Method of treating artherosclerosis and other cardiovascular and inflamatory diseases

The present invention relates to a process for the preparation of a pharmaceutical composition for the treatment of a disease mediated by VCAM-1, i.e. a composition for the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

The adhesion of leukocytes (white blood cells) to the endothelium is a basic, early phenomenon in a wide variety of inflammations, including atherosclerosis, autoimmune diseases, and bacterial and viral infections. Recruitment of leukocytes into the endothelium begins when the inducible receptors of the adhesion molecule on the surface of the endothelial cells react with their corresponding anti-receptors on immune cells. Which types of leukocytes (monocytes, lymphocytes or neutrophils) are recruited are determined by vascular endothelial cells by selective expression of specific adhesion molecules, such as vascular cell adhesion molecule - 1 (VCAM-1), intercellular adhesion molecule -1 (ICAM-1) and E-selectin. At the earliest stage of atherosclerotic injury, local expression of VCAM-1 on the endothelium and selective recruitment of mononuclear leukocytes expressing the VLA-4 integrin anti-receptor. Because selective VLA-4 expression occurs on monocytes and lymphocytes, but not on neutrophils, VCAM-1 plays an important role in regulating the selective adhesion of mononuclear leukocytes. As a result of the subsequent transformation of leukocytes into frothy macrophages, the synthesis of various inflammatory cytokines, growth factors and chemical attracting factors, which allow the spread of leukocyte and platelet recruitment, the proliferation of smooth muscle cells, activation of endothelial cells and extracellular matrix synthesis, characteristic of atherosclerotic plaque maturation.

VCAM-1 is expressed in the culture of human vascular endothelial cells after activation with lipopolysaccharide (LPS) and cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF-α). These factors are not selective for activating expression of cellular adhesion molecules. Scheme I depicts the process of cytokine activation of the YCAM-1 expression gene in vascular endothelial cells.

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Diagram I

l. Cytokine binds with its rprsntnrpni to ·?; - and ··, ·.

2. Activated t production receptor \. intracellular signal

3. Activation of the transcriptional regulatory roller

4. Displaced to the nucleus

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9. Posttranslational processing / 5. Specific binding / element strengthens] ΐ) ΝΑ on the promoter with VCAM-I

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10. Incorporation of VCAM-I into the membrane

Protein Translation

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.Treatment

-RNA

Cell membrane

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Regulation of cytokine activation of the gene expression cell vascular adhesion molecule - 1 (VCAM-1) by regulatory factors sensitive to oxidation and reduction reactions (redox reactions), such as - NF - kp, in vascular endothelial cells (IkB is an inhibitory subunit; NF - kβ is a nuclear factor - kB; NH3 is the amino terminus of the protein, and RNA Pol II is RNA polymerase II).

The molecular analysis of regulatory elements on the human VCAM-1 gene that controls its expression suggests that an important role in redox-sensitive VCAM-1 gene expression is played by the nuclear factor - kB (NF - kp), transcriptional regulatory factor, or NF - k β - similar binding protein. Transcription factors are proteins that activate (or inhibit) the expression of a gene inside a cell's nucleus by attaching to specific DNA sequences, called 'enhancers', usually located next to a region of the gene called the 'promoter' from which RNA synthesis begins. The nuclear factor - kB is a widely expressed multi-subunit transcription factor, activated in several types of cells by a wide and diverse group of inflammatory factors such as TNF-α and L-1p, bacterial endotoxins and RNA viruses. It plays a key role in mediating inflammatory and other stress signals to the nuclear regulatory system. Although unknown biochemical signals activating NF.-kB are unknown, this transcription factor can reduce to a single molecular pathway numerous risk factors and "causative" factors of atherosclerosis such as hyperlipidemia, smoking, hypertension and diabetes.

Importantly, the activation of NF-kB in vascular endothelial cells by various signals can be specifically inhibited by antioxidants such as N-acetylcysteine ​​and pyrrolidine dithiocarbamate (see USSN 07/969 934). The discovery of this phenomenon led to the hypothesis that oxygen radicals play an important role in the activation of NF-kB through an unspecified redox mechanism. Because the NF-kB-like enhancer also regulates the transcription of the VCAM-1 promoter in a manner related to redox sensitivity, oxidative stress in atherosclerotic injury may play a role in regulating the expression of the VCAM-1 gene through this transcriptional regulatory protein, sensitive to redox reactions.

It is believed that the modification of low density lipoprotein (LDL) to oxidatively modified LDL (ox-LDL) by reactive oxygen species is the main phenomenon,

180 874 causing atherosclerosis. Steinberg et al. N. Engl. J. Med. 1989; 320: 915-924. Oxidized LDL is a complex structure formed of at least several chemically distinct oxidized materials, each of which, alone or in combination, is capable of modulating the expression of a cytokine activated gene of an adhesive molecule. Fatty acid hydroxides, such as linoleyl hydroxide (13-HPODE), are produced from free fatty acids by the action of lipoxygenases and are an important component of oxidized LDL.

It is claimed that the generation of oxidized lipids is produced by the action of the cellular lipoxygenase system and that the oxidized lipids are then transferred to LDL. There is therefore a spreading reaction inside the LDL in the medium, catalyzed by transition metals and / or sulfhydryl compounds. Previous studies have shown that the modification of fatty acids in endothelial cell cultures can alter their susceptibility to oxidative trauma. The addition of saturated or monounsaturated fatty acids to endothelial cell culture reduces their susceptibility to oxidative injury, while the addition of polyunsaturated fatty acids (PUFA) increases this susceptibility to oxidative injury.

Using reverse phase HPLC analysis of native and saponified LDL lipid extracts, 13-HPODE was shown to be the dominant oxidized fatty acid in LD oxidized by activated human monocytes. Chronic exposure to oxidized LDL is an oxidative signal to vascular endothelial cells, probably via specific fatty acid hydroxide, which selectively increases the expression of the gene induced by the VCAM-1 cytokine.

Through a vague yet defined mechanism, the areas of the vascular wall, predisposed to atherosclerosis, preferentially capture circulating LDL. Through a completely unknown pathway, smooth muscle endothelial cells and / or inflammatory cells then cause LDL to ox-LDL. Unlike LDL, which is captured by the LDL receptor, monocytes greedily capture ox-LDL via a "scavenger" receptor, whose expression, unlike the LDL receptor, is not inhibited as intracellular lipid levels increase. Thus, monocytes still capture ox-LDL and transform into lipid-filled foam macrophage cells, producing a fat band.

Given that cardiovascular disease is currently the leading cause of death in the United States, and that 90% of cardiovascular disease is now recognized as atherosclerosis, there is a clear need to invent new methods and pharmaceuticals for treating these diseases. To achieve this goal, it is important to identify and manipulate specific oxidized biological compounds that act as selective regulators of the expression of inflammatory mediators, in particular, VCAM-1. The more general goal is to identify selective ways of inhibiting the expression of redox sensitive genes or activating redox sensitive genes that are inhibited.

The present invention relates to a process for the preparation of a pharmaceutical composition for the treatment of a disease mediated by VCAM-1 containing a known carrier and / or excipient that (i) it is determined whether this substance inhibits the oxidation of polyunsaturated fatty acid and then the degree of oxidation of polyunsaturated fatty acid is evaluated and then (ii) the active substance is combined, which inhibits the oxidation of polyunsaturated fatty acid in step (i) with a pharmaceutically acceptable carrier and / or substance auxiliary for oral, parenteral, subcutaneous, topical or local administration, the active substance which prevents or minimizes the oxidation of polyunsaturated fatty acid is present in an amount of 0.5 -500 mg / kg body weight.

This method determines a substance that selectively inhibits VCAM-1.

The composition according to the invention finds use in the treatment of a disease or disorder occurring in humans in which expression or inhibition of a gene sensitive to redox reactions is involved, and thus for the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

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Polyunsaturated fatty acids ("PUFA") and their hydroxides ("oxPUFA"), which are important components of oxidatively modified low density lipoproteins (LDL), have been found to increase VCAM-1 expression but do not increase expression of intracellular adhesive molecule-1 (ICAM) -1) neither E-selectin in human aortic endothelial cells, through a mechanism mediated by neither cytokines nor other non-cytokine signals. This is the basic discovery of an important and previously unknown biological pathway of immune responses mediated by VCAM-1.

Non-limiting examples of compounds that increase the expression of the VCAM-1 cell surface gene, but not ICAM-1 or E-selectin, are linoleic acid, linolenic acid, peanut acid, linoleyl hydroxide (13-HPODE) and arachidonic hydroxide (15-HPETE). Saturated fatty acids (such as stearic acid) and monounsaturated fatty acids (such as oleic acid) do not induce the expression of VCAM-1, ICAM-1 or E-selectin.

The induction of VCAM-1 by PUFA and their fatty acid hydroxides is inhibited by the antioxidant pyrrolidine dithiocarbamate (PDTC). This indicates that the mediation of induction is an oxidized signal molecule, and that induction does not occur when the oxidation of this molecule is blocked (i.e., oxidation will not occur), inverted (i.e., the signal molecule is reduced) or when interactions of this molecule are prevented. signal, modified by redox reactions, with its regulatory purpose.

Cells chronically exposed to higher than normal levels of polyunsaturated fatty acids or their oxidized counterparts may give rise to an abnormal immune response disproportionate to the current threat, leading to a disease state. Excessive sensitization of vascular endothelial cells to PUFA and ox-PUFA can accelerate the formation of, for example, atherosclerotic plaque.

Based on these findings, a method of treating atherosclerosis, angioplasty restenosis, coronary artery disease, angina pectoris and other cardiovascular diseases, as well as inflammatory diseases not involving the cardiovascular system, mediated by VCAM-1, is provided, this method being involves removing, reducing the concentration or preventing the production of oxidized polyunsaturated fatty acids, such as (but not limited to) oxidized linoleic acid (C₁₈ Δ⁹’¹²), linolenic (C18 Δ⁶·⁹·¹²), arachidonic (C20 δ⁵’8&gt;^π·1⁴) and eicosatriene (C₂₀ Δ ^⁴⁴).

Non-limiting examples of non-cardiovascular inflammatory diseases mediated by VCAM-1 are rheumatoid arthritis, osteoarthritis, bronchial asthma, dermatitis and multiple sclerosis.

This method is a significant advance in the treatment of cardiovascular diseases, because it goes beyond the currently used methods aimed only at inhibiting the progression of the disease, and with its proper use provides the opportunity to conservatively "cure" atherosclerosis by preventing the formation of new plaques and causing the disappearance of plaques already formed.

In an alternative embodiment, there is provided a method of inhibiting expression of a redox-sensitive gene or activating a gene inhibited in a redox-responsive pathway, comprising administering an effective amount of a substance that prevents signal oxidation, and typically, oxidation of polyunsaturated fatty acids. Exemplary redox-sensitive genes associated with the presentation of an immune response are, but are not limited to, genes expressing cytokines involved in initiating an immune response (e.g., IL-1 β), chemical attractants, facilitating the migration of inflammatory cells into damage sites (e.g. MCP-1), growth factors (e.g. IL-6 and thrombin receptor) and adhesion molecules (e.g. VCAM-1 and E-selectin).

Screening methods for detecting disorders mediated by VCAM-1 or a gene sensitive to redox reactions by quantifying surrogate disease markers are also provided. In one embodiment, the level of oxidized polyunsaturated fatty acid or other appropriate markers is assessed; in tissue or blood, for example, the host, as a means of assessing the "oxidative environment" of the host and

180 874 host sensitivity to diseases mediated by a gene sensitive to redox or VCAM-1 reactions.

In another embodiment, the level of VCAM-1 or other appropriate marker circulating or on the surface of the cell is quantified and its effect on the level of administration of the appropriate antioxidant.

In yet another embodiment, the sensitization of host vascular endothelial cells to polyunsaturated fatty acids or their oxidized counterparts is assessed. This can be achieved, for example, by exposing the host to PUFA or ox-PUFA and comparing the obtained concentration of VCAM-1 on the surface of the cell or circulating, or other surrogate marker with the population norm. In yet another embodiment, in vivo models for atherosclerosis or other heart disease or inflammatory diseases provided by VCAM-1 are mediated by administering excessive amounts of PUFA or oxidized polyunsaturated fatty acid to host animals to cause the disease. These animals can be used in clinical studies to further understand these disorders.

In yet another embodiment of the invention, the compounds are evaluated for their ability to treat disorders mediated by VCAM-1 based on their ability to inhibit oxidation of polyunsaturated fatty acid, or PUFA or ox-PUFA interactions with the target protein.

This can be achieved by exposing the host, for example a human or animal, e.g. mouse, to a high level of PUFA or ox-PUFA and then assessing the therapeutic effectiveness of the test compound based on its ability to reduce the concentration of VCAM-1 circulating or deposited on the surface of the cells. Instead, an in vitro screening test based on the ability of the test compound to prevent PUFA oxidation or PUFA or ox-PUFA interaction with the target protein may be used in the presence of an oxidizing substance such as a metal, e.g. copper, or an enzyme such as peroxidase, lipoxygenase, cyclooxygenase or cytochrome P-450.

In another embodiment, the vascular endothelial cells are exposed to TNF-α or other VCAM-1 inducing material for an appropriate period of time, and then damaged by appropriate means, e.g., sonication or freezing-thawing. Cytosol and membrane compartments are isolated. The radiolabeled PUFA is added to the specified number of compartments. The fluid's ability to convert PUFA to ox-PUFA is tested in the presence or absence of test compound. Instead of the damaged cell system, intact cells can be used.

Oral administration of pyrrolidine dithiocarbamate (PDTC) at a dose of 25-50 mg / kg / day dramatically inhibited atherosclerotic fat band formation, monocyte-macrophage inflammation of the arteries, expression of endothelial VCAM-1 and basically normalized endothelium-dependent relaxation function in diet-induced hypercholesterol rabbits cholesterol level was above 1000 mg / dl. At the same doses, other putative therapeutic agents, such as the antioxidants probucol and vitamin E, did not affect plaque formation in this model.

In an experimental atherosclerosis model, endothelium-dependent arterial relaxation is restored by administration of PDTC. In the rabbit model with diet-induced hypercholesterolemia, oral administration of PDTC at a dose of 25-50 mg / kg / day resulted in a return of endothelium-dependent vascular reactivity. This was determined by testing with aortic contraction ring taken from control animals and test animals. In patients with atherosclerosis, this phenomenon manifests as normalization of peripheral vascular responsiveness in response to congestion, as measured by non-invasive Doppler flow testing. This is a standardized, widely available and easy-to-perform study that can be used to select oral doses to achieve effective blood levels. PDTC acts as an anti-ischemic drug, quickly normalizing the pathological reduction of endothelium-dependent arterial vasodilatation characteristic of cardiovascular disease and atherosclerosis. This improvement in vascular blood flow is seen as an improvement in symptom and ischemia-restricted exercise capacity, and provides a non-invasive assessment of vascular protection. Other clinical signs of endothelium-dependent vasodilatation disorders include impotence.

The molecular regulatory system controlling the transcription of the VCAM-1 gene is a new transcription factor system formed of the p65 and p50 subunits of the NF-kB / Rel family, cross-linked with the c-fos and c-jun subunits of the AP-1 family. Both structure and functional studies have shown that these AP-1 factors play an important role in regulating the VCAM-1 promoter, which is likely to be crucial in the therapeutic regulation of VCAM-1 gene expression. For the first time, the functional role of this cross-linked transcription system in the regulation of endogenous gene has been demonstrated.

Short description of the drawings

Figure 1 is a plot of cell surface expression (OD 450 nm) of VCAM-1 in human aortic endothelial cells versus the number of hours of exposure to the TNF-α cytokine (painted circle); linoleic acid (painted triangle); and linoleyl hydroxide (13-HPODE, square painted); and in the absence of exposure to these substances (control, unpainted square).

Figure 2 is a plot of cell surface expression (OD 450 nm) of VCAM-1 in human aortic endothelial cells when exposed to linoleic acid (painted triangle); and linoleyl hydroxide (13-HPODE, square painted) depending on the concentration of fatty acid (μΜ).

Figure 3 is a column graph of cell surface expression (OD 450 nm) of VCAM-1, ICAM-1 and E-selectin in human aortic endothelial cells when exposed to TNF-α, stearic acid, oleic acid, linoleic acid; linolenic acid and arachidonic acid.

Figure 4 is a column graph of cell surface expression (OD 450 nm) of VCAM-1 in human aortic endothelial cells when exposed to linoleic acid, 13-HPODE, arachidonic acid, arachidonic acid hydroxide (15-HPETE) from (columns painted black) ) or without (hatched columns) with antioxidant pyrrolidine dithiocarbamate.

Figure 5 is an autoradiogram showing acute induction of VCAM-1 mRNA by linoleic acid and 13-HPODE. HAEC has been exposed or not to linoleic acid (7.5 μM), 13-HPODE (7.5 μM) or TNF-α (100 U / ml). Total RNA was isolated and fractionated by size denaturation, 1% agarose-formaldehyde gel electrophoresis, nitrocellulose transfer, and hybridization to labeled human³²P cDNA A) VCAM-1 - specific B) β-actin - specific. After washing, the filters were exposed to X-ray film at -70 ° C with one intensifying screen for 24 hours. Identification of bands: 1) control 2) linoleic acid (impact exposure, 8 hours) 3) linoleic acid (exposure 48 hours) 4) 13-HPODE (impact exposure, 8 hours) and 5) TNF-α (100 U / ml, 4-hour exposure).

Figure 6 is an autoradiogram showing that the induction of VCAM-1 mRNA by polyunsaturated fatty acids is not dependent on cellular protein synthesis. HAEC was exposed to linoleic acid (7.5 μM) or arachidonic acid (7.5 (M) in the presence or absence of cycloheximide (10 μg / ml) for 4 hours and then treated as described in Figure 5 .

Figure 7 is an autoradiogram showing that linoleic acid induces transcriptional activation of the VCAM-1 promoter via redox-sensitive NF-kB-like factor. HAEC was cleaved in such a way as to obtain about 60% confluence in 100 mm tissue culture plates. HAEC was transfected with 30 μg of the plasmid VCAMCAT p288, VCAMCAT p85 or pSV2CAT using calcium phosphate co-precipitation technology using standard methods. After a 24-hour regeneration period, HAEC was or not pretreated with 50 μM PDTC and after 30 minutes of exposure to linoleic acid (7.5 μΜ) or TNF-α (100 U / ml), added directly to the plates. After 18 hours, cell 8 extracts were made

180 874 EC by rapid freeze-thaw in 0.25 M Tris, pH 8.0. Proteins from individual cell extracts were tested for chloramphenicol acetyltransferase (CAT) activity as previously described (Ausubel, 1989) (Ac, acetylated, N, non-acetylated chloramphenicol).

Figure 8 shows an acrylamide gel plate showing that polyunsaturated fatty acids activate NF-kB - similar DNA binding activity blocked by the PDTC antioxidant. Confluent HAEC in medium containing 4% FBS (as described in Figure 1) was or was not subjected to PDTC pre-treatment (50 pM) for thirty minutes followed by three hours exposure to linoleic acid (7.5 pM), respectively. oleic acid (7.5 pM) or TNF-α (100 U / ml). Five micrograms of the nuclear extract were incubated with labeled double-stranded wtVCAM-1 ³²P, fractionated by size on a 4% native acrylamide gel, and exposed to an autoradiographic film at -70 ° C for 18 hours. Two bands A and C were determined, corresponding to NF-kB-like activity. Weak B-band was observed in control (untreated) cells.

Figures 9A and 9b are column charts of relatively thiabarbituric acid reactive substances (OD 532 nm) of arachidonic acid and 15-HPETE in the presence or absence of PDTC. The thiabarbituric acid (TBARS) reactivity test measures the oxidative capacity of a material in a cell-free, nutrient-free environment.

Figure 10 shows an mRNA autoradiogram obtained as described below hybridized to 32p labeled VCAM-1 specific cDNA (Plate A), E-selectin specific cDNA (ELAM-1) (Plate B) or ICAM-1 specific cDNA (Plate C). After a 30-minute pretreatment of 50pM sodium pyrrolidine dithiocarbamate (PDTC), HUVE cells (human umbilical vein cells) were treated with IL-1b (10 U / ml) in the constant presence of 50 pM PDTC. Total RNA was isolated and fractionated at the designated time points due to the size of 20 pg of material by denaturing electrophoresis on a 1% agarose-formaldehyde gel, transfer to nitrocellulose, hybridization as described above, and visualization by autoradiography. Band 1 hour 0, Band 2,4,6, 8, - only OL-1 for 2,4, 8 and 24 hours respectively. Bands 3, 5, 7.9 - IL-1 and PDTC for 2, 4, 8 and 24 hours, respectively.

Figure 11 shows an autoradiogram of mRNA obtained as described above hybridized to labeled ³2P, VCAM-1 specific cDNA (Column A), E-selectin specific cDNA (ELAM-1) (Column B) or ICAM-1 specific cDNA (Column C). HUVE cells were pretreated with the indicated PDTC concentrations followed by IL-1b in the presence of PDTC for four hours, and tested for accumulation of VCAM-1 mRNA by filter hybridization using Northem. Band 1-control, band 2-IL-1 (10 u / ml), band 3-IL-1b + PDTC (0.05 pM), band 4-IL-1 LB + PDTC (0.5 pM), band 5- IL-1b + PDTC (5.0 pM), band 6 - IL-1b + PDTC (50.0 pM), band 7 - IL-1b + PDTC (100 pM).

Figure 12 is an autoradiogram of mRNA obtained as described above hybridized to 32p labeled, VCAM-1 specific cDNA (Column A), E-selectin specific cDNA (ELAM-1) (Column B) or ICAM-1 specific cDNA ( Column C). HUVE cells were pretreated as described in Figure 9, 50 pM PDTC, exposed for four hours to the agents indicated below, and tested for the accumulation of mRNAVCAM-1 (Column A) and ICAM-1 (Column B). Band 1 - TNFa (100 U / ml), band 2 - TNFa + PDTC, band 3 - lipopolysaccharide (LPS) (100 ng / ml), band 4 - LpS + PdTc, band 5 - poly (I: C) (100 mg / ml), band 6 - poly (I: C) + PDtC.

Figure 13 is a graph of the relative expression of VCAM-1 and ICAM-1 on the cell surface in the presence (dark columns) or absence (light columns) of PDTC and in the presence of various types of inducing stimuli. Confluent HUVE was or not pretreated (cTl only) for 30 minutes with 50 pM PDTC, and then exposed for specific periods to specific agents in the presence or absence (CTL only) of PDTC. Cell surface expression was determined by primary binding to mouse monoclonal antibodies specifically directed against VCAM-1 (4B9) and ICAM-1 (84H10) followed by binding.

180 874 secondary with horseradish peroxidase-labeled goat anti-mouse (IgG) antibodies. Quantification was performed by calorimetric conversion at 450 nm TMB. Figure 13 indicates that multiple regulatory signals by induction of VCAM-1, but not ICAM-1, in human vascular endothelial cells via a common dithiocarbamate-sensitive pathway.

Figure 14 is a graph of the relative expression of VCAM-1 cell surface (OD 595 nm) in human umbilical vein endothelial cells activated by TNFα versus the concentration of various antioxidants. (PDTC is the sodium salt of N-pyrrolidine dithiocarbamate; DETC is sodium N, N-diethyl-N-carbodithiolate, also called sodium diethyldithiocarbamate, NAC is N-acetylcysteine, and DF is desferoximin).

Figure 15 is a graph of the relative expression of cell surface VCAM-1 (OD 595 nm) in human umbilical vein endothelial cells activated by TNFα in the presence of a specified amount of various antioxidants. (PDTC is the sodium salt of N-pyrrolidine dithiocarbamate; DIDTC is the sodium N, N-diethyl-N-carbodithiolate, SarDTC is the sodium N-methyl-N-carboxymethyl-N-carbodithiolate; IDADTC is the N, N-di (carboxymethyl) -N trisodium carbodithiolate, MGDTC is sodium N-methyl-D-glucamino-N-carbodithiolate, MeOBGDTC is sodium N- (4-methoxybenzyl) -D-glucamine-N-carbodithiolate; DEDTC is sodium, N-diethyl-N-carbodithiolate ; Di-PDTC is sodium N, N-diisopropyl-N-carbodithiolate, NAC is N-acetylcysteine).

Figure 16 is a graph of the percentage of Molt-4 cells binding to HUVE cells after or without stimulation with TNFα (100 U / ml) for six hours in the presence or absence of PDTC.

Figure 17 illustrates the chemical structure of the following active dithiocarbamates: sodium pyrrolidine-N-carbodithiolate, N-methyl-N-carboxymethyl-N-carbodithiolate, N, N-di (carboxymethyl) -N-trisodium trisodium, N-methyl-D-glucamino Sodium N-carbodithiolate, sodium N, N-diethyl N-carbodithiolate (sodium diethyldithiocarbamate) and sodium N, N-diisopropyl N-carbodithiolate.

Figure 18 is a column graph of the effect of PDTC on the formation of BSA and 13-HPODE fluorescent addition compounds, as measured in fluorescent units, relative to micromolar PDTC concentration. One micromole of 13-HPODE was incubated with 200 BSA microns in the presence of PDTC for six days. Fluorescence was measured at 430-460 nm with excitation at 330-360 nm.

Figure 19 is a graph of the effect of PDTC on the formation of BSA and ox-PUFA fluorescent addition compounds as a function of wavelength (nm) and PDTC concentration. As the PDTC increases, the content of fluorescent addition compounds decreases.

Figure 20 is a graph of the effect of PDTC on LDL oxidation by horseradish peroxidase (HRP), measured by an increase in OD (234 nm) versus time (minutes) for varying PDTC concentration. It was found that after the incubation period, PDTC inhibits LDL oxidation by HRP in a concentration-dependent manner.

Figure 21 is a graph of the effect of PDTC on cytokine-induced ox-PUFA formation in human aortic endothelial cells. As indicated, both TNF-α and IL-1B cause oxidation of linoleic acid to oxylinolic acid. PDTC clearly reduces oxidants.

Detailed description of the invention

I. Definitions

As used herein, the term "polyunsaturated fatty acid" (also called "PUFA") means fatty acid (typically C₈ to C.₂₄) having at least two alkenyl bonds; Linoleic acid belongs to this group, but is not limited to them (Cj8 A⁹’¹²), linolenic (C18 A^6&gt;9¹²), arachidonic (C28 ^^5,8’^11,14) and eicosatriene (C₂q A8^{i1&gt; 14}). The term "oxidized polyunsaturated fatty acid" means an unsaturated fatty acid in which at least one alkenyl bond has been converted to peroxide. Non-limiting examples are:

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13-HPODE

ΌΟΗ

15 HPET

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OOH '' ΠΟΗ

The term "alkyl" as used herein, unless otherwise defined, means saturated hydrocarbon C_tto C.₁₀, straight, branched or cyclic (in the case of C hydrocarbons₅ or higher) (or lower alkyl, e.g. C to C₅; this group includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutynyl ). The alkyl group may be optionally substituted at any of the carbon atoms with one or more residues selected from the group consisting of hydroxy, amino and amino with one or two substituents, wherein the substituent is independently alkyl, aryl, alkoaryl or aroalkyl; aryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonium acid, phosphate, or phosphonate, unprotected or protected, if necessary, as known to those skilled in the art, and as described in, for example, Greene et al., "Protective Groups in Organic Synthesis ”, John Wiley and Sons, 2nd Edition, 1991.

The term "alkenyl", as used herein, unless otherwise specified, means hydrocarbon C₂ to C ^, with a straight, branched or cyclic chain, with at least one double bond.

The term "alkynyl", as used herein, unless otherwise specified, means hydrocarbon C₂ to C.₁₀, with a straight or branched chain, with at least one triple bond.

The term "aralkyl" means an aryl group with at least one alkyl substituent.

The term "alkaryl" means an alkyl group with at least one aryl substituent.

The term "halogen" (alkyl, alkenyl or alkynyl) means an alkyl, alkenyl or alkynyl group in which at least one of the hydrogen atoms in the group has been replaced by a halogen atom.

The term "aryl" as used herein, unless otherwise defined, means a phenyl, biphenyl or naphthyl group, preferably a phenyl group. The aryl group may be optionally substituted with one or more residues selected from the group to which the alkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate or phosphonate, CO2H or its group a pharmaceutically acceptable salt, CO2 (alkyl, aryl, alkaryl or aroalkyl) or glucosamine, unprotected or protected as necessary, as is known to those skilled in the art, and as described, for example, by Greene et al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991.

The term "alkoxy" as used herein, unless otherwise defined, means a -O-alkyl structure particle.

The term "acyl", as used herein, means a group of formula C (O) R 'wherein R' is an alkyl, aryl, alkoaryl or aroalkyl group.

The term "heteroaryl or heteroaromatic group," as used herein, means an aromatic moiety containing at least one sulfur, oxygen or nitrogen atom in the aromatic ring. The non-limiting, non-limiting, the non-limiting, the group is defined for the benzyl, carbozolil, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isoxazolyl, pyrrolyl, pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, quinoxalinyl, xantinyl, hypoxantinyl, pteridinyl, 5-azacytidinyl, 5-azauracylyl, triazolopyridinyl, imidazolopyridinyl, pyrinopyridinyl, pyrrolidinyl⁶-alkylpurine, N⁶-benzylpurine, N6-halogenopurine, N6-vinylpurine, N6-acetylene purine, N6-acylpurine, N6-hydroxyalkylpurine, N6-thioalkylpurine, thymine, cytosine, 6-azapyrimidine, 1-mercaptopyr^s-alkylpyrimidine, N⁵-benzylpyrimidine, N5-halogenopyrimidine, N5-vinylpyrimidine, N5-acetylenepyrimidine, N5-acylpyrimidine, N⁵-hydroxyalkylpurine and N6-thioalkylpurine, and isoxazolyl. The heteroaromatic group may be optionally substituted as described above for an aryl group. The heteroaromatic ring may be fully or partially hydrogenated if it is preferred. Instead of pyridine, e.g. dihydropyridine may be used, this example is not limiting. Oxygen and nitrogen functional groups can be protected, if necessary or preferred, during the reaction sequence. Suitable protecting groups are well known to those skilled in the art and are trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl, tritylmethyl, alkyl, acyl, e.g. acetyl or propionyl, methylsulfonyl and

The term "hydroxyalkyl", as used herein, means a C, to C6 alkyl group in which at least one of the hydrogen atoms attached to any carbon atom has been replaced by a hydroxyl group.

The term "thiol antioxidant" means a sulfur-containing oxidation retarding compound.

The term "pharmaceutically acceptable derivative" means a derivative of an active compound which, when administered to the body, is capable of releasing, directly or indirectly, the parent compound, or which has activity by itself.

The term "pharmaceutically acceptable cation" means a positively charged organic or inorganic particle that can be administered in conjunction with a pharmaceutical agent, for example, as a salt countercation. Pharmaceutically acceptable cations are known to those skilled in the art and are, but are not limited to, sodium, potassium and quaternary amines.

The term "physiologically cleavable leaving group" means a particle that can be cleaved in vivo from the molecule to which it is attached and is, but is not limited to, an organic or inorganic anion, an acyl group (including but not limited to limited to it, (alkyl) C (O), e.g. acetyl, propionyl and butyryl), alkyl, phosphate, sulfate or sulfonate.

The term "enantiomerically enriched composition or compound" means a composition or compound containing at least 95%, preferably at least 97, 98, 99 or 100% by weight of a single enantiomer of a compound.

The term "amino acid" means synthetic and naturally occurring amino acids, including, but not limited to, alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl serinyl threoninyl, cysteinyl glutaminyl, aspartoyl glutaoyl, lysinyl, argininyl and histidinyl.

The term "connecting moiety" as used herein, means any divalent group connecting two chemical residues, including, but not limited to, an alkyl, alkenyl, alkynyl aryl, polyalkyleneoxy (e.g. - [(CH₂)_nABOUT-]_n-), -C, _6alkoxy-C,.,₀alkyl-, C.₁.6alkilotio-C.,₀alkyl-, -NR³-, and - ^ (C'HOH)_nCH₂OH, in which n can be independently 0, 12, 3, 4, 5 or 6.

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II. Identification of kdec ^ ioutycl · and non-uenuinated oolylons; nc to be used as direct mediators of VCAM-1 expression

To determine whether PUFAs or oxidized PUFAs act as a direct immunomodulator of expression of the endothelial cell gene, early-passaged human aortic endothelial cells (HAEC) were cultured for eight hours in media and serum and exposed to saturated fatty acids (stearic acid), monounsaturated (oleic acid) and polyunsaturated (linoleic and arachidonic acid); as well as the hydroxides of linoleic (13-HPODE) or arachidonic (15-HPETE) fatty acids. HAEC has also been exposed instead to the tumor necrosis factor-α cytokine.

HAEC was exposed to linoleic acid or 13-HPODE for various periods of time, up to 48 hours, and then tested for V CAM-1 expression on the cell surface by ELISA. The results were compared with those obtained with HAEC exposure to the TNF-α cytokine (100 U / ml) for the same periods of time. Expression of VCAM-1 in HAEC incubated with linoleic acid or 13-HPODE is temporarily increased. Expression peaks after about 8-9 hours, significant expression is found after 24 hours, and then decreases after 48 hours. The kinetics of VCAM-1 induction by both linoleic acid and 13-HPODE is very similar to TNF-α kinetics, and therefore the mechanisms by which polyunsaturated fatty acids induce VCAM-1 appear to be similar to TNF-α mechanisms.

A dose response study of linoleic acid and 13-HPODE on VCAM-1 gene expression after 8 hours was also performed. 7.5 μM was found to be the lowest peak dose at which linoleic acid and 13-HPODE significantly induce VCAM-1 gene expression.

It was then examined whether short-term incubation of endothelial cells with polyunsaturated fatty acids also induces the expression of ICAM-1 and E-selectin. Polyunsaturated linoleic and arachidonic fatty acids have been found to induce gene expression on the cell surface to a value of about 59% of TNF-induced VCAM-1 gene expression. However, it was found that neither ICAM-1 nor E-selectin were induced by these fatty acids. In contrast, saturated fatty acid - stearic acid - and monounsaturated fatty acid - oleic acid - did not induce the expression of VCAM-1, ICAM-1 or E-selectin. VCAM-1 gene expression was also observed after 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, induces VCAM-1 through a mechanism sensitive to redox reactions, HAEC has been pretreated with antioxidant pyrithinium dithiocarbamate (PDTC, 50 μM) for 30 minutes, and then cells were incubated independently with linoleic acid, arachidonic acid, 13-HPODE and 15-HPETE (7.5 (M) each for 8 hours). It was found that PDTC inhibited the expression of the VCAM-1 gene induced by polyunsaturated fatty acids and their oxidized counterparts. This indicates that the mediation of induction is an oxidized signal molecule, and that induction does not occur when the oxidation of the molecule is blocked (i.e., oxidation does not occur), inverted (i.e., the signal molecule is reduced) or when it is prevented from interacting with it target protein, perhaps via the redox system. To determine whether the selective induction of VCAM-1 by PUFA and their oxidized metabolites occurs at the level of mRNA, HAEC was incubated with linoleic acid or 13-HPODE. Linoleic acid and 13-HPODE induced the accumulation of VCAM-1 mRNA, similar to that induced by TNF-α. In contrast, no induction of ICAM-1 gene expression or E-selectin at the level of mRNA in HAEC incubated with linoleic acid or 13-HPODE was found. These results are similar to those observed at the cell surface level. They indicate that pre-transcriptional regulatory mechanisms act as a mediator for the induction of VCAM-1 gene expression by polyunsaturated fatty acids and their oxidized metabolites. It was also necessary to determine whether polyunsaturated fatty acids act as a primary signal or via a cytokine regulatory protein

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IL-4 in induction of VCAM-1 gene expression. To investigate whether newly produced proteins, such as IL-4, are involved in the production and expression of the PUFA-induced VCAM-1 gene, such as linoleic acid, HAEC was incubated with 13-HPODE (7.5 pM) and exposed to protein synthesis inhibitor, cycloheximide. No inhibition of VCAM-1 mRNA accumulation by cycloheximide was found in HAEC incubated with 13-HPODE. IL-4 production by HAEC incubated with linoleic or arachidonic acids and their oxidized metabolites was also measured by ELISA. No increase in IL-4 secretion by HAEC was incubated with these PUFAs or their oxidized metabolites.

Previous studies have shown, through studies on deletion and heterologous promoters, that cytokines and non-cytokines activate the expression of the VCAM-1 gene in endothelial cells at least partly in a transcriptional way through two NF-kB - similar DNA binding elements. PDTC has also been shown to inhibit the expression of the VCAM-1 gene by a redox-sensitive factor NF-kB - like. To determine whether polyunsaturated fatty acids induce transcriptional activation of the human VCAM-1 promoter in a similar mechanism, transient transfection of the chimeric p288 VCAM-CAT reporter gene containing coordinates 288 to +22 of the human VCAM-1 promoter was performed. The addition of linoleic acid (7.5 pM) increased the activity of the VCAM-1 promoter, which was twice the control activity and was about 60% of the maximum signal induced by TNF-α. Similar results were obtained using the minimal cytokine-inducible VCAM-1 gene promoter (p85 VCAM-CAT) containing -77 and -63 bp-like sites. Neither linoleic acid nor TNF-α showed any effect on the activity using the constitutively expressed pSV sequence₂ CAT. PDTC inhibited the transcriptional activation of both VCAM-1 promoter sequences induced by linoleic acid. Data indicate that, analogously to TNF-α, polyunsaturated fatty acids such as linoleic acid induce transcriptional activation of VCAM-1 through a redox-sensitive NF-kB-like mechanism.

To determine whether polyunsaturated fatty acids and their oxidized metabolites regulate the activity of the VCAM-1 promoter through NF-kB - a similar transcriptional regulatory factor, HAEC nuclear extracts were tested for DNA binding activity to a double-stranded oligonucleotide containing NF -kB - similar promoter elements V CAM-1, located at -77 and -63. As shown in fig. 7, two bands A and C, showing NF-kB-like activity, were induced in response to three-hour exposure to linoleic acid (7.5 pM). Similar results were obtained after exposure to the TNF-α cytokine (100 U / ml). Weak band B was found in control (untreated) cells. No induction of NF-kB binding - similar to monounsaturated fatty acid - oleic acid was found. An initial thirty-minute treatment of PDTC cells inhibited the binding activity of the DNA complex A and C after activation with linoleic acid. These results are similar to previously published observations that PDTC blocks the activation of VCAM-1 gene expression in HUVEC by inhibiting the activation of these NF-kB - similar DNA binding proteins.

Example 1. 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 seeded in a 96-well plate and incubated with linoleic acid (7.5 pM), 13-HPODE (7.5 pM) or TNF-α (100 U / ml) at five different time points to 48 hours. HAEC, obtained from Clonetics (Boston, mA), were cultured on Medium 199 medium supplemented with 20% fetal bovine serum (FBS), 16 U / ml heparin, 10 U / ml epidermal growth factor, 50 jogml of endothelial growth supplement, 2 mM L-glutamine, 100 U / ml penicillin, and 100 pg / ml streptomycin. One day before the experiment, the cells were placed in medium containing 4% FBS. Confluent HAEC was incubated for up to 48 hours with TNF-α (100 U / ml), or stearic, oleic, linoleic, linolenic or arachidonic (7.5 pM) acids. A similar experiment was conducted with different doses of linoleic acid or 13-HPODE for 8 hours (1-60 pM) (Figure 2). Quantification was done by determining the colorimetric conversion at 450 nm TMB. The experiments were carried out in triplicate (n = 3 for each experience value14)

180 874 total). * - value different (p &lt;0.05) from Control. As shown in Figure 1, both linoleic acid and 13-HPODE induced VCAM-1 expression. Ten hours after exposure, the amount of VCAM-1 on the cell surface induced by linoleic acid and 13-HPODE was above half the value induced by the TNF-α cytokine.

As shown in Figure 2, the induction of VCAM-1 by linoleic acid and 13-HPODE is concentration dependent. At a concentration of these compounds from 2 to 10 pM, a sharp increase in the amount of VCAM-1-induced cell surface is observed, which then remains approximately constant to a concentration of at least 100 pM. It should be noted that the PUFA concentration indicated in Figure 2 is added to the endogenous concentration found in HAEC.

Example 2. Polyunsaturated fatty acids induce the expression of the VCAM-1 gene, but not ICAM-1 or E-selectin

The expression of VCAM-1, ICAM-1 and E-selectin on the HAEC cell surface was measured by ELISA. HAEC obtained from Clonetics (Kahfomia) were grown in Medium 199 with the addition of 20% fetal bovine serum (FBS), 16 U / ml heparin, 10 U / ml epidermal growth factor, 50 pg / ml growth additive for endothelial cells, 2 mM L -glutamine, 100 U / ml penicillin, and 100 pg / ml streptomycin. One day before the experiment, the cells were placed in medium containing 4% FBS. Confluent HAEC was incubated or not incubated for 8 hours with TNF-α (100 U / ml), or one of the acids; stearic, olein, linoleic, linolenic or arachidonic (7.5 pM). Expression on the cell surface of A) VCAM-1B) ICAM-1 and C) E-selectin was determined by primary binding to murine antibodies specifically directed against VCAM-1, ICAM-1 and E-selectin, followed by secondary binding to labeled peroxidase horseradish goat anti-mouse antibodies (IgG).

Quantification was performed by determining the colorimetric conversion at 450 nm TMB. The experiments were carried out in triplicate (n = 4 for each experimental value). * - value different (p &lt;0.05) from Control.

As shown in Figure 3, linoleic acid, linolenic acid and arachidonic acid significantly induced VCAM-1 expression but did not induce cell surface expression of ICAM-1 or E-selectin. Neither stearic acid nor oleic acid induced the expression of VCAM-1, ICAM-1 or E-selectin '. TNF-α strongly induced the expression of all three cell surface molecules.

Example 3. The antioxidant PDTC inhibits VCAM-1 induction induced by polyunsaturated fatty acids and their oxidized metabolites

Confluent HAEC was pretreated in the presence or absence of PDTC (sodium pyrrolidine dithiocarbamate, 50pM) for thirty minutes. Cells were then incubated for eight hours with TNF-α (100 U / ml), linoleic or arachidonic acid (7.5 pM); or 13-HPODE (7.5 pM) or 15-HPETE (7.5 pM) fatty acid hydroxides. Cell surface expression of VCAM-1 was measured by ELISA, as described in Example 1. The experiments were carried out in triplicate (n = 4 for each experimental value). * - value different (p &lt;0.05) from Control.

As shown in Figure 4, PDTC inhibits VCAM-1 induction by linoleic acid, 13-HPODE, arachidonic acid and 15-HPETE.

Example 4. Acute induction of VCAM-1 mRNA by linoleic acid and 13-HPODE

HAEC was exposed to linoleic acid (7.5 pM) or 13-HPODE (7.5 pM). Total RNA was isolated, 20 pg divided into fractions by size by denaturation, electrophoresis on a 1% agarose-formaldehyde gel, transfer to nitrocellulose and hybridization to human, labeled³²P cDNA A) VCAM-1 - specific B) β-actin - specific, and visualization by autoradiography was performed. After washing, the filters were exposed to X-ray film at -70 ° C with one intensifying screen for 24 hours. Identification of bands: 1) control 2) linoleic acid (impact exposure, 8 hours) 3) linoleic acid (exposure 48 hours) 4) 13-HPODE (impact exposure, 8 hours) and 5) TNF-α (100 U / ml), 4-hour exposure).

180 874

As shown in Figure 5, both linoleic acid and 13-HPODE induced VCAM-1 mRNA production for eight hours. After 48 hours, linoleic acid no longer induced VCAM-1 mRNA.

Example 5. The induction of VCAM-1 mRNA by PUFA does not depend on the synthesis of bi but k cells

HAEC was exposed to linoleic or arachidonic acid (7.5 pM) in the presence or absence of cycloheximide (10 pg / ml) for a period of 4 hours. Total RNA was isolated, 20 pg divided into fractions by size by denaturation, electrophoresis on a 1% agarose-formaldehyde gel, transfer to nitrocellulose and hybridization to human, labeled³2P cDNA A) VCAM-1 - specific B) β-actin - specific, and visualization by autoradiography was performed. After washing, the filters were exposed to X-ray film at -70 ° C with one intensifying screen for 24 hours.

As shown in Figure 6, induction of VCAM-1 by linoleic and arachidonic acids does not depend on cellular protein synthesis.

Example 6. Linoleic acid induces transcriptional activation of the VCAM-1 promoter through redox sensitive NF-kB factor - similar

HAEC was cleaved in such a way as to obtain about 60% confluence in 100 mm tissue culture plates. HAEC was transfected with 30 pg of the plasmid VCAMCAT p288, VCAMCAT p85 or pSV2CAT using calcium phosphate co-precipitation technology using standard methods. After a 24-hour recovery period, HAEC was pretreated with 50 pM PDTC and after 30 minutes exposure to linoleic acid (7.5 pM) or TNF-α (100 U / ml), added directly to the plates. After 18 hours, cell extracts were prepared by rapid freeze-thaw in 0.25 M Tris, pH 8.0. Proteins from individual cell extracts were tested for chloramphenicol acetyltransferase (CAT) (Ac, acetylated, N, non-acetylated chloramphenicol). Fig. 7 shows the results of this experiment. Linoleic acid induces transcriptional activation of the VCAM-1 promoter through a redox-sensitive factor NF-kB - similar. These results are similar to those observed when the VCAM-1 promoter is activated by cytokines such as TNF-α. This suggests that PUFAs act through the oxidized intermediate, which is also a mediator of V CAM-1 cytokine activation.

Example 7. Polyunsaturated fatty acids activate NF-kB - similar DNA binding activity blocked by the PDTC antioxidant

Confluent HAEC in medium containing 4% FBS (as described in Example 1) was pretreated with PDTC (50 pM) for thirty minutes, followed by three hours of exposure to linoleic or oleic acid (7.5 pM), or TNF-α (100 U / ml). Five micrograms of the nuclear extract were incubated with 32V labeled double-stranded VCAM-1, fractionated by size on a 4% native acrylamide gel, and exposed to autoradiographic film at -70 ° C for 18 hours. Two bands A and C were determined, corresponding to NF-kB-like activity. Weak band B was observed in control (untreated) cells. Figure 8 shows that linoleic acid induces NF-kB - similar binding activity to the VCAM-1 promoter in a manner sensitive to redox reactions. This is analogous to the TNF-α cytokine and suggests a similar mechanism of action. TNF-α probably induces VCAM-1 through a mechanism mediated by ox-PUFA.

Example 8. Oxidation in a cell-free, nutrient-free system by non-oxidized and oxidized (15-HPETE) arachidonic acid

Figures 9A and 9B are column charts of relatively thiabarbituric acid reactive substances (OD 532 nm) of arachidonic acid and 15-HPETE in the presence or absence of PDTC. The thiabarbituric acid (TBARS) reactivity test measures the oxidative capacity of a material in a cell-free, nutrient-free environment. As shown in the figures, both arachidonic acid and 15-HPFTE exhibit pronounced TBARS activity, inhibited by PDTC.

180 874

III. The method of lacerations with mediators which is OCAM-1

The discovery that polyunsaturated fatty acids and their oxidized metabolites are selective, redox-sensitive immunomodulators provides the basis for the treatment of disorders mediated by VCAM-1 or genes sensitive to redox reactions.

A method is provided for the treatment of atherosclerosis, post-angioplasty restenosis, coronary artery disease, angina pectoris and other cardiovascular diseases as well as inflammatory diseases not involving the cardiovascular system mediated by VCA'M-1, which method involves the removal of , reducing the concentration or preventing the production of oxidized polyunsaturated fatty acids, e.g. (but not limited to) oxidized linoleic, linolenic and arachidonic acid. In an alternative embodiment, there is provided a method of treating these diseases by preventing the interaction of PUFA or ox-PUFA with a protein or peptide mediating the expression of VCAM-1.

Inhibition of VCAM-1 expression can be achieved in a variety of ways, including by administering an antioxidant that prevents oxidation of polyunsaturated fatty acid, by modifying ib vivo metabolism of PUFA to ox-PUFA as detailed below. l. podewebir counterweight

Any ox-PUFA reducing or PUFA inhibiting compound that is relatively non-toxic and bioavailable, or which can be modified to ensure its bioavailability can be used in this method of treatment. The skilled person will easily determine by standard methods whether a given compound reduces ox-PUFA or inhibits PUFA oxidation.

Pkzód and zlobiedzie eitiokekńokcylebowr

Dithiocarboxylates have been found to have beneficial effects 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 inhibit the expression of a redox sensitive gene or to activate a inhibited gene on a redox sensitive pathway.

At least one of these compounds, pyrrolidine dithiocarbamate (PDTC) inhibits the expression of the VCAM-1 gene at less than 1.0 micromolar. These compounds also show beneficial toxicity to proliferating or abnormally dividing vascular smooth muscle cells. Another dithiocarbamate, N-methyl-N-carboxymethyl-N-carbodithiolate, also inhibits VCAM-1 expression, but does not show beneficial toxicity against abnormally dividing vascular smooth muscle cells.

Pyrrolidine dithiocarbamate has not been found to significantly inhibit ICAM-1 or ELAM-1 expression, so treatment with this compound does not adversely affect aspects of the inflammatory response mediated by ELAM-1 or ICAM-1. Thus, generalized immunosuppression is avoided. In this way, systemic complications that result from the generalized inhibition of adhesion molecules in a number of other cells known to express these molecules are avoided. Other pharmaceutically acceptable salts of PDTC are also effective agents for the treatment of cardiovascular and inflammatory diseases.

Dithiocarbamates are transition metal hepatics used in the clinic for heavy metal poisoning. Baselt, RC, FWJ Sunderman et al. (1977), "Comparisons from antidotal efficacy of sodium diethyldithiocarbamate, D-penicillamine and triethylenetetramine upon acute toxicity of nickel carbonyl in rats." Res Commun Chem Pathol Pharmacol 18 (4): 677-88; Menne, T. and K. Kaaber (1978), "Treatment of pompholyx due to nickel allergy with chelating agents". Contact Dermatitis (4 (5): 289-90; Sunderman, FW (1978), "Clinical response to therapeutic agents in poisoning from mercury vapor" Ann Clin Lab Sci 8 (4): 259-69; Sunderman, FW (1979) , "Efficacy of sodium diethylodithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning." Ann Clin Lab Sci 9 (1): 1-10; Gale, GR, AB Smith et al., (1981), "Diethylodithiocarbamate in treatment of acute cadmium poisoning ", Ann Clin Lab Sci 11 (6): 476-83; Jones, MM and MG Cherian (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 diethylodit 180,874 hiocarbamate and EDTA as antidotes for acute cadmium intoxication", Res Commun Chem Pathol Pharmacol 38 (2): 271-8; Pages, A., JS Casas et al. (1985), "Dithiocarbamates in heavy metal poisoning: complexes of N, N-di (1-hydroxyethyl) dithiocarbamate with Zn (II), Cd. (II), Hg (II), CH₃Hg (II) and C.₆H₅And Ig (II). : J. Inorg Biochem 25 (1): 35-42; Tandon, SK, NS Hashmi et al. (1990), "The lead-chelating effects of substituted dithiocarbamates". Biomed Environ Sei 3 (3): 299-305.

Dithiocarbamates are also used as an additional agent in cisplatin chemotherapy to prevent nephrotoxicity. Hacker, MP, WB Ershler et al (1982), "Effect of disulfiram (tetraethylthiuram disulfide) and diethyldithiocarbamate 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 reactions in vivo - an overview". Radiat. Environ. Biophys. 29 (4): 249-62.

The dithiocarbamate currently used to treat alcohol abuse is disulfiram, diethyldithiocarbamate dimer. Disulfiram inhibits aldehyde dehydrogenase in the liver. Inoue, K. and Fukunaga, et al., (1982) "Effect of disulfiram and its reduced metabolite, diethyldithiocarbamate on aldehyde dehydrogenase of human erythrocytes", Life Sci 30 (5): 419-24.

There are reports that dithiocarbamates inhibit HIV replication as well as accelerate the maturation of specific T cell subpopulations. This discovery gave rise to clinical trials of dithiocarbamate in the AIDS population. Reisinger, E. et al. (1990), "Inhibition of HIV progression by dithiocarb.", Lancet 335: 679.

Dithiocarboxylates are compounds of formula A-SC (S) -B, belonging to the general class of compounds known as thiol antioxidants, and are otherwise known as carboditiols or carboditiolates. The -SC (S) particle appears to be essential for therapeutic activity, and that A and B may be any group that does not adversely affect the efficacy or toxicity of the compound.

In an alternative embodiment, one or both sulfur atoms of the dithiocarbamate are replaced with a selenium atom. Replacing sulfur with selenium may reduce the toxicity of the molecule in some cases, making it better tolerated by the patient. The skilled person will select A and B so as to obtain favorable compound characteristics, including size, charge, toxicity, and degree of stability (including stability in an acidic environment, such as the stomach, or alkaline, such as distal gastrointestinal tract). The choice of A and B also has a significant effect on tissue distribution and pharmacokinetics of the compound. In general, in the treatment of cardiovascular diseases, it is preferred that the compound accumulate, or localize, in the intimal layer of the vessel in which the vascular endothelial cells are located. Urinary excreted compounds are preferred.

An advantageous feature with the pharmaceutical administration of dithiocarboxylates is that they appear not to undergo enzymatic cleavage in vivo by thioesterases and may therefore have a long in vivo half-life.

In a preferred embodiment, A is a hydrogen atom or a pharmaceutically acceptable cation, such as, but not limited to, sodium, potassium, calcium, magnesium, aluminum, zinc, bismuth, barium, copper, cobalt, nickel, or cadmium; salt-forming organic acid, typically a carboxylic acid, such as, but not limited to, acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, acid polyglutamic acid, naphthalene sulfonic acid, naphthalene disulfonic acid, or polygalacturonic acid; or a cation formed from ammonia or other nitrogen base, such as, but not limited to, a heterocyclic nitrogen group, or a particle of formula NR⁴R⁵R⁶R7, in which R⁴, R⁵, R⁶ and R7 are, independently of each other, a hydrogen atom, a C1 linear, branched, branched, or (in the case of C4.6) cyclic, hydroxy (C₁.₆6 alkyl (in which one or more hydroxyl groups are present on any carbon atom), or aryl, N, N-diphenylethylenediamine, D-glucosamine, choline, tetraethylammonium, or ethylenediamine.

In another embodiment, A may be a physiologically cleavable leaving group that can be cleaved in vivo from the molecule to which it is attached, such as, for example,

180 874 but not limited to, acyl (including acetyl, propionyl and butyryl), alkyl, phosphate, sulfate or sulfonium groups.

In one embodiment, B is an alkyl, alkenyl, alkynyl, alkaryl, aroalkyl, haloalkyl, haloalkenyl, haloalkynyl, aryl, alkoaryl, hydrogen atom, a C 1 -C 1 -oxyoxy-C 1-1 group₍₎C 1-4 C 1-4 alkyl-C 1-6 alkyl-alkyl²R³, - (CHOH)_nCH2OH, in which the value of n is 0,1,2, 3,4, 5 or 6, - (CH2)_nCO2Ri, including alkylacetyl, alkylpropionyl and alkylbutyryl, or hydroxy (C1-6) alkyl- (in which one or more hydroxyl groups are present at any of the carbon atoms).

In another embodiment, B is No. 2³in which R2 and R³ are independently alkyl groups; - (CHOHjnC ^ OH, in which the value of n is 0,2,2,3,4,5 or 6, - (CH2) nCO2Ri, - (CH2)_nCO2R4; hydroxy (Ci-6) alkyl-; alkenyl (e.g., but not limited to, vinyl, allyl, and CH₃CH = CH-CH₂CH2); alkyl (CO2H), alkenyl (CO2H), alkynyl (CO2H), or aryl, in which the aryl group may have a substituent as described above, especially, for example, NO2, CH3, t-butyl, CO2H, halogen, or p-OH group; or R2 and R3 can together form a bridge such as - (CH2)_m- wherein the value of m is 3, 4, 5 or 6, and wherein R4 is an alkyl, aryl, alkaryl or aroalkyl group, for example, but not limited to, acetyl, propionyl and butyryl.

In yet another embodiment, B may be a heterocyclic or alkycloheterocyclic group. The heterocyclic group may be optionally partially or completely hydrogenated. Non-limiting examples are the groups mentioned above, including the phenazine, phenothiazine, pyridine and dihydropyridine groups.

In yet another embodiment, B is a residue of a pharmaceutically active compound or drug. The term "drug", as used herein, means any substance for internal or external use as a means to treat or prevent a disease or disorder.

Non-limiting examples are drugs for the treatment or prevention of cardiovascular diseases such as antioxidants, e.g. probucol; nicotinic acid; agents that prevent platelets from sticking together, such as aspirin; anticoagulants such as coumarin; calcium channel blockers like verapamil, diltiazem and nifedipine; angiotensin converting enzyme (ACE) inhibitors like captopril and enalapril; β-blockers, like propanolol, terbutalol and labetalol; non-steroidal anti-inflammatory drugs like ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, sulindac, or corticosteroids. The - C (S) SA group can be directly attached to the drug, or attached via any suitable linking particle.

In another embodiment, the dithiocarbamate is an amino acid derivative of formula AO₂C-R9-NR^1o-C (S) SA, wherein R9 is a divalent B particle, a linking particle, or an internal residue of any of naturally occurring amino acids (e.g., CH₃CH for alanine, CH2 for glycine, CH (CH2) 4NH2 for lysine, etc.), and Rⁱⁿ is a hydrogen atom or a lower alkyl group.

B may also be a polymer to which one or more dithiocarbamate groups are attached, either directly or via any counter-diatomic linkage. The dithiocarbamate is preferably released from the polymer in vivo at a time appropriate to provide a beneficial therapeutic effect. In a preferred embodiment, the polymer itself is also degraded in vivo. The term "biodegradable", as used herein, means a polymer that dissolves or degrades within the acceptable time period for the desired use (usually in vivo therapy), usually less than five years, and preferably less than one year when exposed to physiological solution at pH 6-8 at 25 and 37 ° C. In a preferred embodiment, the polymer degrades over a period of time from 2 hours to several weeks, depending on the application.

Many degradable polymers are known. Non-limiting examples are peptides, proteins, nucleoproteins, lipoproteins, glycoproteins, synthetic and natural polypeptides and polyamino acids, for example, but not limited to, polymers and copolymers of lysine, arginine, asparagine, aspartame acid, cysteine, cystine, glutamic acid, glutamine, hydroxylysine, serine, threonine, and tyrosine; polyorthoesters, such as poly (α-hydroxy acids), for example, polylactic acid, polyglycolic acid, poly (lactide co-180 874

-glycolide), polyanhydrides, albumin or collagen, polysaccharides containing sugar units such as lactose, and polycaprolactone. The polymer may be an irregular or block copolymer.

B may also be a group that increases the solubility of dithiocarbamate in water, e.g., lower -O-R8 alkyl, in which R⁸ is -PO2 (OH) M⁺ OR PO3 (M⁺)₂wherein M + is a pharmaceutically acceptable cation; -C (O) (CH2)₂CO2-M + or -SO₃- M + - lower alkylcarbonyl lower; -carboxy-lower alkyl; - lower alkylamino-lower alkyl, N, N-disubstituted amino lower alkyl, in which the substituents are independently of each other a lower alkyl group; pyridyl-lower alkyl, imidazolyl-lower alkyl, imidazolyl-Y-lower alkyl wherein Y is a thiol or amino group; morpholinyl lower alkyl, pyrrolidinyl lower alkyl; thiazolinyl lower alkyl, piperidinyl lower alkyl, morpholinyl lower hydroxyalkyl; N-pyrrolyl; piperosinyl lower alkyl, N-substituted piperazinyl lower alkyl wherein the substituent is a lower alkyl group; triazolyl lower alkyl; tetrazolyl lower alkyl, tetrazolyl lower alkyl; or thiazolyl lower alkyl.

In an alternative embodiment, a Toki dimer such as BC (S) S-SC (S) -B may be administered.

Non-limiting examples of dithiocarbamates are compounds having the formula:

1) Aliphatic substrate

HR

<img file="PL180874B1_D0007.tif" />

2) amino acid

COOH • C.

AND

S ·

COOH

IR'H

COOH, RH

Lys-Lys' I.

NR I c = p

AND

I rn

AND

C = S.

AND

S '

C = S

And p^-

<img file="PL180874B1_D0008.tif" />

and

3’

ABOUT

B

Rpodstawniki

No J

CH₃ or a t-butyl group

COOH p-OH

AscB

K * s SH * ua

BCSSCB

RCSCR Na * Tioester

Ca **

NH /

Choline * and quaternary amines

mg **

Al.⁺⁺⁺

180 874 changed. The compound should not accumulate in low metabolic rates, such as adipose tissue. In a preferred embodiment for the treatment of cardiovascular diseases, the pharmacokinetics of the compound should not be significantly changed in congestive heart failure or renal failure.

For topical administration, for the treatment of inflammatory skin diseases, the selected compound should be prepared as a formulation absorbed through the skin in an amount sufficient to achieve a therapeutic effect at the lesion site.

The dithiocarboxylate must be physiologically acceptable. Generally, compounds with a therapeutic index of at least 2, and at least 5 or 10 are allowed. The therapeutic index is defined as EC₅₀/ IC_s0when the EC50 is the concentration of the compound that inhibits VCAM-1 expression by 50%, while the IC50 is the concentration of the compound that is toxic to 50% of the target cells. Cellular toxicity can be measured by direct cell counting, trypan blue exclusion method, or various studies on metabolic activity, e.g., 3H-thymidine incorporation method, as known to those skilled in the art. The therapeutic index PDTC in cell culture in HUVE cells, measured as the quotient of cell toxicity and the ability to inhibit TNFα activated VCAM-1 expression, is above 100. Preliminary studies on rapidly dividing HT-18 human glioblastoma cells showed no toxicity at concentrations of 100 - times higher than therapeutic concentration. Disulfiram, the orally used form of diethyldithiocarbamate, used to treat alcohol abuse, does not usually show any clinically significant toxic effect when properly used.

Several dithiocarbamates with gene damaging (genotoxic) effects are known. These compounds are not within the scope of the present invention, which is limited to the administration of physiologically acceptable materials. An example of genotoxic dithiocarbamate is zinc dimethyldithiocarbamate. In addition, the anticholinesterase properties of some ditircarbamates may lead to neurotoxicity. Miller, D. (1982), "Neurrerxiciey of the pesticidal carbamates. Neurobehav. Toxicol. Tetratol. 4 (6): 779-87.

The term "dithiocarboxylate" as used herein means, but is not limited to, dithiocarbamates with the formulas:

R¹ SC (S) NR2R³ or R2R³N (S) CS-SC (S) NR2R³wherein R1 is a hydrogen atom or a pharmaceutically acceptable cation, such as, but not limited to, sodium, potassium, or nR⁴R⁵R⁶R⁷in which R⁴, R5, R⁶ and R⁷ they are, irrespective of each other hydrogen, a straight-chain, branched or cyclic C1.6 alkyl group, a hydroxy xalkylalkyl group of Sw which, at the donor stage, has a vaginal hydroxyl group or a hydroxyl group), or an aryl group, and

R2 and R.³ they form, independently of each other, group C_INstraight, branched or cyclic alkyl; - (CHOH)_n(CH2)_nOH, in which n may be 0, 12, 3, 4, 5 or 6; - (CH2)_nCO2R1, - (CH2) nCO2R4; a hydroxyalkyl group, or R2 and R3 together form a bond, for example - (CH2) o-, in which the value of m is 3-6, and in which R4 is an alkyl, aryl, alkyaryl, or aroalkyl group, including acetyl, propissinyl butyryl.

Detailed examples of preferred dithiocarbamates, shown in Fig. 15, are the sodium salt of N-pyrrolidine diethiocarbamate, N-methyl-N-carbocymethyl-N-carbidi diethylate, N, N-two (carboxymethyl) -N-methyl trisodium N-carbodithiolate sodium glucamine-N-carbidithiol; Sodium N, N-diethyl-N-carbodithiolate (sodium dicyyl Zithiocarbamate) and sodium N, N-diisopropyl-N-carbidithiolate.

Active Zithiocarboxylates, and in particular dithiocarbamates, are commercially available or can be prepared by methods known in the art.

II. Biological activity

The ability of dithiocarbylate to inhibit VCAM-1 expression can be measured in a variety of ways, for example, in the methods described in detail below in Examples 9-15. For

180 874, Examples 9-11 and 14-15 describe the evaluation of the biological activity of N-pyridinyl carbodithiolate sodium (also called PDTC). The purpose of these examples is not to limit the scope of the invention, which includes the use of any of the compounds described above for the treatment of atherosclerosis and other types of inflammatory and cardiovascular diseases mediated by VCAM-1. Instead of PDTC, any of the compounds described above can easily be used and evaluated in a similar manner.

Examples 12 and 13 give comparative data on the ability of some dithiocarbamates to inhibit VCAM-1 gene expression. The following examples show that the dithiocarbamates of the present invention specifically block the ability of VCAM-1 to express in vascular endothelial cells in response to many signals known to be active in atherosclerosis and in the inflammatory response.

Experimental procedures

Cell cultures

Isolation from catheterized human veins of HUVE umbilical cells, rinsing with Hanks solution to remove blood, and then incubating with 1% collagenase for 15 minutes at 37 ° C. After collagenase removal, cells were cultured in M199 medium supplemented with 20% fetal bovine serum (HyClone), 16 pg / ml heparin (ESI Pharmaceuticals, Cherry Hill, NJ), 50 (g / ml endothelial cell growth additive (Collaborative Reasearch Incorporates, Bedford) MA), 25 mM Hepes Buffer, 2 mM L-glutamine, 100 pg / ml penicillin and 100 pg / ml streptomycin, at 37 ° C in tissue culture plates coated with 0.1% gelatin. Confluent cells were passaged by 1: 4 cleavage. Cells were used in the first 8 passages.

Incubation with cytokines and other reagents

Confluent HUVE cells were washed with phosphate buffered saline and then placed in fresh medium. 30 minutes before adding cytokines, the indicated PDTC concentrations were added. Cytokines and other inducers were directly added to the medium at the time points and concentration indicated for each experiment. Human recombinant IL-1b was a gift from the Upjohn Company (Kalamazoo, Michigan). TNFa was obtained from Boehringer Ingelheim. Bacterial lipopolysaccharide (LPS), polyinosinic acid: polycytidilic acid (Polyl I: C) and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma Chemical (St. Louis, MO). All other reagents were used as reagent grade.

RNA isolation

Total cellular RNA was isolated by single extraction using an acid mixture of thiocyanate-phenol-chloroform. Cells were washed with physiological saline / phosphate buffered saline followed by lysis with 2 ml guanidine isothiocyanate. The solution was acidified with 0.2 mL sodium acetate (pH 4.0), followed by extraction with 2 mL phenol and 0.4 mL chloroform: isoamyl alcohol (24: 1). RNA ethanol precipitation was carried out twice before being used for Northem blot analysis.

Northem blot analysis

Total RNA (20 pg) was isolated and sized according to size using 1% agarose-formaldehyde gel in the presence of 1 pg / ml of ethylene bromide. RNA was transferred to a nitrocellulose filter and covalently combined with ultraviolet radiation using a Stratlinker UV cross-binding device (Stratagene, La Jolla, CA). Hybridization was carried out at 42 ° C for 18 hours in 5X SSC (1X = 150 mM NaCl, 15 mM Na citrate), 1% sodium dodecyl sulfate, 5X Denhardt's solution, 50% formamide, 10% dextran sulfate and 100 pg / ml cut denatured salmon sperm DNA. About 1-2X 10 was used⁶ cpm / ml labeled probe (specific activity&gt; 108 cpm / pg DNA) for hybridization. After hybridization, the filters were finally rinsed with 0.2X SSC at 55 ° C. Before rehybridization with other probes

180 874 nitrocellulose was removed with boiled water. Autoradiography was performed with an intensifying screen at -70 ° C.

32Sondy

DNA probes labeled with 32p were generated by randomly selecting an oligonucleotide primer. The ICAM-1 probe was an EcoRI fragment of human cDNA. The ELAM-1 probe was a 1.85 kb Hind III human cDNA fragment. The VCAM-1 probe was a Hind III - Xho I fragment of human cDNA formed from nucleotides 132-1814.

Enzyme Immunosorption Test (ELISA)

HUVE cells were seeded into 96-well plates 48-72 hours prior to testing. Primary antibodies in M199 with 5% FBS were added to each well and incubated for one hour at 37 ° C. Cells were then washed and incubated for one hour with IgG peroxidase labeled goat anti-mouse (Bio Rad) diluted 1/500 in M199 with 5% FBS. The wells were then washed and detection of antibody binding was performed by adding 100 μΐ 10 mg / ml 3, 3, 5, 5'-tetramethylbenzidine (Sigma) with 0.003% H2O2. The reaction was stopped by adding 25 μΐ 8N sulfuric acid. Plates were read on an ELISA reading device (Bio Rad) at OD 450 nm after plugging on rows stained only with second stage antibody. Data represent the average of three replicates.

antibodies

Monoclonal antibody (MAb) 4B9 that recognizes the vascular cell adhesion molecule - 1 (VCAM-1) was a gift from Dr. John Harlan (University of Washington). MAbs recognizing the endothelial cell adhesion molecule (ELAM-1) were a gift from Dr. Swerlick (Emory University). Hybridomas producing MAb 84H10 recognizing the intracellular adhesive molecule (ICA.M-1) were grown routinely in our laboratory and the antibody was used as the cell culture supernatant.

Example 9. PDTC blocks induction of VCAM-1 HUVEC mRNA accumulation mediated by IL-1B, but does not block ICAM-1 or ELAM-1 induction

To determine whether the oxidative state of the endothelial cell could alter the basal or induced expression of the cell adhesion molecule gene, human vascular endothelial cells were exposed to induction cytokine IL-1b (10 U / ml) in the presence or absence of thiolated metal chelating antioxidant, dithiocarbamate pyrrolidine (PDTC, 50 pM) for up to 24 hours. As shown in fig. 10, IL-1b alone (bands 2, 4, 6, 8) induces the expected rapid and transient induction of VCAM-1 mRNA accumulation (Plate A), E-selectin (ELAM-1, Plate B) and ICAM-1 (Plate C ), all of which reach their peak after four hours. However, in the presence of PDCT, the induction of VCA.M-1 mRNA accumulation by IL-1b is rapidly inhibited by more than 90% (Plate A, bands 3.5.1 and 9). In contrast, although ELAM-1 induction mediated by IL-1b is slightly inhibited after 2 and 24 hours (see bands 2 and 3), 8 and 9, Plate B), PDCT does not inhibit induction after 4 and 8 hours (bands 5 and 7, Plate B). Induction of ICAM-1 mRNA accumulation mediated by IL-1b is not affected (Plate B, bands 3, 5, 7 and 9). Indeed, a slight increase in IL-1b induction of ICAM-1 mRNA accumulation (-30%) is observed (see bands 4 and 5, Plate B). An equal amount of RNA transferred to nitrocellulose per band was confirmed by ethidium bromide staining and visualization.

A dose-response analysis was performed to determine if PDTC inhibits IL-1b induction of VCAM-1 gene expression in a dose-dependent manner. As shown in Figure 11, PDTC inhibits the induction of VCAM-1 gene expression mediated by IL-1b, a steep dose response curve (Figure 11, Plate A) with calculated EC5_ABOUT of about 10 pM, while PDTC does not inhibit the induction of ELAM-1 expression mediated by IL-1b at these concentrations (Figure 11, Plate B). The induction of ELAM-1 mRNA accumulation, whose media path is IL-1b, is increased by PDTC at a concentration above 0.5 μM (Figure 2, see band 2 and band 4-7, Plate C).

These data show that IL-1b uses a dithiocarboxylate sensitive step, and in particular dithiocarbamate, as part of its signaling mechanism for the induction of VCAM-1 gene expression. The data also seems to indicate that this dithiocarbamate sensitive step does not play a significant role in the induction of ELAM-1 or ICAM-1 gene expression mediated by IL-1b.

Example 10. PDTC blocks the induction of VCAM-1HUVEC mRNA accumulation by a variety of stimuli

In order to determine whether other known activators of VCAM-1 gene expression also utilize the PDTC sensitive stage, three separate classes of activators were tested: another classic inducer that mediates the receptor (TNFa), an inducer whose mediator is not the receptor (lipopolysaccharide - LPS), and the recently described new inducer (double-stranded RNA, poly (I: C). In all three cases, PDTC significantly inhibited the induction of VCAM-1 HUVEC mRNA accumulation after four hours (Figure 12, VCAM-1 Plate A A mRNA). Although the expression of the ELAM-1 gene mediated by TNF? Is somewhat inhibited (Fig. 12 lanes 1 and 2, Plate B), the accumulation of ELAM-1 mRNA mediated by LPS and poly (I: C) does not change ( Fig. 12, bands 3-6, Plate B). These data indicate that structurally different inducing agents, acting through separate pathways, have a common regulatory step specific to the induction of VCAM-1 gene expression.

Example 11.

PDTC blocks VCAM-1 expression on the HUVE cell surface induced by various stimuli

To determine whether the induction of VCAiM-1 endothelial cell surface protein expression, like its mRNA, is inhibited by PDTC, an ELISA using monoclonal antibodies was performed to quantitatively assess the induction of VCAM-1 and ICAM-HUVE cell surface culture cells. As shown in fig. 13, many classes of activating agents, in the absence of PDTC (-PDTC), induce rapid and transient accumulation of VCAM-1 (upper left plate) at the peak of the cell surface after six hours. In the presence of PDTC (+ PDTC, upper plate on the right), the induction of all VCAM-1 expression factors on the cell surface is significantly reduced (80-90%) by all examined factors. In contrast, induced ICAM-1 expression on the cell surface under the same conditions does not change (bottom plates left and right).

These data indicate that cell surface expression of VCAM-1, as well as the accumulation of its mRNA, is selectively inhibited by dithiocarbamates and that many classes of activating agents use a similar dithiocarbamate sensitive mechanism to induce VCAM-1 gene expression.

Example 12. Comparison of antioxidant efficacy in blocking TNCα induction of VCAM-1

To determine whether structurally similar or dissimilar antioxidants can also inhibit VCAM-1 gene expression, and to what extent, HUVE cells were exposed to TNFα for six hours in the presence or absence of four different antioxidants at different concentrations. As shown in Figure 14, both diethyldithiocarbamate (DETC) and N-acetylcysteine ​​(NAC) inhibited VCAM-1 expression at a concentration of 5 μM and 30 μM, respectively. In contrast, PDTC (PDTC) showed efficacy at a concentration of 5 to 50 μM. The metal (iron) chelator, desferoxamine, was not active at the concentrations tested.

Example 13. PDTC inhibits TNF induction of adhesion mediated by VCAM-1 / VLA-4

The ability of various antioxidants to inhibit the induction of TNF-α VCAM-1 in HUVE cells was investigated by the methods described in Example 12. Fig. 15 is a graph of the relative expression of VCAM-1 cell surface (OD 595 nm) in human cells

180 874 umbilical vein endothelium, activated with TNFa, depending on the concentration of PDTC (sodium salt of N-pyrrolidine dithiocarbamate), DIDTC (N, N-diethyl-N-carbodithiolate), SarDTC (N-methyl-N-carboxymethyl-N-carbodithiolate) ), IDADTC (N, N-di (carboxymethyl) -N-carbodithiol trisodium), MGDTC (N-methyl-D-glucamino-N-carbodithiolate), MeOBGDTC (\ - (4-methoxybenzyl) -D-glucamino-N sodium carbodithiolate), DEDTC (sodium N, N-diethyl-N-carbodithiolate), Di-PDTC (N, N-diisopropyl-N-carbodithiolate) and NAC (N-acetylcysteine).

Example 13. PDTC inhibits TNF induction of adhesion mediated by VCAM-1 / VLA-4

To determine whether PDTC inhibition of VCAM-1 regulation is associated with functional consequences, the attachment of Molt-4 cells to HUVEC cells, stimulated or not, with TNFα (100 U / ml) for six hours in the presence or absence of PDTC was examined. Molt-4 cells have previously been shown to attach to activated HUVECs via a VCAM-1 dependent mechanism. As shown in fig. 16, the percentage of Molt-4 cells attaching to HUVEC cells was reduced when present in PDTC medium.

Example 14.

PDTC inhibits monocyte binding in the thoracic aorta in cholesterol-treated rabbits

An experiment was conducted to determine whether the thiol antioxidant PDTC would be effective in blocking the first component of atherosclerosis, monocyte binding, in an experimental animal model. One mature New Zealand white rabbit (3.5 kg) received intravenous PDTC (20 mg / kg, which corresponds to 20 mg / ml in PBS) once daily for 5 days. Injections were administered through a lateral ear vein tube, whose patency was maintained by flushing with heparinized saline. The PDTC solution was prepared daily or the other day (stored in a dark place at 4 ° C), and filtered (0.45 mm pore filter) immediately before use. After the first injection, after placing the tube, the drug was administered to a conscious rabbit without obvious discomfort or other adverse effects. On the second day of injections, the rabbit was given food containing 15 (by weight) cholesterol, which was then added for the remainder of the experiment. On the fifth day, the animal was sacrificed, and the thoracic aorta was resected and fixed. After proper preparation, the sample was viewed at a lower stage of the scanning electron microscope equipped with a LaB emitter. Using two-screen imaging and a transparent grid on a CRT screen, 64 adjacent fields were evaluated at a magnification of 62ox, which corresponds to an area of ​​~ 1.3 mm2. The number of adherent leukocytes (WBC) and erythrocytes (RbC) was counted in each field.

Data from aortic arch sample were: 5 WBC and ~ 25 RBC per area

1.3 mm2. This level of WBC adhesion is similar to that observed in control animals given plain feed (approximately 7 per field was observed in hatch and thoracic aortic samples in two "negative control" experiments). "Positive control" rabbits, receiving 1% cholesterol for 4 days and not receiving antioxidant, showed an approximately fivefold increase in adhesion to 38 WBC / 1.3 mm2. A significant amount of cell-size debris was observed with aortic arch specimens. It is not clear whether this material is an artifact created during the preparation of the preparation or 42X. These studies suggest that PDTC injections may effectively block the initial adhesion of monocytes to the aortic endothelium.

Example 15. Inhibition of BSA13-HPODE Addition Compounds by PDTC

Figure 18 is a column graph of the effect of PDTC on the formation of the bSa and 13-HPODE fluorescent addition compounds, as measured in fluorescent units, relative to the micromolar concentration of PDTC. One micromole of 13-HPODE was incubated with 200 micrograms BSA in the presence of PDTC for six days. Fluorescence was measured at 430-460 nm with excitation at 330-360 nm. Test details, see Freebis, J. Parthasarathy, S., Steinberg, D., Proceedings of the National Academy of Sciences 89, 10588-10592, 1992. In a typical reaction

180 874

100 nmol LOOH (produced by lipoxygenase catalyzed oxidation of linoleic acid) is incubated with 100 pg bovine serum albumin for 48-72 hours and after the formation of fluorescent products the fluorescence spectrum with 360 excitation and emission between 390 and 500 nm is measured.

As shown, PDTC reduces the concentration of BSA and 13-HPODE fluorescent addition compounds.

Figure 19 is a graph of the effect of PDTC on the formation of BSA and ox-PUFA fluorescent addition compounds as a function of wavelength (nm) and PDTC concentration. As the PDTC increases, the content of fluorescent addition compounds decreases.

Example 16. Effect of PDTC on LDL oxidation by horseradish peroxidase

Figure 20 is a graph of the effect of PDTC on LDL oxidation by horseradish peroxidase (HRP), measured versus time (minutes) for varying PDTC concentration. After LDL oxidation, the oxidation of LDL fatty acid components was measured, determined by an increase in optical density at 234 nm. When polyunsaturated fatty acid is oxidized, there is a displacement of double bonds, resulting in conjugated dienes absorbing at 234 nm. The segment of the onset and spreading curve (latency period) is considered a measure of LDL's ability to oxidize. Typically, 100 pg human LDL is incubated with 5 pM H2O2 and an increase in absorption is then observed at 234 nm. It was found that after the incubation period, PDTC inhibits LDL oxidation by HRP in a concentration-dependent manner.

Example 17. Effect of PDTC on cytokine-induced ox-PUFA production

Figure 21 is a graph of the effect of PDTC on cytokine-induced ox-PUFA formation in human aortic endothelial cells. As indicated, both TNF-α and IL-1B cause oxidation of linoleic acid to oxylinolic acid. PDTC significantly reduces oxidation.

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

By modifying the PUFA to ox-PUFA conversion, inhibition of VCAM-1 expression can be obtained. For example, many enzymes that oxidize unsaturated compounds are known, such as peroxidases, lipoxygenases, cyclooxygenases, or cytochrome P-450. Inhibition of these enzymes may prevent PUFA oxidation in vivo. PUFAs can also be oxidized by metal-dependent non-enzymatic substances.

IV. A method of modifying the expression of a redox sensitive gene

In an alternative embodiment, there is provided a method of inhibiting expression of a redox responsive gene or activating a inhibited gene in a redox responsive pathway, comprising administering an effective amount of an anti-oxidant substance, and typically, oxidation of a polyunsaturated fatty acid. Exemplary redox-sensitive genes associated with the presentation of an immune response are, but are not limited to, genes expressing cytokines involved in initiating an immune response (e.g., IL-1 β), chemical attractants, facilitating the migration of inflammatory cells into damage sites (e.g. MCP-1), growth factors (e.g. IL-6 and thrombin receptor) and adhesion molecules (e.g. VCAM-1 and E-selectin).

Considering the above, a specialist will be able to screen various antioxidants for their ability to inhibit the expression of a redox sensitive gene or to activate a gene inhibited in the redox sensitive pathway. All such embodiments are within the scope of the present invention.

Based on the results of such screening, nucleic acid molecules containing 5'-end regulatory sequences of genes sensitive to redox reactions can be identified to regulate or inhibit gene expression in vivo. Vectors can be used to express a specific recombinant 5 'side sequence gene sequence in cells, including plasmids and eukaryotic viral vectors, depending on the preferences and judgment of the specialist (cf. e.g. Sambrook et al., Chapter 16). In addition, a number of viral and non-viral vectors are being developed to enable sequence incorporation

180 874 nucleic acids in vivo (cf. e.g., Mulligan, 1993, Science, 260, 926-932; US Patent No. 4,988,286; US Patent No. 4,868,116, incorporated herein by reference). Recently, a transport system has been developed in which the nucleic acid is encapsulated in cationic liposomes that can be injected intravenously into a mammal. This system is used to introduce DNA into the cells of various tissues of adult mice, including endothelium and bone marrow (see, e.g., Zhu et al., 1993 Science, 261, 209-211, incorporated herein by reference).

The 5 'side sequences of the redox sensitive gene can be used to inhibit expression of the redox sensitive gene. For example, antisense RNA of all or part of the 5 'side region of a redox sensitive gene can be used to inhibit gene expression in vivo. Expression vectors (e.g. retroviral expression vectors) are already known in the art and can be used to produce the antisense RNA of a selected DNA sequence that is expressed in a cell (cf. e.g. United States Patent No. 4,868,116, United States Patent No. 4,988,286).

Accordingly, it is possible to introduce DNA containing all or part of the 5 'side sequence sequences of a gene into a suitable expression vector, so that when transcribed into the cell, transcription of the introduced DNA produces antisense RNA complementary to the transcription product of the mRNA of the gene normally found in the cell . This transcription product of the antisense RNA introduced with dNa can then be paired with bases with the correct mRNA transcription product present in the cell and thereby prevent mRNA translation. It is of course necessary to select a 5 'side region sequence towards the 3' end from the start of transcription for the gene sensitive to redox reactions to ensure that the antisense RNA contains complementary sequences present on the mRNA. Antisense mRNA can also be produced in vitro, and then introduced into cells. Oligonucleotides can be produced in an automatic synthesis device (e.g., Model 8700 automatic synthesis device from Milligen-Biosearch, Burlington, MA, or ABI, Model 380 B). Antisense deoxyoligonucleotides have also been proven to effectively inhibit gene transcription and viral replication (cf. e.g. Zamecnik et al., 1978, Proc. Notl. Acad. Sci USA 75, 280-284; Zamecnik et al., 1986, Proc. Natl. Acad. Sci. 83, 4143-4146; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85.1028-1032; Crooke 1993 FASEB J. 7.533-539. In addition, recent studies have shown that it is possible to improve the inhibition of gene expression by antisense oligonucleotides if the antisense oligonucleotides contain modified nucleotides (see, e.g., Offensperger et al., 1993 EMBO J. 12,1257-1262 (inhibition of in vivo replication of viral hepatitis B virus in ducks and expression of its gene by phosphorothiolone antisense oligonucleotides); Rosenberg et al., PCT WO 93/01286 (production of sulfate thiol oligonucleotides); Agrawal et al., 1988 Proc. Natl. Acad. Sci. USA 85, 7079-7083 (production of antisense phosphoramidones and oligonucleoside phosphorothiolates to inhibit replication of human immunodeficiency virus-1); Sarin et al., 1989, Proc. Natl. Acad. Sci. USA 85, 7448-7794 (production of antisense methyl phosphonate oligonucleotides); Shaw et al., 1991 Nucleic Acid Res. 19, 747-750 (production of 3'-exonuclease resistant oligonucleotides containing 3'-terminal phosphoramidate modifications); the above publications are incorporated herein by reference.

The 5 'side sequence sequences of the redox sensitive gene can also be used in triple helix (triplex) therapy. Oligonucleotides complementary to the promoter gene sequences on one of the DNA strands have been proven to bind to the promoter and regulatory sequences to form locally triple nucleic acid helices that block gene transcription (cf. e.g. 1989 Maher et al., Science 245,725-730; Orson and et al., 1991 Nucl. Acids Res. 19, 3435-3441; Postal et al., 1991 Proc. Natl. Acad. Sci. USA 88, 8227-8231; Cooney et al., 1988 Science 241,456-459; Young et al., 1991 Proc. Natl. Acad. Sci. USA 88, 10023-10026; Dwal-Yolentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504-508; 1992 Blume et al., Nucl. Acids Res. 20, 1777-1784; 1992 Grigoriev et al., J. Biol. Chem. 267, 3389-3395.

180 874

Recently, theoretical calculations and empirical findings have been presented to facilitate the design of oligonucleotides for use in directed oligonucleotide production of triple helix to inhibit gene expression. For example, oligonucleotides should generally be greater than 14 nucleotides in length to ensure target specificity (cf. e.g. Maher et al. (1989); Grigoriev et al. (L992)). In addition, many cells greedily capture oligonucleotides less than 50 nucleotides in length (cf. e.g. Orson et al., (2991); Holt et al., 1988 Mol. Cell. Biol. 8,963-973; Wickstromiwsp., L988 Proc. Natl. Acad. Sei. USA 85, 1028-1032). To reduce susceptibility to intracellular degradation, e.g. by 3'-exonucleases, a free amine can be introduced into the 3'-terminal hydroxyl group of oligonucleotides without losing sequence binding specificity (Orson et al., 1991). In addition, triplex stability increases with methylation of cytokines present in the oligonucleotide, as well as when the resolving agent, e.g. an acridine derivative, is covalently attached to the 5'-terminal phosphate (e.g., via a pentamethylene bridge); again, without loss of sequence binding specificity (Maher et al. (1989); Grigoriev et al. (1992).

Methods for producing oligonucleotides are well known to those skilled in the art. Such methods vary from standard enzymatic digestion and subsequent isolation of a nucleotide fragment (cf. Eg Sambrook et al. Chapters 5 and 6) up to purely synthetic methods, e.g. by cyanoethyl phosphoamidine using a Milligen or Beckman DNA synthesis device The iPlus system (see also Ikuta et al., Ann. Rev. Biochem., 2984 53, 323-356 (phosphoto and phosphite registry methods); Narang et al., Methods Enzymol, 65,610-620 (1980) (ester phosphate triple method). Accordingly, the DNA sequences of the 5 'side region of the redox sensitive gene described above can be used to design and produce oligonucleotides containing DNA sequences formed essentially from at least 15 consecutive nucleotides ·', with (or without) base modification, with (or without) resolving derivative factors, for use in the production of triple helices, particularly in the 5 'side region of a redox sensitive gene to inhibit expression of that gene.

In some cases, it may be beneficial to incorporate enhancer agents or multiple copies of regulatory sequences into the expression system to facilitate screening of methods and reagents for manipulation of expression.

V Models and methods of screening

Screening methods for detecting disorders mediated by VCAM-1 or a gene sensitive to redox reactions by quantifying surrogate disease markers are also provided.

In one embodiment, the level of oxidized polyunsaturated fatty acid or other appropriate markers, in tissue or blood, for example, of the host is assessed as a means of assessing the host's "oxidative environment " and host sensitivity to diseases mediated by a redox sensitive gene or VCAM-1.

In yet another embodiment, the sensitization of host vascular endothelial cells to polyunsaturated fatty acids or their oxidized counterparts is assessed. This can be achieved, for example, by exposing the host to PUFA or ox-PUFA and comparing the obtained concentration of VCAM-1 on the surface of the cell or circulating, or other surrogate marker with the population norm. In yet another embodiment, in vivo models for atherosclerosis or other heart disease or inflammatory diseases provided by VCAM-1 are mediated by administering excessive amounts of PUFA or oxidized polyunsaturated fatty acid to the host animals to cause the disease. These animals can be used in clinical studies to further understand these disorders.

In yet another embodiment of the invention, the compounds are evaluated for their ability to treat disorders mediated by V CAM-1, based on their ability to inhibit the oxidation of polyunsaturated fatty acid, or the interaction of PUFA or ox-PUFA with the target protein.

180 874

This can be achieved by exposing the host, for example a human or animal, e.g. mouse, to a high level of PUFA or ox-PUFA and then assessing the therapeutic effectiveness of the test compound based on its ability to reduce the concentration of VCAM-1 circulating or deposited on the surface of the cells. Instead, an in vitro screening test based on the ability of the test compound to prevent PUFA oxidation or PUFA or ox-PUFA interaction with the target protein may be used in the presence of an oxidizing substance such as a metal, e.g. copper, or an enzyme such as peroxidase, lipoxygenase, cyclooxygenase or cytochrome P-450.

In another embodiment, the vascular endothelial cells are exposed to TNF-α or other VCAM-1 inducing material for an appropriate period of time, and then damaged by appropriate means, e.g., sonication or freezing-thawing. Cytosol and membrane compartments are isolated. The radiolabeled PUFA is added to the specified number of compartments. The fluid's ability to convert PUFA to ox-PUFA is tested in the presence or absence of test compound. Instead of the damaged cell system, intact cells can be used.

III. Pharmaceutical preparations

It is possible to treat humans, horses, dogs, cattle and other animals, in particular mammals, suffering from cardiovascular diseases and other inflammatory diseases mediated by VCAM-1 or a gene sensitive to redox reactions, by administering to the patient a compound that causes removal. reducing the concentration or preventing the production of oxidized polyunsaturated fatty acids, such as (but not limited to) oxidized linoleic acid (C₁₈ AND⁹'2), linolenic (C₁₈ Α69Ί2), peanut (C2₀ A5 ^&gt;^11J4) and eicosatriene (C20 A^8,11'14); other oxidative signals; or other active compound, or a pharmaceutically acceptable derivative or salt thereof in a pharmaceutically acceptable carrier or solvent. The active substances can be administered by any preferred route, e.g. orally, parenterally, intravenously, intradermally, subcutaneously or locally.

As used herein, the term "pharmaceutically acceptable salts or complexes" means salts or complexes that retain the beneficial biological activity of the aforementioned compounds and exhibit minimal undesirable toxic effects. Non-limiting examples of such salts are: (a) acid addition salts, prepared from inorganic acids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and salts made from 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, naphthalene sulfonic acid, naphthalene disulfonic acid, or polygalacturonic acid; (b) basic addition salts, made from a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium etc., or from an organic cation made from N, N-dibenzylethylenediamine, D-glucosamine, ammonia, tetraethylammonium, or ethylenediamine or (c) combinations of (a) and (b), e.g. zinc taninate etc.

The active compound or mixture of compounds is administered in any suitable manner, for example, but not limited to these methods, orally and intravenously. The general dosage range for the disorders mentioned above will be from 0.5 to 500 mg / kg body weight, administered with a frequency of once every two days to several times a day. Daily doses of from about 1 to 3000 mg / patient / day are preferred, more preferably, from about 5 to 500 mg / patient / day, and most preferably, from about 25 to 500 mg / patient / day.

The active ingredient should be administered in such a way that a peak plasma concentration of the active compound of about 0.1-100 μM is achieved, preferably about 1-10 μM. This can be achieved, e.g., by intravenous injection of a solution or preparation of the active ingredient, optionally in physiological saline or in an aqueous medium, or by administration of a bolus of the active ingredient.

180 874

The compounds can also be administered directly into the vascular wall using balloon balloon perfusion catheters, after or instead of coronary angioplasty or other arterial vessels. For example, 2-5 ml of a physiologically acceptable solution containing about 1-500 pM of compound or mixture of compounds is administered at a pressure of 1-5 atmospheres. Then, over the next six months, during the period of greatest risk of restenosis, the active compounds are administered by appropriate routes in the appropriate dosage regimen.

To cause shrinkage of coronary artery disease that cannot be treated with angioplasty and surgery, relatively short-term treatment with the active compound is used. A non-limiting example of short-term treatment is administration of the compound for a period of two to six months at a dose of 0.5 to 500 mg / kg body weight, with a frequency of once every two days to three times a day.

Long-term treatment can be used to prevent the development of advanced lesions in high-risk patients. Long-term treatment can last for many years, at doses from 0.5 to 500 mg / kg body weight, with a frequency of once every two days to three times a day.

The active compounds can also be administered during periods immediately before and after coronary angioplasty to reduce or eliminate the abnormal proliferative and inflammatory response that currently leads to clinically significant restenosis.

The active compounds can be administered with other drugs for the treatment of cardiovascular diseases, including lipid lowering agents such as probucol and nicotinic acid; agents that inhibit platelet aggregation, such as aspirin; anticoagulants such as coumarin; calcium channel blockers like verapamil, diltiazem and nifedipine; angiotensin converting enzyme (ACE) inhibitors like captopril and enalapril and β-blockers like propranolol, terbutalol and labetalol. The compounds can also be administered together with non-steroidal anti-inflammatory drugs, such as ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, sulindac. The compound can also be administered with corticosteroids.

The concentration of active compound in the formulation will depend on the extent of absorption, distribution, inactivation and excretion of the drug, as well as other factors known to those skilled in the art. It should be noted that the dosage will also depend on the severity of the disease. It should be understood that the dosage regimen should be selected appropriately for a given patient over a given period, depending on the individual needs and professional judgment of the person administering or supervising the administration of the preparations, and that the concentration range provided herein is exemplary only and does not limit the scope of the present invention . The active ingredient may be used in one dose or in divided doses administered at different time intervals. Oral preparations will usually contain an inert diluent or edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. The active compound intended for therapeutic oral administration can be combined with excipients and used in the form of tablets, troches or capsules. The formulation may also include pharmaceutically suitable binders and / or additives.

Tablets, pills, capsules, troches, etc. they may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a wetting agent such as magnesium stearate or Sterotes; a glidant like colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; flavoring agent, peppermint, methyl salicylate, or orange flavor. In the case of capsules, they may contain, in addition to the substances discussed above, a liquid carrier, e.g. an oil. In addition, particular types of preparations may contain various other substances that modify the physical form of the preparation, e.g., sugar, shellac or other enteric coatings.

The active compound or a pharmaceutically acceptable salt or derivative thereof can be administered as a component of an elixir, suspension, syrup, cachet, chewing gum, etc. The syrup may, in addition to the active compounds, contain sucrose as a sweetening agent, and some preservatives, dyes and flavors.

180 874

The active compound or pharmaceutically acceptable derivatives or salts thereof may also be administered with other active substances which do not impair the beneficial effect, or with substances which supplement the beneficial effect, such as antibiotics, anti-fungal, anti-inflammatory or anti-viral agents.

Solutions or suspensions for parenteral, intradermal, subcutaneous or topical use may contain the following components: a sterile diluent, such as water for injections, saline, vegetable 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; buffers such as acetates, citrates or phosphates and isotonic agents such as sodium chloride or dextrose. The preparation for oral administration can be prepared in the form of ampoules, disposable syringes or glass or plastic multidose vials.

Suitable carriers for topical applications are known, such as lotions, suspensions, ointments, creams, gels, tinctures, sprays, powders, pastes, transdermal patches, release form, bronchial aerosols, and suppositories for rectal, vaginal use. and on the mucosa of the nose or mouth.

Thickening, softening and stabilizing agents can be used for the preparation of topical preparations. Examples of thickeners are petroleum jelly, beeswax, xanthan gum and polyethylene glycol, humectants such as sorbitol, softeners such as mineral oils, lanolin and its derivatives, or squalene. Numerous solutions and ointments are commercially available.

Natural or synthetic flavors or sweeteners can be used to improve the taste of topical mucosal administration. Inert dyes may be added, in particular for preparations intended for application to the surface of the oral mucosa.

The active compounds can be combined with carriers that protect the compound against rapid release, e.g., in the form of sustained release formulations, including implants and microcapsule release systems. Biologically suitable and biodegradable polymers can be used, such as ethylene vinyl acetate, polyhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods have been patented for the preparation of such preparations which are generally known to those skilled in the art.

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

The active compound can also be administered in a transdermal patch. Methods for making transdermal patches are known to those of skill in the art, Por, e.g., Brown, L. and Langer, R., "Transdermal Delivery of Drugs, Annual Review ofMedicine, 39: 221-229 (1988), which are incorporated herein by reference.

In another embodiment, the active ingredients are combined with carriers that protect the compound against rapid removal from the body, e.g. in the form of sustained release formulations, including implants and microcapsule release systems. Biologically suitable and biodegradable polymers can be used, such as ethylene vinyl acetate, polyhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Methods for making such preparations are obvious to those skilled in the art. Materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc.

Pharmaceutically acceptable carriers can also be liposomal suspensions. They can be prepared by methods known to those skilled in the art, described, e.g., in US Patent No. 4,522,811 (which is incorporated herein by reference in its entirety). For example, liposomal preparations can be prepared by dissolving the corresponding lipid (s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidylcholine, arachidoyl phosphatidylcholine and cholesterol) in an inorganic solvent, which is then evaporated, leaving a thin layer of dried lipid on the surface of the dish. Then, an aqueous solution of the active compound or its monophosphate, diphosphate and / or triphosphate derivative is introduced into the preparation. The vessel is then centrifuged manually to release lipid substances from the sides of the vessel and to disperse liquid aggregates, thereby producing a lipytimal suspension.

Modifications and variations of the present invention resulting from the detailed description above will be apparent to those skilled in the art. Such modifications and variations are within the scope of the present invention.

180 874

INFLUENCE OF LINOLIC ACID AND 13-HPODE ON THE VCAM-1 GENE EXPRESSION KINETICS

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180 874

Effect of linoleic acid and 13-HPODE (dose response nd) on VCAM-1 gene expression

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Fig. 2

180 874

Cell surface expression (OD 450nm)

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· Fig. 3

180 874

VCAM-1

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180 874

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180 874

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180 874

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LINOLIC ACID (7.5 flM) Li LINOLIC ACID (7.5 µM)

180 874

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180 874

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180 874

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180 874

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180 874

Relative Relative ICAM-1 accumulation VCAM- accumulation

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180 874

Relative VCAM-1 expression on the cell surface

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180 874% MOLT-4 binding Relative VCAM expression

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180 874

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180 874

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180 874

Impact of PDTC on the production of fluorescent addition compounds from BSA and LOOH

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180 874

EFFECT OF PDTC ON LDL OXIDATION BY HRP

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Time (minutes)

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180 874

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FIG.21

UP Department of Publications. Circulation of 70 copies Price PLN 6.00.