.

The term hydroxyalkyl, as used here, refers to a C ₁ -C ₆ alkyl group in which at least one of the hydrogens attached to any of the carbon atoms is substituted with a hydroxy group.

The term thiol antioxidant refers to a sulfur-containing compound that inhibits oxidation.

The term pharmaceutically acceptable derivative refers to a derivative of an active compound which, when administered to a recipient, is capable of creating, directly or indirectly, a basic compound or which exhibits its activity.

The term "pharmaceutically acceptable cation" refers to an organic or inorganic radical that carries a positive charge and which can be introduced in association with a pharmaceutical agent, for example, as a counter cation in a salt. Pharmaceutically acceptable cations are known to those skilled in the art and include, but are not limited to, sodium, potassium, and quaternary amine.

The term “physiologically cleavable leaving group” refers to a radical that can be cleaved in vivo from the molecule to which it is attached, and includes, but is not limited to, an organic or inorganic anion, a pharmaceutically acceptable cation, acyl (including, but not limited to , (alkyl) C (O), including acetyl, propionyl and butyryl), alkyl, phosphate, sulfate and sulfonate.

The term "enantiomerically enriched composition or compound" refers to a composition or compound that comprises at least 95%, and preferably at least 97, 98, 99, or 100% by weight of an individual enantiomer of the compound.

The term amino acid includes synthetic or naturally occurring amino acids, including, but not limited to, for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, traptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartyl , glutaoyl, lysine, argenin and histidine.

A “binder radical,” as used herein, is any divalent group that binds two chemical moieties, including, but not limited to, alkyl, alkenyl, alkynyl, aryl, polyalkyleneoxy (for example, - [(CH ₂ ) _n O-] _n -), -C _1-6 alkoxy-C _1-10 alkyl-, -C _1-6 alkylthio-C _1-10 alkyl-, NR ³ - and - (СНОН) _n СН ₂ ОН, where n independently, is 0 , 1, 2, 3, 4, 5, or 6.

II. Identification of oxidized and unoxidized polyunsaturated fatty acids as direct mediators of VCAM-1 expression

To determine whether PUFA or oxidized PUFA act as a direct immunomodulator of gene expression in endothelial cells, early passages of human aortic endothelial cells (HAEC) were cultured for eight hours in serum medium and kept with saturated (stearic), monounsaturated (oleic) and polyunsaturated (linoleic and arachidonic) fatty acids; as well as hydroperoxides of linoleic (13-HPODE) or arachidonic (15-HPETE) acids. NAEC was also alternatively maintained with a cytokine - tumor necrosis factor α.

NAEC was incubated with linoleic acid or 13-HPODE for various times, up to 48 hours, and then the expression of VCAM-1 on the cell surface was determined by ELISA. The results were compared with HAEC aged with the TNF-α cytokine (100 units / ml) over the same period. Expression of VCAM-1 in NAES, maintained with either linoleic acid or 13-HPODE, was briefly induced. Peaks of expression were observed at approximately 8–9 hours with significant expression, at 24 hours, and then decreased at 48 hours. The kinetics of induction of VCAM-1 under the action of both linoleic acid and 13-HRODE reflects that for TNF-α, and thus the mechanism by which polyunsaturated fatty acids induce VCAM-1, therefore, appears similar to that for TNF-α.

A dose-dependent study of VCAM-1 gene expression under the influence of linoleic acid and 13-HRODE was also conducted. It was found that 7.5 μM is the lowest peak dose at which linoleic acid and 13-HOPDE significantly induces the expression of the VCAM-1 gene.

A study was conducted to determine whether short-term incubation of endothelial cells with polyunsaturated fatty acids induces the expression of both ICAM-1 and E-selectin. It was determined that polyunsaturated fatty acids linoleic and arachidonic acids induce gene expression on the cell surface to 59% of THF-induced gene expression of VCAM-1. As it turned out, neither ICAM-1 nor E-selectin was induced by these fatty acids. Conversely, saturated fatty acid — stearic acid and monounsaturated fatty acid — oleic acid do not induce the expression of VCAM-1, ICAM-1, or E-selectin. VCAM-1 gene expression was also observed upon incubation of HAEC with oxidized metabolites of linoleic acid (13-HPODE) and arachidonic acid (15-HPETE).

To investigate whether oxidative stress in endothelial cells caused by polyunsaturated fatty acids and their oxidized metabolites actually induces VCAM-1 via an oxidation-reduction-sensitive mechanism, HAEC was pretreated with antioxidant pyrrolidine dithiocarbamate (PDTC, 50 μM) for 30 minutes, and then the cells were independently incubated with linoleic acid, arachidonic acid, 13-HPODE and 15-HPETE (all 7.5 μM) for 8 hours. It was determined that PDTC inhibits the expression of the VCAM-1 gene induced by polyunsaturated fatty acids and their oxidized derivatives. This shows that induction is mediated by an oxidized signal molecule, and that induction is prevented when the oxidation of the molecule is blocked (i.e., no oxidation) is reversed (i.e., the signal molecule is reduced), or its interaction with the protein target is prevented, possibly through a redox complex.

To determine whether the selective induction of VCAM-1 under the influence of PUFAS and its oxidized metabolites is observed at the mRNA level, HAEC was incubated with linoleic acid or 13-HPODE. The accumulation of VCAM-1 mRNA induced by linoleic acid and 13-HRODE was close to the levels that TNF-α induced. In contrast, the induction of VCAM-1 and E-selectin gene expression at the mRNA and HAEC levels incubated with linoleic acid or 13-HOPDE was not observed. The data obtained correspond to the data obtained in the study of the cell surface. The results show that pre-translational regulatory mechanisms mediate the induction of VCAM-1 gene expression under the action of polyunsaturated fatty acids and their oxidized metabolites.

It was also necessary to determine whether polyunsaturated fatty acids actually act as a primary signal or act through a regulatory protein involved in the induction of VCAM-1 gene expression by IL-4. To study whether newly synthesized proteins, such as IL-4, are involved in the synthesis and expression of VCAM-1 induced by PUFAS, such as linoleic acid, HAEC was incubated with 13-HPODE (7.5 μM) and kept with a protein synthesis inhibitor cycloheximide. Inhibition of VCAM-1 mRNA accumulation by cycloheximide in NAES incubated with 13-HRODE was not observed. The amount of IL-4 produced in NAES incubated with linoleic acid or arachidonic acid and their oxidized metabolites was also determined by ELISA. An increase in the yield of IL-4 from NAES incubated with these PUFAS and their oxidized metabolites was not observed.

It was previously discovered, by studying fission and a heterologous promoter, that cytokines and non-cytokines activate the expression of the VCAM-1 gene in endothelial cells, at least partially at the transcription level, by means of two NF-kB-like DNA binding elements. PDTC has also been shown to inhibit the expression of the VCAM-1 gene through an oxidation-reduction sensitive NF-kB-like factor. To determine whether polyunsaturated fatty acids actually induce transcriptional activation of the human VCAM-1 promoter by the same mechanism, HAEC was temporarily transfected with the chimeric reporter p288 V CAM-CAT gene containing coordinates from -288 to +22 human VCAM-1 promoter. The addition of linoleic acid (7.5 μM) induced the activity of the VCAM-1 promoter, which was more than double that in the control, and approximately 60% of the maximum signal induced by TNF-α. Similar results were obtained with the minimal cytokine-inducible promoter of the VCAM-1 gene (p85 VCAM-CAT) containing -77 and -63 base pairs of NF-kB-like sites. Neither linoleic acid nor THF-α had any effect on activity when using the organically expressed pSV ₂ CAT construct. PDTS inhibits the transcriptional activation of both structural linoleic acid induced VCAM-1 promoters. These data indicate that, similarly to THF-α, polyunsaturated fatty acids, such as linoleic acid, induce transcriptional activation of VCAM-1 via NF-kB like an oxidation-reduction-sensitive mechanism.

To determine whether polyunsaturated fatty acids and their oxidized metabolites actually regulate the activity of the VCAM-1 promoter by means of NF-kB-like transcriptional regulatory factor, the activity of DNA binding to a double-stranded nucleotide containing NF-kB elements of the similar VCAM-1 promoter was studied in HAEC nuclear extracts located at positions -77 and -63. As shown in FIG. 8, two bands A and C, representing NF-kB similar activity, were induced in response to three hours of exposure with linoleic acid (7.5 μM). A similar detection was observed when incubated with the cytokine TNF-α (100 units / ml). A weak band was observed in control (untreated) cells. No NF-kB similar binding to oleic acid, a monounsaturated fatty acid, was observed. Cell pretreatment for thirty minutes with PDTC inhibited the activity of DNA binding to A and C, after activation with linoleic acid. Detection data are close to previously obtained data showing that PDTC blocks the activation of VCAM-1 gene expression in HUVEC by inhibiting the activation of these NF-kB-like DNA-binding proteins.

Example 1

The effect of oxidized and non-oxidized polyunsaturated fatty acids on the kinetics of activation of VCAM-1 gene expression

Human aortic endothelial cells (HAEC) were applied to a 96-well microtiter plateau and incubated with linoleic acid (7.5 μM), 13-HOPODE (7.5 μM) or TNF-α (100 u / ml) for five different time intervals, up to 48 hours. NAECs obtained from Clonetics (Boston, MA) were cultured in medium 199 supplemented with 20% fetal bovine serum (FBS), 16 units / ml heparin, 10 units / ml epidermal growth factor, 50 μg / ml supplement for growth of endothelial cells, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin. One day before the experiment, the cells were placed in a medium containing 4% FBS. Confluent HAECs were incubated for up to 48 hours with TNF-α (100 u / ml) or stearic, oleic, linoleic, linolenic or arachidonic acids (7.5 μM). Similar studies were carried out with various doses of linoleic acid or 13-HPODE over an 8-hour period (1-60 μM) (Fig. 3). The calculation was carried out by determining the calorimetric transition color TMB at 450 nm. Studies were performed three times (n = 4, for each experimental value) * - the value differs from the control (p <0.05).

As shown in FIG. 2, both linoleic acid and 13-HRODE induced the expression of VCAM-1. Ten hours after aging, the amount of linoleic acid and 13-HOPDE induced surface-cell VCAM-1 was more than half that TNF-α cytokine induced.

As shown in figure 3, the induction of VCAM-1 under the action of linoleic acid and 13-HOPDE is sensitive to concentration. At a concentration of these compounds between 2 and 10 μM, there is a sharp increase in the amount of VCAM-1 induced on the cell surface, which then remains approximately constant, up to a concentration of at least 100 μM. It should be noted that the PUFA concentration noted in FIG. 3 is complementary to that which is endogenously detected in HAEC.

Example 2

Polyunsaturated fatty acids induce expression of the VCAM-1 gene, but not ICAM-1 or E-selectin

Expression of VCAM-1, ICAM-1, and E-selectin on the NAEC cell surface was determined by ELISA. NAECs obtained from Clonetics (California) were cultured in 199 medium supplemented with 20% fetal bovine serum (FBS), 16 units / ml heparin, 10 units / ml epidermal growth factor, 50 μg / ml endothelial cell growth supplement, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin. One day before the experiment, the cells were placed in a medium containing 4% FBS. Confluent HAECs were incubated for 8 hours or not with TNF-α (100 u / ml) or stearic, oleic, linoleic, linolenic or arachidonic acids (7.5 μM). The expression of A) VCAM-1, B) ICAM-1 and C) E-selectin on the cell surface was determined by primary binding to mouse antibodies specific for VCAM-1, specific for ICAM-1 and specific for E-selectin, then secondary binding to goat anti-mouse (IgG) bound to horseradish peroxidase labeled. The calculation was carried out by determining the calorimetric transition TMB at 450 nm. The study was carried out three times (n = 4, for each experimental value) * - the value differs from the control (p <0.05).

As shown in FIG. 4, linoleic acid, linolenic acid and arachidonic acid significantly induced the expression of VCAM-1, but not the expression of ICAM-1 and E-selectin on the cell surface. Neither stearic acid nor oleic acid induced the expression of VCAM-1, ICAM-1 and E-selectin. TNF-α strongly induced the expression of all three molecules on the cell surface.

Example 3

PDTC antioxidant inhibits the induction of VCAM-1 by polyunsaturated fatty acids and their oxidized metabolites

Confluent HAECs were pretreated in the presence or absence of PDTC (sodium pyrrolidine dithiocarbamate, 50 μM) for thirty minutes. Cells were then incubated for eight hours with TNF-α (100 u / ml), linoleic or arachidonic acid (7.5 μM) or 13-HRODE (7.5 μM) or 15-HPETE (7.5 μM) fatty acid hydroperoxides ) The expression of VCAM at NAES on the cell surface was determined by ELISA, as described in example 1. The studies were performed in triplicates (n = 4, for each experimental value), * - the value differs from the control (p <0.05).

As noted in FIG. 5, PDTC inhibits the induction of VCAM-1 by linoleic acid, 13-HPODE, arachidonic acid and 15-HPETE.

Example 4

Rapid induction of VCAM-1 mRNA by linoleic acid and 13-HPODE

NAEC was incubated with linoleic acid (7.5 μM) or 13-HRODE (7.5 μM). Total RNA was isolated and 20 μg was size fractionated by denaturing electrophoresis in a 1.0% agarose formaldehyde gel, transferred to nitrocellulose and hybridized with ³² P-labeled human DNA specific for either A) VCAM-1 or specific for B) β-actin , and visualized by autoradiography. After washing, the filters were kept with an x-ray film at -70 ° C with one intensifying screen for 24 hours. Line Identification:

1) control;

2) linoleic acid (acute, 8 hours exposure);

3) linoleic acid (48 hours exposure);

4) 13-НРОДЕ (acute, 8 hours exposure); and

5) TNF-α (100 u / ml, 4 hours exposure).

As shown in FIG. 6, both linoleic acid and 13-HPODE induce mRNA production for VCAM-1 after 8 hours. After 48 hours, linoleic acid no longer induced the induction of VCAM-1 mRNA.

Example 5

PUFA induction of VCAM-1 mRNA is independent of cell protein synthesis

NAEC was incubated with either linoleic or arachidonic acid (7.5 μM) in the presence or absence of cycloheximide (10 μg / ml) for a 4-hour period. Total RNA was isolated and 20 μg was size fractionated by denaturing electrophoresis on 1.08 agarose formaldehyde gel, transferred onto nitrocellulose and hybridized with specific A) ³² P-labeled VCAM-1 or B) β-actin cDNA and visualized by autoradiography. After washing, the filters were kept with an x-ray film at -70 ° C with one intensifying screen for 24 hours.

As noted in FIG. 7, the induction of VCAM-1 by linoleic and arachidonic acids is independent of cell protein synthesis.

Example 6

Linoleic acid induces transcriptional activation of the VCAM-1 promoter via NF-kB-like oxidation-reduction sensitive factor

NAES was applied to 100 mm growth plates in the ratio necessary to obtain 60% confluency. HAECs were transfected with either 30 μg p288 VCAMCAT, p85 VCAMCAT, or with the plasmid pSV ₂ CAT by the method of coprecipitation with calcium phosphate, using the usual technique. After a 24-hour recovery period, HAEC was pretreated with 50 μM PDTS and after 30 minutes with linoleic acid (7.5 μM) or TNF-α (100 u / ml), directly applied to the plates. After 18 hours, cell extracts were prepared by rapid freezing-thawing in 0.25 M Tris, pH 8.0. To determine the activity of chloramphenicol acetyl transferase (CAT), as previously described [Ausubel, 1989], a protein was determined for each cell extract (Ac, acetylated; N, non-acetylated chloramphenicol).

Fig. 8 illustrates the results of this experiment. Linoleic acid induces transcriptional activation of the VCAM-1 promoter via NF-kB, a similar oxidation-reduction sensitive factor. These results are close to those obtained by activation of the VCAM-1 promoter under the action of cytokines, such as TNF-α. This confirms that PUFAS act through oxidized intermediates that also mediate cytokine activation of VCAM-1.

Example 7

Polyunsaturated fatty acids activate NF-kB-like, DNA-binding activity, which is inhibited by PDTC antioxidant

Confluent HAECs in a medium containing 4% FBS (as described in Example 1) were pretreated with PDTC (50 μM) for 30 minutes and then incubated for 3 hours with linoleic acid or oleic acid (7.5 μM), or TNF-α (100 units / ml). Five micrograms of core extract was incubated with a ³² -strand ³² P-labeled wtVCAM, fractionated by size on a 4% acrylamide gel without additives and kept with an autoradiographic film at -70 ° C for 18 hours. Two bands A and B were isolated, representing NF-kB similar binding activity. A weak band B in the control (untreated) cells was observed).

Fig. 9 illustrates that linoleic acid induces NF-kB binding activity with a VCAM-1 promoter in an oxidation-reduction sensitive manner. This, similar to the cytokine TNF-α, confirms the proximity of the mechanism of action. TNF-α induces VCAM-1, apparently through a mechanism that is mediated by the action of ox-PUFA.

Example 8

Oxidation under cell-free, medium-free conditions, under the action of unoxidized and oxidized (15-HPPE) arachidonic acid

10A and 10B are graphical diagrams of the reactivity of the compounds (O.P. 532 nm) of arachidonic acid and 15-HPETE, with respect to thiobarbituric acid, in the presence or absence of PDTC. The study of reactivity of thiobarbituric acid (TBARS) allows you to determine the oxidative ability of a material in a cell-free environment, in a free environment. As can be seen from the figures, both arachidonic acid and 15-HPETE show marked TBARS activity, which is inhibited by PDTC.

III. A method for the treatment of disorders mediated by VCAM-1

The discovery that polyunsaturated fatty acids and their oxidized metabolites are selective, oxidation-reduction-sensitive immunomodulators, provides a basis for the treatment of diseases that are mediated by VCAM-1 or oxidation-reduction-sensitive genes.

Describes a method for the treatment of atherosclerosis, postangioplastic restenosis, diseases of the coronary arteries, angina pectoris and other cardiovascular diseases, as well as non-cardiovascular inflammatory diseases that are mediated by VCAM-1, which includes eliminating, reducing the concentration or preventing the formation of oxidized polyunsaturated fatty acids, including, but not limited to, oxidized linoleic, linolenic, and arachidonic acids. In an alternative embodiment, a method for treating these diseases is described, which comprises preventing the interaction of PUFA or ox-PUFA with proteins or peptides that mediate the expression of VCAM-1.

Inhibition of VCAM-1 expression can be accomplished in a variety of ways, including by administering an antioxidant that prevents oxidation of the polyunsaturated fatty acid by modifying in vivo metabolism of PUFAS to ox-PUFAS, as described in detail below.

1. The introduction of antioxidants

Any compound that reduces ox-PUFA or which inhibits the oxidation of PUFA and which is relatively non-toxic and bioavailable or that can be modified to make it bioavailable can be used in this therapy. An ordinary laboratory technician, using conventional techniques, can easily determine if a compound is capable of reducing ox-PUFA or inhibiting PUFA oxidation.

Dithiocarboxylant Antioxidants

Dithiocarboxylates have been found to be useful in the treatment of atherosclerosis and other cardiovascular and inflammatory diseases. Dithiocarboxylates, including dithiocarbamates, can be used to block the ability of cells, including endothelial cells, to express VCAM-1 or to suppress expression of a redox-sensitive gene or to activate a gene that is suppressed by a redox-sensitive trace.

At least one of the compounds, perrolidindithiocarbamate (PDTK), inhibits the expression of the VCAM-1 gene at a concentration of less than 1.0 micromolar. These compounds also exhibit preferred toxicity to proliferative or pathologically dividing smooth muscle vessel cells. Another dithiocarbamate, N-methyl-N-carboxymethyl-N-carbotithioate sodium, also inhibits the expression of VCAM-1, without a noticeable effect on ICAM-1, but does not exhibit preferred toxicity to pathologically dividing smooth muscle blood vessels.

It was found that pyrrolidine dithiocarbamate did not noticeably block the expression of ELAM-1 or ICAM-1, and therefore treatment with this compound did not adversely affect the anti-inflammatory response mediated by ELAM-1 or ICAM-1. Thus, the generalization of immunosuppression is avoided. This avoids systemic complications caused by generalized inhibition of adhesive molecules in many other cell types in which expression is known. Other pharmacologically acceptable salts of PDTC are also effective in the treatment of cardiovascular and inflammatory diseases.

Dithiocarbamates are transition metal chelators used in the clinic for heavy metal intoxication. Baselt, RC, FWJ Sunderman, et al. (1997), "Comparisons of antidotal efficacy of sodium diethyldithiocarbamat, D-penicillamine and triethylenetetramine upon acute toxicity of nickel carbonyl in rats." Res Commun Chem Pathol Pharmacol 18 (4): 677-88; Henne, T. and K. Kaaber (1978), "Treatment of pompholyx due to nickel allergy with chelating agents." Contact Dermatitis 4 (5): 289-90; Sunderman, FV (1978), "Clinical response to therepeutic agents in poisoning from mercury vapor". Ann clin lab sci 8 (4): 259-69; Sunderman, FV (1979), "Efficacy of sodium diethyldithiocarbamate (dithiocarb) in acute nickel carbonil poisoning." Ann clin lab sci 9 (1): 1-10; Gale, GR, AB Smith, et al. (1981), "Diethilditiocarbamat in treatment of acute cadmium poisoning." Ann clin lab sci 11 (6): 476-83; Jons, MM and MG Cheriman (1990), "The search for chelate antagonists for chronic cadmium intoxication." Toxicology 62 (1): 1-25; Jones, SG, MA Basinger, et al. (1982), "A comparison of diethyldithiocarbamate and EDTA as antidotes for acure cadmium intoxication." Res Commun Chem Pathol Pharmacol 38 (2): 271-8; Pades, A., JS Casas, et al. (1985), "Diethyldithiocarbamates in heavy metal poisoning: complexis of N, N, -di (1-hydroxiethyl) dithiocarbamate with Zn (II), Cd (II), Hg (II), CH ₃ Hg (II) and C ₆ H ₅ Hg (II), J. Inorg Biochem 25 (I): 35-42; Tandon SK, NS Hashmi, et al. (1990), "The lead-chelatinq effects of substituted dithiocarbamates." Biomed Environ Sci 3 (3 ): 299-305.

Dithiocarbamates have also been used as adjuvants in cis-platinum chemotherapy to prevent renal toxicity. Hacker, M.R., W.B. Ershler, et al. (1982). "Effect of disulfiram (tetraethylthiuram disulfide) and diethyldithiocarbamat on the bladder toxicity and antitumor activity of cyclophosphamide in mice." Cancer Res 42 (11): 4490-4. Bodenner, 1986 # 733; Saran, M. and Bors, W. (1990). "Radical reaction in vivo- -an overview." Radiat. Environ. Biophys. 29 (4): 249-62.

A recently used dithiocarbamate in the treatment of alcoholism is disulfiram, a diethyldithiocarbamate dimer. Disulfiram inhibits liver aldehyde dehydrogenase. Inoue, K. and Fukunada, et al., (1982). "Effect of diculfiram and its reduced metabolite, diethyldithiocarbamate on aldehyde dehydroqenase of human erythrocytes." Life Sci 30 (5): 419-24.

Dithiocarbamates have been reported to inhibit HIV virus replication and also accelerate the maturation of specific T-cell subpopulations. This has been shown in clinical trials of diethyldithiocarbamate in AIDS patients. Reisinger, E., et al., (1990). "Inhibition of HIV progression by dithiocarb." Lancet 335: 679.

Dithiocarboxylates are compounds of structure A-SC (S) -B, which are members of a general class of compounds known as thiol antioxidants, and which are alternatively referred to as carbodithiols or carbodithiolates. It seems that the radical -SC (S) - is essential for the manifestation of therapeutic activity and that A and B can be any group that does not adversely affect the effectiveness or toxicity of the compound.

In an alternative embodiment, one or both of the dithiocarbamate sulfur atoms is replaced by a selenium atom. Substitution of sulfur for selenium can reduce the toxicity of the molecule in some cases and, thus, may be more tolerant to the patient.

A and B may be selected by one of ordinary skill in the art to impart desired properties to the compound, including size, charge, toxicity, and degree of stability (including stability in an acidic environment, such as in the stomach, or in a basic medium, such as in the intestinal tract ) The choice of A and B will also be of great importance for the distribution in the tissues and pharmacokinetics of the compound. Typically, for the treatment of cardiovascular disease, it is desirable that the compound accumulate or localize in the intima of the artery containing vascular endothelial cells. The compound is preferably removed by excretion by the kidneys.

An advantage of the pharmaceutical use of dithiocarboxylate is that it does not appear to undergo enzymatic degradation in vivo and therefore may exhibit a prolonged half-life in vivo.

In a preferred embodiment, A is hydrogen or a pharmaceutically acceptable cation, including, but not limited to, sodium, potassium, calcium, magnesium, aluminum, zinc, bismuth, barium, copper, cobalt, nickel or cadmium; a salt forming an organic acid, usually a carboxylic acid, including, but not limited to, acetic acid, oxalic acid, grape acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, on acid, naphthalene disulfonic acid or polygalacturonic acid; or a cation formed by ammonium or another nitrogen base, including, but not limited to, a nitrogen containing heterocycle or a radical of the formula NR ⁴ R ⁴ R ⁵ R ⁶ R ⁷ , where R ⁴ , R ⁵ , R ⁶ and R ⁷ are independently hydrogen C _1-6 linear branched or (in the case of C _4-6 ) cyclic alkyl, hydroxy (C _1-6 ) alkyl (where one or more hydroxyl groups are on any carbon atom) or aryl, N, N-dibenzylethylenediamine, D -glucosamine, choline, tetraethylammonium or ethylenediamine.

In another embodiment, A may be a physiologically cleavable leaving group that may be cleaved in vivo from the molecule to which it is attached and includes, but is not limited to, acyl (including acetyl, propionyl and butyryl), alkyl, phosphate, sulfate or sulfonate .

In one embodiment, B is alkyl, alkenyl, alkynyl, alkaryl, aralkyl, haloalkyl, haloalkenyl, haloalkynyl, aryl, alkaryl, hydrogen, C _1-6 alkoxy-C _1-11 alkyl, C _1-6 alkylthio-C _1-10 alkyl , NR ² R ³ , - (CHOH) _n CH ₂ OH, where n is 0, 1, 2, 3, 4, 5 or 6, - (CH ₂ ) _n CO ₂ R ¹ , including alkylacetyl, alkpropionyl and alkylbutyryl, or hydroxy (C _1-6 ) alkyl- (where one or more hydroxyl groups are located on any of the carbon atoms).

In another embodiment, B is NR ² R ³ , where R ² and R ³ are independently alkyl; - (CHOH) _n CH ₂ OH, where n is 0, 1, 2, 3, 4, 5, or 6; - (CH ₂ ) _n CO ₂ R ¹ , - (CH ₂ ) _n CO ₂ R ⁴ , hydroxy (C _1-6 ) alkyl-; alkenyl, including, but not limited to, vinyl, allyl and CH ₃ CH = CH — CH ₂ CH ₂ ; alkyl (CO ₂ H), alkenyl (CO ₂ H), alkynyl (CO ₂ H) or aryl, where the aryl group may be substituted as described above, in particular, for example, substituted by NO ₂ -, CH ₃ , tert-butyl , CO ₂ H, halogen or p-OH group; or R ² and R ³ can together form a bridge, such as - (CH ₂ ) m-, where m is 3, 4, 5 or 6 and where R ⁴ is alkyl, aryl, alkaryl or aralkyl, including acetyl, propionyl and butyryl .

In yet another embodiment of the invention, B may be a heterocyclic or alkyl heterocyclic group. The heterocycle may optionally be partially or fully hydrogenated. Non-limiting examples are those listed above, including phenazine, phenothiazine, pyridine and dithiopyridine.

In yet another embodiment, B is a residue of a pharmaceutically active compound or drug. The term drug, as used here, refers to any substance for internal or external use as a therapeutic drug for the treatment, treatment or prevention of a disease or disease.

Non-limiting examples are drugs for treating or preventing a cardiovascular disease, including antioxidants such as probucol; a nicotinic acid; agents that prevent blood platelets from sticking together, such as aspirin; antiplatelet agents such as coumadin; calcium channel blockers such as veropamil, diltiazem and nifedipine; angiotensin converting enzyme (ACE) inhibitors, such as captopril and inaloprine, β-blockers, such as propanolol, terbutalol and labetalol, non-steroidal anti-inflammatory drugs, such as ibuprofen, indomethacin, phenoprofen, mefenamic acid, flufenofenoids, corfufenamide acid, flufenofenides. The group —C (S) SA can be directly attached to the drug or attached via any suitable linking radical.

In another embodiment, the dithiocarbamate is a derivative of the amino acid structure AO ₂ CR ⁹ —NR ¹⁰ —C (S) SA, where R ⁹ represents a divalent radical B, a linking radical, or an internal residue of any natural amino acids (for example, CH ₃ CH for alanine, CH ₂ for glycine, CH (CH ₂ ) ₄ NH ₂ for lysine, etc.) and R ¹⁰ is hydrogen or lower alkyl.

It may also be a polymer to which one or more dithiocarbomate groups are attached either directly or via some suitable binder radical. Dithiocarbamate is preferably released from the polymer under in vivo conditions for a suitable period of time to achieve a therapeutic effect. In a preferred embodiment, the polymer itself is also capable of degrading in vivo. The term biodegradable or biodegradable, as used here, refers to a polymer that dissolves or breaks down within a period of time that is acceptable for the intended use (usually in vivo therapy), usually less than five years, and preferably less than one year, when maintaining in physiological saline with a pH of 6-8, having a temperature between 25 and 37 ° C. In a preferred embodiment, the polymer breaks down between one hour and several weeks, in accordance with the method of use.

Many degradable polymers are known. Non-limiting examples are peptides, proteins, nucleoproteins, lipoproteins, glycoproteins, synthetic and natural polypeptides and polyamino acids, including, but not limited to, polymers and copolymers of lysine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, hydrosylamine serine, threonine and tyrosine; polyorthoesters, including poly (α-hydroxy acids), for example polylactic acid, polyglycolic acid, poly (lactide-soglycolide), polyanhydrides, albumin or collagen, polysaccharide-containing sugar units, such as lactose, and polycaprolactone.

The polymer may be any or block copolymer.

B may also be a group that increases the solubility of dithiocarbamate in water, for example, lower alkyl-OR ⁸ , where R ⁸ is —PO ₂ (OH) ^- M ⁺ or PO ₃ (M ⁺ ) ₂ , where M ⁺ is a pharmaceutically acceptable cation ; -C (O) (CH ₂ ) $\genfrac{}{}{0ex}{}{\text{-}}{\text{2}}$ M ⁺ or -SO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ M ⁺ ; lower alkylcarbonyl lower alkyl; -carboxy lower alkyl; lower alkylamines lower alkyl; N, N-disubstituted amino lower alkyl— wherein the substituents each independently represent lower alkyl; pyridyl lower alkyl-; imidazolyl lower alkyl; imidazolyl-Y-lower alkyl wherein Y is thio or amino; morpholinyl lower alkyl; pyrrolidinyl lower alkyl; thiazolinyl lower alkyl-; piperidinyl lower alkyl; morpholinyl lower hydroxy alkyl; N-pyrryl; piperazinyl lower alkyl; N-substituted piperazinyl lower alkyl- wherein the substituent is lower alkyl; triazolyl lower alkyl; tetrazolyl lower alkyl; tetrazolylamino lower alkyl or thiazolyl lower alkyl.

In an alternative embodiment, a dimer may be introduced, such as B-C (S) S-SC (S) -B. Non-limiting examples of dithiocarbamates are dithiocarbamates of the following structure:

For use in the treatment of atherosclerosis and other cardiovascular and anti-inflammatory diseases, dithiocarbamates should be selected that possess the necessary lipophilicity in order to localize at the site of action. The compound should not penetrate into areas of low metabolism, such as body fat. In a preferred embodiment for the treatment of cardiovascular disease, the pharmacokinetics should not be significantly affected by chronic heart failure and renal failure.

For external use in the treatment of inflammatory skin diseases, the selected compound should be included in the preparation absorbed by the skin in sufficient quantities to provide a therapeutic effect on the affected area.

Dithiocarboxylate should be physiologically acceptable. Compounds with a therapeutic index of at least 2 and preferably at least 5 or 10 are generally acceptable. The therapeutic index is defined as EC ₅₀ / IC ₅₀ , where EC ₅₀ is a concentration of a compound that inhibits 50% expression of VCAM-1 , and IC ₅₀ is the concentration of the compound that is 50% toxic to target cells. Cellular toxicity can be measured by direct counting of cells excluded by trepan blue, or by various studies of metabolic activity, such as the incorporation of 3H-thymidine, known to those skilled in the art. The therapeutic PDTK index in tissue culture is greater than 100, as determined by cellular toxicity divided by the ability to inhibit TNFa-activated VCAM-1 expression in HUVE cells. Initial studies on the rapidly growing human cell type glioma NT ₁₈ did not demonstrate toxicity at concentrations 100 times the therapeutic concentration.

Disulfiram, an orally prescribed form of diethyldithiocarbamate used in the treatment of alcoholism, usually does not exhibit primarily clinical toxicity when administered appropriately.

A small number of dithiocarbamates are known which are genotoxic; these compounds are not included in the scope of the present invention, which is limited by the use of physiologically acceptable substances. An example of a genotoxic dithiocarbamate is zinc dimethyldithiocarbamate fungicide. In addition, the anticholinesterase properties of certain dithiocarbamates can lead to neurotoxic effects. Miller D. (1982). Neurotoxisity of the pesticidal carbamates. Neurobehav. Toxicol. Teratol. 4 (6): 779-87.

The term dithiocarboxylate, as used here, especially includes, but is not limited to, dithiocarbamates of the formulas: R ¹ SC (S) NR ² R ³ or R ² R ³ N (S) CS-SC (S) NR ² R ³ , where R ¹ is H or a pharmaceutically acceptable cation, including, but not limited to, sodium, potassium or NR ⁴ R ⁵ R ⁶ R ⁷ , where R ⁴ , R ⁵ , R ⁶ and R ⁷ are independently hydrogen, C _1-6 linear, branched or cyclic alkyl, hydroxy (C _1-6) alkyl (wherein one or more hydroxyl groups are located at any carbon atom) or aryl, and R ² and R ³ independently are C _1-10 straight, branched yl cyclic alkyl; - (CHOH) _n CH ₂ OH, where n is 0, 1, 2, 3, 4, 5, or 6; - (CH ₂ ) _n CO ₂ R ¹ , - (CH ₂ ) _n CO ₂ R ⁴ , hydroxy (C _1-6 ) alkyl-; or R ² and R ³ can together form a bridge, such as - (CH ₂ ) _m -, where m is 3-6 and where R ⁴ is alkyl, aryl, alkaryl or aralkyl, including acetyl, propionyl and butyryl.

Specific examples of the dithiocarbamates used shown in FIG. 16 include, but are not limited to, sodium pyrrolidine-N-carbo-dithioate, N-methyl-N-carboxymethyl-N-carbo-dithioate sodium, N, N-di (carboxymethyl) -N-carbo-dithioate trisodium, N-methyl-D-glucamine-N-sodium carbodithioate, N, N-diethyl-N-carbodithioate sodium (sodium diethyl dithiocarbamate) and N, N-diisopropyl-N-carbodithioate sodium.

Active dithiocarboxylates, and in particular dithiocarbamates, are either commercially available or can be prepared by known methods.

II. Biological activity

The ability of dithiocarbamates to inhibit the expression of VCAM-1 can be determined in various ways, including the method described in detail below, in examples 9 to 15. For convenience, examples 9-11 and 14-15 describe the assessment of the biological activity of sodium pyrrolidine-N-carbodithioate (also designated as PDTC). These examples do not limit the scope of the invention, but specifically include the use of the above compounds for the treatment of atherosclerosis and other types of inflammatory and cardiovascular diseases mediated by VCAM-1. Any of the compounds described above can be replaced in place of PDTC and evaluated in a similar manner.

Examples 12 and 13 describe comparative data on the ability of certain dithiocarbamates to inhibit the expression of the VCAM-1 gene. The examples below establish that the claimed dithiocarbamates specifically inhibit the ability of VCAM-1 to be expressed by vascular endothelial cells in response to many known signals active in atherosclerosis and inflammatory diseases.

Experimental methods

Cell culture. HUVE cells were isolated from the umbilical veins into which the cannulas were inserted, perfused with Hanks's solution to remove blood, and then incubated with 1% collagenase for 15 minutes at 37 ° C. After removing collagenase, the cells were cultured in M199 medium supplemented with 20% fetal bovine serum (HyC1ope), 16 μg / ml heparin (ESI Pharmaceuticals, Cherry Нill, nj), 50 μg / ml endothelial cell growth supplement (Collaborative Research Jncorporated, Bedford, MA), 25 mM Heps buffer, 2 mM L-glutamine, 100 μg / ml penicillin and 100 μg / ml streptomycin, and grown at 37 ° C. in cell growth plates coated with 0.1% gelatin. Cells were transplanted in a confluent state by a 1: 4 distribution. The cells of the first 8 passages were used.

Incubation with cytokines and other reagents

HUVE confluent cells were washed with phosphate buffer in saline and then fresh medium was added. Marked concentrations of PDTC were added as pretreatment before cytokines were added. Cytokines and other inducers were directly added to the medium at a specific time and in the concentrations indicated for each experiment. Recombinant human IL-2b represented the amiable gift of Upiohn Сompany (Kalamazoo, Michigan). TNF-α was obtained from Boehringer Engelheim). Bacterial lipopolysaccharide (LPS), polyninosinic acid: polycytidic acid (Poly I: C) and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma Chemical (St. Zouis, MO). All other reagents represented an appropriate degree of purity for the reagents.

RNA isolation. Total cellular RNA was isolated by extraction using an acidified mixture of guanidine thiocyanate-phenol-chloroform. Cells were washed with phosphate buffer in physiological saline and then lysed with 2 ml of guanidine isothiocyanate. The solution was acidified with 0.2 ml of sodium acetate (pH 4.0) and then extracted with 2 ml of phenol and 0.4 ml of a mixture of chloroform: isoamyl alcohol (24: 1). RNA was subjected to two ethanol precipitation before use for Northern blot analysis.

Northern blot analysis. Total cell DNA (20 μg) was size fractionated using 1% agarose formaldehyde gel in the presence of 1 μg / ml ethidium bromide. RNA was transferred onto a nitrocellulose filter and covalently sutured by UV irradiation using a Stratlinker UV crosslinker (Siratagene, Za Jolla, CA).

Hybridization was carried out at 42 ° C for 18 hours in 5X (multiple) SSC (1X = 150 mM NaCl, 15 mM Na citrate), 1% sodium dodecyl sulfate, 5X Denhardt solution, 50% formamide, 10% dextran sulfate and 100 μg / ml chopped denatured salmon sperm DNA. About 1-2 · 10 ⁶ cpm / ml labeled probe was used for hybridization (specific activity> 108 cpm / μg DNA). After hybridization, the filters were washed with a final solution of 0.2X ionic strength at 55 ° C. Cellulose was washed using boiling water before re-hybridization with other samples. Autoradiography was performed with an intensifying screen at -70 ° C.

³² Probes. ³² P labeled DNA probes were prepared using the random oligonucleotide primer method. The ICAM-1 probe was an Eco R1 fragment of a human cDNA. The ELAM-1 probe was a 1.85 kb Hind III fragment of human cDNA. The VCAM-1 probe was a fragment of Hind III-Xho I human cDNA, including nucleotides from 132 to 1814.

Enzyme immunosorbent test

(ELISA). HUVE cells were placed on a microtiter plateau with 96 cells for 48-72 hours before the study. Primary antibody was added in M199 with 5% FBS to each well and incubated for one hour with a goat anti-mouse IqG conjugate with peroxidase (Bio Rad) diluted 1/500 in M199 with 5% FBS. Then the cells were washed and antibody binding was determined by adding 100 μl of a solution of 10 mg / ml 3,3,5,5'-tetramethylbenzidine (Sigma) in the presence of 0.003% H ₂ O ₂ . The reaction was stopped by adding 25 μl of 8 N sulfuric acid. The cells were read in a reader (reader) ELISA (Bio Rad) at O.P. 450 nm, after determining the blank in a row stained only with antibody in the second stage. Data represent the average of triplicates.

Antibodies A monoclonal antibody (Mab) 4B9 recognizing a cell adhesion molecule-1 molecule (VCAM-1) was supplied by Dr. John Harlan (University of Washington). Antibodies that recognize the endothelial cell adhesion molecule (ELAM-1) were supplied by Dr. Schwerlick (Emory University) Hybridomas producing mAB 84H10, recognizing the intracellular adhesion-1 molecule (ICAM-1), were usually grown in the laboratory and cell culture supernatant was used in as an antibody.

Example 9

PDTS blocks 1L-2b-mediated induction of VCAM-1 in HUVEC, but not the accumulation of ICAM-1 or ELAM-1 mRNA.

To determine whether the oxidized state of enothelial cells can alter the basal and induced gene expression of the cell adhesion molecule, cultured human vascular endothelial cells were maintained with IL-1b induction cytokine (10 units / ml) in the presence or absence of antioxidants chelating a thiolated metal, pyrrolidine dithiocarbamate (PDTC, 50 μM) up to 24 hours. As shown in FIG. 11, one 1L-2b (lines 2, 4, 6, 8) induces the expected fast and transient induction of VCAM-1 mRNA (Panel A), E-selectin (ELAM-1, Panel B) and ICAM- 1 (Panel C), all peaks for four hours. However, in the presence of PDTC, the induction of accumulation of VCAM-1 mRNA by 1L-2b is dramatically inhibited by more than 90% (Panel A, lines 3, 5, 7, 9). In contrast, although 1L-2b mediated induction of ELAM-1 is slightly inhibited at 2 and 24 hours (compare line 2 and 3, 8 and 9, Panel B), PDTC does not inhibit induction at 4 and 8 hours (line 5 and 7 , Panel B), 1L-2b, indirect induction of ICAM-1 mRNA accumulation does not change (panel B, lines 3, 5, 7, 9). Undoubtedly, there is moderate inhibition of the induction of ICAM-1 mRNA accumulation under the influence of 1L-2b (-30%) (compare lines 4 and 5, panel B). Equivalent amounts of RNA transferred to cellulose per line are confirmed by ethidium bromide staining and visualization.

To determine whether PDTS actually inhibits the induction of VCAM-1 gene expression under the influence of 1L-2b in a dose-dependent manner, a dose-dependent analysis was performed. As shown in FIG. 12, PDTC inhibits 1L-2b mediated induction of VCAM-1 gene expression in a sequential dose-dependent curve (FIG. 12, panel A) with an estimated EC _{50 of} approximately 10 μM, although PDTC does not inhibit 1L-2b mediated induction of expression of ELAM-1 at the same concentrations (Fig. 12, panel B). 1L-2b mediated induction of ICAM-1 mRNA accumulation is enhanced by PDTC at concentrations higher than 0.5 μM (Fig. 13, compare line 2 and line 4-7, panel C).

These data show that 1L-2b uses dithiocarboxylate and, in particular, the dithiocarbamate-sensitive stage as its signaling mechanism for inducing expression of the VCAM-1 gene. The data, apparently, also show that this dithiocarbamate sensitive stage does not play a significant role in IL-2b mediated induction of gene expression of ELAM-1 or ICAM-1.

Example 10

PDTS blocks the induction of VCAM-1 HUVEC mRNA accumulation under the influence of multiple stimuli

Three different classes of activators were tested to determine whether other well-described VCAM-1 gene expression activators also use the PDTC-sensitive stage: classic, receptor-mediated other inducing agent (TNF-α), receptor-mediated inducer (lipopolysaccharide (LPS)) and the recently described new inducer (double-stranded RNA, poly (I: C). In all three cases, PDTC inhibited the induction of accumulation of VCAM-1 mRNA in HUVEC after four hours (Fig. 13, panel A). Despite the fact that TNF-α is mediated gene expression VCAM-1 is suppressed to the same degree (Fig. 13, line 1 and 2, panel B), mediated by LRS and poly (I: C) accumulation of VCAM-1 mRNA was unchanged (Fig. 13, line 3-6, panel B ). Induction of ICAM-1 mRNA accumulation was unchanged (Fig. 13, Panel C). These data show that structurally different inducible genes acting through different pathways share a common regulatory stage specific for inducing VCAM-1 gene expression.

Example 11

PDTS blocks the expression of VCAM-1 on the cell surface of HUVE induced by multiple stimuli

To determine whether, like its mRNA, the induction of expression on the surface of the endothelial cell of VCAM-1 protein can also be inhibited by PDTC, monoclonal antibodies were used in ELISA to quantify the induction on the surface of VCAM-1 and ICAM-1 cells in HUVE cells in culture. As shown in FIG. 14, multiple classes of activating agents in the absence of PDTC (-PDTS) induce fast and temporary accumulation of VCAM-1 (top left panel) on the cell surface with a maximum of six hours. In the presence of PDTS (+ PDTS, top right panel), the induction of VCAM-1 expression on the cell surface under the influence of all tested agents is dramatically inhibited (80-90%). In contrast, the induced expression of ICAM-1 on the cell surface does not change under identical conditions (bottom left and right panels).

These data indicate that, like the accumulation of intrinsic mRNA, VCAM-1 expression on the cell surface is selectively suppressed by the action of dithiocarbamates and that multiple classes of activating agents use a similar dithiocarbamate-sensitive mechanism of induction of VCAM-1 gene expression.

Example 12

Comparative efficacy of antioxidants in blocking the induction of VCAM-1 by TNF-α

To determine whether structurally similar or dissimilar antioxidants could also inhibit VCAM-1 gene expression and with what efficiency, HUVE cells were incubated with TNF-α for six hours in the presence or absence of different concentrations of four different antioxidants. As shown in FIG. 15, both diethyldithiocarbamate (DETC) and N-acetylcysteine (NAC) inhibited VCAM-1 expression at concentrations of 5 μM and 30 μM, respectively. In contrast, PDTC (PDTC) was effective between 5 and 50 μM. The iron metal chelator, desferroximin, did not have an effect in the concentrations studied.

Example 13

PDTS inhibits TNF-induced VCAM-1 / VLA-4 mediated adhesion

The ability of various antioxidants to inhibit the induction of VCAM-1 in HUVE cells under the influence of TNF-α was determined by the method described in example 12. Fig.16 is a graph of the relative expression of VCAM-1 on the cell surface (O.P. 595 nm) in HUVE cells, activated TNF-α, relative to the concentrations of PDTC (sodium N-pyrrolidine dithiocarbamate), DIDTC (sodium N, N-diethyl-N-carbodithioate), SarDTC (sodium N-methyl-N-carboxymethyl-N-carbodithioate), IDADC (trisodium N , N-di (carboxymethyl) -N-carbodithioate), MGDTC (sodium N-methyl-D-glucamine-N-carbodithioate), MeOBGDTC (sodium N- (4-methoxybenzyl) -D -glucamine-N-carbodithioate), DEDTS (sodium N, N-diethyl-N-carbothioate), Di-PDTC (sodium N, N-diisopropyl-N-carbodithioate), and NAC is (N-acetylcysteine).

Example 13

PDTC inhibits induction by TNF-mediated adhesion of VCAM-1 / VLA-4

In order to determine whether the inhibition of regulation of VCAM-1 by PDTC is associated with a functional sequence of events, the binding of Molt-4 cells to HUVEC cells, either not stimulated or stimulated by TNFa (100 u / ml) for six hours was investigated the presence or absence of PDTC. Molt-4 cells, as previously shown, bind to activated HUVEC via a VCAM-1 dependent mechanism. As shown in FIG. 17, the percentage of binding of Molt-4 to HUVEC cells decreased when PDTC was present in the medium.

Example 14

PDTS inhibits monocyte binding to the abdominal aorta of rabbits on a cholesterol diet

The experiment was carried out on animals in an experimental model to determine whether the thiol antioxidant PDTC could be effective in blocking the binding of the primary component of monocytes in atherosclerosis. An adult New Zealand white rabbit (3.5 kg) received intravenous injections of PDTS (20 mg / kg, as a solution, concentration of 20 mg / ml in PBS) daily for 5 days. Injections were performed through an implanted cannula into the marginal ear vein, which was kept unclogged by washing with heparinized saline. The PDTC solution was prepared either daily or on the previous day (stored protected from light at 4 ° C) and filtered (0.45 mm filter with pores) immediately before use. Through the implanted cannula, after the first injection, the drug was administered to the rabbit in a healthy state in the absence of discomfort or other painful effect. On the second day of injection, the rabbit received a diet containing 1% cholesterol by weight, which continued to be given for the remainder of the experiment. On the fifth day, the animal was sacrificed, the abdominal aorta was excised and fixed. After appropriate preparation, the samples were imaged at a low stage on an ISI-130 electron scanning microscope equipped with a LaB emitter. Using images on two screens and a transparent grid on the CRT screen, 64 adjacent fields were examined at 620x magnification, covering an area of 1.3 mm ² . Inside each field, the number of adherent leukocytes (WBC) and erythrocytes (RBC) was counted and recorded.

The data from the arch sample are as follows: 5 WBC and approximately 25 RBC per 1.3 mm ² field. This level of WBC adhesion is close to control animals receiving normal nutrition (about 7 cases were found on the field in samples of the arch and abdominal region from 2 'negative control' experiments). The 'positive control' rabbits receiving 1% cholesterol for 4 days but not receiving an antioxidant showed a 5-fold increase in adhesion to 38 WBS / 1.3 mm ² . Basically, a large number of cell-sized fibers adhered to each arch sample were observed. It is unclear whether this material is indeed a preparation artifact or was present in vivo and, if so, whether this is related to the administration of PDTC. These studies confirm that the administration of PDTC can effectively block the initial adhesion of monocytes to the aortic endothelium.

Example 15

Inhibition of BSA 13-HOPODE Addition Products with PDTC

Fig. 19 is a graphical diagram of the effect of PDTC on the formation of fluorescent BSA addition products with 13-HPODE as determined in fluorescence units depending on the micromolar concentration of PDTC. One micromole of 13-HPODE was incubated with 200 micrograms of BSA in the presence of PDTS for six days. Fluorescence was measured at 430-460 nm with excitation at 330-360 nm. For details of the determination, see Freebs, J., Parthasarathy, S., Steinberg, D., Prosedings of the National Academy of Sciences 89, 10588-10592, 1992. In a typical reaction, 100 nmoles of LOOH (obtained by oxidation-catalyzed lipoxygenase) linoleic acid) was incubated with 100 μg of bovine serum albumin for 48-72 hours and the subsequent formation of fluorescent products was measured by determining the fluorescence spectrum upon excitation at 360 nm and emission between 390 and 500 nm.

As noted, PDTC reduces the concentration of fluorescent BSA and 13-HPODE adducts.

FIG. 20 is a graph of the effect of PDTC on the formation of fluorescent BS A and ox-PUFA addition products as a function of wavelength (nm) and PDTC concentration. As the concentration of PDTC increases, the amount of fluorescent adducts decreases.

Example 16

The effect of PDTC on LDL oxidation under the influence of horseradish peroxidase

Fig is a graph of the effects of PDTC on LDL oxidation under the influence of horseradish peroxidase (HRP), measured in time (minutes) at various concentrations of PDTC. LDL oxidation was carried out by measuring the fatty acid components of LDL by determining the increase in optical density at 234 nm. When polyunsaturated fatty acids are oxidized, a shift in the double bond is observed, leading to the formation of conjugated dienes, which exhibit absorption at 234 nm. The delay in the initiation and development of the curve (lag phase) confirms that there is a measurement of the oxidative ability of lipids. A longer lag phase, therefore, greater stability of LDL to oxidation. Typically, 100 μg of human LDL is incubated with 5 μM H ₂ O _2, and then an increase in absorption at 234 nm follows.

It was found that after the incubation period, PDTC inhibits LDL oxidation under the influence of HRP in a concentration-dependent manner.

Example 17

Effect of PDTC on Cytokine-Induced Ox-PuFA Formation

Fig. 22 is a graph of the effect of PDTS on cytokine-induced production of ox-PUFA in human aortic endothelial cells. As noted, both TNF-α and IL-2b cause oxidation of linoleic acid to oxidized linoleic acid. Oxidation is significantly arrested by PDTC.

2. Modification of the synthesis and metabolism of PUFA and ox-PUFA

Inhibition of VCAM-1 expression can be completed by modifying the metabolism of PUFA in ox-PUFA. Many known enzymes oxidize unsaturated materials, including, for example, peroxidases, lipoxygenases, cyclooxygenases, and cytochrome P450. Inhibition of these enzymes can prevent in vivo oxidation of PUFA. PUFAS can also be oxidized by metal-dependent non-enzymatic materials.

IV. The method of modifying the expression of genes sensitive to oxidation-reduction

In an alternative embodiment, a method is described for suppressing the expression of oxidation-reduction sensitive genes or activating a gene that is suppressed by an oxidation-reduction sensitive pathway, which comprises administering an effective amount of a substance that prevents oxidation of the oxidizable signal, and typically, oxidation of a polyunsaturated fatty acid . Typical oxidation-reduction-sensitive genes that are involved in the development of the immune response include, but are not limited to those expressed cytokines involved in the initiation of the immune response (e.g., IL-1B), chemoattractants that promote the migration of inflammatory cells to the point of damage (e.g. , MCP-1), growth factors (IL-6, thrombin receptor) and adhesion molecules (e.g., VCAM-1 and E-selectin).

With the present invention, a skilled worker will be able to test a wide variety of antioxidants by their ability to inhibit the expression of a redox sensitive gene or the activation of a gene that is suppressed via a redox sensitive pathway. All these embodiments are included in the scope of the present invention.

Based on the results of this test, nucleic acid molecules containing 5'-regulatory sequences of redox sensitive genes that can be used to regulate or inhibit gene expression in vivo can be identified. Vectors including both the plasmid and eukaryotic viral vectors can be used for expression of a particular construct of the 5'-flanking region of the recombinant gene in cells depending on the preference and judgment of a qualified cell udnika (see., e.g., Sambrook et al., Chapter 16). Moreover, many viral and non-viral vectors have been developed that are capable of being introduced in vivo in a nucleic acid sequence (see, for example, Mulligan, 1993, Science, 260, 926-932; US Patent No. 4,980,286; US Patent No. 4,868,116, incorporated here as a reference). A delivery system has recently been developed in which a nucleic acid is encapsulated in cationic liposomes that can be intravenously administered to a mammal. This system was used to introduce DNA into the cells of many tissues of an adult mouse, including the endothelium and bone marrow (see Zhu et al., 1993, Scientific 261, 209-211, incorporated herein by reference).

The 5’ flanking sequences of the redox sensitive gene can be used to inhibit the expression of the redox sensitive gene. For example, the antisense RNA of all or part of a 5'-flanking region of a redox sensitive gene sequence can be used to inhibit gene expression in vivo. Expression vectors (eg, retroviral expression vectors) are already available in the art, can be used to produce antisense RNA, a selected DNA sequence that is expressed in a cell (see, for example, US Patent No. 4868116; US Patent No. 4980286). Accordingly, DNA containing the entire portion of the sequence of the 5 ′ flanking region of the gene can be introduced into a suitable expression vector such that when introduced into the cell, transcription of the inserted DNA gives an antisense RNA that is complementary to the mRNA transcript of a gene of a commonly found gene. This transcript of antisense RNA from the inserted DNA can then form base pairs with the normal mRNA transcript found in the cells, and thus interfere with the mRNA to be translated. Of course, you need to select the 5’ sequence of the flanking region, which is located in the direction from the transcriptional start site for the oxidation-reduction sensitive gene to confirm that the antisense RNA contains complementary sequences present in the mRNA. Antisense RNA can also be obtained in vitro and then introduced into cells. Oligonucleotides can be synthesized using an automatic synthesizer (for example, Model 8700 Milligen-Biosearch automatic synthesizer, MA or ABI Model 380B). In addition, antisense deoxynucleotides have been shown to be effective in inhibiting gene transcription and viral replication (see, for example, Zamecnik et al., 1978, Proc. Natl. Acad. Sci. USA, 75, 280-284; Zamecnik et al. , 1986, Proc. Natl. Acad. Sci. USA, 83, 4143-4146; Wickstrom et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 1028-1032; Crooke, 1993, FASEB J. 7 533-539 Moreover, recent work has shown that enhanced inhibition of gene expression by antisense nucleotides is possible if antisense oligonucleotides contain modified nucleotides (see Offensperger et al., 1993 EMBO J, 12, 1257-1262 (inhibition of hepatitis B replication and gene expression in vivo by antisense phosphorothioate oligonucleotides); Rosenberg et al., PST WO 93/01286 (synthesis of serotioate oligonucleotides); Agrawal et al., 1988, Pr. Natl. Acad. Sci, USA 85, 7079 -7083 (synthesis of antisense phosphoramidate phosphorothioate nucleotides to inhibit the replication of human immunodeficiency virus-1); Sarin et al., 1989, Proc. Natl. Acad. Sci. 85, 7448-7794 (synthesis of antisense methylphosphonate oligonucleotides); Show et al., 1991, Nisleis Acids Res, 19, 747-750 (synthesis of exonuclease-resistant 3'-oligonucleotides containing 3'-final phosphoamidate modifications); included here as links).

The sequence of the 5’-flanking region of the oxidation-reduction sensitive gene can also be used in tri-helical (triplex) gene therapy. It was shown that oligonucleotides complementary to the promoter sequence of a gene of one of the DNA strands bind the promoter and the regulatory sequence to form a triplex nucleic acid that blocks gene transcription (see, for example, 1989, Maher et al., Science 245, 725-730; Orson et al., 1991 Nucl. Asids Res. 19, 3435-3441; Postal et al., 1991 Proc. Natl. Acad. Sci. USA 88, 8227-8231; Cooney et al., 1988 Sciece 241, 456-459; Young et al., 1991 Proc. Natl. Acad. Sci. USA 88, 10023-10026; Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504-508; 1992 Blume et al., Nucl. Acids Res. 20, 1777-1784; 1992, J. Bioi. Chem. 267, 3389-3395.

Recently, theoretical calculations and empirical findings have been reported that provide guidance for the construction of oligonucleotides whose triple helix inhibits gene expression. For example, oligonucleotides with a total length greater than 14 nucleotides are preferred to ensure the specificity of the target sequence (see, for example, Maher et al., (1989); Grigoriev et al., (1992)). Many cells also greedily capture oligonucleotides that are less than 50 nucleotides in length (see, for example, Orson et al., (1991); Holt et al., 1988, Pr. Natl, Acad. Sci., USA, 85, 1028-1032). To reduce the susceptibility to intracellular destruction, for example, when exposed to 3'-exonucleases, you can introduce a free amine into the 3'-terminal hydroxyl group of nucleotides, without violating the specific binding of the sequence (Orson et al., 1991). In addition, more stable triplexes are formed if any cytosines that may be in the oligonucleotide are methylated, as well as if an intercalating agent, such as an acridine derivative, is covalently linked to a 5'-terminal phosphate (for example, via a pentamethylene bridge); again without loss of specificity by sequence (Maher et al., (1989); Grigoriev et al., (1992).

Methods for preparing or synthesizing oligonucleotides are well known in the art. Similar methods can be arranged in order from conventional enzymatic digestion followed by isolation of nucleotide fragments (see, for example, Sambrook, et al., Parts 5, 6) to purely synthetic methods, for example, using the cyanoethyl phosphoramidate method using Milligen or Weightman System 1 Plus DNA synthesizers (see also Jkuta et al., In Ann. Rev. Biochem. 1984, 53, 323-356 (phosphotriether and phosphite-triester methods); Nrang et al., In Methods Enzymol., 65, 610-620 (1980) (phosphotriether method). Accordingly, the DNA sequence of the 5'-flanking region of the gene oxidation reduction described herein can be used to construct oligonucleotides comprising a DNA sequence consisting of at least 15 consecutive nucleotides, with or without modified bases or derivatives of intercalating agents, to produce triple helices specifically within 5 ' -flanking portion of a gene sensitive to oxidation-reduction to inhibit gene expression.

In some cases, it may be preferable to introduce an enhancer or multiple copies of regulatory sequences to facilitate the selection of methods and reagents for expression control.

V. Models and kits

For diseases mediated by VCAM-1 or a gene that is sensitive to oxidation-reduction, kits are also described that include the counting of replacement disease markers. In one embodiment, for example, the level of oxidized polyunsaturated fatty acids or other suitable markers in the host tissue or blood is assessed as the average of the assessments of the “oxidative state of the environment” of the host and the susceptibility of the host to VCAM-1 mediated or oxidation-reduction-sensitive gene diseases .

In another embodiment, the level of circulating VCAM-1 on the surface of the cell or other suitable marker and the effect of the administration of the necessary antioxidant on this level are quantified.

In another analysis, the sensitivity of the host endothelial cells to polyunsaturated fatty acids and their oxidized derivatives was evaluated. This can be accomplished, for example, by replacing the host with PUFA or ox-PUFA and comparing the final concentration of circulating VCAM-1 on the cell surface or another replacement marker with a normal population.

In another embodiment, in an in vivo model of atherosclerosis or other cardiac or inflammatory diseases that are mediated by VCAM-1, for inducing the disease, it may be provided by administering to the host animal an excessive amount of PUFA or polyunsaturated fatty acids. These animals can be used for clinical studies to further research these diseases.

In another embodiment of the invention, the compounds can still be evaluated for their ability to treat VCAM-1 mediated diseases based on their ability to inhibit the oxidation of polyunsaturated fatty acids, or the interaction of PUFA or ox-PUFA with a protein target.

This can be done by replacing the host, for example a human or animal, such as a mouse, with a high level of PUFA or ox-PUFA, and then determining the therapeutic efficacy of the test compounds by their ability to reduce the concentration of circulating or cell-surface VCAM-1. Alternatively, in vivo testing can be used, which is based on the ability of the compounds to prevent the oxidation of PUFAs, or the interaction of PUFAs or ox-PUFAs with a protein target, in the presence of oxidizing compounds such as metal, such as copper, or enzymes such as peroxidase, lipoxygenase, cyclooxygenase or cytochrome P450.

In another embodiment, the vascular endothelial cells are incubated with TNF-α or other VCAM-1 inducing material for an appropriate time, then destroyed, for example, by sound or by freezing-thawing. The cytosolic and membrane fractions were isolated. To determine the number of fractions, radioactively labeled PUFA was added. The ability of a liquid to convert PUFA to ox-PUFA in the presence or absence of a test compound was investigated. You can use intact cells instead of a destroyed cellular system.

III. Pharmaceutical Compositions

People, horses, dogs, cattle and other animals, in particular mammals, suffering from cardiovascular and other inflammatory diseases mediated by VCAM-1 and the redox-sensitive gene, can receive treatment by introducing the patient an effective amount of the compound, which leads to remove, reduce concentration or protect against the formation of oxidized polyunsaturated fatty acids, including, but not limited to, linoleic (C ₁₈ Δ ^{9, 12} ), linolinic (C ₁₈ , Δ ^{6, 9, 12} ), arachidonic (C ₂₀ Δ ^{5 , 8, 11, 14} ) and ecosatriene (c ₂₀ Δ ^{8, 11, 14} ) acids; other oxidation signals or a pharmaceutically acceptable derivative or salt thereof with a pharmaceutically acceptable carrier or diluent. The active substances may be administered by any suitable route, for example, orally, parenterally, intravenously, intradermally, subcutaneously or externally.

As used here, the term pharmaceutically acceptable salts or complexes refers to salts or complexes that retain the desired biological activity of the above compounds and exhibit minimal undesirable toxic effects. Non-limiting examples of such salts are:

(a) acid addition salts formed by inorganic acids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like), and salts formed by organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalene disulfonic acid and ligalacturonic acid;

(b) base addition salts formed by polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium and the like, or with an organic cation formed from N, N-dibenzylethylenediamine, D-glucosamine, ammonia, tetraethylammonium or ethylenediamine; or

(c) combination of (a) and (b); for example, a zinc tinnate salt or the like.

The active compound or mixture of compounds is administered in any suitable manner, including but not limited to the oral or intravenous routes. Typical dose levels for any of the above conditions will be from 0.5 to 500 mg / kg body weight with a dosage regimen varying from 1 every other day to several times a day. Preferred daily doses are in the range of from about 1 to 3000 mg / patient / day, more preferably between about 5 to 500 mg / patient / day, and even more preferably between about 25 to 500 mg / patient / day.

The active ingredient should be administered until peak plasma concentrations of the active substance are from about 0.1 to 100 μM, preferably about 1-10 μM. This can be achieved, for example, by intravenous injection of a solution or preparation of the active ingredient, optionally in physiological saline or in an aqueous medium, or by administering the active ingredient as a bolus.

The compound can also be introduced directly into the vascular wall using perfusion balloon catheters after or instead of coronary or other arterial angioplasty. As an example, 2-5 ml of a physiologically acceptable solution containing approximately 1-500 μM of the compound or mixture of compounds is administered at 1-5 atmospheric pressure. Since that time, during the course of the next six months, during the period of maximum danger of restenosis, the active compounds are administered by other suitable ways and regimens.

Treatments for a relatively short period of time with active joint are used to cause the contraction of the diseased coronary artery that cannot be treated either angioplastically or surgically. A non-limiting example of treatment for a short period of time is two to six months of treatment with doses of 0.5 to 500 mg / kg body weight given from once every second day to three times a day.

Treatments over a longer period of time can be used to prevent the development of progressive lesions in patients with an increased risk of disease. Treatment over a long period of time can be a treatment for several years in doses of 0.5 to 500 mg / kg body weight, administered at intervals of once every second day to three times a day.

The active compound can also be administered immediately or after coronary angioplasty as a means that reduces or eliminates the abnormal proliferative and inflammatory response, which often lead to clinically significant restenosis.

Active compounds may be prescribed in combination with other drugs used in the treatment of cardiovascular disease, including lipid lowering agents such as probucol and nicotinic acid; platelet blocking inhibitors such as aspirin; antiplatelet agents such as coumadin; calcium channel blockers such as veropamil, diltiazem and nifedipine; angiotensin converting enzyme (ACE) inhibitors, such as captopril and inaloprine, and β-blockers, such as propanolol, terbutalol and labetalol. The compounds can also be administered in combination with non-steroidal anti-inflammatory drugs, such as ibuprofen, indomethacin, phenoprofen, mefenamic acid, flufenamic acid, sulindac. The compounds may also be administered in combination with corticosteroids.

The concentration of the active compound in the drug composition will depend on the absorption, distribution, inactivation and excretion routes of the drug, as well as other factors known to those skilled in the art. It should also be noted that dosage sizes will also vary depending on the severity of the condition that needs to be alleviated. Further, it should be noted that for each specific subject, specific treatment regimens should be established for a period of time corresponding to individual needs and in accordance with the professional judgment of the person prescribing or observing the prescription of the compositions, and that the concentration ranges indicated here are only an example and not are intended to limit the field of practical application of the claimed composition. The active ingredient may be administered immediately or may be divided into smaller doses for administration at various time intervals.

Oral compositions typically include an inert diluent or food carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated into excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents and / or excipients may be included as part of the composition.

Tablets, dragees, capsules, lozenges and the like contain any of the following ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose, tragacanth resin or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel or corn starch; a lubricant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the unit dosage form is a capsule, it may contain, in addition to the substances of the above types, a liquid carrier such as fatty oil. In addition, unit dosage forms may contain various other substances that modify the physical form of the dosage unit, for example, coatings with sugar, shellac or other coating agents.

The active compound or a pharmaceutically acceptable salt or derivative thereof may be administered as a component of an elixir, suspension, syrup, cachet, chewing gum or the like. The syrup may contain, in addition to the active compound, sucrose as a sweetening agent and some preservatives, paints and dyes, flavorings. The active compound or its pharmaceutically acceptable salts or derivatives may also be administered with other active substances that do not interfere with the desired effect, or with substances that support the desired effect, such as antibiotics, antifungal, anti-inflammatory or antiviral compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous or external use may include the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates or phosphates; and isotonic agents such as sodium chloride or dextrose. The parenteral preparation may be placed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Suitable solvents or vehicles for external use are known and include lotions, suspensions, ointments, creams, gels, tinctures, sprays, powders, pastes, slow release subcutaneous lozenges, asthma aerosols and suppositories for application to rectal, vaginal, nasal or oral mucous membranes .

To obtain compositions for external use, thickeners, emollients and stabilizers can be used. Examples of thickeners include petroleum jelly, beeswax, xanthan gum or polyethylene glycol, humectants such as sorbitol, emollients such as mineral oil, lanolin and its derivatives or squalene. A large number of solutions and ointments are commercially available.

Natural and synthetic flavors or sweeteners can be added to improve the taste of external preparations used for local action on mucosal surfaces. Inert paints or dyes may be added, in particular in the case of preparations intended for application to the surface of an oral mucosa.

Active compounds can be prepared with carriers that prevent the compounds from being rapidly released, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art.

For intravenous administration, preferred carriers are saline or phosphate buffered saline (PBS).

The active compound may also be administered via a transdermal plate. Methods for producing a transdermal plate are known to those skilled in the art. For example, see Brown, Z. and Zanger, R., Transdermal Delivery of Drugs, Annual Review of Medicine, 39: 221-229 (1988), incorporated herein by reference.

In another embodiment, the active compounds are prepared with carriers that prevent the compounds from being rapidly excreted from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such formulations are known to those skilled in the art. Substances may also be commercially available from Alza Corporacion and Nova Pharmaceuticals, Jnc.

Liposomal suspensions may also be pharmaceutically acceptable. They can be obtained in accordance with methods known to specialists in this field, for example as described in US patent 4522811 (which is incorporated herein by reference in its entirety). For example, liposome formulations can be prepared by dissolving the corresponding lipid (s) (such as stearoylphosphatidylethanolamine, stearoylphosphatidylcholine, arachadoylphosphatidylcholine) and cholesterol in an inorganic solvent, which is then evaporated to give a thin film of dry lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate and / or triphosphate derivative is then placed in this container. The container is then subjected to manual agitation to separate the lipid substance from the walls of the container and to disperse the lipid aggregates, thereby obtaining a liposomal suspension. Modifications and variations of the present invention from the foregoing detailed description of the invention will be apparent to those skilled in the art. Such modifications and variations are included in the scope of the attached claims.

Claims (37)

1. A method of treating a disease that is mediated by VCAM-1, comprising administering to a mammal a substance that prevents or minimizes the oxidation of a polyunsaturated fatty acid and thereby inhibits the expression of VCAM-1. 2. The method according to claim 1, characterized in that the polyunsaturated fatty acid is linoleic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{9,}}{}$ ¹² ), linolenic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{6,}}{}$ ^{9, 12} ), arachidonic (С ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{5,}}{}$ ^{8, 11, 14} ) or eicosatriene (C ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{8,}}{}$ ^{11, 14} ) an acid or its hydroperoxide or oxidized form. 3. The method according to claim 1, characterized in that said disease is a cardiovascular disease. 4. The method according to claim 3, characterized in that the cardiovascular disease is atherosclerosis. 5. The method according to claim 3, characterized in that the cardiovascular disease is postangioplastic restenosis. 6. The method according to claim 3, characterized in that the cardiovascular disease is a disease of the coronary arteries. 7. The method according to claim 3, characterized in that the cardiovascular disease is angina pectoris. 8. The method according to claim 1, characterized in that said disease is an inflammatory disease. 9. The method according to claim 8, characterized in that said inflammatory disease is rheumatoid arthritis. 10. The method of claim 8, wherein said inflammatory disease is osteoarthritis. 11. The method according to claim 8, characterized in that said inflammatory disease is asthma. 12. The method according to claim 8, characterized in that the indicated inflammatory disease is dermatitis. 13. The method according to claim 8, characterized in that said inflammatory disease is multiple sclerosis. 14. A method of treating a disease that is mediated by expression of a redox-sensitive gene, comprising administering to a mammal a substance that prevents or minimizes the oxidation of a polyunsaturated fatty acid and thereby inhibits the expression of a redox-sensitive gene. 15. The method according to 14, characterized in that the polyunsaturated fatty acid is linoleic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{9,}}{}$ ¹² ), linolenic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{6,}}{}$ ^{9, 12} ), arachidonic (С ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{5,}}{}$ ^{8, 11, 14} ) or eicosatriene (C ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{8,}}{}$ ^{11, 14} ) acid. 16. The method according to 14, characterized in that said gene mediates the expression of MCP-1. 17. The method according to 14, characterized in that the gene mediates the expression of IL-6. 18. A method for evaluating a test compound for its ability to treat a VCAM-1 mediated disease, comprising the steps of: (a) exposing a polyunsaturated fatty acid with a protein and test compound in vitro in solution in the presence of a substance known for its ability to oxidize a polyunsaturated fatty acid in the absence of a test compound; and (b) assessing the degree of interaction between the polyunsaturated fatty acid or the oxidized polyunsaturated fatty acid and protein. 19. A method for evaluating a test compound for its ability to treat a VCAM-1 mediated disease, comprising assessing the ability of this compound to inhibit the interaction of an oxidized polyunsaturated fatty acid with a protein by the following steps: (a) exposing the oxidized polyunsaturated fatty acid with the protein and the test in vitro solution compound; and (b) assessing the degree of interaction between the oxidized polyunsaturated fatty acid and the protein. 20. The method according to p. 18, characterized in that said polyunsaturated fatty acid is linoleic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{9,}}{}$ ¹² ), linolenic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{6,}}{}$ ^{9, 12} ), arachidonic (С ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{5,}}{}$ ^{8, 11, 14} ) or eicosatriene (C ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{8,}}{}$ ^{11, 14} ) acid. 21. A method for evaluating a test compound for its ability to treat a VCAM-1 mediated disease, comprising assessing the ability of this compound to inhibit the oxidation of a polyunsaturated fatty acid by the following steps: (a) exposing the polyunsaturated fatty acid with the test compound in vitro in solution in the presence of a substance that is known for its ability to oxidize a polyunsaturated fatty acid in the absence of a test compound; and (b) an assessment of the oxidation state of the polyunsaturated fatty acid. 22. The method according to item 21, wherein the specified oxidizing substance is selected from the group consisting of metal and enzyme. 23. The method according to item 22, wherein said oxidizing agent is an enzyme selected from peroxidase, lipoxygenase, cyclooxygenase and cytochrome P450. 24. The method according to item 22, wherein the specified oxidizing substance is copper. 25. A method for evaluating in vitro sensitization of vascular endothelial cells of a host to polyunsaturated fatty acids or their oxidized derivatives, comprising stimulating the host with a polyunsaturated fatty acid or an oxidized polyunsaturated fatty acid and comparing the resulting concentration of cell surface-bound or circulating VCAM-1 or other inflammatory mediator expressed by a redox-sensitive gene upon exposure to a polyunsaturated fatty acid or an oxidized polyunsaturated fatty acid slot, with that characterizing the population norm. 26. The method according A.25, characterized in that the specified polyunsaturated fatty acid is linoleic (C ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{9,}}{}$ ¹² ), linolenic (С ₁₈ Δ $\genfrac{}{}{0ex}{}{\text{6,}}{}$ ^{9, 12} ), arachidonic (С ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{5,}}{}$ ^{8, 11, 14} ) or eicosatriene (C ₂₀ Δ $\genfrac{}{}{0ex}{}{\text{8,}}{}$ ^{11, 14} ) acid. 27. The method according A.25, characterized in that the specified owner is a person. 28. The method according A.25, characterized in that the specified owner is an animal. 29. The method according A.25, characterized in that the specified host is a mouse. 30. The method according A.25, characterized in that the mediator is selected from the group consisting of IL-1β, MCP-1 and IL-6. 31. The method according A.25, characterized in that said mediator is a cytokine that initiates an immune response. 32. The method according A.25, characterized in that said mediator is a chemoattractant, which causes a promotion of the migration of inflammatory cells in the lesion. 33. The method according A.25, characterized in that the specified mediator is a growth factor. 34. The method according A.25, characterized in that said mediator is an adhesion molecule. 35. The method according A.25, characterized in that said mediator is a thrombin receptor. 36. The method according A.25, characterized in that it includes stimulating the host with an oxidized polyunsaturated fatty acid, and the oxidized polyunsaturated fatty acid is hydroperoxide or linoleic acid. 37. The method according A.25, characterized in that it includes stimulating the host with an oxidized polyunsaturated fatty acid, and the oxidized polyunsaturated fatty acid is arachidonic acid hydroperoxide.

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