AU733198B2 - Treatment for atherosclerosis and other cardiovascular and inflammatory diseases - Google Patents

Description

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AUSTRALIA

Patents Act 1990 Emory University

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Treatment for atherosclerosis and other cardiovascular and inflammatory diseases The following statement is a full description of this invention including the best method of performing it known to us:- 1A Treatment for Atherosclerosis and Other Cardiovascular and Inflammatory Diseases Background of the Invention This application is in the area of methods and compositions for the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

Adhesion of leukocytes to the endothelium represents a fundamental, early event in a wide variety of inflammatory conditions, including atherosclerosis, autoimmune disorders and bacterial and viral infections. Leukocyte recruitment to the endothelium is started when inducible adhesion 10 molecule receptors on the surface of endothelial cells interact with counterreceptors on immune I cells. Vascular endothelial cells determine which type of leukocytes (monocytes, lymphocytes, or neutrophils) are recruited, by selectively 15 expressing specific adhesion molecules, such as vascular cell adhesion molecule-i (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and E-selectin. In the earliest stage of the atherosclerotic lesion, there is a localized endothelial expression of VCAM-1 and selective recruitment of mononuclear leukocytes that express the integrin counterreceptor VLA-4. Because of the selective expression of VLA-4 on monocytes and lymphocytes, but not neutrophils, VCAM-1 is important in mediating the selective adhesion of mononuclear leukocytes. Subsequent conversion of leucocytes to foamy macrophages results in the synthesis of a wide variety of inflammatory cytokines, growth factors, and chemoattractants that help propagate the leukocyte and platelet recruitment, smooth muscle cell proliferation, endothelial cell activation, and extracellular matrix synthesis characteristic of maturing atherosclerotic plaque.

-2- VCAM-2. is expressed in, cultured human vascular endothelial cells after activation by lipopolysaccharide (LPS) and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF-a) These factors are not selective for activation of cell adhesion molecule expression.

Scheme 1 illustrates the process of cytokine activation of VCAM-l gene expression in vascular endothelial cells.

to wa 3-pt- Qww~lp 1Ia hu 6( VCAM- I la. LhA Moum Ragwatm7 Pluw.

Post.

Tr~das I A&AAAAAh. r Scheme I Regulatory schemes for -cytokine -activation of vascular cell adhesion molecule-i (VCAM-1) gene expression, through redox sensitive regulatory factors such as NF-kfS, in vascular endothelial cells (IkB is an inhibitory subunit; NF-kB is nuclear factor-kB; .NH 3 refers to the amino terminus of the protein and RNA Pol -Il is RNA polymerase

II).

Molecular analysis of the regulatory elements on the human VCAM-. gene that control its expression suggests an important role for nuclear factor-kB (NF-kB) a transcriptional regulatory factor, or an NF-kS like binding protein in oxidation-reductionsensitive regulation of VCAM-1 gene expression.

Transcriptional factors are proteins that activate (or repress) gene expression within the cell nucleus by binding to specific DNA sequences called "enhancer elements" that are generally near the region of the gene, called the "promoter," from which RNA synthesis is initiated. Nuclear factorkB is a ubiquitously expressed multisubunit transcription factor activated in several cell types by a large and diverse group of inflammatory agents such as TNFa, IL-1S, bacterial endotoxin, and RNA viruses. It plays a key role in mediating inflammatory and other stress signals to the 15 nuclear regulatory apparatus. Although the precise biochemical signals that activate NF-kB are unknown, this transcriptional factor may integrate into a common molecular pathway many of the risk factors and "causative" signals of atherosclerosis, 20 such as hyperlipidemia, smoking, hypertension, and diabetes mellitus.

Importantly, the activation of NF-k3 in vascular endothelial cells by diverse signals can be specifically inhibited by antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate (see U.S.S.N. 07/969,934, now allowed). This has led to the hypothesis that oxygen radicals play an important role in the activation of NF-kB through an undefined oxidation-reduction mechanism.

Because an NF-kB-like enhancer element also regulates the transcription of the VCAM-1 promoter in an oxidation-reduction-sensitive manner, oxidative stress in the atherosclerotic lesion may play a role in regulating VCAM-1 gene expression through this oxidation-reduction-sensitive transcriptional regulatory protein.

-4- It has been hypothesized that modification of low-density lipoprotein (LDL) into oxidatively modified LDL (ox-LDL) by reactive oxygen species is the central event that initiates and propagates atherosclerosis. Steinberg, et al., N. Engl. J.

Med. 1989; 320:915-924. Oxidized LDL is a complex structure consisting of at least several chemically distinct oxidized materials, each of which, alone or in combination, may modulate cytokine-activated adhesion molecule gene expression. Fatty acid hydroperoxides such as linoleyl hydroperoxide (13- HPODE). are produced from.free fatty acids by lipoxygenases and are an important component of oxidized LDL.

15 It has been proposed that a generation of oxidized lipids is formed by the action of the cell lipoxygenase system and that the oxidized lipids are subsequently transferred to LDL. There is thereafter a propagation reaction within the LDL in 20 the medium catalyzed by transition metals and/or sulfhydryl compounds. Previous investigations have demonstrated that fatty acid modification of cultured endothelial cells can alter their susceptibility to oxidant injury. Supplementation 25 of saturated or monounsaturated fatty acids to cultured endothelial cells reduces their susceptibility to oxidant injury, whereas supplementation with polyunsaturated fatty acids (PUFA) enhances susceptibility to oxidant injury.

Using reverse-phase HPLC analysis of native and saponified lipid extracts of LDL, it has been demonstrated that 13-HPODE is the predominant oxidized fatty acid in LDL oxidized by activated human monocytes. Chronic exposure to oxidized LDL provides an oxidative signal to vascular endothelial cells, possibly through a specific fatty acid hydroperoxide, that selectively augments cytokine-induced VCAM-1 gene expression.

Through a mechanism that is not well defined, areas of vessel wall predisposed to atherosclerosis preferentially sequester circulating LDL. Through a poorly understood pathway,- endothelial, :smooth muscle, and/or inflammatory cells then convert LDL to ox-LDL. In contrast- toLDL, which:is taken up through the LDL receptor, monocytes-avidly take up ox-LDL through a "scavenger" receptor whose expression, unlike the LDL receptor, is not inhibited as the content of intracellular lipid rises. Thus, monocytes continue to take up ox-LDL and become lipid-engorged macrophage-foam cells 15 that form the fatty-streak.

Given that cardiovascular disease is currently the leading cause of death in-the United States, and ninety percent ofccardiovascular disease is presently diagnosed as atherosclerosis, there is a 20 strong need to identify new methods and pharmaceutical agents for its:treatment. Important to this goal is-the identification and manipulation of the specific oxidized biological compounds that act as selective regulators of the expression of 25 mediators of-the inflammatory-process,: and in particular, VCAM-1. A'more'general goal is to identify selective methods for suppressing the expression of redox sensitive genes or activating redox sensitive'genes that are suppressed.

It is therefore an object of the present S invention to provide a treatment for atherosclerosis and other cardiovascular and inflammatory diseases.

It is another object of the present invention to provide a method for the selective inhibition of VCAM-1.

-6- It is still another object of the present invention to provide a method for the treatment of a human disease or disorder that is mediated by the expression or suppression of a redox sensitive gene.

It is another.object of the present invention to provide pharmaceutical compositions for the treatment.of.atherosclerosis and other cardiovascular and inflammatory diseases.

Summary of the Invention It has been discovered that polyunsaturated fatty acids ("PUFAs") and their hydroperoxides ("ox-PUFAs"), which are-important components of oxidatively modified low density lipoprotein (LDL), 15 induce the expression of VCAM-1, but not intracellular .adhesion molecule-1 (ICAM-1) or Eselectin in human aortic endothelial cells, through a mechanism that is not mediated by cytokines or other noncytokine.signals. This is a fundamental 20 discovery of an important and previously unknown biological pathway in VCAM-1 mediated immune responses.

As nonlimiting examples, linoleic acid, linolenic acid, arachidonic acid; linoleyl hydroperoxide (13-HPODE).and arachidonic hydroperoxide (15-HPETE) induce-cell-surface gene expression of VCAM-1 but not ICAM-1 or E-selectin.

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 PUFAs and their fatty acid hydroperoxides is suppressed by the antioxidant pyrrolidine dithiocarbamate (PDTC).

This indicates that the induction is mediated by an -7oxidized signal molecule, and that the induction is prevented when the oxidation of the molecule is blocked the oxidation does not occur), reversed the signal molecule is reduced), or when the redox modified.signal is.otherwise prevented from interacting with its regulatory target.

Cells that are. chronically.exposed to higher than normal levels of polyunsaturated fatty acids or their oxidized counterparts can initiate an immune response that is not normal and which is out of proportion to the.threat presented, leading to a diseased state. ,The oversensitization of vascular endothelial cells to .PUFAS and ox-PUFAS can 15 accelerate the formation, for example, of atherosclerotic plaque.

Based on these, discoveries, a method for the S. treatment of atherosclerosis,. post-angioplasty restenosis, coronary artery diseases, angina, and 20 other cardiovascular diseases, as well as noncardiovascular. inflammatory diseases that are mediated by VCAM-1, is provided.that includes the removal, decrease.in.the concentration of, or prevention of the formation of oxidized S. 25 polyunsaturated fatty .acids including but not limited to oxidized linoleic .(Cs A 91 2 linolenic

(C,

8 arachidonic (Ca, A 5 8"11.4) and icosatrienoic (C A 8 4 acids.

Nonlimiting examples of noncardiovascular inflammatory diseases that are mediated by VCAM-1 include rheumatoid and osteoarthritis, asthma, dermatitis, and multiple sclerosis.

This method represents a significant advance in treating cardiovascular disease, in that it goes beyond the current therapies designed simply to inhibit the progression of the disease, and when used appropriately, provides the possibility to -8medically "cure" atherosclerosis by preventing new lesions from developing and causing established lesions to regress.

In an alternative embodiment, a method is provided for suppressing'the-expression of a redoxsensitive gene or activating a gene that is suppressed through a redox-sensitive pathway, that includes administering-an effective amount of a substance that prevents the oxidation of the oxidized signal, and-typically, the oxidation of a polyunsaturated fatty acid. Representative redoxsensitive genes that are involved in the presentation of an immune response include, but are not limited to, those expressing cytokines involved 15 in initiating the immune response IL-1S) chemoattractants that promote the migration of inflammatory cells to a point of injury MCP-l),- growth factors IL-6 and the thrombin receptor), and adhesion molecules 20 VCAM-1 and E-selectin) Screens for disorders mediated by VCAM-1 or a redox-sensitive gene are also provided that include the quantification of surrogate markers of the disease. In one embodiment, the level of oxidized 25 polyunsaturated fatty acid, or other appropriate markers, in the tissue or blood, for example, of a host is evaluated as-a means of assessing the "oxidative environment" of the host and the host's susceptibility to VCAM-i or redox-sensitive gene mediated disease.

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

In yet another assay, the sensitization of a host's vascular endothelial cells to polyunsaturated fatty acids or their oxidized -9counterparts is evaluated. .This can be accomplished, for example, by challenging a host with a PUFA or ox-PUFA and comparing the resulting concentration of cell-surface or circulating VCAM-1 or other surrogate marker to a population norm.

In:another embodiment, :in vivo.models of atherosclerosis or other heart or. inflammatory diseases that are mediated by VCAM-1 can be provided by administering to a host animal an excessive amount of PUFA or oxidized polyunsaturated fatty acid to induce disease.

These animals.can be used-in clinical research to further the understanding of these. disorders.

.In yet another embodiment of the invention, 15 compounds can be assessed for their ability to treat disorders mediated by VCAM-1 on the basis of their ability to inhibit the.oxidation of a polyunsaturated fatty acid, or the interaction of a PUFA or.ox-PUFA:with a protein target.

20 This can be accomplished by challenging a host, for example, a human:or an animal such as a mouse, to a high level of PUFA or ox-PUFA and then determining the therapeutic efficacy of a test compound-based on-its ability to decrease 25 circulating or.cell surface ,VCAM-l concentration.

Alternatively, an in vitro screen can be used that is based on the .ability of the test compound to prevent the oxidation of a PUFA, or the interaction of a PUFA or ox-PUFA with a protein target in the presence of-an oxidizing substance such as a metal, for example, copper:, or an enzyme. such as a peroxidase, lipoxygenase,-cyclooxygenase, or cytochrome P450.

In another embodiment, vascular endothelial cells are exposed to TNF-a or other VCAM-1 inducing material- for an appropriate time and then broken by any appropriate means, for example by sonication or freeze-thaw. The cytosolic and membrane compartments are isolated. Radiolabeled PUFA is added to defined amounts of the compartments. The ability of the liquid to convert PUFA to ox-PUFA in the presence or absence of a test compound isassayed. Intact cells can be used in place of the broken-cell system.

Pyrrolidine dithiocarbamate. (PDTC), orally delivered at 25-50mg/kg/day;, dramatically inhibited atherogenic fatty streak'formation, arterial monocyte-macrophage inflammation,: endothelial VCAM- 1 expression and essentially normalized endothelium dependent relaxation function :in diet induced hypercholesterolemic rabbits with serum cholesterol levels over 1000 mg/dl. At the-same doses, other putative therapeutic agents, such as the antioxidants probucol and vitamin E, had no .effect on lesion formation in this model.

Endothelial dependent arterial relaxation is 20 restored in experimental atherosclerosis by administration of PDTC. In the diet induced hypercholesterolemic rabbit model, orally delivered (25-50mg/kg/day) PDTC restored endothelial dependent vasoreactivity. This was determined by 25 ring-contraction studies of excised aorta from control and test animals... In patients with atherosclerosis, this manifests itself as normalization of peripheral vascular reactivity in response-to hyperemia as measured by non-invasive -Doppler flow'studies This is a.standardized, commonly available and easily:administered test that can be used to titrate functional drug levels to oral doses. The PDTC functions as an antiischemic therapy by rapidly normalizing the pathological loss of endothelial derived arterial vasodilation characteristic of cardiovascular diseases and atherosclerosis. This improvement in -11vascular blood flow is manifested as an improvement in symptom and ischemia-limited exercise function and provides a non-invasive assessment of vascular protection. Other clinical indications of abnormalities in endothelial derived vasorelaxation include impotence.

The molecular regulator factory that controls VCAM-1 gene transcription is a novel transcription factor complex consisting of the p65 and subunits of the NF-kB/Rel family cross-coupled to the c-fos and the c-jun subunits of the AP-1 family. By both structural and functional studies, it has been established that these AP-1 factors i play an important role in the regulation of the VCAM-1 promoter that likely are central to therapeutic regulation of VCAM-1 gene expression.

This is the first demonstration of a functional role of this cross-coupled transcription complex in the regulation of an endogenous gene.

20 Brief Description of the Figures Figure 1 is a graph of the cell-surface expression 450 nm) of VCAM-1 as a function of hours in human aortic endothelial cells on exposure to the cytokine TNF-a (closed circle); linoleic acid (closed triangle); and linoleyl hydroperoxide (13-HPODE, closed square); and in the absence of exposure to these substances (control, open square).

Figure 2 is a graph of the cell-surface expression 450 nm) of VCAM-1 in human aortic endothelial cells on exposure to linoleic acid (closed triangle) and linoleyl hydroperoxide (13- HPODE, closed square) as a function of the concentration of fatty acid (AM).

-12- Figure 3 is a bar chart graph of the cellsurface expression 450 nm) of VCAM-1, ICAM-1 and E-selectin in human aortic endothelial cells on exposure to the cytokine TNF-a, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid.

Figure 4 is a bar chart graph of the cellsurface expression 450 nm) f VCAM-1 in human aortic endothelial cells on exposure to linoleic acid, 13-HPODE, arachidonic acid, and arachidonic acid hydroperoxide (15-HPETE), with (solid black) or without (hatched lines) the antioxidant pyrrolidine dithiocarbamate.

Figure 5 is an illustration of an autoradiogram 15 indicating the acute induction of VCAM-1 mRNA by linoleic acid and 13-HPODE. HAEC were exposed or not to linoleic acid (7.5 MM), 13-HPODE (7.5 AM) or TNF-a (100 U/ml). Total RNA was isolated and 20 Ag was size-fractionated by denaturing 20 agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose, and hybridized to either 3 P-labeled human A) VCAM-1 specific or B) S-actin specific cDNA. After washing, the filters were exposed to X-ray film at -70 0 C with one 25 intensifying screen for 24 hours. Identification of lanes: i) control; 2) linoleic acid (acute, 8-hour exposure); 3) linoleic acid (48-hour exposure); 4) 13-HPODE (acute, 8-hour exposure); and 5) TNF-a (100 U/ml, 4-hour exposure).

Figure 6 is an illustration of an autoradiogram that indicates that induction of VCAM-1 mRNA by polyunsaturated fatty acids is independent of cellular protein synthesis. HAEC were exposed to either linoleic (7.5 pM) or arachidonic (7.5 MM) acid in the presence or absence of cycloheximide Mg/ml) for a 4-hour period, and then treated as described in Figure -13- Figure 7 is an illustration of an autoradiogram that indicates that linoleic acid induces transcriptional activation of the VCAM-1 promoter by a redox-sensitive NF-kB like factor. HAEC were split at the ratio to give approximately confluence in 100-mm tissue culture plates. HAEC were transfected with either 30 Ag of p288 VCAMCAT, VCAMCAT, or pSV 2 CAT plasmid by the calcium phosphate coprecipitation technique using standard techniques. After a 24-hour recovery period, HAEC were pretreated or not with 50 MM PDTC and after minutes exposed to linoleic acid (7.5 AM) or TNF-a (100 U/ml) directly added to the plates. After 18 hours, cell extracts were prepared by rapid 15 freeze-thaw in 0.25 M Tris, pH 8.0. The protein of each cell extract was assayed for chloramphenicol acetyl transferase (CAT) activity, as previously described [Ausubel, 1989] (Ac, acetylated; N, nonacetylated chloramphenicol) 20 Figure 8 is an illustration of an acrylamide gel slab that indicates that polyunsaturated fatty acids activate NF-kB-like DNA binding activities that are blocked by the antioxidant PDTC.

Confluent HAEC in media containing 4% FBS (as 25 described in Figure 1) were pretreated or not with PDTC (50 MM) for thirty minutes and then exposed for three hours to linoleic acid (7.5 MM, oleic acid (7.5 MM), or TNFa (100 U/ml), respectively.

Five micrograms of nuclear extract was incubated with a double-stranded "P-labeled wtVCAM, size fractionated on 4t native acrylamide gels, and exposed to autoradiography film at -70 0 C for 18 hours. Two bands A and C, representing NF-kB like binding activity are designated. A weak band B was observed in control (untreated) cells.

Figures 9A and 9B are bar chart graphs of the relative thiabarbituric acid reactive substances -14- 532 nm) of arachidonic acid and 15-HPETE in the presence or absence of PDTC. The thiobarbituric acid reactivity assay (TBARS) measures the oxidation ability of a material in a cell-free, media-free environment.

Figure 10 is an illustration of an autoradiogram of mRNA, obtained as described below, hybridized to either 32P-labeled human VCAM-1 specific cDNA (Panel E-selectin (ELAM-1) specific cDNA (Panel or ICAM-1 specific cDNA (Panel Following pre-treatment for 30 minutes with 50 M of sodium pyrrolidine dithiocarbamate (PDTC), HUVE (human umbilical vein) cells were exposed to IL-lb U/ml) in the continuous presence of 50 AM PDTC.

15 Parallel controls were performed identically except in the absence of PDTC. At the indicated times, o total RNA was isolated and 20 Ag of material sizefractionated by denaturing 1.0% agaroseformaldehyde gel electrophoresis, transferred to 20 nitrocellulose, hybridized as described above, and visualized by autoradiography. Lane 1-0 hour; Lanes 2,4,6,8 OL-1 alone for 2, 4, 8 and 24 hours, respectively; Lanes 3,5,7,9 IL-1 with PDTC for 2,4,8 and 24 hours, respectively.

25 Figure 11 is an illustration of an autoradiogram of mRNA, obtained as described below, hybridized to either 32P-labeled human VCAM-1 specific (Panel A), E-selectin (ELAM-1) specific cDNA (Panel or ICAM-1 specific cDNA (Panel HUVE cells were pretreated with the indicated concentrations of PDTC, and then exposed to IL-lb in the presence of PDTC for four hours and assayed for VCAM-1 mRNA accumulation by Northern filter hybridization analysis. Lane 1 control, lane 2 IL-1 (10u/ml), lane 3 IL-lb PDTC (0.05 AM), lane 4 IL-1 LB PDTC (0.5 MM), lane 5 IL-lb PDTC AM), lane 6 IL=lb PDTC (50.0 AM), lane 7 IL-lb PDTC (100 Figure 12 is an illustration of an autoradiogram of mRNA, obtained as described below, hybridized to either 32P-labeled human VCAM-1 specific cDNA (Panel E-selectin (ELAM-1) specific cDNA (Panel or ICAM-1 specific cDNA (Panel HUVE cells were pretreated:as described in Figure 9 with 50 M PDTC, exposed for fourhours to..the agents indicated below, and assayed for VCAM-1 (Panel A) and ICAM-1 (Panel B) mRNA accumulation. Lane 1 TNFa .(1QOU/ml), lane 2 TNFa PDTC, lane 3 lipopolysaccharide (LPS) :(100ng/ml), lane 4 LPS PDTC, lane 5 poly(I:C) (100mg/ml), lane 6 poly(I:C) PDTC.

15 Figure 13.,isa graph of relative cell surface expression of VCAM-1 and ICAM-1 in the presence (dark bars) or absence (white bars) of PDTC and in the presence of:multiple types of inducing stimuli.

Confluent HUVECs were pretreated or not pretreated 20 (CTL only) for. 30 minutes with 50 IM PDTC, and then exposed for the indicated times to the indicated agents in the presence or absence (CTL only) of PDTC.. Cell surface expression was determined by primary binding with VCAM-1 specific (4B9) and 25 ICAM-1 -specific (84H10) mouse monoclonal antibodies followed by secondary binding with a horse-radish peroxidase tagged goat anti-mouse (IgG).

Quantitation was performed by determination of calorimetric conversion at 450 nm of TMB. Figure 13 indicates that multiple regulatory signals induce VCAM-1 but not ICAM-1 through a common, dithiocarbamate-sensitive pathway in human vascular endothelial cells.

Figure 14 is a graph of the relative VCAM-1 cell surface expression 595 nM) in human umbilical vein endothelial cells, activated by TNFa, versus concentration of various antioxidants. (PDTC is ~-16sodium N-pyrrolidine dithiocarbamate; DETC is sodium N,N-diethyl-N'-carbodithiolate, also referred to as sodium diethyldithiocarbamate; MAC is Nacetyl cysteine; and DF is desferroximine) Figure 15 is a graph of-the relative VCAm-1 cell surface expression 595 n14) in human umbilical vein endothelial cells, activated by TNF-cy, .in the presence of the specified amount of antioxidant.

(PDTC is sodium N-pyrrolidine dithiocarbamate; DIDTC is sodium N,N-diethyl-N-carbodithioate; SarDTC is sodium N-methyl-N-carboxymethyl-Ncarbodithioate;- IDADTC i-strisodium N,N-' di(carboxymethyl)-N-carbodithioate; MGDTC is sodium N-methyl -D-glucamine-N-carbodithioate; MeOBGDTC is sodium N- (4-methoxybenzyl) -D-glucamine-Ncarbodithioate; DEDTC is sodium N,N-diethyl-Ncarbodithioate; Di-PDTC i~s sodium N,N-diisopropyl- N-carbodithioate; -NAC is N-acetyl cysteine.) Figure 16 is a graph of the percentage of Molt-4 cells binding to HUVE cells either unstimulated or stimulated with TNFa (100 U/mi) for six hours in the presence or absence of PDTC.

Figure 17is An illustration' of the chemical' structures of the following Active dithiocarbamfates: 'Sodium pyrrolidine-Ncarbodi -thioate', sodium N-ffithyl-N-carboxymethyl-Ncarbodithicate, trisodium. N, N-di (carboxymethyl) -Nca rbodithioate, sodium N-methyl-D-glucamine-Ncarbodithioate, sodium N, N-diethyl -N-carbodithioate (sodium diethyldithiocarbamate) and sodium N,Ndiisopropyl-N-:c'arbodithioate.' Figure 18 is a bar chart graph of the effect of PTflC on the formation of fluorescent adducts of BSA and 13-HPODE, as measured in fluorescent units versus micromolar concentration of PDTC. One micromolar of 13-HPODE was incubated with 200 micrograms of BSA in the presence of PDTC for six -17days. Fluorescence was measured at 430-460 nm with excitation at 330-360 nm.

Figure 19 is a graph of the effect of PTDC on the formation of fluorescent adducts of BSA and ox- PUFA as a function of wavelength (nm) and concentration of PDTC. As the concentration of PDTC increases, the quantity of fluorescent adducts decrease.

Figure 20 is a graph of the effect of PDTC on the oxidation of LDL by horseradish peroxidase (HRP), as measured by the increase in O.D. (234 nm) versus time (minutes) for varying concentrations of PDTC. It is observed that after an incubation period, PDTC inhibits the oxidation of LDL by HRP 15 in a manner that is concentration dependent.

Figure 21 is a chart of the effect of PDTC on the cytokine-induced formation of ox-PUFA in human aortic endothelial cells. As indicated, both TNF-t and IL-1B causes the oxidation of linoleic acid to 20 ox-linoleic acid. The oxidation is significantly prevented by PDTC.

Detailed Description of the Invention I. Definitions As used herein, the term polyunsaturated fatty acid (also referred to herein as a "PUFA") refers to a fatty acid (typically C, to C 24 that has at least two alkenyl bonds, and includes but is not limited to linoleic (C,8-A- 1 2 i.linolenic (C, 1

A

6 9 .1 2 arachidonic (C 20

A

5 4 and eicosatrienoic (C AL8.". 14) acids.

The term oxidized polyunsaturated fatty acid refers to an unsaturated fatty acid in which at least one of the alkenyl bonds has been converted to a hydroperoxide. Nonlimiting examples are: s

-,COOH

SOH 13

-HODE

"OOH

-18-

COOH

"OOH

The term alkyl, as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic (in the case of Cs or greater) hydrocarbon of C, to CIo (or lower alkyl, C, to which specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, *oo *isohexyl, cyclohexyl, cyclohexylmethyl, 3methylpentyl, 2,2-dimethylbutyl, and 2,3o 10 dimethylbutyl. The alkyl group can be optionally substituted on any of the carbons with one or more moieties selected from the group consisting of hydroxyl, amino, or mono- or disubstituted amino, wherein the substituent group is independently alkyl, aryl, alkaryl or aralkyl; aryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught "20 in Greene, et al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991.

The term alkenyl, as referred to herein, and unless otherwise specified, refers to a straight, branched, or cyclic hydrocarbon of C 2 to CI 0 with at least one double bond.

The term alkynyl, as referred to herein, and unless otherwise specified, refers to a C 2 to CIO straight or branched hydrocarbon with at least one triple bond.

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

PCT/U 95/05880 -19- The term alkaryl refers to an alkyl group that has at least one aryl substituent.

The term halo (alkyl, alkenyl, or alkynyl) refers to an alkyl, alkenyl, or alkynyl group in which at least one of the hydrogens in the group has been replaced with a halogen atom.

The term aryl, as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group can be optionally substituted with one or more moieties selected from the group consisting of alkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or 15 phosphonate, CO 2 H, or its pharmaceutically acceptable salt, C0 2 (alkyl, aryl, alkaryl or aralkyl), or glucamine, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et 20 al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991.

The term alkoxy, as used herein, and unless otherwise specified, refers to a moiety of the structure -0-alkyl.

25 The term acyl, as used herein, refers to a group of the formula wherein R' is an alkyl, aryl, alkaryl or aralkyl group.

The term heteroaryl or heteroaromatic, as used herein, refers to an aromatic moiety that includes at least one sulfur, oxygen, or nitrogen in the aromatic ring. Nonlimiting examples are phenazine, phenothiazine, furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, morpholinyl, carbozolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl, pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, auinoxalinyl, -xanthinyl, hypoxanthinyl, pteridinyl, S 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine, N 6 -alkylpurines, N6benzylpurine, N6-halopurine, N'-vinylpurine, N'acetylenic purine, N6-acyl purine, N'-hydroxyalkyl purine, N6-thioalkyl purine, thymine, cytosine, 6-azapyrimidine, 2mercaptopyrmidine, uracil, NI-alkylpyrimidines, ;*Ni s-benzylpyrimidines, N'-halopyrimidines,

N

5 -vinylpyrimidine, Ns-acetylenic pyrimidine, NI-acyl pyrimidine, N'-hydroxyalkyl purine, and N6-thioalkyl purine, and isoxazolyl. The heteroaromatic group can be optionally substituted as described above for aryl. The heteroaromatic can be'partially or totally hydrogenated as desired. As a nonlimiting example, dihydropyridine can be used in place of pyridine. Functional oxygen and nitrogen groups on the heterocyclic base can be protected as necessary or desired during the reaction sequence.

Suitable protecting groups are well known to those 25 skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, tritylmethyl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyl, and p-toluylsulfonyl.

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

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

The term pharmaceutically acceptable derivative refers to a derivative of the active compound that -21upon administration to the recipient, is capable of providing directly or indirectly, the parent compound, or that exhibits activity itself.

The term "pharmaceutically acceptable cation" refers to an organic or inorganic moiety that carries a positive charge and that can be administered in association with a pharmaceutical agent, for example, as a countercation in a salt.

Pharmaceutically acceptable cations are known to those of skill in the art, and include but are not limited to sodium, potassium, and quaternary amine.

The term "physiologically cleavable leaving .group" refers to a moiety that can be cleaved in vivo from the molecule to which it is attached, and 15 includes but is not limited to an organic or inorganic anion, a pharmaceutically acceptable cation, acyl (including but not limited to (alkyl)C(O), including acetyl, propionyl, and butyryl), alkyl, phosphate, sulfate and sulfonate.

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

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

A "linking moiety" as used herein, is any divalent group that links two chemical residues, including but not limited to alkyl, alkenyl, alkynyl, aryl, polyalkyleneoxy (for example -C,.alkoxy-C.o 10 alkyl-, -22-

-C

1 alkylthio-C.o 0 alkyl-, -NR 3 and -(CHOH),CHOH, wherein n is independently 0, 1, 2, 3, 4, 5, or 6.

II. Identification of Oxidized and Unoxidized Polyunsaturated Fatty Acids as Direct Mediators of VCAM-1 Expression To establish whether a PUFA or oxidized PUFA acts as a direct immunomodulator of endothelial cell gene expression, early passaged human aortic endothelial cells (HAEC) were cultured for eight hours in media and serum and exposed to saturated (stearic), monounsaturated (oleic), and •polyunsaturated (linoleic and arachidonic) fatty S: acids; as well as with the fatty acid Shydroperoxides of linoleic (13-HPODE) or 15 arachidonic (15-HPETE) acids. HAEC were also alternatively exposed to the cytokine tumor necrosis factor-a.

HAEC were exposed to linoleic acid or 13-HPODE for varying times up to 48 hours and then assayed for cell surface VCAM-1 expression by ELISA.assay.

The results were compared to HAEC exposed to the cytokine TNF-a (100 U/ml) for the same time periods. VCAM-1 expression in HAEC incubated with either linoleic acid or 13-HPODE is transiently induced. The expression peaks at approximately 8-9 hours with significant expression at 24 hours and then decreases by 48 hours. The kinetics of VCAM-1 induction by both linoleic acid and 13-HPODE mirror that of TNF-a, and thus the mechanisms by which polyunsaturated fatty acids induce VCAM-1 thus appear to be similar to that of TNF-a.

Dose-response studies of linoleic acid and 13-HPODE on VCAM-1 gene expression at 8 hours were also conducted. It was observed that 7.5 iM is the lowest peak dose by which linoleic acid and -23- 13-HPODE induces significant VCAM-1 gene expression.

It was then explored whether short term incubation of endothelial cells with polyunsaturated fatty acids induces ICAM-1 and E-selectin expression as well. It was determined that the polyunsaturated fatty acids linoleic and arachidonic acids induced cell-surface gene expression to approximately 59% of TNF-induced gene expression of VCAM-1. Strikingly, neither ICAM-1 nor E-selectin were induced by these fatty acids.

Conversely, the saturated fatty acid stearic acid and the monounsaturated fatty acid oleic acid did not induce the expression of VCAM-1, ICAM-1, or E-selectin. VCAM-1 gene expression was also '"00 observed by incubation of HAEC with the oxidized metabolites of linoleic acid (13-HPODE) and arachidonic acid To investigate whether oxidative stress in 20 endothelial cells provided by polyunsaturated fatty acids and their oxidized metabolites induces VCAM-1 through a redox-sensitive mechanism, HAEC were pretreated with the antioxidant pyrrolidine dithiocarbamate (PDTC, 50 MM) for 30 minutes and 25 then the cells were independently incubated with linoleic acid, arachidonic acid, 13-HPODE, and (all 7.5 AM) for 8 hours. It was determined that PDTC suppressed the gene expression of VCAM-1 induced by the polyunsaturated fatty acids and their oxidized counterparts. This indicates that the induction is mediated by a oxidized signal molecule, and that the induction is prevented when the oxidation of the molecule is blocked the oxidation does not occur), reversed the signal molecule is reduced), or its interaction with a target protein prevented, perhaps through a redox complex.

-24- To determine whether the selective induction of VCAM-1 by PUFAs and their oxidized metabolites is observed at the mRNA level, HAEC were incubated with linoleic acid or.13-HPODE. Linoleic acid and 13-HPODE induced VCAM-1 mRNA accumulation that was similar to levels induced by TNF-a. In contrast, there was no induction of ICAM-1 or E-selectin gene expression at the mRNA level in HAEC incubated with linoleic acid or 13-HPODE. The findings mimic those found at the cell-surface level. These results indicate that pretranslational regulatory mechanisms mediate induction of VCAM-1 gene •expression by polyunsaturated fatty acids and their oxidative metabolites.

15 It was also desired to determine whether polyunsaturated fatty acids work as a primary Ssignal or operate through a regulatory protein involving the cytokine IL-4 in inducing VCAM-1 gene expression. To investigate whether newly 20 synthesized proteins such as IL-4 are involved in the synthesis and gene expression of VCAM-1 induced by PUFAs such as linoleic acid, HAEC were incubated with 13-HPODE (7.5 AM) and exposed to the protein synthesis inhibitor, cycloheximide. There was no 25 inhibition of mRNA accumulation of VCAM-1 by cycloheximide in HAEC incubated with 13-HPODE. The production of IL-4 by HAEC incubated with linoleic or arachidonic acids and their oxidative metabolites, as determined by ELISA was also measured. There was no increase in IL-4 output by HAEC incubated with these PUFAs or their oxidized metabolites.

Previous investigations have demonstrated through deletion and heterologous promoter studies that cytokines and non-cytokines activate VCAM-1 gene expression in endothelial cells at least in part transcriptionally through two NF-kB-like DNA binding elements. It has also been demonstrated that PDTC inhibits VCAM-1 gene expression through a redox-sensitive NF-kB like factor. To determine whether polyunsaturated fatty acids induce transcriptional activation of the human VCAM-1 promoter via a similar mechanism, the chimeric reporter gene p288 VCAM-CAT, cortaining coordinates -288 to +22 of the human VCAM-1 promoter, was transiently transfected into HAEC. The addition of linoleic acid (7.5 AM) induced VCAM-1 promoter.

The addition of linoleic acid (7.5 AM) induced VCAM-I promoter activity that was over two fold that of the control and approximately 60% of the maximum signal induced by TNF-a. Similar results 15 were obtained with the minimal cytokine-inducible promoter of the VCAM-1 gene (p85 VCAM-CAT), containing the -77 and -63 bp NF-kB-like sites.

Neither linoleic acid nor TNF-a had any effect on activity using a constitutively expressed pSV 2

CAT

20 construct. PDTC inhibited the transcriptional activation of both VCAM-1 promoter constructs induced by linoleic acid. The data indicate that analogous to TNF-.a, polyunsaturated fatty acids such as linoleic acid induce the transcriptional 25 activation of VCAM-1 through an NF-kB-like redox-sensitive mechanism.

To determine whether polyunsaturated fatty acids and their oxidative metabolites regulate VCAM-1 promoter activity through an NF-kB-like transcriptional regulatory factor, nuclear extracts from HAEC were assayed for DNA binding activity to a double-stranded oligonucleotide containing the VCAM-1 NF-kB-like promoter elements located at -ositions -77 and -63. As shown in Figure 7, two bands A and C, representing NF-kB-like activity were induced in response to a three hour exposure to linoleic acid (7.5 AM). Similar findings were -26observed on exposure to the cytokine TNF-a (100 U/ml). A weak band B was observed in control (untreated) cells. No induction of NF-kB-like binding was observed with the monounsaturated fatty acid oleic acid. Pretreatment of the cells for thirty minutes with PDTC inhibited the A and C complex DNA binding activity after linoleic acid activation. These findings are similar to previously reported findings that PDTC blocks the activation of VCAM-1 gene expression in HUVEC by inhibiting the activation of these NF KB-like DNA binding proteins.

Example 1 Effect of Oxidized and Unoxidized Polyunsaturated Fatty Acids on the 15 Kinetics of the Activation of VCAM-1 Gene Expression *Human aortic endothelial cells (HAEC) were plated in 96 well plates and incubated with linoleic acid (7.5 AM), 13-HPODE (7.5 AM), or TNF-a (100 U/ml) at five different time points up to 48 *hours. HAEC, obtained from Clonetics (Boston, MA), were cultured in Medium 199 supplemented with fetal bovine serum (FBS), 16 U/ml heparin, 10 U/ml epidermal growth factor, 50 g/ml endothelial cell growth supplement, 2 mM L-glutamine, 100 U/ml penicillin, and 100 Ag/ml streptomycin. One day before the experiment, cells were-placed in a medium containing 4% FBS. Confluent HAEC were incubated for up to 48 hours with TNF-C (100 U/ml), or stearic, oleic, linoleic, linolenic, or arachidonic acids (7.5 MM). Similar studies were performed with differing doses of linoleic acid or 13-HPODE for an 8 hour period (1-60 AM) (Figure Quantitation was performed by determination of colorimetric conversion at 450 nm of TMB. Studies were performed in triplicate (n=4 -27for each experimental value). *-value differs (p<0.05) from Control.

As shown in Figure 1, both linoleic acid and 13- HPODE induced the expression of VCAM-1. At ten hours after exposure, the amount of cell surface VCAM-1 induced by linoleic acid and 13-HPODE was greater than half that induced by the cytokine TNF-a.

As shown in Figure 2, the induction of VCAM-1 by linoleic acid and 13-HPODE is concentration sensitive. At a concentration of between 2 and AM of these compounds, there is a sharp increase in the amount of induced cell surface VCAM-1, which then remains approximately constant up to a 15 concentration of at least 100 AM. It should be observed that the PUFA concentration indicated in Figure 2 is in addition to that found endogenously in HAEC.

20 Example 2 Polyunsaturated Fatty Acids Induce Gene Expression of VCAM-1 but not ICAM-1 or E-selectin.

The cell surface expression of VCAM-1, ICAM-1, and E-selectin was measured in HAEC by ELISA.

HAEC, obtained from Clonetics (California), were cultured in Medium 199 supplemented with 20% fetal bovine serum (FBS), 16 U/ml heparin, 10 U/ml epidermal growth factor, 50 pg/ml endothelial cell growth supplement, 2 mM L-glutamine, 100 U/ml penicillin, and 100 Ag/ml streptomycin. One day before the experiment, cells were placed in a medium containing 4% FBS. Confluent HAEC were incubated or not for 8 hours with TNF-a (100 U/ml), or stearic, oleic, linoleic, linolenic, or arachidonic acids (7.5 AM). Cell-surface expression of A) VCAM-1, B) ICAM-1, and C) E-selectin was determined by primary binding with VCAM-1 specific, ICAM-1 specific, and E-selectin -28specific mouse antibodies followed by secondary binding with a horseradish peroxidase-tagged goat anti-mouse (IgG). Quantitation was performed by determination of colorimetric conversion at 450 mm of TMB. Studies were performed in triplicate (n=4 for each experimental value) *-value differs (p<0.05) from Control.

As shown in Figure 3, linoleic acid, linolenic acid, and arachidonic acid significantly induced the expression of VCAM-1, but did not induce the 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-a strongly induced the expression of all three cell- 15 surface molecules.

Example 3 The Antioxidant PDTC Suppresses VCAM-1 Induction by Polyunsaturated Fatty Acids and their Oxidative Metabolites.

Confluent HAEC were pretreated in the presence or absence of PDTC (sodium pyrrolidine dithiocarbamate, 50 AM) for thirty minutes. The *cells were then incubated for eight hours with TNFa (100 U/ml), linoleic or arachidonic acid or the fatty acid hydroperoxides 13-HPODE AM) or 15-HPETE (7.5 AM). The cell surface expression of VCAM-1 was measured in HAEC by ELISA, as described in Example 1. Studies were performed in triplicate (n 4 for each experimental value).

*-value differs (p<0.05) from control.

As indicated in Figure 4, PDTC suppresses the induction of VCAM-1 by linoleic acid, 13-HPODE, arachidonic acid and Example 4 Acute Induction of VCAM-1 mRNA by Linoleic Acid and 13-HPODE.

HAEC were exposed to linoleic acid (7.5 AM) or 13-HPODE (7.5 Total RNA was isolated and -29- Ag size-fractionated by denaturing agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose, and hybridized to either P-ilabeled human A) VCAM-1 specific or B) G-actin specific cDNA and visualized by autoradiography. After washes, filters were exposed to X-ray film at -70°C with one intensifying screen for 24 hours. Identification of lanes: 1) control; 2) linoleic acid (acute, 8-hour exposure); 3) linoleic (48-hour exposure); 4) 13-HPODE (acute, 8-hour exposure); and 5) TNF-a S" (100 U/ml, 4-hour exposure).

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

Example 5 Induction of VCAM-1 mRNA by PUPAs is Independent of Cellular Protein Synthesis.

20 KAEC were exposed to either linoleic or arachidonic acid (7.5 AM) in the presence or absence of cycloheximide (10 Ag/ml) for a 4-hour eriod. Total RNA was isolated and 20 pg was size-fractionated by denaturing agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose, and hybridized to A) 32P-labeled human VCAM-1 or B) S-actin specific cDNA and then visualized by autoradiography. After washes, filters were exposed to X-ray film at -700C with one intensifying screen for 24 hours.

As indicated in Figure 6, the induction of VCAM- 1 by linoleic and arachidonic acids are independent of cellular protein synthesis.

Example 6 Linoleic acid induces transcriptional activation of the VCAM-1 promoter by a redox-sensitive NF-kB like factor.

HAEC were split at the ratio to give approximately 60% confluence in 100-mm tissue culture plates. HAEC were transfected with either pg of p288 VCAMCAT, p85 VCAMCAT, or pSV 2

CAT

plasmid by the calcium phosphate coprecipitation technique using standard techniques. After a 24-hour recovery period, HAEC were pretreated with AM PDTC and after 30 minutes exposed to linoleic acid (7.5 AM) or TNF-a (100 U/ml) directly added to he plates. After 18 hours, cell extracts were prepared by rapid freeze-thaw in 0.25 M Tris, pH 15 8.0. Protein of each cell extract was assayed for chloramphenicol acetyl transferase (CAT) activity (Ac, acetylated; N, nonacetylated chloramphenicol).

Figure 7 illustrates the results of this experiment. Linoleic acid induces transcriptional activation of the VCAM-1 promoter by a redox-sensitive NF-kB like factor. These results are similar to those observed by the activation of VCAM-1 promotor by cytokines such as TNF-a. This suggests that PUFAs act through an oxidized intermediate that also mediates the cytokine activation of VCAM-1.

Example 7 Polyunsaturated Fatty Acids Activate NF-kB-like DNA Binding Activities that are Blocked by the Antioxidant PDTC.

Confluent HAEC in media containing 4% FBS (as described in Example 1) were pretreated with PDTC uM) for 30 minutes and then exposed for 3 hours to linoleic acid or oleic acid (7.5 AM), or TNF-a (100 U/ml). Five micrograms of nuclear extract was incubated with a double-stranded "P-labeled wtVCAM, size fractionated on 4% native acrylamide gels, and exposed to autoradiography film at -700C for 18 -31hours. Two bands A and C, representing NF-kB like binding activity are designated. A weak band B was observed in control (untreated) cells.

Figure 8 illustrates that linoleic acid induces NF-kS binding activity to VCAM-1 promotor in a redox-sensitive manner. This is analogous to cytokine TNF-a and suggests a similar mechanism of action. TNF-a probably induces VCAM-1 through a mechanism that is mediated by an ox-PUFA.

Example 8 Oxidation in a cell-free, media-free setup, by both unoxidized and oxidized (15-EPETE) arachidonic acid *Figures 9A and 9B are bar chart graphs of the Srelative thiabarbituric acid reactive substances 15 532 nm) of arachidonic acid and 15-HPETE in the presence or absence of PDTC. The thiobarbituric acid reactivity assay CTBARS) measures the oxidation ability of a material in a cell-free, media-free environment. As indicated in the Figures, both arachidonic acid and showed significant TBARS activity that was inhibited by PDTC.

III. Method for the Treatment of VCAM-1 Mediated Disorders The discovery that polyunsaturated fatty acids and their oxidized metabolites are selective, redox-sensitive immunomodulators provides a basis for the therapy of disorders that are mediated by VCAM-1 or by redox-sensitive genes.

A method for the treatment of atherosclerosis, post-angioplasty restenosis, coronary artery diseases, angina, and other cardiovascular diseases, as well as noncardiovascular inflammatory diseases that are mediated by VCAM-1 is provided that includes the removal, decrease in the -32concentration of, or prevention of the formation of oxidized polyunsaturated fatty acids, including but not limited to oxidized linoleic, linolenic, and arachidonic acids. In an alternative embodiment, a method for the treatment of these diseases is provided that includes the prevention of the interaction of a PUFA or ox-PUFA with a protein or peptide that mediates VCAM-1 expression.

Inhibition of the expression of VCAM-1 can be accomplished in a number of ways, including through the administration of an antioxidant that prevent the oxidation of a polyunsaturated fatty acid, by in vivo modification of the metabolism of PUFAs into ox-PUFAs, as described in more detail below.

1. Administration of Antioxidants Any compound that reduces an ox-PUFA or which inhibits the oxidation of PUFA, and which is relatively nontoxic and bioavailable or which can be modified to render it bioavailable, can be used in this therapy. One of ordinary skill in the art can easily determine whether a compound reduces an ox-PUFA or inhibits the oxidation of PUFA using standard techniques.

Dithiocarboxylate Antioxidants It has been discovered that dithiocarboxylates are useful in the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

Dithiocarboxylates, including dithiocarbamates, can be used to block the ability of cells, including endothelial cells, to express VCAM-1 or to suppress the expression of a redox-sensitive gene or activate a gene that is suppressed through a redoxsensitive pathway.

-33- At least one of the compounds, pyrrolidine dithiocarbamate (PDTC), inhibits VCAM-1 gene expression at a concentration of less than micromolar. This compounds also exhibits preferential toxicity to proliferating or abnormally dividing vascular smooth muscle cells.

Another dithiocarbamate, sodium N-methyl-Ncarboxymethyl-N-carbodithioate, also inhibits the expression of VCAM-1, without significant effect on ICAM-1, but does not exhibit preferential toxicity to abnormally dividing vascular smooth muscle cells. Another dithiocarbamate, sodium N-methyl-Ncarboxymethyl-N-carbodithioate, also inhibits the expression of VCAM-1, without significant effect on 15 ICAM-1, but does not exhibit preferential toxicity to abnormally dividing vascular smooth muscle cells.

It has been discovered that pyrrolidine dithiocarbamate does not significantly block ELAM-1 or ICAM-1 expression, and therefore treatment with this compound does not adversely affect aspects of the inflammatory response mediated by ELAM-1 or ICAM-1. Thus, a generalized immunosuppression is avoided. This may avoid systemic complications from generalized inhibition of adhesion molecules in the many other cell types known to express them.

Other pharmaceutically acceptable salts of PDTC are also effective agents for the treatment of cardiovascular and inflammatory disorders.

Dithiocarbamates are transition metal chelators clinically used for heavy metal intoxication.

Baselt, F.W.J. Sunderman, et al. (1977), "Comparisons of 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 -34- (1978), "Treatment of pompholyx due to nickel allergy with chelating agents." Contact DermaiitIs 289-90; Sunderman, F.W. (1978), "Clinical response to therapeutic agents in poisoning from mercury vapor" Ann Clin Lab Sci 259-69; Sunderman, F.W. (1979), "Efficacy of sodium diethyldithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning." Ann Clin Lab Sci 1-10; Gale, A.B. Smith, et al. (1981), "Diethyldithiocarbamate in treatment of acute cadmium poisoning." Ann Clin Lab Sci 11(6): 476- 83; Jones, M.M. and M.G. Cherian (1990), "The search for chelate antagonists for chronic cadmium intoxication." Toxicolooy 62 1-25; Jones, 15 M.A. Basinger, et al. (1982), "A comparison q ~of diethyldithiocarbamate and EDTA as antidotes for acute cadmium intoxication." Res Commun Chem SPathol Pharmacol 38(2): 271-8; Pages, J.S.

Casas, et al. (1985), "Dithiocarbamates in heavy metal poisoning: complexes of N,N-di(1hydroxyethyl)dithiocarbamate with Zn(II), Cd(II), Hg(II), CH3Hg(II), and C6H5Hg(II).: J. Inorg Biochem 25(1): 35-42; Tandon, N.S. Hashmi, et al. (1990), "The lead-chelating effects of substituted dithiocarbamates. Biomed Environ Sci 299-305.

Dithiocarbamates have also been used adjunctively in cis-platinum chemotherapy to prevent renal toxicity. Hacker,

W.B.

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 4733; Saran, M. and Bors, W. (1990) "Radical reactions in vivo--an overview." Radiat. Environ. Biohvs.

29(4):249-62.

A dithiocarbamate currently used in the treatment of alcohol abuse is disulfiram, a dimer of diethyldithiocarbamate. Disulfuram inhibits hepatic aldehyde dehydrogenase. Inoue, and Fukunaga, et al., (1982). "Effect of disulfiram and its reduced metabolite, diethyldithiocarbamate on aldehyde dehydrogenase of hurin erythrocytes." Life Sci 30(5): 419-24.

It has been reported that dithocarbamates inhibit HIV virus replication, and also enhance the maturation of specific T cell subpopulations. This has led to clinical trials of diethyldithiocarbamate in AIDs patient populations. Reisinger, et al., (1990). "Inhibition of HIV progression 15 by dithiocarb." Lancet 335: 679.

*Dithiocarboxylates are compounds of the structure A-SC(S)-B, which are members of the general class of compounds known as thiol S.antioxidants, and are alternatively referred to as 20 carbodithiols or carbodithiolates. It appears that the moiety is essential for therapeutic activity, and that A and B can be any group that does not adversely affect the efficacy or toxicity of the compound.

25 In an alternative embodiment, one or both of the sulfur atoms in the dithiocarbamate is replaced with a selenium atom. The substitution of sulfur for selenium may decrease the toxicity of the molecule in certain cases, and may thus be better tolerated by the patient.

A and B can be selected by one of ordinary skill in the art to impart desired characteristics to the compound, including size, charge, toxicity, and degree of stability, (including stability in an acidic environment such as the stomach, or basic environment such as the intestinal tract). The selection of A and B will also have an important effect on the tissue-distribution and pharmacokinetics of the compound. In general, for treatment of cardiovascular disease, it is desirable that the compound accumulate, or localize, in the arterial intimal layer containing the vascular endothelial cells. The compounds are preferably eliminated by renal excretion.

An advantage in administering a dithiocarboxylate pharmaceutically is that it does not appear to be cleaved enzymatically in vivo by thioesterases, and thus may exhibit a prolonged halflife in vivo.

In a preferred embodiment, A is hydrogen or a pharmaceutically acceptable cation, including but 15 not limited to sodium, potassium, calcium, e magnesium, aluminum, zinc, bismuth, barium, copper, cobalt, nickel, or cadmium; a salt-forming organic acid, typically a carboxylic acid, including but not limited to acetic acid, oxalic acid, tartaric 20 acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, or polygalacturonic acid; or a cation formed from ammonia or other 25 nitrogenous base, including but not limited to a nitrogenous heterocycle, or a moiety of the formula

NR

4

RR

6 R7, wherein R 4

R

5

R

6 and R 7 are independently hydrogen, C 14 linear, branched, or (in the case of C4) cyclic alkyl, hydroxy(C, 4 )alkyl (wherein one or more hydroxyl groups are located on any of the carbon atoms), or aryl, N,N-dibenzylethylenediamine, D-glucosamine, choline, tetraethylammonium, or ethylenediamine.

In another embodiment, A can be a physiologically cleavable leaving group that can be cleaved in vivo from the molecule to which it is attached, and includes but is not limited acyl -37acetyl, propionyl, and 'butyry1) alkyl, zhospohate, sulfate or sulfonate.

in one embodiment, B is alkyl, alkenyl, alkynyl, alkar-yl, aralkyl, haloalkyl, haloalkenyl, ha2.oalkynyl, aryl, alkaryl, hydrogen, C1_ alkoxy-c 1 10 alkyl, C1_ alkylthio-C.

10 alkyl, NR 2

R

3 -CCHOH),CHt,OH, wherein n is 0, 1, 2, 3, 4, 5, or 6, (CH 2 ICo 2

R,

including alkylacetyl, alkyipropionyl, and alkylbutyryl, or hydroxy(C 1 4 6)alkyl- (wherein one or more hydroxyl groups are located on any of the carbon atoms).

In another embodiment, B is N'RR, wherein R 2 and R 3 are independently alkyl; wherein :n is 0, 1, 2, 3, 4, 5, or 6; -(CH 2 ).COR1,

(CH

2 "COR4 hydroxy (C 1 alkyl- alkenyl (including but not limited to vinyl, allyl, and

CH

3 CH C-C.

2 CH,) al1kyl1 (CO 2 H) alk e nyl1 (CO, H), alkynyl (C0 2 or aryl, wherein the aryl group can be substituted as described above, notably, for example, with a CH 3 t-butyl, CO,H, halo, or D- OH group); or R 2 and R1 can together constitute a bridge such as _(C12)m_1 wherein m is 3, 4, 5, or 6, and wherein R' is alkyl, aryl, alkaryl, or aralkyl, including acetyl, propionyl, and butyryl.

4425 In yet another embodiment, B can be a heterocyclic or alkylheterocyclic: group. The heterocycle can be optionally partially or totally hydrogenated. Nonlimiting examples are those listed above, including phenazine, phenothiazine, pyridine and dihydropyridine.

In still another embodiment, B is the residue of a pharmaceutically-active compound or drug. The term drug, as used herein, refers to any substance used internally or externally as a medicine fozr tne treatment, cure, or prevention of a disease or disorder.

-38- Nonlimiting examples are drugs for the treatment or prevention of cardiovascular disease, including antioxidants such as probucol; nicotinic acid; agents that prevent platelets from sticking, such as aspirin; antithrombotic agents such as coumadin; calcium channel blockers such as varapamil, diltiazem, and nifedipine; angiotensin converting enzyme (ACE) inhibitors such as captopril and enalopril, fi-blockers such as propanalol, terbutalol, and labetalol, nonsteroidal antiinflammatories such as ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, sulindac, or corticosteriods. The -C(S)SA group can be directly attached to the drug, or attached 15 through any suitable linking moiety.

In another embodiment, the dithiocarbamate is an amino acid derivative of the structure AO 2

C-R

9

-NR'

0 C(S)SA, wherein R 9 is a divalent B moiety, a linking moiety, or the internal residue of any of the 20 naturally occurring amino acids (for example, CH 3

CH

for alanine, CH 2 for glycine, CH(CH 2 z)NH 2 for lysine, etc.), and R' is hydrogen or lower alkyl.

B can also be a polymer to which one or more dithiocarbamate groups are attached, either directly, or through any suitable linking moiety.

The dithiocarbamate is preferably released from the polymer under in vivo conditions over a suitable time period to provide a therapeutic benefit. In a preferred embodiment, the polymer itself is also degradable in vivo. The term biodegradable or bioerodible, as used herein, refers to a polymer that dissolves or degrades within a period that is acceptable in the desired application (usually in vivo therapy), usually less than five years, and preferably less than one year, on exposure to a physiological solution of pH 6-8 having a temperature of between 25 and 37 0 C. In a preferred -39embodiment, the polymer degrades in a period of between 1 hour and several weeks, according to the application.

A number of degradable polymers are known.

Nonlimiting examples are peptides, proteins, nucleoproteins, lipoproteins, glycoproteins, synthetic and natural polypeptides and polyamino acids, including but not limited to polymers and copolymers of lysine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, hydroxylysine, serine, threonine, and tyrosine; polyorthoesters, including poly(a-hydroxy acids), for example, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polyanhydrides, 15 albumin or collagen, a polysaccharide containing sugar units such as lactose, and polycaprolactone.

The polymer can be a random or block copolymer.

B can also be a group that enhances the water solubility of the dithiocarbamate, for example, 20 -lower alkyl-O-R 8 wherein R 8 is -P0 2 (OH)M or PO3(M") 2 wherein M' is a pharmaceutically acceptable cation; (CH2) 2 CO M or -S0 3 -lower alkylcarbonyllower alkyl; -carboxy lower alkyl; -lower alkylamino-lower alkyl; N,N-di-substituted amino lower alkyl-, wherein the substituents each independently represent lower alkyl; pyridyl-lower alkyl-; imidazolyl-lower alkyl-; imidazolyl-Y-lower alkyl wherein Y is thio or amino; morpholinyl-lower alkyl; pyrrolidinyl-lower alkyl; thiazolinyl-lower alkyl-; piperidinyl-lower alkyl; morpholinyl-lower hydroxyalkyl; N-pyrryl; piperazinyl-lower alkyl; Nsubstituted piperazinyl-lower alkyl, wherein the substituent is lower alkyl; triazolyl-lower alkyl; tetrazolyl-lower alkyl; tetrazolylamino-lower alkyl; or thiazolyl-lower alkyl.

In an alternative embodiment, a dimer such as B- C(S)S-SC(S)-B can be administered.

NonliLmiting examples of. dithiocarbamates are those of the structure: V I) Aliphafic Subscrate 2) Amino Acid

HR/COOH

S-C

R CH.,-CH

CH

1

-CH-CH

Polyamino acid COCH COOH

N

Lys-Lys-Lys I I I R'N NR' NR c=U=si C=S

R

N NS Substiruencs

NO

2 CH3 or t-Buryl

COOH

p-OH Na" Ca S .0 11 N R -c -S-C -R Thioester wH Choline& and quaternary amnines Mg ,Ad+-

S

I1 A -S -C-B S S II1 11 -C-S -S-C-B -41- Dithiocarboxylaces should be chosen for use in treating atherosclerosis and other cardiovascular and inflammatory diseases that have the proper lipophilicity to locate at the affected cite. The compound should not compartmentalize in low turnover regions such as fat deposits. In a preferred embodiment for treatment of cardiovascular disease, the pharmacokinetics of the compound should not be dramatically affected by congestive heart failure or renal insufficiency.

For topical applications for the treatment of inflammatory skin disorders, the selected compound should be formulated to be absorbed by the skin in a sufficient amount to render a therapeutic effect to the afflicted site.

The dithiocarboxylate must be physiologically acceptable. In general, compounds with a therapeutic index of at least 2, and preferably at least 5 or 10, are acceptable. The therapeutic 20 index is defined as the EC, 5 /ICs 0 wherein EC, is the concentration of compound that inhibits the expression of VCAM-1 by 50% and IC is the concentration of compound that is toxic to 50% of the target cells. Cellular toxicity can be measured by direct cell counts, trypan blue exclusion, or various metabolic activity studies such as 3H-thymidine incorporation, as known to those skilled in the art. The therapeutic index of PDTC.in tissue culture is over 100 as measured by cell toxicity divided by ability to inhibit VCAM-1 expression activated by TNFa, in HUVE cells.

Initial studies on the rapidly dividing cell type HT-18 human glioma-demonstrate no toxicity at concentrations 100-fold greater than the therapeutic concentration. Disulfiram, an orally administered form of diethyldithiocarbamate, used in the treatment of alcohol abuse, generally -42elicits no major clinical toxicities when administered appropriately.

There are a few dithiocarbamates that are known to be genotoxic. These compounds do not fall within the scope of the present invention, which is limited to the administration of physiologically acceptable materials. An example of a genotoxic dithiocarbamate is the fungicide zinc dimethyldithiocarbamate. Further, the anticholinesterase properties of certain dithiocarbamates can lead to neurotoxic effects.

Miller, D. (1982). Neurotoxicity of the pesticidal S'carbamates. Neurobehav. Toxicol. Teratol. 4(6): 15 The term dithiocarboxylate as used herein specifically includes, but is not limited to, *:dithiocarbamates of the formulas:

R'SC(S)NR

2

R

3 or R 2

R

3

N(S)CS-SC(S)NRR

wherein R' is H or a pharmaceutically acceptable 20 cation, including but not limited to sodium, potassium, or NR 4

RSR

6

R

7 wherein R 5

R

6 and R 7 are independently hydrogen, C.4 linear, branched, or cyclic alkyl, hydroxy(C, 4 )alkyl (wherein one or more hydroxyl groups are located on any of the carbon atoms), or aryl, and

R

2 and R 3 are independently C.o linear, branched or cyclic alkyl; wherein n is 0, 1, 2, 3, 4, 5, or 6; -(CH2),CO 2

-(CH

2

)CO

2 R4; hydroxy(C 1 4 )alkyl-, or R 2 and R 3 together constitute a bridge such as wherein m is 3-6, and wherein R' is alkyl, aryl, alkaryl, or aralkyl, including acetyl, propionyl, and butyryl.

Specific examples of useful dithiocarbamates, illustrated in Figure 15, include sodium pyrrolidine-N-carbodithioate, sodium N-methyl-Ncarboxymethyl-N-carbodithioate, trisodium N,Ndi(carboxymethyl)-N-carbodithioate, sodium N- -43methyl-D-glucamine-N-carbodithioate, sodium N,Ndiethyl-N-carbodithioate (sodium diethyldithiocarbamate), and sodium N,N-diisopropyl-Ncarbodithioate.

The active dithiocarboxylates and in particular, dithiocarbamates are either commercially available or can be prepared using known methods.

II. Biological Activity The ability of dithiocarboxylates to inhibit the 10 expression of VCAM-1 can be measured in a variety S' of ways, including by the methods set out in detail below in Examples 9 to 15. For convenience, Examples 9-11 and 14-15 describe the evaluation of the biological activity of sodium pyrrolidine-N- 15 carbodithioate (also referred to as PDTC). These examples are not intended to limit the scope of the invention, which specifically includes the use of any of the above-described compounds to treat atherosclerosis, and other types of inflammation and cardiovascular disease mediated by VCAM-1. Any of the compounds described above can be easily substituted for PDTC and evaluated in similar fashion.

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

Experimental Procedures Cell Cultures HUVE cells were isolated from human umbilical veins that were cannulated, -44perfused with Hanks solution to remove blood, and then incubated with 1% collagenase for 15 minutes at 37 0 C. After removal of collagenase, cells were cultured in M199 medium supplemented with 20% fetal bovine serum (HyClone), 16 Ag/ml heparin (ESI Pharmaceuticals, Cherry Hill, NJ), 50 &g/ml endothelial cell growth supplement (Collaborative Research Incorporated, Bedford MA), 25 mM Hepes Buffer, 2 mM L-glutamin, 100 gg/ml penicillin and 100 Ag/ml streptomycin and grown at 37 0 C on tissue culture plates coated 0.1% gelatin. Cells were passaged at confluency by splitting 1:4. Cells were used within the first 8 passages.

Incubation with Cvtokines and Other Reagents 15 Confluent HUVE cells were washed with phosphate buffered saline and then received fresh media. The indicated concentrations of PDTC were added as pretreatment 30 minutes before adding cytokines.

Cytokines and other inducers were directly added to 20 medium for the times and at the concentrations indicated in each experiment. Human recombinant IL-lb was the generous gift of Upjohn Company (Kalamazoo, Michigan). TNFa was obtained from Bohringer Engelheim. Bacterial lipopolysaccharide (LPS), polyinosinic acid: polycitidilic acid (Poly and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma Chemical (St. Louis, MO). All other reagents were of reagent grade.

RNA Isolation: Total cellular RNA was isolated by a single extraction using an acid guanidium thiocyanate-phenol-chloroform mixture. Cells were rinsed with phosphate buffered saline and then lysed with 2 ml of guanidium isothiocyanate. The solution was acidified with 0.2 ml of sodium acetate (pH 4.0) and then extracted with 2 ml phenol and 0.4 ml chloroform:isoamyl alcohol The RNA underwent two ethanol precipitations prior to being used for Northern blot analysis.

Northern Blot Analysis: Total cellular RNA Ag) was size fractionated using 1% agarose formaldehyde gels in the presence of 1 ug/ml ethidium bromide. The RNA was transferred to a nitrocellulose filter and covalently linked by ultraviolet irradiation using a Stratlinker UV crosslinker (Stratagene, La Jolla, CA).

Hybridizations were performed at 42 0 C for 18 hours in 5X SSC (1X=150 mM NaC1, 15 mM Na citrate), 1% sodium dodecyl sulfate, 5X Denhardt solution, formamide, 10% dextran sulfate and 100 ug/ml of sheared denatured salmon sperm DNA. Approximately 15 1-2X 106 cpm/ml of labeled probe (specific activity> 108 cpm/ug DNA) were used per hybridization.

Following hybridization, filters were washed with a final stringency of 0.2X SSC at 55 0 C. The nitrocellulose was stripped using boiled water 20 prior to rehybridization with other probes.

Autoradiography was performed with an intensifying screen at -70 0

C.

nProbes: "P labeled DNA probes were made using the random primer oligonucleotide method. The ICAM-1 probe was an Eco R1 fragment of human cDNA.

The ELAM-1 probe was a 1.85 kb Hind III fragment of human cDNA. The VCAM-1 probe was a Hind III-Xho I fragment of the human cDNA consisting of nucleotide 132 to 1814.

Enzyme Linked Immunosorbent Assay (ELISA): HUVE cells were plated on 96-well tissue culture plates 48 to 72 hours before the assay. Primary antibodies in M199 with 5% FBS were added to each well and incubated one hour at 370C. The cells were then washed and incubated for one hour with peroxidase conjugated goat anti-mouse IgG (Bio Rad) diluted 1/500 in M199 with 5% FBS. The wells were -46then washed and binding of antibody was detected by the addition of 100 Al of 10 mg/ml 3,3,5,5'tetramethyl-benzidine (Sigma) with 0.003% H 2 02. The reaction was stopped by the addition of 25 Al of 8N sulfuric acid. Plates were read on an ELISA reader (Bio Rad) at OD 450 nm after blanking on rows stained only with second step antibody. Data represent the means of triplicate.

Antibodies: Monoclonal antibody (MAb) 4B9 recognizing vascular cell adhesion molecule-i (VCAM-1) was the generous gift of Dr. John Harlan (University of Washington). MAb E9A1F1 recognizing endothelial cell adhesion molecule (ELAM-1) was the generous gift of Dr. Swerlick (Emory University).

15 Hybridomas producing mAb 84H10 recognizing intercellular adhesion molecule 1 (ICAM-1) are routinely grown in our laboratory and antibody was used as tissue culture supernatant.

Example 9 PDTC Blocks IL-lb Mediated Induction of HUVEC VCAM-1, but not ICAM-1 or ELAM-1, mRNA Accumulation To determine whether the oxidative state of the endothelial cell can alter the basal or induced expression of cell adhesion molecule genes, cultured human vascular endothelial cells were exposed to the inducing cytokine, IL-lb (10 U/ml) in the presence or absence of the thiolated metal chelating antioxidant, pyrrolidine dithiocarbamate (PDTC, 50 AM) for up to 24 hours. As shown in Figure 10, IL-lb alone (lanes 2, 4, 6, 8) induces the expected rapid and transient induction of VCAM- 1 (Panel E-selectin (ELAM-1, Panel B) and ICAM- 1 (Panel C) mRNA accumulation, all of which peak at four hours. However, in the presence of PDTC, IL- Ib induction of VCAM-1 mRNA accumulation is dramatically inhibited by over 90% (panel A, lanes 3, 5, 7, In contrast, although IL-lb mediated -47induction of ELAM-1 is slightly inhibited at 2 and 24 hours (compare lane 2 and 3, 8 and 9, panel B), PDTC does not inhibit the induction at 4 and 8 hours (lane 5 and 7, panel IL-lb mediated induction of ICAM-1 mRNA accumulation is not affected (panel B, lanes 3, 5, 7, Indeed, a mild augmentation of IL-lb induction of ICAM-1 mRNA accumulation is observed (compare lanes 4 and 5, panel Equivalent amounts of nitrocellulose transferred RNA per lane was confirmed by ethidium bromide staining and visualization.

A dose-response analysis was performed to determine whether PDTC inhibits the induction of 15 VCAM-1 gene expression by IL-lb in a dose dependent manner. As shown in Figure 11, PDTC.inhibits IL-lb mediated induction of VCAM-1 gene expression with a steep dose-response curve (Figure 11, panel A) with a calculated ECs 0 of approximately 10 AM, while PDTC 20 does not inhibit IL-lb mediated induction of ELAM-1 ,expression with these concentrations (Fig. 11, panel The IL-lb mediated induction of ICAM-1 mRNA accumulation is enhanced by PDTC with the concentration higher than 0.5 AM (Fig. 2, compare lane 2 and lane 4-7, panel C) These data demonstrate that IL-lb utilizes a dithiocarboxylate, and in particular, a dithiocarbamate sensitive step as part of its signaling mechanism in the induction of VCAM-1 gene expression. The data also appear to indicate that this dithiocarbamate sensitive step does not play a significant role in the IL-lb mediated induction of ELAM-1 or ICAM-1 gene expression.

Example 10 PDTC Blocks Induction of HUVEC VCAM-1 mRNA Accumulation by Multiple Stimuli To determine whether other well-described activators of VCAM-1 gene expression also utilize a -48- PDTC sensitive step, three distinct classes of activators were tested: another classic receptor mediated inducing agent (TNFa), a non-receptor mediated inducer (lipopolysaccharide (LPS)) and a recently described novel inducer (double stranded RNA, poly(I:C)). In all three cases, PDTC dramatically inhibited the induction of VCAM-1 mRNA accumulation in HUVECs after four hours (Figure 12, Panel Although the TNFa mediated ELAM-1 gene expression is suppressed to some extent (Fig. 12 lane 1 and 2, panel LPS and poly(I:C) mediated ELAM-1 mRNA accumulation was unaffected (Fig. 12 lane 3-6, panel The induction of ICAM-1 mRNA accumulation was unaffected (Figure 12, Panel C).

15 This data indicates that structurally distinct inducing agents, acting through distinct pathways, share a common regulatory step specific for the induction of VCAM-1 gene expression.

Example 11 PDTC Blocks HUVE Cell Surface Expression of VCAM-1 Induced by Multiple Stimuli To determine whether, like its mRNA, the induction of endothelial cell surface protein expression of VCAM-1 could also be inhibited by PDTC, monoclonal antibodies were used in an ELISA assay to quantitate the induction of cell surface VCAM-1 and ICAM-1 in cultured HUVE cells. As shown in Figure 13, multiple classes of activating agents, in the absence of PDTC (-PDTC), induce the rapid ahd transient accumulation of VCAM-1 (top left panel) at the cell surface peaking at six hours. In the presence of PDTC (+PDTC, top right panel), the induction of cell surface expression of VCAM-1 by all agents tested is dramatically inhibited In contrast, the induced expression of cell surface ICAM-1 is unaffected -49under identical conditions (bottom left and right panels) These data demonstrate that, like its mRNA accumulation, cell surface VCAM-1 expression are selectively inhibited by dithiocarbamates and that multiple classes of activating agents utilize a similar, dithiocarbamate sensitive mechanism to induce VCAM-1 gene expression.

Example 12 Comparative Effectiveness of Antioxidants in Blocking TNFa Induction of VCAM-1 To determine whether structurally similar or dissimilar antioxidants could also inhibit VCAM-1 gene expression, and with what potency, HUVE cells 15 were exposed to TNFa for six hours in the presence or absence of different concentrations of four different antioxidants. As shown in Figure 14, both diethyldithiocarbamate (DETC) and N-acetyl cysteine (NAC) inhibited VCAM-1 expression at 20 concentrations of 5 AM and 30 AM, respectively. In contrast, PDTC (PDTC) was effective between 5 and AM. The iron metal chelator, desferroximine, had no effect at the concentrations tested.

Example 13 PDTC Inhibits TNF Induction of VCAM- 1/VLA-4 Mediated Adhesion The ability of a variety of antioxidants to inhibit TNF-a induction of VCAM-1 in HUVE cells was evaluated by the method set out in Example 12.

Figure 15 is a graph of the relative VCAM-1 cell surface expression 595 nM) in TNF-c activated HUVE cells versus concentrations of PTDC (sodium Npyrrolidine dithiocarbamate), DIDTC (sodium N,Ndiethyl-N-carbodithioate), SarDTC (sodium N-methyl- N-carboxymethyl-N-carbodithioate), IDADTC (trisodium N,N-di(carboxymethyl)-N-carbodithioate), MGDTC (sodium N-methyl-D-glucamine-Ncarbodithioate), MeOBGDTC (sodium N-(4methoxybenzyl)-D-glucamine-N-carbodithioate), DEDTC (sodium N,N-diethyl-N-carbodithioate), Di-PDTC (sodium N,N-diisopropyl-N-carbodithioate), and NAC is (N-acetyl cysteine).

Example 13 PDTC Inhibits TNF Induction of VCAMl/VLA-4 Mediated Adhesion In order to define whether PDTC inhibition of VCAM-1 regulation is associated with functional consequences, the binding of Molt-4 cells to HUVEC cells either unstimulated or stimulated with TNFa (100U/ml) was examined for six hours in the presence or absence of PDTC. Molt-4 cells have 15 been previously shown to bind to activated HUVEC via a VCAM-1 dependent mechanism. As shown in Figure 16, the percentage of Molt-4 binding to HUVEC cells decreased when PDTC was present in the media.

20 Example 14 PDTC Inhibits Monocyte Binding to the Thoracic Aorta of Cholesterol Fed Rabbits An experiment was performed to determine whether the thiol antioxidant PDTC would be efficacious in blocking the first monocyte binding component of atherosclerosis in an experimental animal model.

One mature New Zealand white rabbit (3.5 Kg) received an intravenous injection of PDTC mg/Kg, as a concentration of 20 mg/ml in PBS) once daily for 5 days. Injections were given via an indwelling cannula in the marginal ear vein, which as kept patent by flushing with heparinized saline solution. The PDTC solution was mixed fresh daily or on alternate days (stored light-protected at and filtered (0.45 mm pore filter) just prior to use. After the first injection, when the -51cannula was placed, the drug was administered with the rabbit in the conscious state without apparent discomfort or other ill effect. On the second day of injections, the rabbit was given chow containing 1% cholesterol by weight, which was continued throughout the remainder of the experiment. On the fifth day, the animal was euthanized and the thoracic aorta was excised and fixed. After appropriate preparation, the sample was imaged on the lower stage of an ISI DS-130 scanning electron microscope equipped with a LaB emitter. Using dual-screen imaging and a transparent grid on the CRT screen, 64 adjacent fields at a 620x magnification were assessed, to cover an area of 15 -1.3 mm:. Within each field, the number of adherent leukocytes (WBC) and erythrocytes (RBC) were counted and recorded.

The data from the arch sample are as follows: WBC and -25 RBC per 1.3 mm 2 field. This level of WBC adhesion is similar to control animals fed regular chow (about 7 per field have been seen in arch and thoracic samples from 2 'negative control' experiments). 'Positive control' rabbits fed 1% cholesterol for 4 days but not given antioxidant show about a 5-fold increase in adhesion, to 38 WBC/1.3 mm 2 A considerable amount of mostly cellsized debris was observed adherent to each arch sample. It is unclear whether this material is an artifact of preparation, or was present in vivo, and if so, whether it is related to PDTC administration. These studies suggest that PDTC infusions can effectively block initial monocyte adhesion to the aortic endothelium.

Example 15 Inhibition of BSA 13-HPODE Adducts with PDTC Figure 18 is a bar chart graph of the effect of PTDC on the formation of fluorescent adducts of BSA -52and 13-HPODE, as measured in fluorescent units versus micromolar concentration of PDTC. One micromolar of 13-HPODE was incubated with 200 micrograms of BSA in the presence of PDTC for six days. Fluorescence was measured at 430-460 nm with excitation at 330-360 nm. For details of the assay, see Freebis, Parthasarathy, S., Steinberg, D, Proceedings of the National Academy of Sciences 89, 10588-10592, 1992. In a typical reaction 100 nmols of LOOH (generated by the lipoxygenase catalyzed oxidation of linoleic acid) in incubated with 100 Ag of bovine serum albumin .for 48 to 72 hours and the formation of fluorescent "products are followed by measuring the fluorescent 15 spectrum with excitation at 360 nm and emission between 390 and 500 nm.

As indicated, PDTC decreases the concentration of fluorescent adducts of BSA and 13-HPODE.

Figure 19 is a graph of the effect of PTDC on the formation of fluorescent adducts of BSA and ox- PUFA as a function of wavelength (nm) and concentration of PDTC. As the concentration of PDTC increases, the quantity of fluorescent adducts decrease.

Example 16 Effect of PDTC on the oxidation of LDL by horseradish peroxidase Figure 20 is a graph of the effect of PDTC on the oxidation of LDL by horseradish peroxidase (HRP), as measured over time (minutes) for varying concentrations of PDTC. The oxidation of LDL was followed by measuring the oxidation of the fatty acid components of LDL as determined by the increase in optical density at 234 nm. When a polyunsaturated fatty acid is oxidized, there is a shift of double bonds resulting in the formation of conjugated dienes which absorb at 234 nm. The -53intercept of the initiation and propagation curve (lag phase) is suggested to be a measure of the oxidizability of LDL. Higher the lag phase, more resistant is the LDL to oxidation. Typically 100 Mg of human LDL is incubated with 5 MM HO 2 and the increase in absorption of 234 nm is followed.

It is observed that after an incubation period, PDTC inhibits the oxidation of LDL by HRP in a manner that is concentration dependent.

Example 17 Effect of PDTC on the cytokine-induced formation of ox-PUFA Figure 21 is a chart of the effect of PDTC on the cytokine-induced formation of ox-PUFA in human aortic endothelial cells. As indicated, both TNF-a S: 15 and IL-1B causes the oxidation of linoleic acid to ox-linoleic acid. The oxidation is significantly prevented by PDTC.

2. Modification of the Synthesis and Metabolism of PUFAs and ox-PUFAs Inhibition of the expression of VCAM-1 can be "accomplished via a modification of the metabolism of PUFAs into ox-PUFAs. For example, a number of enzymes are known to oxidize unsaturated materials, including peroxidases, lipoxygenases, cyclooxygenases, and cytochrome P450. The inhibition of these enzymes may prevent the oxidation of PUFAs in vivo. PUFAs can also be oxidized by metal-dependent nonenzymatic materials.

IV. Method for Modifying the Expression of a Redox-Sensitive Gene In an alternative embodiment, a method is provided for suppressing the expression of a redoxsensitive gene or activating a gene that is -54suppressed through a redox-sensitive pathway, that includes administering an effective amount of a substance that prevents the oxidation of the oxidized signal, and typically, the oxidation of a polyunsaturated fatty acid. Representative redoxsensitive genes that are involved in the presentation of an immune response include, but are not limited to, those expressing cytokines involved in initiating the immune response IL-1), chemoattractants that promote the migration of inflammatory cells to a point of injury MCP-1), growth factors (IL-6, thrombin receptor), and adhesion molecules VCAM-1 and E-selectin).

15 Given this disclosure, one of ordinary skill in the art will be able to screen a wide variety of antioxidants for their ability to suppress the expression of a redox-sensitive gene or activate a gene that is suppressed through a redox-sensitive pathway. All of these embodiments are intended to fall within the scope of the present invention.

Based on the results of this screening, nucleic acid molecules containing the 5' regulatory sequences of the redox-sensitive genes can be used to regulate or inhibit gene expression in vivo can be identified. Vectors, including both plasmid and eukaryotic viral vectors, may be used to express a particular recombinant 5' flanking region-gene construct in cells depending on the preference and judgment of the skilled practitioner (see, e.g., Sambrook et al., Chapter 16) Furthermore, a number of viral and nonviral vectors are being developed that enable the introduction of nucleic acid sequences in vivo (see, Mulligan, 1993 Science, 260, 926-932; United States Patent No.

4,980,286; United States Patent No. 4,868,116; incorporated herein by reference). Recently, a delivery system was developed in which nucleic acid is encapsulated in cationic liposomes which can be injected intravenously into a mammal. This system has been used to introduce DNA into the cells of multiple tissues of adult mice, including endothelium and bone marrow (see, Zhu et al., 1993 Science 261, 209-211; incorporated herein by reference) The 5' flanking sequences of the redox-sensitive gene can be used to inhibit the expression of the redox-sensitive gene. For example, an antisense RNA of all or a portion of the 5' flanking region of the redox-sensitive gene can be used to inhibit expression of the gene in vivo. Expression vectors 15 retroviral expression vectors) are already available in the art which can be used to generate an antisense RNA of a selected DNA sequence which is expressed in a cell (see, U.S. Patent No.

4,868,116; U.S. Patent No. 4,980,286).

Accordingly, DNA containing all or a portion of the sequence of the 5' flanking region of the gene can **be inserted into an appropriate expression vector so that upon passage into the cell, the transcription of the inserted DNA yields an antisense RNA that is complementary to the mRNA transcript of the gene normally found in the cell.

This antisense RNA transcript of the inserted DNA can then base-pair with the normal mRNA transcript found in the cell and thereby prevent the mRNA from being translated. It is of course necessary to select sequences of the flanking region that are downstream from the transcriptional start sites for the redox-sensitive gene to ensure that the antisense RNA contains complementary sequences present on the mRNA. Antisense RNA can be generated in vitro also, and then inserted into cells. Oligonucleotides can be synthesized on an -56automated synthesizer Model 8700 automated synthesizer of Milligen-Biosearch, Burlington,

MA

or ABI Model 380B). In addition, antisense deoxyoligonucleotides have been shown to be effective in inhibiting gene transcription and viral replication (see Zamecnik et al., 1978 Proc. Natl. 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.

Furthermore, recent work has shown that improved inhibition of expression of a gene by antisense oligonucleotides is possible if the antisense ~oligonucleotides contain modified nucleotides (see, 15 Offensperger et. al., 1993 EMBO J. 12, 1257- 1262 (in vivo inhibition of duck hepatitis B viral a replication and gene expression by antisense phosphorothioate oligodeokynucleotides); Rosenberg et al., PCT WO 93/01286 (synthesis of sulfurthioate oligonucleotides); Agrawal et al., 1988 Proc. Natl.

Acad. Sci. USA 85, 7079-7083 (synthesis of antisense oligonucleoside phosphoramidates and phosphorothioates to inhibit replication of human :immunodeficiency virus-1); Sarin et al., 1989 Proc.

Natl. Acad. Sci. USA 85, 7448-7794 (synthesis of antisense methylphosphonate oligonucleotides); Shaw et al., 1991 Nucleic Acids Res 19, 747-750 (synthesis of 3' exonuclease-resistant oligonucleotides containing 3' terminal phosphoroamidate modifications); incorporated herein by reference).

The sequences of the 5' flanking region of the redox-sensitive gene can also be used in triple helix (triplex) gene therapy. Oligonucleotides complementary to gene promoter sequences on one of the strands of the DNA have been shown to bind promoter and regulatory sequences to form local -57triple nucleic acid helices which block transcription of the gene (see, 1989 Maher et al., Science 245, 725-730; Orson 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; Duval- Valentin 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.

Recently, both theoretical calculations and empirical findings have been reported which provide guidance for the design of oligonucleotides for use 15 in oligonucleotide-directed triple helix formation to inhibit gene expression. For example, oligonucleotides should generally be greater than 14 nucleotides in length to ensure target sequence specificity (see, Maher et al., (1989); Grigoriev et al., (1992)). Also, many cells avidly take up oligonucleotides that are less than nucleotides in length (see Orson et al., (1991); Holt et al., 1988 Mol. Cell. Biol. 8, 963i 973; Wickstrom et al., 1988 Proc. Natl. Acad. Sci.

USA 85, 1028-1032). To reduce susceptibility to intracellular degradation, for example by 3' exonucleases, a free amine can be introduced to a 3' terminal hydroxyl group of oligonucleotides without loss of sequence binding specificity (Orson et al., 1991). Furthermore, more stable triplexes are formed if any cytosines that may be present in the oligonucleotide are methylated, and also if an intercalating agent, such as an acridine derivative, is covalently attached to a 5' terminal phosphate via a pentamethylene bridge); again without loss of sequence specificity (Maher et al., (1989); Grigoriev et al., (1992).

-58- Methods -c produce or synthesize cligonucleotides are well known in the ar:. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see Sambrook et al., Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System 1Plus DNA synthesizer (see also, Ikuta et al., in Ann. Rev. Biochem. 1984 53, 323-356 (phosphotriester and phosphite-triester methods); Narang et ai., in Methods Enzvmol., 65, 610-620 (1980) (phosphotriester method). Accordingly,

DNA

sequences of the 5' flanking region of the redoxsensitive gene described herein can be used' to 15 design and construct oligonucleotides including a DNA sequence consisting essentially of at least consecutive nucleotides, with or without base modifications or intercalating agent derivatives, for use in forming triple helices specifically within the 5' flanking region of a redox-sensitive gene in order to inhibit expression of the gene.

In some cases it may be advantageous to insert enhancers or multiple copies of the regulatory sequences into an expression system to facilitate screening of methods and reagents for manipulation of expression.

V. Models and Screens Screens for disorders mediated by VCAM-1 or a redox-sensitive gene are also provided that include the quantification of surrogate markers of the disease. In one embodiment, the level of oxidized polyunsaturated fatty acid, or other appropriate markers, in the tissue or blood, for example, of a host is evaluated as a means of assessing the "oxidative environment" of the host and the host's -59susceptibility to VCAM-. or redox-sensitive gene mediated disease.

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

In yet another assay, the sensitization of a host's vascular endothelial cells to polyunsaturated fatty acids or their oxidized counterparts is evaluated. This can be accomplished, for example, by challenging a host with a PUFA or ox-PUFA and comparing the resulting concentration of cell-surface or circulating VCAM-I or other surrogate marker to a population norm.

In another embodiment, in vivo models of atherosclerosis or other heart or inflammatory diseases that are mediated by VCAM-1 can be provided by administering to a host animal an .":'.excessive amount of PUFA or oxidized polyunsaturated fatty acid to induce disease.

These animals can be used in clinical research to .urther the understanding of these disorders.

In yet another embodiment of the invention, compounds can be assessed for their ability to treat disorders mediated by VCAM-1 on the basis of their ability to inhibit the oxidation of a polyunsaturated fatty acid, or the interaction of a PUFA or ox-PUFA with a protein target.

This can be accomplished by challenging a host, for example, a human or an animal such as a mouse, to a high level of PUFA or ox-PUFA and then determining the therapeutic efficacy of a test compound based on its ability to'decrease circulating or cell surface VCAM-. concentration.

Alternatively, an in vitro screen can be used that is based on the ability of the test compound to prevent the oxidation of a PUFA, or the interaction of a PUFA or ox-PUFA with a procein :argec in the presence of an oxidizing substance such as a metal, for example, copper, or an enzyme such as a peroxidase, lipoxygenase, cyciooxygenase, or cytochrome P450.

In another embodiment, vascular endothelial cells are exposed to TNF-a or other VCAM-1 inducing material for an appropriate time and then broken by any appropriate means, for example by sonication or freeze-thaw. The cytosolic and membrane compartments are isolated. Radiolabeled PUFA is added to defined amounts of the compartments. The ability of the liquid to convert PUFA to ox-PUFA in the presence or absence of a 15 test compound is assayed. Intact cells can be used in place of the broken cell system.

III. Pharmaceutical Compositions S. Humans, equine, canine, bovine and other animals, and in particular, mammals, suffering from 20 cardiovascular disorders, and other inflammatory conditions mediated by VCAM-1 or a redox sensitive gene can be treated by administering to the patient an effective amount of a compound that causes the removal, decrease in the concentration of, or prevention of the formation of an oxidized polyunsaturated fatty acids, including but not limited to oxidized linoleic A 9 1 linolenic

A

69 1 2 arachidonic (C 0 AS. 8.11.14) and eicosatrienoic A 8 1 1 4 acids; other oxidation signal; or other active compound, or a pharmaceutically acceptable derivative or salt thereof in a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, -Slparenceraliy, intravenously, in:radermally, subcutaneously, or topically.

As used herein, the term pharmaceutically acceptable salts or complexes refers to salts or complexes that retain the desired biological activity of the above-identified compounds and exhibit minimal undesired toxicological effects.

Nonlimiting examples of such salts are acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such •as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic 15 acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; base addition salts formed with -polyvalent metal cations such as zinc, calcium, 20 bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the *like, or with an organic cation formed from N,Ndibenzylethylene-diamine, D-glucosamine, ammonium, cetraethylammonium, or ethylenediamine; or (c) S" 25 combinations of and a zinc tannate salt or the like.

The active compound or a mixture of the compounds are administered in any appropriate manner, including but not limited to orally and intravenously. General range of dosage for any of the above-mentioned conditions will be from 0.5 to 500 mg/kg body weight with a dose schedule ranging from once every other day to several times a day.

Preferred daily dosages are between approximately 1 and 3000 mg/patient/day, more preferably between approximately 5 and 500 mg/patienc/day, and even -62more preferably, between approximately 25 and 500 mg/patient/day.

The active ingredient should be administered to achieve peak plasma concentrations of the active compound of about 0.1 to 100 M, preferably about 1-10M. This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient.

The compounds can also be administered directly to the vascular wall using perfusion balloon catheters following or in lieu of coronary or other *.0 arterial angioplasty. As an example, 2-5 mL of a 15 physiologically acceptable solution that contains approximately 1 to 500 AM of the compound or mixture of compounds is administered at atmospheres pressure. Thereafter, over the course of the next six months during the period of maximum 20 risk of restenosis, the active compounds are administered through other appropriate routes and dose schedules.

'Relatively short term treatments with the active compounds are used to cause the "shrinkage" of 25 coronary artery disease lesions that cannot be treated either by angioplasty or surgery. A nonlimiting example of short term treatment is two to six months of a dosage ranging from 0.5 to 500 mg/kg body weight given at periods ranging from once every other day to three times daily.

Longer term treatments can be employed to prevent the development of advanced lesions in high-risk patients. A long term treatment can extend for years with dosages ranging from 0.5 to 500 mg/kg body weight administered at intervals ranging from once every other day to three times daily.

-63- The active compounds can also be administered in the period immediately prior to and following coronary angioplasty as a means to reduce or eliminate the abnormal proliferative and inflammatory response that currently leads to clinically significant re-stenosis.

The active compounds can be administered in conjunction with other medications used in the treatment of cardiovascular disease, including lipid lowering agents such as probucol and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents such as coumadin; calcium channel blockers such as varapamil, diltiazem, and nifedipine; angiotensin 15 converting enzyme (ACE) inhibitors such as captopril and enalopril, and S-blockers such as propanalol, terbutalol, and labetalol. The compounds can also be administered in combination with nonsteroidal antiinflammatories such as 20 ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, sulindac. The compound can o also be administered with corticosteriods.

The concentration of active compound in the drug composition will depend on absorption, 25 distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the -64claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.

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

15 The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a 20 disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such 25 as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.

In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

The active compound or pharmaceutically acceptable salt or derivative thereof can be administered as a component cf an elixir, suspension, syrup, wafer, chewing gum or the like.

A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable derivatives or salts thereof can also be administered with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, or antiviral compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline 15 solution, fixed oils, polyethylene glycols, glycerine, 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 20 such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or 25 multiple dose vials made of glass or plastic.

Suitable vehicles or carriers for topical application are known, and include lotions, suspensions, ointments, creams, gels, tinctures, sprays, powders, pastes, slow-release transdermal patches, aerosols for asthma, and suppositories for application to rectal, vaginal, nasal or oral mucosa.

Thickening agents, emollients, and stabilizers can be used to prepare topical compositions.

Examples of thickening agents include petrolatum, beeswax, xanthan gum, or polyethylene glycol, humectants such as sorhitol, emollients such as mineral oil, lanolin and its derivatives, or squalene. A number of solutions and ointments are commercially available.

Natural or artificial flavorings cr sweeteners can be added to enhance the taste of topical preparations applied for local effect to mucosal surfaces. Inert dyes or colors can be added, particularly in the case of preparations designed for application to oral mucosal surfaces.

The active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the 20 art.

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

The active compound can also be administered through a transdermal patch. Methods for preparing transdermal patches are known to those skilled in the art. For example, see Brown, and Langer, Transdermal Delivery of Drugs, Annual Review of Medicine, 39:221-229 (1988), incorporated herein by reference.

In another embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, 57 polyglycolic acid, collagen, colyor:hoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving S.appropriate lipid(s) (such as stearoyl phosphatidyl 15 ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of 20 the active compound or its monophosphate, diphosphate, and/or triphosphate derivatives are then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid 25 aggregates, thereby forming the liposomal suspension.

Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to come within the scope of the appended

Claims (40)

1. A method for supressing the expression of VCAM-1 comprising administering an effective amount of a substance that prevents or minimizes the oxidation of a polyunsaturated fatty acid.

2. A method for suppressing the expression of a redox-sensitive gene comprising administering an effective amount of a substance that prevents or minimizes the oxidation of a polyunsaturated fatty acid.

3. A method for activating a gene that is suppressed by the oxidation of a polyunsaturated fatty acid, comprising administering an effective o .oo amount of a substance that prevents or minimizes the oxidation of a polyunsaturated fatty acid.

4. A method for suppressing the expression of VCAM-1 comprising administering an effective amount of a substance that prevents the interaction between a polyunsaturated fatty acid and a protein that mediates the expression of VCAM-1.

5. The method of claims 1-4, wherein the polyunsaturated acid is selected from the group consisting of oxidized linoleic (C 18 A-' 2 linolenic A 6 9 1 2 arachidonic (C20 A 8. 1. 14) and eicosatrienoic (C 2 0 As 8 1 4 acid.

6. The method of claim 2 or 3, wherein the redox-sensitive gene is selected from the group consisting of those expressing cytokines involved in initiating the immune-response IL-1S) chemoattractants that promote the migration of inflammatory cells to a point of injury MCP- growth factors IL-6 and the thrombin receptor), and adhesion molecules VCAM-1 and E-selectin).

7. The method of claims 1-4, wherein the substance is pyrrolidine dithiocarbamate, or its pharmaceutically acceptable salt.

8. A method for the prediction or assessment of disorders mediated by VCAM-1 in vivo, comprising quantifying the level of oxidized polyunsaturated fatty acid in the tissue or blood.

9. A method for the prediction or assessment of redox-sensitive gene mediated disease in vivo, comprising quantifying the level of oxidized polyunsaturated fatty acid in the tissue or blood.

11. A method for the prediction or assessment of disorders mediated by VCAM-1 in vivo, comprising quantifying a surrogate marker for the level of ~oxidized polyunsaturated fatty acid in the tissue or blood.

12. A method for the prediction or assessment Sof redox-sensitive gene mediated disease in vivo, comprising quantifying a surrogate marker for the level of oxidized polyunsaturated fatty acid in the tissue or blood.

13. The method of claim 11, wherein the surrogate marker is circulating or cell-surface VCAM-1.

14. A method for the evaluation of the sensitization of a host's vascular endothelial cells to polyunsaturated fatty acids or their oxidized counterparts, comprising challenging a host with a PUFA or ox-PUFA and comparing the resulting concentration of cell-surface or circulating VCAM-1 or other surrogate marker to a population norm. A method to screen compounds for their ability to treat disorders mediated by VCAM-1 comprising evaluating the ability of the compound to inhibit the oxidation of a polyunsaturated fatty acid.

16. A method :o screen compounds for their abili:y to treat disorders mediated by VCAM-1 comprising evaluating the ability of the compound to inhibit the interaction of a PUFA or ox-PUFA with a protein target.

17. A method for the treatment of a cardiovascular disease in humans comprising administering an effective amount of a dithiocarbamate of the formula A-SC(S)-B; wherein A selected from the group consisting of hydrogen, a pharmaceutically acceptable cation, and a physiologically cleavable leaving group; and B is selected from the group consisting of alkyl, alkenyl, alkynyl, alkaryl, aralkyl, haloalkyl, haloalkenyl, haloalkynyl, aryl, alkaryl, hydrogen, C- alkoxy-C 1 0 alkyl, C 1 6 alkylthio-Ci. 10 alkyl, NRR', -(CHOH),CHOH, wherein n is 0, 1, 2, 3, S4, 5, or 6, including alkylacetyl, alkylpropionyl, and alkylbutyryl, and hydroxy(C l 6) alkyl-.

18. The method of claim 17, wherein A is hydrogen or a pharmaceutically acceptable cation selected from the group consisting of sodium, potassium, calcium, magnesium, aluminum, zinc, bismuth, barium, copper, cobalt, nickel, or cadmium.

19. The method of claim 17, wherein A is a salt-forming organic acid. The method of claim 19, wherein A is selected from the group consisting of choline, acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid.

22.. The method of claim 17, wherein A is a cation formed from ammonia or other nitrogenous base-. 22. The method of claim 21, wherein A is a nitrogenous heterocycle, or a moiety of the formula NR'R 5 R 6 R 7 wherein i R 6 and A 7 are independently hydrogen, C1. alkyl, hydroxy(Cl.6)alkyl, aryl, N, N -dibenzyl ethylene -diamine, D-glucosamine, tetraethylammoniun, or ethylenediamine.

23. The method of claim 17, wherein A is a physiologically cleavable leaving group.

24. The method of claim 17, wherein A is an acyl group. The method of claim 17, wherein B is NR 2 R 3 wherein R 2 and R 3 are selected f rom the group *consisting of alkyl; (CHQH), (CH 2 wherein n is 0, 1, 2, 3, 4, 5, or 6; (CM 2 1COOR, -(CH2)),Co 2 R4; hydroxy (CI6) alkyl alkenyl; alkyl (COH), alkenyl (COH) alkynyl (C 2 or aryl, or R 2 and R3 can together constitute a bridge of the formula CI)- wherein mn is 3, 4, 5, or 6, and wherein Z 4 is selected from the group consisting of aryl, alkaryl, or aralkyl, including acetyl, propionyl, and butyryl.

26. The method of claim 17, wherein B is a heterocyclic or alkyiheterocyclic group.

27. The method of claim 26, wherein the heterocycle is partially or totally hydrogenated.

28. The method of claim 17, wherein B is the residue of a pharmaceuticailly- active compound or drug which is directly linked to A-SC(S)- or linked through a divalent linking moiety.

29. The method of claim 17, wherein B is selected from the group) consisting of probucol, nicotinic acid, aspirin, coumadin, varapamil, diltiazem, nifedimine, captopril, enalopril, crooanalo'L, terbuctalol, labetalol, ibuprofen, -72- indomethacin, fenoprofen, mefenamic acid, flufenamic acid, sulindac, and a corticosteriod. The method of claim 17, wherein the dithiocarbamate is an amino acid derivative of the structure AOC-R 9 -NR'-C(S)SA, wherein R, is B or the internal residue of an amino acid and R'O is hydrogen or lower alkyl.

31. The method of claim 17, wherein B is a polymer to which one or more dithiocarbamate groups are attached, either directly, or through any suitable linking moiety.

32. The method of claim 17, wherein the polymer is biodegradable.

33. The method of claim 32, wherein the polymer is selected from the group consisting of peptides, proteins, nucleoproteins, lipoproteins, glycoproteins, synthetic and natural polypeptides and polyamino acids, polyorthoesters, poly(a- hydroxy acids), polyanhydrides, polysaccharides, and polycaprolactone.

34. The method of claim 1, wherein B-C(S)S- is pyrrolidine-N-carbodithioate. The method of claim 17 wherein the cardiovascular disease is atherosclerosis.

36. The method of claim 17, wherein the cardiovascular disease is post-angioplasty restenosis.

37. The method of claim 17, wherein the cardiovascular disease is coronary artery disease.

38. The method of claim 17, wherein the cardiovascular disease is angina.

39. The method of claim 17, wherein the cardiovascular disease is a small vessel disease. The method of claim 17, wherein the dithiocarbamate is administered in a dosage of between 0.5 and 500 mg/kg body weight. -73-

41. The method of claim 17, wherein the dithiocarbamate is administered by perfusion balloon catheter.

42. The method of claim 17, wherein the dithiocarbamate is administered in combination with a pharmaceutical agent selected from the group consisting of a lipid lowering agent, a platelet aggregation inhibitor, an antithrombotic agent, a calcium channel blocker, an angiotensin converting enzyme (ACE) inhibitor, a G-blocker, a nonsteroidal antiinflammatory, and a corticosteroid.

43. A method for the suppression of VCAM-1 expression in human cells comprising administering **o*oo an effective amount of the dithiocarbamate described in claim 17.

44. A method for the treatment of an inflammatory skin disease that is mediated by SVCAM-1 comprising administering an effective amount of the dithiocarbamate described in claim 17. "45. A method for the -reatment of a human endothelial disorder that is mediated by VCAM-1 comprising administering an effective amount of the dithiocarbamate described in claim 17. S"

46. The method of claim 45, wherein the disorder is selected from the group consisting of asthma, psoriasis, eczematous dermatitis, Kaposi's sarcoma, multiple sclerosis, and proliferative disorders of smooth muscle cells.

47. A method for the treatment of an inflammatory condition that is mediated by a mononuclear leucocyte comprising administering an effective amount of the dithiocarbamate described in claim 17.

48. The dithiocarbamate disclosed in any of claims 17-34.

74- 49. A pharmaceuzical composition comprising an effective amount to treat cardiovacular disease of a compound disclosed in any of claims 17-34. A pharmaceutical composition comprising an effective amount to treat a disorder mediated by VCAM-1 of a compound disclosed in any of claims 17- 34.