CA2189336A1 - Treatment for atherosclerosis and other cardiovascular and inflammatory diseases - Google Patents

Abstract

Dithiocarboxylates, including dithiocarbamates, block the induced expression of the endothelial cell surface adhesion molecule VCAM-1, and are therefore useful in the treatment of cardiovascular disease, including atherosclerosis, as well as noncardiovascular inflammatory diseases that are mediated by VCAM-1.

Description

2 1 8 9 3 3 6 r ~ J~' Treatment ~or Ath~ B ~l~o~is and other Cardiov~scular and Tn~l ~ tory Di~ea~e~
8ackground of the Invention This application i9 in the area of methods and compositionS for the treatment of atherosclerosis and other cardiovascular and inf lammatory diseases .
Adhesion of leukocytes to the endothelium 5 represents a f1ln~ ti~l, early event in a wide variety of ; n f 1 tery conditions, including atherosclerosis, autoimmune disorders and bacterial and viral infections. Leukocyte recruitment to the endothelium is started when ;n~ c;hle adhesion lO molecule receptors on the surface of endothelial cells interact with counterreceptors on immune cells. Vascular endothelial cells determine which type of leukocytes (monocytes, lymphocyte5, or neutrophils) are recruited, by selectively 15 expressing specific adhesion molecules, such as vascular cell adhesion molecule-l (VCAM-l), intracellular adhesion molecule-l (ICAM-l), and E-selectin. In the earliest stage of the atherosclerotic lesion, there is a localized 20 endothelial expression of VCl~M-l and selective recruitment of ~nl~- lear leukocytes that express the integrin counterreceptor VI A-4 . Because of the selective expression of VLA-4 on monocytes and lymphocytes, but not neutrophils, VC~M-l is 25 important in ~;Atin~ the selective adhesion of ~ n~ ear leukocytes. Subsequent conversion of leucocytes to foamy macrophages results in the synthesis of a wide variety of ; nfl i ~ ry cytokines, growth factors, and chemoattractants

3 0 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 1 ~9336 WO 951304~5 VCAM-l i8 expressed in cu~tured human vaseular endothelial cells after activation by lipopolysaccharide (~PS) and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor 5 (TNF-a) . These factors are not selective for activation of eell adhesion moleeule expression.
Scheme 1 illustrates the proeess of eytokine activation of VCAM-1 gene expression in vaseular endothelial eells.
1. Cr~l_ B~.
"'! ~/ VC~W.I iDlt l.h . A~i~
r _ ~ , r ~ / ~~
< T~_Ut~ Na~/
9~c DNA ~ ~' ~PI_ ~ ~
Seheme 1 Regulatory sehemes f or eytokine aetivation of vaseular eell adhesion moleeule-1 (VCAM-1) gene expression, through redox sensitive regulatory faetors sueh as NF-ki~;, in vaseular endothelial 15 eells ( IkB is an inhibitory subunit; NF-kB is nuelear faetor-kB; NH3 refers to the amino terminus of the protein and RNA Pol II is RNA polymerase II) .
Moleeular analysis of the regulatory elements on 2 0 the human VCAM- 1 gene that eontrol its expression suggests an important role for nuelear faetor-kB
(NF-kB), a transeriptional regulatory factor, or an `~ 2189 Wo 95/30415 6 p~""
NF-k~ like binding protein in oxidation-rF~ tinn-sensitive regulation of VCAM-l gene expression.
Transcriptional factors are proteins that activate (or repress) gene expression within the cell 5 nucleus by binding to specif ic DNA sequences called ~f.nh~n~,or elements~ that are generally near the region of the gene, called the "promoter, " from which RNA synthesis is initiated. Nuclear factor-kB is a ubiquitously expressed multisubunit lO transcription factor activated in several cell types by a large and diverse group of ;nfl t~ry agents such as TNF~, IL-13, bacterial endotoxin, and RNA viruses. It plays a key role in m~;;3t;
;nfl tory and other stress signals to the l5 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 rli ~hetf~q mellitus .
Importantly, the activation of NF-ks in vascular endothelial cells by diverse signals can be ~pe~ ;fi~-~lly inhibited by ~nt;r~ ntc such as 25 N-acetylcysteine and pyrrolidine dithio~ ~rh~mate (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 r--^h~n; ~
30 Because an NF-kB-like ~nh~n~r element also regulates the transcription of the VCAM-l promoter in an oxidation-reduction-sensitive manner, oxidative stress in the atherosclerotic lesion may play a role in regulating VCAM-l gene expression 35 through this n~ tirm-reduction-sensitive transcriptional regulatory protein.

~i 8q33 WO95130415 6 r~l",~

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 ~Lu~clyaLes 5 atherosclerosis. Steinberg, et al., N. Engl. 1:.
Med. 1989; 320: 915-924 . ~ ; 7C.Cl 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 10 adhesion molecule gene expression. Fatty acid hydroperoxides such as linoleyl llydLu~eLu~Lide (13-HPODE) are produced from free fatty acids by lipoxygenases and are an important c ,,nnont of oxidized LDL.
It has been proposed that a generation of n~ ; 70d lipids is formed by the action of the cell lipu~y~e~c-se system and that the n~r; ~; 70~i lipids are subsequently transferred to LDL. There is thereafter a propagation reaction within the LDL i~
the medium catalyzed by transition metals and/or sulfhydryl ~ u~.ds. Previous investigations have demonstrated that ~atty acid modif ication of cultured endothelial cells can alter their susceptibility to oxidant injury. supplomontAt; nn of saturated or monounsaturated fatty acids to cultured endothelial cells reduces their susceptibility to oxidant injury, whereas suppl~ tAt;nn with polyunsaturated fatty acids (PUFA) enhances susceptibility to oxidant injury.
Using reverse-phase HPLC analysis of native and .3Arnn;f;ed lipid extracts of LDL, it has been demonstrated that 13-HPODE is the ~L~ ' nAnt n~ ; 70~1 fatty acid in LDL oxidized by activated human monocytes. Chronic e~u~uLe to n~ ; 70d LDL
provides an oxidative signal to vascular endothelial cells, possibly through a specific Wo 95/30415 2 ~ 8 9 3 3 6 . ~
fatty acid hydroperDxide, that selectively A1l~^J--^ntS
cytokine-induced V~ l gene expre3 5 ion.
Through a r~ ^h^n;~^~ that is not well defined, areas of vessel wall predisposed to atherosclerosis preferentially seyuester cir^-1lAtin~ LDL. Through a poorly understood pathway, endothelial, smooth muscle, and/or ;nfl; tory cells then convert LDL
to ox-LDL. In contrast to LDL, 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 that form the fatty streak.
Given that cardiovascular disease is currently the leading cause of death in the United States, and ninety percent of cardiovascular disease is presently diagnosed as atherosclerosis, there is a strong need to identify new methods and pharmaceutical agents for its treatment. Important to this goal is the identification and manipulation of the specific ~ l; 7ed biological In~q that act as selective reyulators of the expression of mediators of the ;nfli tory process, and in particular, V~AM-l. 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 invention to provide a treatment for atherosclerosis and other cardiovascular and ;nfli tory diseases.
It is another obj ect of the present invention to provide a method for the selective inhibition of V~M- l .

Wo95/30415 i 9336 1 .,, - ~
It i8 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 5 gene. =~
It is another object of the present invention to provide pharmaceutical compositions for the treatment of atherosclerosis and other cardiovascular and ;nfli tory diseases.
Sun~nary of the Invent~on It has been discovered that polyunsaturated f atty acids ( " PUFAs " ) and their hydroperoxides ("ox-PUFAs"), which are important ~ ^~tR of oxidatively modified low density lipoprotein (LDL), 15 induce the expression of VCAM- 1, but not intracellular A~hPS~n molecule-1 (ICAM-1) or E-selectin in human aortic endothelial cells, through a m~rhAn; Pm that is not mediated by cytokines or other noncytokine signals. This is a flln~ A
20 discovery of an important and previously unknown biological pathway in VCAM-1 1; At~d immune responses .
As nonlimiting examples, linoleic acid, linolenic acid, arArh; ~rn; C acid, linoleyl 2 5 I'YdL u~eL u~Lide ( 13 - HPODE ) and arA rh i ~-~n; c 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 30 not induce the expression of VCAM-1~ M-1, or E-selectin .
The ;n~llrt;~m of VCAM-1 by PUFAs and their fatty acid hydroperoxides is suppressed by the Ant; r,~; ~Ant pyrrolidine dithiocarbamate (PDTC) .
3 5 This indicates that the induction is mediated by an - ~ 2 1 89336 WO 95/30415 r n~;~;7ec~ signal molecule, ancl that the ;n~llctinn is prevented when the n~;rli~t;on of the molecule is blocked (i.e., the oxidation does not occur), reversed (i.e., the signal molecule i8 reduced), or 5 when the redox modif ied signal is otherwise prevented from interacting with its regulatory target .
Cells that are chronically exposed to higher than normal levels of polyunsaturated fatty acids 10 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 oversensit; 7at; nn 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 treatment of atherosclerosis, post-angioplasty restenosis, coronary artery diseases, angina, and 20 other cardiovascular diseases, as well as noncardiovascular ;nfl: tory ~l;RPi~RPR that are ted by VCAM- 1, is provided that includes the removal, decrease in the cnnrPntl-ation of, or prevention of the formation of n~;~l; 7Pd 25 polyunsaturated fatty acids including but not limited to n~ ; 7ed linoleic (Cl8 ~9~12), linolenic (CI8 t~6~9~l2), ara(-h;rinn;r (c20 ~5-8-11~14) and -icosatrienoic (C20 ~8, ll. l4) acids .
Nonlimiting examples of noncardiovascular 30 ;nfli -tory diseases that are r ';i~tPd by VCAM-1 include rheumatoid and osteoarthritis, asthma, dermatitis, and multiple sclerosis.
This method represents a signif icant advance in treating cardiovascular disease, in that it goes 35 beyond the current therapies designed simply to inhibit the progression of the disease, and when used appropriately, p~ovides the possibility to wo 95l30415 P~"'~
_ ~ _ medically ~cure~ atherosclerosis by preventing new lesions from developing and causing est~hl; qh~
lesions to regress.
In an alternative embodiment, a method is provided for suppressing the expression of a redox-sensitive 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 0 n~ i7~ gignal, and typically, the ~ tirn of a polyunsaturated fatty acid. Repr~q~nt~t;ve redox-sensitive genes that are involved in the presentation of an immune response include, but are not limited to, those expressing cytokines involved in ;n;t;~tin~ the immune response (e.g., IL-1~
chemoattractants that promote the migration of ;nf~. tory cells to a point of injury ( e . g ., MCP - 1 ), growt h f actors ( e . g ., IL - 6 and the thrombin receptor), and ~h~q;~n molecules (e.g., VCAM-1 and E-g~le~rt;n) .
Screens for-disorders 1 ';~t~1 by VCAM-1 or a redox-sensitive gene are also provided that include the quantification of surrogate markers of the disease. In one ~ ;rnl~nt~ the level of ~ ;7C~
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' 8 susceptibility to VCAM-1 or redox-sensitive gene 3 0 mediated disease .
In ano~her embodiment, the level of circ~ t; n~
or cell-surfac~ VCAM-1 or other appropriate marker and the effect on that level of administration of an appropriate antioxidant is quantif ied .
In yet another assay, the sensitization of a host~ 8 vascular endothelial cells to polyun8aturated f atty acids or their oxidized 2 t ~9336 _g_ counterparts is evaluated. This can be accomplished, for example, by challenging a host with a PUFA or ox-PUFA and comparing the resulting -~77rPntration of cell-surface or cir~ 1 C7t;n~ VCAM-l or other surrogate marker to a population norm.
In another: ' ~'; ~, in vivo models of atherosclerosis or other heart or ;nfl. tory ~7; C~';7ReC that are mediated by VCAM-l can be provided by administering to a host animal an excessive amount of PUFA or ~ 7; 7er7 polyunsaturated f atty 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, c1 ~71n~7,c can be i7cspRRed for their ability to treat disorders mediated by VC~M-l on the basis of their ability to inhibit the f7~ 7;7t;nn of a polyunsaturated fatty acid, or the interaction of a PUFA or ox-PUFA with a protein target.
This can be accomplished by rh~7llpn~ing 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 ef f icacy of a test compound based on its ability to decrease 2~ cir~ t;n~ or cell surface VCAM-l c~7n~ ntration.
Alternatively, an in vitro screen can be used that is based on the ability of the test c ' 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 ~7~ 7;7;ng substance such as a metal, for example, copper, or an enzyme such as a peroxidase, lipoxygenase, cyclooxygenase, or cytochrome P4 5 0 .
In another embodiment, vascular endothelial cells are exposed to T.~F-a or other VC~M-l ; n-7ll~ in~ material for an appropriate time and then broken by any ~Lu~Liate means, for example by WO 95/30415 ~2 1 8 9 3 3 6 sonication or ~reeze-thaw. The cytosolic and membrane compartments are isolated. ~i~rl; nl ~heled PUFA is added to defined amounts of the compartments. The ability of the liquid to convert 5 PUEA to ox-PUEA in the presence or absence of a test olln~l is assayed. Intact cells can be used in place of the broken cell system.
Pyrrolidine dithior~rh~r~te ~PDTC), orally delivered at 25-SOmg/kg/day, dramatically inhibited 10 atherogenic fatty streak formation, arterial monocyte-macrophage inf l t i nn ~ endothelial VCAM-1 eYpression and essentially normalized endothelium dependent relaYation function in diet induced hypercholesterolemic rabbits with serum cholesterol 15 levels over 1000 mg/dl. At the same doses, other putative therapeutic agents, such as the antinY;ts~nts probucol and vitamin E, had no effect on lesion formation in this model.
Endothelial dependent arterial rPl ;~Y ~t i nn is 20 restored in eYperimental atherosclerosis by administration of PDTC. In the diet induced hypercholestPrnl Pm; c rabbit model, orally delivered (25-50mg/kg/day) PDTC restored endothelial lPrPn~Pnt vasoreactivity. This was determined by 25 ring-contraction studies of eYcised aorta from control and test animals. In patients with atherosclerosis, this manifests itself as norm~ l; 7~t; on of peripheral vascular reactivity in response to hyperemia as measured by non-invasive 30 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 anti-ischemic therapy by rapidly norm-l; 71n~ the 35 pathological loss of endothelial derived arterial vasodilation characteristic of cardiovascular .1;.qP~CPC and atherosclerosis. This i~ v~ in ,~ WO95130415 2 1 8 9336 ~ -vascular blood flow is manifested as an; ~ ~v~
in symptom and ischemia-limited exercise function and provides a non-invasive asses5ment of vascular protection. Other clinical indications of 5 abnormalities in endothelial derived vasor~lA~Ati~,n include impotence.
The molecular regulator f actory that controls VCAM-1 gene transcription is a novel transcription factor complex consisting of the p65 and p50 10 subunits of the NF-kB/Rel family cross-coupled to the c-fos and the c-jun subunits of the AP-1 family. ;3y both structural and functional studies, it has been estAhl; '2h~ that these AP-1 factors play an important role in the regulation of the 15 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.
Brief Description of the Figurcs Figure 1 is a graph of the cell-surface expression (O.D. 450 nm~ of VCAM-1 as a function of hours in human aortic endothelial cells on exposure to the cytokine TNF-~ (closed circle); linoleic 25 acid (closed triangle); and linoleyl l.ydLu~e~ ~J~ide (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 30 expression (O.D. 450 nm) of VCAM-1 in human aortic endothelial cells on exposure to linoleic acid (closed triangle) and linoleyl hydroperoxide (13-EIPODE, closed square) as a function of the crnr,ontration of fatty acid (~LM) .

-Wo 95~3041~ q 3 3~6 ~ l~u~

Figure 3 is a bar chart graph of the cell-surface expression (O.D. 450 nm) of VCAM-l, ICAM-l and E-5electin in human aortic endothelial cells on ~o~uLe to the cytokine TNF-a, stearic acid, oleic acid, linoleic acid, I ;nnlPn; c acid, and arArh; ~r~n; r acid.
Figure 4 is a bar chart graph of the cell-surface expression (O.D. 450 nm) of VCAM-l in human aortic endothelial cells on exposure to linoleic acid, 13-HPODE, ar~rh;~rn;c acid, and ArArh;~rn;r acid h~ eLu~ide (15-HPETE), with (solid black) or without (hatched lines) the antioxidant pyrrolidine dithior~rh~--te.
Figure 5 is an illustration of an autoradiogram indicating the acute induction of VCAM-l mRNA by linoleic acid and 13-HPODE. HAEC were exposed or not to linoleic acid (7.5 ~M), 13-HPODE (?.5 ~LM) or TNF-~Y (100 U/ml) . Total RNA was isolated and 20 ILg was size-fractionated by denaturing 1. 0~
agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose, and hybridized to either 32P-labeled human A) VCAM-l specific or B) i~-actin specific cDNA After washing, the filters were exposed to X-ray film at -70CC with one intensifying screen for 24 hours. Identification of lanes: 1) control; 2) linoleic acid (acute, 8-hour exposure); 3 ) linoleic acid (48-hour exposure); 4 ) 13-HPODE (acute, 8-hour exposure);
and 5) TNF-cr (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 ;n~ r/~n~ n~ of rF~ 1 Ar protein gynthesis . HAEC were exposed to either linoleic (7.5 ILM) or arArh;r~r~n;c (7.5 ~LM) acid in the presence or abgence of cyrl~lhf~;m;~l~
(10 ~g/ml) for a 4-hour period, and then treated as described in Figure 5.

~ Wo 95/304l5 2 1 ~ 9 3 3 6 Figure 7 i8 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 apprn~;r~t~ly 6096 confluence in 100-mm tissue culture plates. XAEC
were transfected with either 30 ~g of p288 VCAMCAT, p85 VCAMCAT, or pSV2CAT plasmid by the calcium phosphate coprecipitation technique using standard techniques. After a 24-hour recovery period, H~EC
were pretreated or not with 50 ~M PDTC and after 30 minutes exposed to linoleic acid (7 . 5 /~M) or TNF-~Y
(100 U/ml) directly added to the plates. After 18 hours, cell extracts were prepared by rapid freeze-thaw in 0.25 M Tris, pE~ 8Ø The protein of each cell extract was assayed for chl~L ~ ; col acetyl transferase (CAT) activity, as previously described [Ausubel, 1989] (Ac, acetylated; N, nonacetylated chloramphenicol).
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 antin~ nt PDTC.
Confluent XAEC in media cnnt~;nin~ 49~ PBS (as described in Figure 1) were pretreated or not with PDTC (50 ~lM) for thirty minutes and then exposed for three hours to linoleic acid (7 . 5 ~M, oleic acid (7.5 ILM), or TNFo~ (100 U/ml), respectively.
Five miC:LUyL - of nuclear extract was incubated with a double-stranded 32P-labeled wtVCAM, size fractionated on 4~ native acrylamide gels, and exposed to autoradiography film at -70OC 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 tlli ~h~rhituric acid reactive substances ~: 21~9336 WO 95/30415 ~

(O.D. 532 nm) of ar~ h;fl~n;c acid and 15-HPETE in the presence or absence of PDTC. The thiobarbituric acid reactivity assay (TBARS) measures the oxidation ability of a -~t~r; ~1 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 A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1 specific cDNA (Panel C). Following pre-treatment for 30 minutes with 50 ~M of sodium pyrrolidine dithio~-~rh---~te (PDTC), HWE (human umbilical vein~ cells were exposed to IL-lb (10 U/ml) in the ~ nnt; nl~r~ presence of 50 llM PDTC.
Parallel controls were performed identically except in the absence of PDTC. At the i~dicated times, total RNA was isolated and 20 ~g of material size-fractionated by denaturing 1.0~ agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose, hybridized as described above, and visualized by autoradiography. Lane 1-0 hour;
Lanes 2, 4, 6, 8 - OL- 1 alone f or 2, 4, 8 and 24 hours, respectively; Lanes 3,5,7,9 - IL-1 with PDTC
for 2,4,8 and 24 hours, respectively.
Figure 11 is an illustration of an autoradiogram of mRNA, obtained as described below, hybridized to either 32P-labeled human VCAM-1 Sro-;f;- (Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1 specific cDNA (Panel C) . HWE cells were 3 0 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
~cllm~ t;on by Northern filter hybr;~;;7~tion analysis. Lane 1 - colltrol, lane 2 - IL-1 (lOu/ml), lane 3 - IL-lb + PDTC (0.05 ~M), lane 4 - IL-1 LB + PDTC (0.5 ~LM), lane 5 - IL-lb + PDTC
(5.0 ~lM), lane 6 - IL=lb + PDTC (50.0 IlM), lane 7 - IL-lb + PDTC (100 jlM) .
Figure 12 is an illustration of an autoradiogram of mRNA, obtained as described below, hybridized to either 32P-labeled human VCAM-l specific cDNA
5 (Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-l specific cDNA (Panel C). HUVE cells were pretreated as described in Figure 9 with 50 ~M
PDTC, exposed for four hours to the agents indicated below, and assayed for VCAM-l (Panel A) 10 and ICAM-l (Panel B) mRNA ;3c~ lAt;~n. Lane 1 -TNFa llOOU/ml), lane 2 - TNFa + PDTC, lane 3 -lipopolysaccharide (LPS) (lOOng/ml), lane 4 - LPS +
PDTC, lane 5 - poly(I:C) (lOOmg/ml), lane- 6 -poly ( I: C) + PDTC .
Figure 13 is a graph of relative cell surface expression of VCAM-l and ICAM-l in the presence (dark bars) or absence (white bars) of PDTC and in the presence of multiple types of ; nrlllr; n~7 stimuli .
Confluent HUVECs were pretreated or not pretreated 20 (CTL only) for 30 minutes with 50 ~LM 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-l specific (4B9) and 25 ICAM-l specific (84H10) mouse monoclonal ~nt;ho~;es followed by secondary binding with a horse-radish peroxidase tagged goat anti-mouse ( IgG) .
Quantitation was performed by detPrm;nAt;nn of calorimetric conversion at 450 nm of TMB. Figure 30 13 indicates that multiple regulatory signals induce VCAM-l but not ICAM-1 through a common, dithiomArh~r~te-sensitive pathway in human vascular endothelial cells.
Figure 14 is a graph of the relative VCAM-l cell 35 surface expression (O.D. 595 nM) in human umbilical vein endothelial cells, activated by TNFa, versus concPntration of various antin~ Ants. (PDTC is ~ 8~336 Wo 9513~415 P~l/IJ~

sodium N-pyrrolidine dithiocarbamate; DETC is sodium N,N-diethyl-N-carbodithiolate, also referred to as sodium diethyldithiocarbamate; NAC is N-acetyl cysteine; and DF is desferroximine) .
Figure 15 is a graph of the relative VCAM-1 cell surface expression (O.D. 595 nM) in human umbilical vein eIldothelial cells, activated by TNF-~, in the presence of the specified amount of ~ntin~ nt.
(PDTC is sodium N-pyrrolidine dithio .~ t~;
DIDTC is sodium N,N-diethyl-N-carbodithioate;
SarDTC is sodium N-methyl-N-.dLL,c,..y~ thyl-N-carbodithioate; ~DADTC is trisodium N, N-di (carboxymethyl) -N-carbodithioate; MGDTC is sodium N-methyl-D-gll-r~m;nP-N-carbodithioate; MeOBGDTC is 15 sodium N- (4-methoxybenzyl) -D-glucamine-N-carbodithioate; DEDTC is sodium N,N-diethyl-N-carbodithioate; Di-PDTC is sodium N,N-diisopropyl-N-carbodithioate; NAC is N-acetyl cysteine. ) Figure 16 is a graph of the percentage of Molt-4 20 cells binding to H~VE cells either unst; 1 ~tPd or 8tir-ll~t~d with TNFa (100 U/ml) for six hours in the presence or absence of PDTC.
Figure 17 is an illustration of the chemical structures of the following active 25 dith;or~rh~r-t~ sodium pyrrolidine-N-carbodithioate, sodium N-methyl-N-carboxymethyl-N-carbodithioate, trisodium N,N-di (~;d~Lu~y, -thyl) -N-r::~rhoA;th;r,:~tP, godium N-methyl-D-glucamine-N-carbodithioate, sodium N,N-diethyl-N-carbodithioate 30 (sodium diethyldithior~rh~r~tP), and sodium N,N-diisopropyl -N-carbodithioate .
Figure 18 is a bar chart graph of the effect of PTDC on the formation of fluorescent adducts of BSA
and 13 -HPODE, as measured in f luorescent units 35 versus micromolar rnnrF~ntration of PDTC. One mi~:L, l~r of 13-HPODE was incubated with 200 miC:LuyLcll.,., of B~A in the presence of PDTC for six o ~5130415 days. Fluorescence was measured at 430-460 nm with excitation at 3 3 0 - 3 6 0 nm .
Figure l9 is a graph of the effect of PTDC on the formation of fluorescent adducts of BSA and ox-5 PUFA as a function of wavelength (nm) and - rnnrPntration of PDTC. As the rrnrpntration 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 (EIRP), as measured by the increase in O.D. (234 nm) versus time (minutes) for varying rnncpntrations Of PDTC. It is observed that after an ;nr1~hatjn~
period, PDTC inhibits the oxidation of LDL by HRP
in a manner that is rnncPntration 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 inrl;r~tP-l~ both TNF-a and IL-lB causes the oxidation of linoleic acid to ox-linoleic acid. The oxidation is significantly prevented by PDTC.
Detailed De~cription o r the Invention I . Def inition~
As used herein, the term polyunsaturated fatty acid (also referred to herein as a "PUFA") refer~
to a fatty acid (typically C8 to C24) that has at least two alkenyl bonds, and includes but is not limited to linoleic (C~ Q9 12), linolenic (C~8 /\6,9,l2), ararh; ~lnn;c (C20 ~5 8 11 1~) and eicosatrienoic (C20 A8 11,14) acid8.
The term n~ ; 7P~l polyunsaturated fatty acid refers to an unsaturated fatty acid in which at least one of the alkenyl bonds has been converted to a 1.y~Lu,ue~u,-ide. Nonlimiting examples are:
/ ~ COOH
~~~ 13-HPO~1E
OOH

WO95/30415 2 i ~933 6 l~HPETE
~COOH
OOH
The term alkyl, as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic ( in the case of C5 or greater) hydrocarbon of C~ to ClO (or lower alkyl, i . e ., C~ to 5 C~), which Rpe~; f;c~lly includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2, 2-dimethylbutyl, and 2, 3-10 dimethylbutyl. The alkyl group can be optionallysubstituted 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 ;nrlPr~ntl~n~ly 15 alkyl, aryl, alkaryl or aralkyl; aryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, rh~srh~te~ or ph~5ph~n~t~ either unprotected, or protected as rl,~c~qq~ry, 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 sp~;f;~rl, refers to a straight, 25 branched, or cyclic hydrocarbon of C2 to C~O with at least one double bond.
The term alkynyl, as referred to herein, and unless otherwise specified, refers to a C2 to ClO
straight or branched hydrocarbon with at least one 3 0 triple bond .
The term aralkyl refers to an aryl group with at least one alkyl substituent.

Wo 95/30415 2 t 8 9 3 3 6 The term alkaryl ref ers 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 5 which at least one of the l~yd~ ugells 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 ~h~5rh(~n;3te, C02H, or its pharmaceutically acceptable salt, CO2(alkyl, aryl, alkaryl or aralkyl~, or glucamine, either unprotected, or protected as nPrPc~ry, as known to those skilled in the art, f or example, as taught in Gree~le, et al., "Protective Groups in Organic Synthesis, "
John Wiley and Sons, Second Edition, l991.
The term alkoxy, as used herein, and unless otherwise specified, refers to a moiety of the structure -O-alkyl .
The term acyl, as used herein, refers to a group of the formula C(O)R', wherein R' i8 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 ~hPn:~7;nP, phenoth' ~ 7; nP, furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, riuinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, b~n7; n~ 7olyl, purinyl, morpholinyl, carbozolyl, oxazolyl, Wo95/30415 21 8q336 r~l,.J.. ~.

thiazolyl, isothiazolyl, l,2,4-th;~S;A--t~lyl, isooxazolyl, pyrrolyl, pyrazolyl, qll;n~-Ql;nyl, pyridazinyl, pyrazinyl, rinnnl;nyl, ~hth~1~7;nyl, qtl;nr,Y~l ;nyl~ xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, ; m; t~ opyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine, N6-alkylpurines, N6-benzylpurine, N6 halopurine, N6 vinylpurine, N5-acetylenic purine, N5-acyl purine, N6-llydLL~ydlkyl purine, N6-thioalkyl purine, thymine, cytosine, 6-azapyrimidine, 2-mercaptopyrmidine, uracil, N5 - alkylpyr; m; tl 1 n P q, N5-benzylpyrim. idines, Ns-halopyrimidines, N5-vinylpyrimidine, N5-acetylenic pyrimidine, N5-acyl pyrimidine, N5-hydroxyalkyl purine, and N6-thioalkyl purine, and; qr~7olyl ~ 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 2û example, dihydropyridine can be used in place of pyridine Functional oxygen and nitrogen groups on the heterocyclic base can be protected as SPrPC5~ry or desired during the reaction sequence.
Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, tritylmethyl, alkyl groups, acyl groups such as acetyl and propionyl, me thyl sul f onyl, and p - toluyl sul f onyl .
The term hydroxyalkyl, as used herein, refers to a C~ to C6 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 rr,nt:q;n;nr compound that retards oxidation.
The term pharmaceutically acceptable derivative refers to a derivatIve of the active rt, t~ that , _ , _ _ ... . . .. . . . .

2~ 89336 Wo 95/30415 P~ 3~ --upon administratior~ to the recipient, i8 capable of providing directly or indirectly, the parent compound, or that exhibits activity itself.
The term ~pharmaceutically acceptable cation"
ref ers to an organic or inorganic moiety that carries a positive charge and that can be administered in association with a pharm-cPvt;c~1 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 qU~tern~ry amine.
The term "physiologically cleavable leaving group" refers to a moiety that can be cleaved n vivo f rom the molecule to which it is attached, and lS includes but is not limited to an organic or inorganic anion, a pharmaceutically acceptable cation, acyl (; nr~ ; nr but not limited to (alkyl) C(0), including acetyl, propionyl, and butyryl), alkyl, rhrR~h~te, sulfate and sulfonate.
The term "~n~ntil -rically enriched composition or compound" reiers to a composition or ,~ d that includes at least 959, and preferably at least 97, 98, 99, or 100% by weight of a single e~antiomer of the, In,l 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 hi s t idinyl .
A "linking moiety" as used herein, is any divalent group that links two chemical residues, 3 S including but not limited to alkyl, alkenyl, alkynyl, aryl, polyalkyleneoxy (for example -[(CH2)c~]n~) I -C~6alkoxy-CI~Oalkyl-, .

WO 95/3041~

-Cl6alkylthio-C~l0 alkyl-, -NR3-, and - (CHOH)~CH20H, wherein n i5 independently 0, 1, 2, 3, 4, 5, or 6.
II. T~n~;f;c~tion of Y;~;~ed ~nd ~ "Y;~;7 Polyunsatur~ ted Fatty Acids as Direct MCI~; Atrlr8 of VCAM-l Expr~ssion To establish whether a PUFA or ~ li 7ed PUFA
acts as a direct; ~ tor of endothelial cell gene expression, early p~R2~ed human aortic endothelial cells (HAEC) were cultured for eight 10 hours in media and serum and exposed to saturated ~stearic),, ~ nf~aturated (oleic), and polyunsaturated (linoleic and arachidonic) fatty acids; as well as with the fatty acid llydLu~eL~ ides of linoleic (13-HPODE) or 15 ar~h;-l~n;r (15-HPETE) acid~. HAEC were also alternatively exposed to the cytokine tumor necros is f actor -~ .
HAEC were exposed to linoleic acid or 13-HPODE
for varying times up to 48 hours and then assayed 20 for cell surfaoe VCAM-1 expression by ELISA assay.
The results were compared to HAEC exposed to the cytokine TNF-al (100 U/ml) for the same time periods . VCAM- 1 expression in HAEC incubated with either linoleic acid or 13-HPODE is transiently 25 induced The expression peaks at appr~;r-tPly 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-~, and thus the mPrh~ni by which 30 polyunsaturated fatty acids induce VCAM-1 thus appear to be similar to that of TNF-om 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 ILM is 35 the lowest peak dose by which linoleic acid and ~ W0 95/304l5 2 1 8 9 3 3 6 13-HPODE induces signi~icant VCAM-1 gene expression .
It was then explored whether short term incubation of endothelial cells with 5 polyunsaturated fatty acids induces ICAM-l and E-selectin expression as well . It was det~rm; n-d that the polyunsaturated f atty acids linoleic and arachidonic acids induced cell-surface gene expression to approximately 59~ of TNF-induced gene ~.
10 expression of VCAM-l. Strikingly, neither ICAM-l nor E-selectin were induced by these fatty acids.
Conversely, the 6aturated fatty acid stearic acid and the mlnq3turated fatty acid oleic acid did not induce the expression of VCAM-1, ICAM-l, or 15 E-selectin. VCAM-1 gene expression was also observed by; nr~1hAr j on of ~IAEC with the ~ 3; 7~d - metabolites of linoleic acid (13-HPODE) and ar~h;~n;c acid (15-HPETE).
To investigate whether oxidative stress in 20 endothelial cells provided by polyunsaturated fatty acids and their oxidized metabolites induces VCAM-l through a redox-sensitive r -h~n; ~m~ HAEC were pretreated with the ~nt; ~ nt pyrrolidine dithiocarbamate (PDTC, 50 IlM) for 30 minutes and 25 then the cells were ;n~l~r~n~Pntly incubated with linoleic acid, arachidonic acid, 13-HPODE, and 15-~PETE (all 7 . 5 ~LM) for 8 hours . It was det~rm;n-~d that PDTC suppressed the gene expression of VCAM-l induced by the polyunsaturated fatty 30 acids and their ~r;~;7~1 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 (i.e., the ~ ;3t;~)n does not occur), 35 reversed (i.e., the signal molecule is reduced), or its interaction with a target protein prevented, perhaps through a redox complex.

w095~304ls ~ ~ 89336 p To determine whether the selective induction of VCAM- 1 by PUFAs and their oxidized metabolites is observed at the mRNA level, HAEC were inrllh~tpd with linoleic acid or 13-HPODE. Linoleic acid and 5 13-HPODE induced VCAM-1 mRNA a~ _ l At j nn that was similar to levels induced by TNF-~Y. In contrast, there was no induction of ICAM-l or E-selectin gene expression at the mRNA level in HAEC incubated with linoleic acid or 13-HPODE. The ~;n~l;nr~ mimic 10 those found at the cell-surface level. These results indicate that pretranslational regulatory ' an; f~- mediate induction of VCAM-1 gene expression by polyunsaturated fatty acids and their oxidative metabolites.
It was also desired to determine whether polyunsaturated f atty acids work as a primary signal or operate through a regulatory protein involving the cytokine IL-4 in inrillr;n~ VCAM-1 gene expression. To investigate whether newly 20 8ynthPR; 7Pfi proteing guch as IL-4 are involved in the synthesis and gene expression of VCAM-1 induced by PUFAs such as linoleic acid, HAEC were; nrllhated with 13 -HPODE (7 . 5 ~LM) and exposed to the protein synthesis inhibitor, cycl~hPxi mi fiP . There was no 25 inhibition of mRNA ~r~ 1 ation of VCAM-1 by cyclr,hPx; mi tlP in HAEC incubated with 13 -HPODE . The production of IL-4 by HAEC; nrlihatpd with linoleic or ar~rh;-~r,n;c acids and their oxidative - - t;~h~l; teg, a8 determined by ELISA was also 30 measured. There was no increase in IL-4 output by HAEC ;nrllh~tP~l with these PUFAs or their oxidized metabolites .
Previous investigations have demonstrated through deletion and heterologous promoter studies 35 that cytokines and non-cytokines activate VCAM-1 gene expression in endothelial cells at least in part transcriptionally through two NF-kR-like DNA

~ WO 9j/304l5 2 1 8 9 3 3 ~

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 f atty acids induce 5 transcriptional activation of the human VCAM-1 - promoter via a similar mechanism, the chimeric reporter gene p288 VCAM-CAT, r~ntA;n;n~ coordinates -238 to +22 of the human VCAM-l promoter, was transiently transfected into HAEC. The addition of 10 linoleic acid (7.5 ~M) induced VCAM-l promoter.
The addition of linoleic acid (7 . 5 ~M) induced VCAM-I promoter activity that was over two fold that of the control and appro~ t~1 y 6096 of the maximum signal induced by TNF-~. Similar results 15 were obtained with the minimal cytokine-;n~ c;hl.o promoter of the VCAM-1 gene (p85 VCAM-CAT), ~nnt~;n;n~ the -77 and -63 bp NF-kB-like sites.
Neither linoleic acid nor TNF-~ had any effect on activity using a constitutively expressed pSV2 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 mr~r h;ln; r-.
To determine whether polyunsaturated fatty acids and their oxidative metabolites regulate VCAM-1 promoter activity through an NF-kB-like 30 transcriptional regulatory factor, nuclear extracts from H~EC were assayed for DNA binding activity to a double-stranded oligonucleotide ~ nf;3;n;nr3 the VCAM-1 NF-kB-like promoter elements located at positions -77 and -63. As shown in Figure 7, two 35 bands A and C, representing NF-kB-like activity were induced in response to a three hour exposure to linoleic acid (7.5 IlM). Similar findings were WO 95/30415 . ~ ,~ 8 ~ 3 3 6 observed on exposure to the cytokine TNF-~Y (100 U/ml). A weak band B was observed in control ~untreated) cells . No; n~lllrt; rn of NF-kB-like binding was observed with the monoun5aturated fatty acid oleic acid. Pretreatment of the cells for thirty minutes with PDTC inhibited the A and C
complex DNA binding activity after l;nnlP;r acid activation . These f indings are similar to previously reported f indings that PDTC blocks the activation of VCAM-l gene expression in HUVEC by inhibiting the activation of these NF K~3-like DNA
binding proteins.
Example 1 Rffect of Oxidized md TTn~Yir~
Polyunsaturated Fatty Acid~ on the l~inetics of the Activation of VCA~-l Gene Expression Human aortic endothelial cells (HAEC) were plated in 96 well plates and; nrllh~tPd with linoleic acid (7.5 ~M), 13-HPODE (7.5 ~M~, or TNF-o~
(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 20~6 fetal bovine serum (FBS), 16 U/ml heparin, 10 U/ml epidermal growth factor, 50 ~g/ml endothelial cell growth supplement, 2 mM L-glut~m;n-o, 100 U/ml penicillin, and 100 ~g/ml streptomycin. One day before the experiment, cells were placed in a medium rr,nt~;n;nrJ 49~ FBS. Confluent HAEC were incubated for up to 48 hours with TNF-~
3 0 ( 1 0 0 U/ml ), or stearic , oleic , linoleic , linolenic , or arArh;~n;C acids (7.5 ~lM). Similar studies were performed with differing doses of linoleic acid or 13-HPODE for an 8 hour period (1-60 ~M) (Figure 2). Quantitation was performed by rl~tprm;n~tirn of colorimetric conversion at 450 nm of TMB. Studies were performed in triplicate (n=4 ~ WO 95~0415 2 1 8 9 3 3 6 I ~
for each experimental value). ~-value differs (pc 0 . 0 5 ) f rom Control .
As shown in Figure 1, both linoleic acid and 13-HPODE induced the expression of VCAM-l. At ten 5 hours af ter exposure, the amount of cell aurf ace VCAM-1 induced by linoleic acid and 13-~IPODE was greater than half that induced by the cytokine TNF-~ .
As shown in Figure 2, the induction of VCAM-1 by 10 linoleic acid and 13 -HPODE is rnnrPnt ration sensitive. At a rnnrPntration of between 2 and 10 IlM of these compounds, there is a sharp increase in the amount of induced cell surface VCAM-l, which then remains approximately constant up to a 15 c^nrPntration of at least 100 /lM. It should be observed that the PUFA rnnrPntration indicated in Figure 2 is in addition to that found endogenously in HAEC.
2 0 3xample 2 Polyunsaturated Patty Acid~ Induce Gene Expre~ion of VCA2~-l but not ICAN-l or 3-~elect~n.
The cell surface expression of VCAM-1, ICAM-1, and E-selectin was measured in HAEC by E:LISA.
25 HAEC, obtained from Clonetics (California~, were cultured in Medium 199 ~upplemented with 20~ fetal bovine serum (FBS), 16 U/ml heparin, 10 U/ml epidermal growth factor, 50 ~lg/ml endothelial cell growth supplement, 2 mM L-glutamine, 100 U/ml 30 penicillin, and 100 ~Lg/ml streptomycin. One day before the experiment, cells were placed in a medium cnnt~in;nr~ 4~ FBS. Confluent HAEC were incubated or not ~or 8 hours with TNF-~ (100 U/ml), or stearic, oleic, linoleic, linolenic, or 35 arachidonic acids (7.5 ~M). Cell-surface expression of A) VCAM-l, B) ICAM-1, and C) E-selectin was de~ermined by primary binding with VCAM-1 specific, ICAM-1 specific, and E-selectin _ _ _ _ _ _ . . . . .. . .. . _ . , . .... . .. _ _ 2 ~ 8q336 Wo 95130415 -28-- .
speci~ic mouse:~AntihQ~l;F q ~ollowed by ~econdary binding with a horseradish peroxidase-tagged goat anti-mouse (IgG) . Quantitation was performed by determination of colorimetric conversion at 450 mm of TM~3. Studies were performed in triplicate (n-4 for each experimental value). *-value differs (p~O . 05) from Control .
As shown in Figure 3, linoleic acid, l;nnl~n;c 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-l, or E-selectin. TNF-~
strongly induced the expression of all three cell-surf ace molecules .
Ex~ple 3 The ~nt;~ nt PDTC S~ ~ eE1~138 VCAN-l Induction by Poly~nrst~ t^~3 Fatty Acid~ and th~ir Oxidativ~ Netabolites.
Conf luent HAEC were pretreated in the presence or absence of PDTC (sodium pyrrolidine dithiocarbamate, 50 IIM) for thirty minutes. The cells were then nr~lh~tP~l for eight hours with TNF-(100 U/ml), linoleic or ~r~rh;~nn;c acid (7.5 IlM), or the fatty acid hydroperoxides 13-HPODE (7.5 ~LM) or 15-HPETE (7.5 ~lM) . The cell surface expression of VCAM- 1 was measured in HAEC by ELISA, as described in Example 1. Studies were peri-ormed in tr;rl ;~ ~tP (n = 4 for each experimental value) .
*-value differs (p~0.05) from control.
3 0 As indicated in Figure 4, PDTC suppresses the ;nrlllrt;nn of VCAM-l by linoleic acid, 13-HPODE, ar~t~h;~nn;c acid and 15-HPETE.
Example 4 Acute Induction of VCAN-1 mRNA by Linoleic Acid ~nd 13-l~PODE.
HAEC were exposed to linoleic acid (7 . 5 ~LM) or 13-HPODE (7.5 ,llM) . Total RNA was isolated and 20 ~ j4 Wo 95/304ls fig size-fractionated by denaturing 1.0~
agarose - f ormaldehyde gel electrophoresis, transferred to nitrocellulose, and hybridized to either 3~P-labeled human A) VCAM-l specific or B) 5 ~-actin specif ic cDNA and visualized by autoradiography. After washes, filters were exposed to X-ray film at -70C with one intensifying scree~ for 24 hours. Identification of lanes: 1) control; 2) linoleic acid (acute, 10 8-hour exposure); 3) linoleic (48-hour exposure);

4) 13-HPODE (acute, 8-hour exposure); and 5) TNF-~Y
( 10 0 U/ml, 4 - hour exposure ) .
As shown in Figure 5, both linoleic acid and 13-HPODE induce the production of mRNA for VCAM-l in 15 eight hours. After 48 hours, linoleic acid no longer induces VCAM- 1 mRNA .
Example 5 Induction of VCAM-l mRN~ by PUFAs is Tn~ r~n~1~nt of ~ Protein Synthesi~ .
HAEC were exposed to either linoleic or arAnh;clnnic acid (7.5 ~M) in the presence or absence of cyrl~h~ (10 ~Lg/ml) for a 4-hour period. Total RNA was isolated and 20 ~g was size-fractionated by denaturing 1. 09~
agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose, and hybridized to A) 32P-labeled human VCAM-l or 3) i3-actin specific cDNA and then visualized by autoradiography . Af ter washes, filters were exposed to X-ray film at -70C
with one intensifying screen for 24 hours.
A5 indicated in Figure 6, the induction of VCAM-1 by linoleic and ar~h;~nn;c acids are in~rQn~nt Of nF~l 1 ,.l ~r protein synthesis .

Wo 95/30415 E~c le 6 T~n~ acid illduc~s trAnscriptional amp _ctivatio~ of the VCAM-l promoter ~y redox-se~sitive NF-kB like factor.
~EC were split at the ratio to give approximately 609,~ rnn~ Pnre in 100-mm tissue culture plates. HAEC were transfected with either 30 ~Lg of p288 VCAMCAT, p85 VCAMCAT, or pSV2CAT
plasmid by the calcium rhnsr)h~te coprecipitation technique using standard techniques . Af ter a 24-hour recovery period, HAEC were pretreated with 50 ~M PDTC and after 30 minutes exposed to linoleic acid (7.5 ~M) or TNF-a (100 U/ml) directly added to the plates. After 18 hours, cell extracts were prepared by rapid freeze-thaw in 0.25 M Tris, pH
8 . 0 Protein of each cell extract was assayed f or chluL , hPn; col acetyl transferase (CAT) activity (Ac, acetylated; N, nonacetylated chluL -n;Cnl)~
Figure 7 illustrates the results of this experiment Li~oleic acid induces transcriptional activation of the VCAM-l promoter by a redox-sensitive NF-kB like factor. These results are similar to those observed by the activation of VCAM-l promotor by cytokines such as TNF-a This suggests that PUFAs act through an nY; ~i 7ed intermediate that also ~ tPA the cytokine activation of VCAM- 1.
Ex~le 7 Polyunsaturated Fatty Acids Activato NF-kB - like DNA Blndi~g Actlvities th~t ~Iro Blocked ~y the 1~n1~ Yi ~Ant PDTC .
Confluent ~EC in media nnnt;:;n;n~ 4~ FBS (as described in Example 1) were pretreated with PDTC
(50 IlM) for 30 minutes and then exposed for 3 hours to linoleic acid or oleic acid (7 5 IIM), or TNF-a (100 U/ml). Five mi~Lu~LalllO of nuclear extract was incubated with a double-stranded 3~P-labeled wtVCAM, size fractionated on 4~ native acrylamide gels, and exposed to autoradiography film at -70C for 18 -21 ~9336 ,~ Wo 95130415 hours. 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

5 NF-ki3 binding activity to VCAM- 1 promotor in a redox-sensitive manner. This is analogous to cytokine TNF-~ and suggests a similar ---h ln; ~-r of action. TNF-a probably induces VCAM-1 through a r-^h~n; Flm that is mediated by an ox-PUFA.
10 Ex lo 8 C~ t;nn in a cell-free, media-free amp ~etup, by both ~ 1 and AY;~
(15-HPETE) A ~/rh;~ln;C ac1d Figures 9A and 9B are bar chart graphs of the relative thi~h~rhituric acid reactive substances (O.D. 532 nm) of ar~-h;dr~nic acid and 15-HPETE in the presence or absence of PDTC. The thiobarbituric acid reactivity assay (TBARS~
measures the oY;ti~t;~rl ability of a material in a cell-free, media-free environment. A5 indicated in 20 the Figures, both ara~ h; ~nn; c acid and 15-HPETE
showed significant TBARS activity that was inhibited by PDTC.
TII. Method for the Treatment of VCAN-1 Mediated D~ sorders The discovery that polyunsaturated fatty acids and their ~Y;~li7Pd metabolites are selective, redox-sensitive; ~ ators provides a basis for the therapy of disorders that are ~ t~ by VCAM-l or by redox-sensitive genes.
A method for the treatment of atherosclerosis, post-angioplasty restenosis, coronary artery ~1; qp~cpc, angina, and other cardiovascular diseases, as well as noncardiovascular ; nf 1: tory ~1; C~cPc that are ~; ~tPd by VCAM-l is provided that includes the removal, decrease in the WO95/30415 : ~' ,-r,n,~on~ation of, or prevention of the fr,~r-til~n of ir?;~od polyunsaturated fatty acids, ;n~ r~;nS but not limited to oxidized linoleic, linolenic, and arachidonic acids. In an alternative ornhQrl;rnont, a 5 method for the treatment of these rl; co~coC is provided that includes the prevention of the interaction of a PUFA or ox-PUFA with a protein or peptide that mediates VCAM-l expression.
Inhibition of the expression of VQM- 1 can be l0 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 ro~hol; r-- of PUFAs into ox- PUFAs, as described in more detail below .
15 l. Administration of Antioxidants Any ~ ~ ~1 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 20 in this therapy. One of ordinary skill in the art can easily rlotorrl; no whether a compound reduces an ox-PUEA or inhibits the ~Y;~l~tir~n of PUFA using standard techniaues .
Dl~h~o~_rl..~yl21te AntIr.Y~ n~
It has been discovered that dithiocarboxylates are useful in the treatment of atherosclerosis and other cardiovascular and ;nl'l tory diseases.
Di~h;s.,..1~ ylates, including dith;o~rh-~-tes, can be used to block the ability of cells, including 30 endothelial cells, to express VCAM-l or to suppress the expression of a redox-sensitive gene or activate a gene that is suppressed through a redox-sensitive pathway.

, ~t WO9513041~ ? ~ 336 P~
At least one of the compounds, pyrrolidine dithiocarbamate (PDTC), inhibits VCAM-l gene expression at a concentration of less than l. 0 micromolar. This ~ n~ also exhibits 5 preferential toxicity to proliferating or - abnormally dividing vascular smooth muscle cells.
Another dithio~rh~r-tP, sodium N-methyl-N-carboxymethyl-N-carbodithioate, also inhibits the expression of VCAM-l, without significant effect on ICAM-l, but does not exhibit preferential toxicity to abnormally dividing vascular smooth muscle cells. Another dithio-~rh~r~te, sodium N-methyl-N-carboxymethyl-N-carbodithioate, also inhibits the expression of VCAM-l, without significant effect on ICAM-l, 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-l or ICAM-l expression, and therefore treatment with this compound does not adversely af f ect aspects of the infl. t~ry response mediated by EI~M-l or ICAM-l. Thus, a ge~eralized immunosuppres~ion 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.
Dithior~rh~r-tes are transition metal chelators clinically used for heavy metal intoxicatio~.
Ba~3elt, R.C., F.W.J. S~n~ n, et al. (1977), "Comparisons of antidotal efficacy of sodium diethyldithiocarbamate, D-penicillamine and triethylenetetramine upon acute toxicity of nickel carbonyl in rats. " Res C~mm~n Chem Pathol ph~rmacol 18(4): 677-88; Menne, T. and K. Kaaber (1978), "Treatment o~ pompholYx due to nickel allergy with rh~1~t;ng agents." Contact Dermatitis 4 (5): 289-90; Sunderman, F.W. (1978), "Clinical response to therapeutic agents in rniqnntn~ from mercury vapor" An~ Clin Lab Sci 8 (4): 259-69;
Sunderman, F.W. (1979), "Efficacy of sodium diethyldith; n~rh~r~te (dithiocarb) in acute nickel carbonyl poisoning. " Ann Clin Lab Sci 9 (1): 1-10;
Gale, G.R., A.B. Smith, et al. (1981), "DiethyldithiorArh~r-t~ in treatment of acute cadmium poisoning. " Ann Clir~ Lab Sci 11 (6): 476-83; Jones, M.M. and M.G. Cherian (1990), "The search for chelate antagonists for chronic cadmium 1ntol~;ration.~ ToxicoloqY 62(1): 1-25; Jones, S.G., M.A. Basinger, et al. (1982), "A comparison of diethyldithiocarbamate and EDTA as antidotes for acute cadmium intoxication. " Res Commurl Chem Pathol Pharmacol 38(2): 271-8; Pages, A., J.S.
Casas, et al. (1985), "Dithiocarbamates in heavy metal poisoning: complexes of N,N-di (1-hydroxYethyl)~;th;nr~rh~r-te with Zn(II), Cd(II), Hg(II), CH3Hg(II), and C6X5Hg(II) .- J. IIlorr~
Biochem 25(1): 35-42; Tandon, S.K., N.S. Hashmi, et al. (1990), "The lead-rh~l~t;nri effects of substituted dith; nr~rh~r-tes . " Biomed Environ Sci 3 (3): 299-305.
Dithiocarbamates have also been used adjunctively in cis-platinum chemotherapy to prevent renal toxicity. Hacker, M.P., W.B.
Ershler, et al. (1982) . "Effect of disulfiram (tetraethylthiuram disulfide) and diethyldithiocarbamate on the bladder toxicity and antitumor activity of cyclophosphamide in mice. "
~nr~r Re8 42 ~11): 4490-4. ~oll~nn~r~ 1986 #733;
Saran, M. and Bors, W. (1990). "Radical reactions in vivo--an overview. " Radiat. Enviro~. BioT:hYs.
29(4) :249-62.

2 t ~9336 Wo 95/3041~
,. _ A dithiocarbamate currently used in the treatment of alcohol abuse is disulfiram, a dimer of diethyldithiocarbamate. Disulfuram inhibits hepatic aldehyde dehydrogenase . Inoue , K ., and 5 Fukunaga, et al., (1932). "Effect of disulfiram and its reduced metabolite, diethyldith;o~rh--~te on aldehyde dehydrogenase of human erythrocytes.
I.ife Sci 30 (5): 419-24 .
It has been reported that dithocarbamates 10 inhibit HIV virus replication, and also enhance the maturation of specific T cell subpopulations. This has led to clinical trials of diethyldithio-r~rh~r-te in AIDs patient population8. R~;c;r~ r, E., et al., (1990). "Inhibition of HIV progression by dithiocarb. " I.ancet 335: 679.
Dithiocarboxylates are compounds of the structure A-SC(S)-B, which are members of the general class of compounds known as thiol ;Int;~.Y;.l~tc~ and are alternatively referred to as carbodithiols or carbodithiolates. It appears that the -SC (S) - 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.
In an alternative embodiment, one or both of the sulfur atoms in the dithio~rh~r-te is replaced with a selenium atom. The substitution of sulfur f or selenium may decrease the toxicity of the molecule in certain cases, and may thus be better 3 0 tolerated by the patient .
A and B can be selected by one of ordinary skill in the art to impart desired characteristics to the , 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 Wo 95/30415 ;~ l ~ q 3 3 6 r~
effect on the tissue-distribution and pharm-rnk;nPtics of the compound. In general, for treatment of cardiovascular disease, it is desirable that the compound i~rrllml~lAte, or 5 localize, in the arterial intimal layer r~nt~;n;n~
the vascular endothelial cells. The ~ R are preferably ol ;m;n~tFd by renal excretion.
An advantage in administering a dithio-carboxylate pharmaceutically is that it does not lO appear to be cleaved enzymatically in vivo by thioesterases, and thus may exhibit a prolonged half lif e in vi~o .
In a prefer~ed ~ ;r t, A is hydrogen or a pharmaceutically acceptable cation, including but 15 not limited to sodium, potassium, calcium, 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, n~hth~l enesulfonic acid, n~rhth~l enedisulfonic 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 NR4R5R6R7, wherein R'l, R5, R6, and R7 are ;n~oron~ontly l~y-l~uyt:ll, Cl6 linear, branched, or ~in the case of C~6) cyclic alkyl, hydroxy(C~6)alkyl (wherein one or 3 0 more hydroxyl groups are located on any of the carbon atoms), or aryl, N,N-dibenzyl-ethylon~ m;no~ D-glucosamine, choline, tetraethylammonium, or ethyl~n~ m; no .
In another ~mhorl;~ t, A can be a 35 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 Wo 95~041~ ~ ~ 8 9 3 3 6 r~." - ^ -(including acetyl, propionyl, and butyryl), alkyl, phosphate, sulfate or sulfonate.
In one embodiment, B is alkyl, alkenyl, alkynyl, alkaryl, aralkyl, haloalkyl, haloalkenyl, haloalkynyl, aryl, alkaryl, hydrogen, Cl~ alkoxy-CIlO
alkyl, Clb alkylthio-C~l0 alkyl, NR~R3~ ~ (CHOH~nCH20H, wherein n i5 0, 1, 2, 3, 4, 5, or 6, - (CH2)~CO2RI, including alkylacetyl, alkylpropionyl, and alkylbutyryl, or hydroxy(CI6)alkyl- (wherein one or more hydroxyl groups are located on any of the carbon atoms).
In another Pmho~i--nt, B is NR2R3, wherein RZ and R3 are independently alkyl; - (CHOH)n(CH2) nOHI wherein n is 0, 1, 2, 3, 4, 5, or 6; - (CH2),C02RI, - ( CH2)nCo2R4; hydroxy(CI6)alkyl-; alkenyl (;nrl~l~;nr~
but not limited to vinyl, allyl, and CHICH=CH-CH~CH2); alkyl (CO2H), alkenyl (CO2H), alkynyl (CO2H), or aryl, wherein the aryl group can be substituted as described above, notably, for example, with a NO2, CH3, t-butyl, CO2H, halo, or p-OH group; or R2 and R3 can together constitute a bridge such as -(CH2)=-, wherein m is 3, 4, 5, or 6, and wherein R4 is alkyl, aryl, alkaryl, or aralkyl, including acetyl, propionyl, and butyryl.
In yet another embodiment, s can be a heterocyclic or alkylheterocyclic group. The heterocycle can be optionally partially or totally hydrogenated. Nonlimiting examples are those listed above, including phenazine, phenothi~7in~
3 o 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 intPrn~lly or externally as a medicine for the treatment, cure, or prevention of a disease or disorder .

WO 95/30415 ;~ ~ 8 9 3 3 6 P~
Nonlimiting examples are drugs for the treatment or prevention of cardiovascular diseage, ;nr~ ;nrJ
Ant;rY;~l~ntR such as probucol; nicotinic acid;
agents that prevent platelets from sticking, such 5 as aspirini antithrombotic agents such as coumadin;
calcium channel blockers such as varapamil, diltiazem, ana nifedipinei angiotensin converting enzyme (ACE) inhibitors such as captopril and enalopril, ~-hlsrk~rs such as propanalol, 10 t~rhUt~lol~ and labetalol, nonsteroidal ;nf1 tories 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 dithior~rh~m-te is an amino acid derivative of the structure AC2C-R9-NR1-C(S) SA, wherein R9 is a divalent B moiety, a linking moiety, or the; ntl~rni~l residue of any of the 20 naturally occurring amino acids (for example, CH3CH
for alanine, C~2 for glycine, CH(CH~)4NH~ for lysine, etc. ), and R10 is hydrogen or lower alkyl.
B can also be a polymer to which one or more dithior~rh~r~te groups are attached, either 25 directly, or through any suitable linking moiety.
The dithior~rh~r-te is preferably released from the polymer under ~in vivo conditions over a suitable time period to provide a therapeutic benefit. In a pref erred embodiment, the polymer itself is also 3 0 degradable in vivo . The term biodegradable or bioerodible, ae 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 f ive years , and 35 preferably less than one year, on exposure to a physiological solution of pH 6 - 8 having a temperature of between 25 and 37C. In a preferred -2 1`~9336 Wo 95130415 r~.,~ 9 embodiment, the polymer degrades in a period of between l hour and several weeks, according to the application .
A number of degradable polymers are known.
5 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, lO aspartic acid, cysteine, cystine, glutamic acid, glutamine, hydroxylysine, serine, threonine, and tyrosine; polyorthoesters, including poly(~Y-hydroxy acids), for example, polylactic acid, polyglycolic ac id , po ly ( l act ide - c o - glyco l i de ), po lyanhydride s , 15 albumin or collagen, a polysaccharide rf~ntA;nin~
sugar units such as lactose, and polycaprt lAct~n~.
The polymer can be a random or block copolymer.
B can also be a group that ~nhAnl-~q the water solubility of the dithio~Arh~r-te, for example, 20 -lower alkyl-O-R~, wherein R8 is -PO2(OH)~M+or PO3(M+) wherein M+ is a pharmaceutically acceptable cation;
-C(o) (CH2)~CO2 M+, or -SO3~+; -lower alkylcarbonyl-lower alkyl; -carboxy lower alkyl; -lower alkylamino-lower alkyl; N,N-di-6ubstituted amino 25 lower alkyl-, wherein the substituents each ; n~l~rf.n~l~nt 1 y 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 30 alkyl-; piperidinyl-lower alkyl; morpholinyl-lower 11yd~ y~lkyl; N-pyrryl; piperazinyl-lower alkyl; N-substituted piperazinyl-lower alkyl, wherein the substituent is lower alkyl; triazolyl-lower alkyl;
tetrA7~lyl-lower alkyl; tetrazolylamino-lower 35 alkyl; or thiazolyl-lower alkyl.
In an alternative ~ ; r-nt, a dimer such as B-C(S)S-SC(S)-B can be administered.
_ _ _ _ _ _ _ _ _ _ _ WO95/30~15 ~?t8~36 - ~
Nonlimiting examples o~ dithiocarbamates are thoEe o~ the structure:
I ) Aliphatic Suhs~ ~N~
~N~ C=S
R- CH.=CH S
SC - S CH~ CH=CH
~) Amino Acid Polyarnino acid HR~ S N~ R'N NR' NR
S' I C=S C=S C2S
~ S,~:S S' S' S
R' S_~,,l;".. , R- Na+ R--C--S--C-R' l NO Ca~ ThioesD~r N--~R' CH~ or t-Butyl R' NH4~
C,, COOH ChoLine+ arld quatemary amines S S p-OH Mg~
Al~
K' A--S--C--El B--C--S--S--C--B H

2~ 8q336 Wo95/30415 p~ "~_,.;/r Dithiocarboxylates should be chosen for ui~e in treating atherosclerosis and other cardiovascular and ;nfl: tnry diseases that have the proper lipophilicity to locate at the affected cite. The compound should not compa~ i tAl; 7e in low turnover regions such as fat deposits. In a preferred pmho~l;r^nt for treatment of cardiovascular disease, the phar--^rk;nPtics of the compound should not be dramatically affected by congestive heart failure or renal insufficiency.
For topical applications for the treatment of I n f l i tory skin disorders, the selected 1- u--d 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, c _ 'q with a therapeutic index of at least 2, and preferably at least S or lO, are acceptable. The therapeutic index is defined as the EC50/IC50, wherein EC50 is the cc ..c~ L c.tion of compound that inhibits the expression of VCAM-l by 50~ and IC50 is the c~^~rlrontration of ~ that is toxic to 50~ of the target cells. Cellular toxicity can be 25 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 iis over lO0 as measured by 30 cell toxicity divided by ability to inhibit VCAM-l expression activated by TNFa, in HUVE cells.
Initial studies on the rapidly dividing cell type HT-18 human glioma demonstrate no toxicity at rrnrontrations lO0-fold greater than the 35 therapeutic rrnr~ntration. Disulfiram, an orally administered form of diethyldithio~rArhiqr~te~ used in the treatment of alcohol abuse, generally _ _ _ _ _ _ _ _ _ _ .

WO 95/30415 ;~ I ~ 9 3 3 6 r~

elicits no mzjor ~l;n;c~l toxicities when administered appropriately.
There are a few dithiQr~rh~r~tes that are known to be genotoxic. These compounds do not fall 5 wlthin the scope of the present invention, which is limited to the administration of physiologically acceptable materials. An example of a genotoxic dith;nc~rhA~-te is the fungicide zinc dimethyldithiocarbamate. Further, the 10 antit h~-l ;n~cterase properties of certain dithio~rh~-~t~R can lead to neurotoxic effects.
Miller, D. (1982) . Neurotoxicity of the pesticidal carbamates . N~llrobehav. Toxicol . Teratol . 4 (6):
779 -87 .
The term dithiocarboxylate as used herein specifically includes, but is not limited to, dithiocarbamates of the formulas:
Rl S C ( S ) NR2R3 or R2R3N ( S ) CS - S C ( S ) NR2R3 wherein Rl is H or a pharmaceutically acceptable 20 cation, including but not limited to sodium, potassium, or NR~R5R6R7, wherein R~, R5, R6, and R7 are independently hydrogen, C~6 linear, branched, or cyclic alkyl, hydroxy(C~6) alkyl (wherein one or more hydroxyl groups are located on any of the carbon 25 atoms), or aryl, and R2 and R3 are ;nll~ron~ntly Cl lO linear, branched or cyclic alkyl; - (CHOH)n(CH2)nOH, wherein n is 0, 1, 2, 3, 4, 5, or 6; - ( CH2) nC2RI ~ ~ (CH2) nCO2R~;
hydroxy(C~6) alkyl-, or R2 and R3 together constitute 30 a bridge such as - (CH2)m-, wherein m is 3-6, and wherein R~ is alkyl, aryl, alkaryl, or aralkyl, including acetyl, propionyl, and butyryl.
Specific ~ 1 Pq of useful dith;or~rh~r~te illustrated in~Figure 15, include sodium 35 pyrrolidine-N-carbodithioate, sodium N-methyl-N-carboxymethyl-N-carbodithioate, trisodium N,N-di (carboxymethyl) -N-c~rbodithioate, sodium N-Wo sst304ls 2 t 8 9 3 3 6 , ~l/L.
methyl-D-glucamine-N-carbodithioate, sodium N,N-diethyl-N-carbodithioate (sodium diethyldithio-carbamate), and sodium N,N-diisopropyl-N-carbodithioate .
The active dith; ori~rhoxylates and in particular, dithiocarbamates are either commercially available or can be prepared using known methods.
II . B~ ~1 o~ Activlty The ability of dith; Dri~rhnYylates to inhibit the expression of VCAM-1 can be measured in a variety of ways, including by the methods set out in detail below in Examples 9 to 15. For convenience, F , lPq 9-11 and 14-15 describe the evaluation of the biological activity of sodium pyrrolidine-N-carbodithioate (also referred to as PDTC). These examples are not ;nt~n~ to limit the scope of the invention, which specifically; n~ c the use of any of the above-described c~r~un~q to treat atherosclerosis, and other types of ;nfli tion and cardiovascular disease ~ ted by VCAM-1. Any of the, ~ ~u~-ds described above can be easily substituted for PDTC and evaluated in similar f ashion .
Examples 12 and 13 provide comparative data on the ability of a number of dithiol-~rhil~ t~q to inhibit the gene expression of VCAM-1. The examples below establish that the claimed dithiocarbamates specif ically 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 infl. tory response .
Exl~erimental Procedures Cell Cultures HUVE cells were isolated from human umbilical veins that were cannulated, ...... . . ... . . . _ _ _ _ _ _ WO 95130415 2 1 8 9 3 3 6 r~
perfused with Hanks solution to remo~e blood, and then incubated with 196 collagenase for 15 minutes at 37C. After removal of collagenase, cells were cultured in Ml99 medium supplemented with 20~ fetal 5 bovine serum (HyClone), 16 ~Lg/ml heparin ~E
Pharmaceuticals, Cherry Hill, NJ), 50 ~g/ml endothelial cell growth supplement (rOl 1 ~horative Research Incorporated, sedford MA), 25 mM Hepes Buffer, 2 mM L-glutamin, 100 ~g/ml ppn;c;llin and 100 ~g/ml streptomycin and grown at 37C on tissue culture plates coated 0.1~ gelatin. Cells were passaged at conf luency by splitting 1: 4 . Cells were used within the first 8 passages.
Incubation with CYtokines and Other Reaqent8 15 Confluent HWE cells were washed with ~hnsph~tf.
buffered saline and then received fresh media. The indicated rrnl-~ntrations of PDTC were added as pretreatment 3 0 minutes before adding cytokines .
Cytokines and other inducers were directly added to 20 medium for the times and at the concentrations ; n~; rated in each experiment . Human recombinant IL-lb was the generous gift of Upjohn Company (Kalamazoo, Michigan). TNFa was oht~;n~ from Bohringer ~n~lhP;m Bacterial lipopolysaccharide 25 ~LPS), polyinosinic acid: polycitidilic acid (Poly I:C), and pyrrnl;~l;nf~ dithior~rh~r=te (PDTC) were obtained from Sigma Chemical (St. Louis, MO). All other reagents were of reagent grade.
RNA Isolation: Total cellular RNA was isolated 30 by a single ex~raction using an acid gll~n; ~ m thiocyanate-phenol-chloroform mixture. Cells were rinsed with phosphate buffered saline and then lysed with 2 ml of guanidium isothiocyanate. The solution was acidif ied with 0 . 2 ml of sodium 35 acetate (pH 4 . 0) and then extracted with 2 ml phenol and 0.4 ml chloroform:isoamyl alcohol (24:1). The R~A underwent two ethanol WO 951'30415 21 ~ q 3 3 6 r precipitations prior to bein~ used f~r Northern blot analysis.
Northern Blot Analvsis: Total cellular RNA (20 ~g) was size fract;on~t~o~ using 1~ agarose 5 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 Str~tl ;nkPr W
crosslinker (Stratagene, La ~olla, CA).
Hybri~1; 7atinnC were performed at 42C for 18 hours in 5X SSC (lX=150 mM NaCl, 15 mM Na citrate), 19c sodium dodecyl sulfate, 5X Denhardt solution, 50~ -formamide, 10~ dextran sulfate and 100 ug~ml of sheared denatured salmon sperm DNA. Apprn~r;r-t~ly 1-2X 106 cpm/ml of labeled probe (specific activity>
108 cpm/ug DNA) were used per hybr;~;7~t;nn Following hybridization, filters were washed with a -final stringency of 0.2X SSC at 55C. The nitrocellulose was stripped~,"u,sing boiled water prior to rehybridization with other probes.
Autoradiography was performed with an; nt~-n~ifying screen at - 7 0 C .
32Probes: 3~p labeled DNA probes were made using the random primer oligonucleotide method. The ICAM-l probe was an Eco Rl ~ _ t of human cDNA.
The ELAM-l probe was a 1. 85 kb Hind III fragment of human cDNA. The VCAM-l probe was a Hind III-Xho I
fragment of the human cDNA consisting of nucleotide 132 to 1814.
~n7:vme T~;nkqd Immunosorbent Assav (EI,ISA): HUVE
cells were plated on 96-well tissue culture plates 48 to 72 hours before the assay. Primary ~ntiho~;es in M199 with 5~ FBS were added to each well and incubated one hour at 37C. The cells were then washed and incubated for one hour with peroxidase conjugated goat anti-mouse IgG (3io Rad) diluted 1/500 in Ml99 with 5~ FBS. The wells were . ~

~ 1 ~93 WO 95/30415 P~

then washed and binding of antibody was detected by the addition of l00 ,ILl of l0 mg/ml 3,3,5,5'-tetramethyl-benzidine (Sigma) with 0 . 003Y~ H O~ . The reaction was stopped by the addition of 25 ~l 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: MrnnrlrnAl antibody (MAb) 4B9 recognizing vascular cell adhesion molecule-l (VCAM-l) was the generous gift of Dr. John E~arlan (University of Washington). MAb E9AlFl recognizing endothelial cell ;l~hPc; on molecule (ELAM-l) was the generous gift of Dr. Swerlick (Emory University) .
Hybridomas producing mAb 84Hl0 rPro~rn;7;n~
intercellular A~hPcjon molecule l (ICAM-l) are routinely grown in our laboratory and antibody was used as tissue culture sUpprnAt~nt.
Ex~ple 9 PDTC Blocks I1- lb Mediated Induction of ~IUVEC VCAN-l, but not ICAM-l or EIAM-l, I:IRNA ~r, 1~ rn 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 ;nr~l1r;nr~ cytokine, IL-lb (l0 U/ml) in the presence or absence of the thiolated metal rhPlAt;nr~ antioxidant, pyrrolidine dithiorArhAr~-te (PDTC, 50 ~LM) for up to 24 hours. As shown in Figure l0, IL-lb alone (lanes 2, 4, 6, 8) induces the expected rapid and transient induction of VCAM-l (Panel A), E-selectin (ELAM-l, Panel B) and ICAM-l (Panel C) mRNA accumulation, all of which peak at four hours. However, in the presence of PDTC, IL-lb induction of VCAM-l mRNA Ar~ 1 ~tion is dramatically inhibited by over 90~ (panel A, lanes 3, 5, 7, 9). In contrast, although IL-lb mediated ~ ~ 2t 89336 Wo 95/30415 - r~-"Jv,~

induction of ELAM-1 i6 slightly inhibited at 2 and 24 hours (compare lane 2 and 3, 8 and 9 , panel B), PDTC does not in~ibit the induction at 4 and 8 - hours (lane S and 7, panel B). IL-lb mediated 5 induction of ICAM-l m~RNA ~r~ l;stion is not affected (panel B, lanes 3, 5, 7, 9). Indeed, a mild ~llrTn~nt~t;~n of IL-lb induction of ICAM-1 mRNA
accumulation (~30~) is observed (compare lanes 4 and 5, panel B). E~uivalent amounts of nitrocellulose transferred RNA per lane was confirmed by ethidium bromide staining and VisllAl i 7~r;r,n, A dose-response analysis was performed to determine whether PDTC inhibits the induction of 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 EC50 of approximately 10 ~M, while PDTC
does not inhibit IL-lb mediated induction of ELAM-l expression with these rrnrPntrations (Fig. 11, panel B). The IL-lb mediated induction of ICAM-1 mRNA acrllmlll ~tion is Pnh~nr~l by PDTC with the concentration higher than 0 . 5 IlM ( Fig . 2, compare lane 2 and lane ~-7, panel C).
These data demonstrate that IL-lb utilizes a dithiocarboxylate, and in particular, a dithior~rhAr-te sensitive step as part of its signaling --^h;3ni~ in the induction of VCAM-1 gene expression. The data also appear to indicate that this dithior~rh~r~te sensitive step does not play a significant role in the IL-lb mediated ;n~llrt;rn Of ELAM-l or ICAM-1 gene expression.
lvex~le 10 PDTC slocks Inductiorl of kuv~C VCAM-l nRNA ~ t; - n by Nultiple Sti~nuli To determine whether other well-described activators of VCAM-1 gene expression also utilize a WO 95/30415 ~ 2 1 ~ 9 3 3 6 P~

PDTC sensitive step, three distinct classes of activators were tested: another classic receptor mediated in~illring agent (TNFa~, a non-receptor mediated inducer ~lipopolysaccharide (LPS) ) and a ~i recently described novel inducer (double stranded RNA, poly~I:C) ) . In all three cases, PDTC
dramatically inhibited the ;n~ rt;nn of VCAM-1 mRNA
Ar~c~ ll Atinn in HUVECs after four hours (Fir,ure 12, Panel A) . Although the TNFa '; Ated ELAM-1 gene expression is suppressed to some extent (Fig. 12 lane 1 and 2, panel B), LPS and poly(I:C) r ';A~ed ELAM-1 mRNA accumulation was unaffected (Fig. 12 lane 3 - 6, panel B ) . The induction of ICAM- 1 mRNA
accumulation was unaffected (Figure 12, Panel C) .
This data indicates that structurally distinct ;nrillr;n~ agents, acting through distinct pathways, share a common regulatory step specific for the induction of VCAM-1 gene expression.
Ex~mple 11 PDTC Blocks ~VE Cell gurface Expre~sion of VCAM-1 Induced by Multiple gtimuli To determine whether, like its mRNA, the ; n~l~rt; nn of endothelial cell surface protein expression of VCAM-1 could also be inhibited by PDTC, monoclonal antibodies were used in an ELISA
assay to r~uantitate 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 and 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 3~ inhibited (80-909~). In contrast, the induced expression of cell surface ICAM-1 is unaffected ~ WO 95130415 2 1 ~ q 3 3 ~

under identical conditions (bottom left and right panels ) .
These data demonstrate that, like its mRNA
~qec1~r-11 qtion, cell surface VCAM-l expression are 5 selectively inhibited by dithiocArh~qrq~Pq and that multiple classes of activating agents utilize a similar, dithiocarbamate sensitive r chqni P~r to induce VCAM-l gene expression.
Ex~ple 12 Comparative Effectivene~s of 1~n~-J~ ~ntn in R~ lr;n~ TNFa Islduction o~ VCAIl-l To determine whether structurally similar or ~;qsiri1qr antioxidants could also inhibit VCAM-l gene expression, and with what potency, HUVE cells were exposed to TNFa for six hours in the presence or absence of different cnnrPntrations of four different antin~iriqntA~ As shown in Figure 14, both diethyldithio~ qrhqr-te (DETC) and N-acetyl cysteine (NAC) inhibited VCAM-l expression at concentrations of 5 ,uM and 30 ~M, respectively. In contrast, PDTC (PDTC) was effective between 5 and 50 /lM. The iron metal ~-hPl~tnr, desferroximine, had no effect at the concentrations tested.
Ex le 13 PDTC Inhibits T~7F Induction of VQl~-amp l/VlA-4 Mediated ~h~ni~In The ability of a variety of antin~ qntP to inhibit TNF-~ induction of VCAM-l in ~UVE cells was evaluated by the method set out in Example 12.
Figure 15 is a graph of the relative VCAM-l cell 30 surface expression (O.D. 595 nM) in TNF-~ activated ~UVE cells versus n on~qntrations of PTDC (sodium N-pyrrolidine di~hiorqrhqr~te), DIDTC (sodium N,N-diethyl-N-carbodithioate), SarDTC (sodium N-methyl-N-carboxymethyl-N-carbodithioate), IDADTC
35 (trisodium N,N-di (carboxymethyl) -N-carbodithioate), .. . _ . . . .

WO 95130415 . 2 1 8 9 3 3 6 r~

MGDTC (sodium N-methyl-D-glucamlne-N-carbodithioate), MeOsGDTC (sodium N- (4-methoxybenzyl)-D-glucamine-N-carbodithioate), D~DTC
(sodium N,N-diethyl-N-carbodithioate), Di-PDTC
5 (sodium N,N-diisopropyl-N-carbodithioate), and NAC
i8 (N-acetyl cysteine).
Example 13 PDTC Inhibit~ T ~F Induction of VCAI~-l/V~A-4 r'-~3tD~ 3h~ nn In order to def ine whether PDTC inhibition of lO VCAM-l regulation is associated with functional conseriuences, the binding of Molt-4 cells to H[iVEC
cells either unstimulated or stimulated with TNFa (lOOU/ml) was f~T~min~cl for six hours in the presence or absence of PDTC. Molt-4 cells have 15 been previously shown to bind to activated X~VEC
via a VCAM-l dependent mechanism. As shown in Figure 16, the percentage of Molt-4 binding to HU~TEC cells decreased when PDTC was present in the media .
0 Example 14 PDTC Inhibits Nonocyte Binding to the Thoracic Aorta of Chole~terol Fed Rabbit~
An experiment was performed to determine whether the thiol antinT;~nt PDTC would be effirar~iollq in 25 blocking the first monocyte binding ~ l. of atherosclerosis in an experimental animal model.
One mature New Zealand white rabbit (3 . 5 Kg) received an intravenous injection of PDTC (20 mg/Kg, as a rnnrrntration of 20 mg/ml in PBS) once 30 daily for 5 days. Injections were given via an indwelling cannula in the marginal ear vein, which as kept patent by f lushing with heparinized saline solution. The PDTC solution was mixed fresh daily or on alternate days (stored light-protected at 35 4C), and filtered (0.45 mm pore filter) just prior to use. After the first injection, when the ~ WO95130~15 ~ ~1 89336 p ~

cannula 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 cnnt~;ni 5 196 cholesterol by weight, which was continued throughout the L~ i n~lPr of the experiment . On the fifth day, the animal was ellth~ni7ed and the thoracic aorta was excised and f ixed . Af ter , iate preparation, the sample was imaged on 10 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 magnif ication were assessed, to cover an area of 15 -1.3 mm2. 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: 5 WBC and ~25 RBC per 1. 3 mm2 f ield . 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!'6 cholesterol for 4 days but not given antioxidant show about a 5-fold increase in adhesion, to 38 WBC/1. 3 mm2. A considerable amount of mostly cell-sized 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 ~h~Rinn to the aortic endothelium.
Example 15 Inhibition of ~SA 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

WO 95130415 2 ~ 8 9 3 3 6 P~
and 13-HPODE, as measured in fluorescent units versus micromolar concentration of PDTC. One micromolar of 13-~PODE was incubated with 200 mivLvyLc~ of BSA in the presence of PDTC for six days. FIuorescence was measured at 430-460 nm with excitation at 330-360 nm. For details of the assay, see Freebis, J., Parthasarathy, S., Steinberg, D, ProcP.-~;n~c of the N~tion~l Academy of Sciences 89, 10588-10592, 1992. In a typical reaction 100 nmols of LOOX (generated by the lipoxygenase catalyzed oxidation of linoleic acid) in; ncllhated with 100 llg of bovine serum albumin for 48 to 72 hours and the formation of fluorescent products are followed by measuring the fluorescent spectrum with excitation at 360 nm and emission between 390 and 500 nm.
As indicated, PDTC decreases the cnncpntration of f luorescent 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 cnnrPntration of PDTC. As the cnnnpntration of PDTC increases, the quantity of flllnr~ct~Pnt adducts decrease .
Exa~nple 16 Effect of PDTC on the oxidntion of LDL
by horseradish peroxid~3e Figure 20 i8 a graph of the ef~ect of PDTC on the oxidation of LDL by horseradish peroxidase (HRP), as measured over time (minutes) for varying rnn~PntratiOng of PDTC. The oxidation of LDL was followed by measuring the oxidation of the fatty acid ~ Pntc of LDL as determined by the increase in optical density at 234 nm. When a polyunsaturated fatty acid is n~ ; zPd, there i9 a shift of double bonds resulting in the formation of conjugated die~res whi~h absorb at 234 nm. The ~ W 0 95/304 l5 2 ~ ~ 9 3 3 6 ~ ~ l / L v, ~ r intercept 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 n~ tirll. Typically lO0 5 ~g of human LDL is incubated with 5 ~M X202 and the increase in absorption of 234 nm is followed.
It is observed that after an incuh~ti~n period, PDTC inhibits the oxidation of LDL by HRP in a manner that is r~nc~nt-^ation dependent.
10 Ex~ple 17 Effect of PDTC on the cytokine-induced fo~-t~o~t 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 15 and IL-lB causes the oxidation of linoleic acid to ox-linoleic acid. The ~ t;nr is significantly prevented by PDTC.
2. Modification of the SYnthesis and Met~holism of pUFAs and ox- PUFAs Inhibition of t~e expression of VCAM-l can be accomplished via a modification of the --t~h.~l; pm of PUFAs into ox-PUFAs. For example, a number of enzymes are known to oxidize unsaturated materials, including peroxidases, lipoxygenases, cyclooxygenases, and cytochrome P~50. The inhibition of these enzymes may prevent the oxidation of PUFAs in vivo. PUFAs can also be oxidized by metal-~p~nt1~nt nonerLzymatic materials.
IV. Method for Modifying the Expression of a Redox-Sensitive Gene In an alternative; ' ~i t, a method is provided for suppressing the expression of a redox-sensitive gene or activating a gene that is WO9S/3041~ ~ l 8q33fj r~
-54- ~--suppre6sed through a redox-sensitive pathway, that includes administering an effective amount of a substance that prevents the nx; rl~t; nn of the nx;rli7~1 signal, and typically, the nX;~jnn of a 5 polyunsaturated fatty acid. Representative redox-sensitive 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 (e.g., IL-li~), 10 chemoattractants that promote the migration of infl~ tnry cells to a point of injury ~e.g., MCP-1), growth factors (IL-6, thrombin receptor), and adhesion molecules (e.g., VCAM-1 and E-selectin) .
Given this disclosure, one of ordinary skill in the art will be able to screen a wide variety of ~nt;nx;fl~nt~ 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--,nnt~;n;nrJ the 5' regulatory sequences of the redox-sensitive genes can be used to regulate or inhibit gene expression in vivo can be irlf~nt; f; ~d. Vectors, including both plasmid and eukaryotic viral vectors, may be used to express a particular rPrrm~;n~nt 5' flanking region-gene collstruct in cells ~,or.-n~l;nrJ 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, e.g., Mulligan, 1993 Science, 260, 926-932; United States Patent No.
4, 980, 286; United States Patent No . 4, 868 ,116;
incorporated herein by rei~erence) . Recently, a Wo 9S/30415 2` 1 8 9 3 3 6 . ~

delivery system was developed in ~hich 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 5 multiple tissues of adult ~ice, including endothelium and bone marrow ( see , e . g ., Zhu et al ., 1993 Science 261, 209-211; incorporated herein by ref erence ) .
The 5~ fl;~nkln~ sequences of the redox-sensitive 10 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 ~ f 1 ;Ink; n~ region of the redox-sensitive gene can be used to inhibit expression of the gene in vivo. Expression vectors 15 (e.g., 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, e.g., U.S. Patent No.
4,868,116; U.S. Patent No. 4,980,286).
20 Accordingly, DNA ~ nt;~;n;n~ all or a portion of the sequence of the 5 ' f lanking region of the gene can be inserted into an appropriate expression vector 80 that upon passage into the cell, the tran3cription of the inserted DNA yields an 25 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 nonnal mRNA transcript f ound in the cell and thereby prevent the mRNA f rom 30 being translated. It is of course nP~ ~cq~ry to select sequences of the 5 ' f lanking region that are downstream from the transcriptional start sites for the redox-sensitive gene to ensure that the ~ntiq~nqe RNA ~ t~;nq complementary sequences 3S present on the mRNA. Antisense RNA can be generated in vitro also, and then inserted into cells. Oligonucleotides can be synthesized on an _ .

WO 95/30415 ~ l 8 9 3 3 6 P~
automated synthe~izer (e.g., Model 8700 A1lt~ ted synthesizer of Milligen-Biosearch, Burlington, MA
or ABI Model 380B) . In addition, antisense deoxyoligonucleotides have been shown to be 5 effective in ;nh;hit;nr~ gene transcription and viral replication (see e.g., 7 -n;k et al., 1978 Proc . Natl . Acad. Sci . USA 75, 280-284; 7~ -n; k et al., 1986 Proc. Natl. Acad. Sci., 83, 4143-4146;
Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA
85, 1028-1032; Crooke, 1993 F~q~ J. 7, 533-539.
Further~ore, recent work has shown that; ~vt:d inhibition of expression of a gene by antisense oligonucleotides is possible if the antisense oligonucleotides contain modified nucleotides (see, e.g., Offensperger et. al., 1993 EMBO J. 12, 1257-1262 (in vivo inhibition of duck hepatitis B viral replication and gene expression by antisense ~h~gFhrrothioate oligodeoxynucleotideg); ~gPnhF.rg et al, PCT WO 93/01286 (synthesis of sulfurthioate ~l;rJ~nllrleotideg); Agrawal et al., 1988 Proc. Natl.
Acad. Sci. USA 85, 7079-7083 (synthesis of antisense oligr,nllrl Pscide rh~sFhrrAm; r~tP~ and phosphorothioates to inhibit replication of human immunodeficiency virus-1); Sarin et al., 1989 Natl. Acad. Sci. USA 85, 7448-7794 (synthesis of antisense methylphosphonate ol; Jr~mllrleotides); Shaw et al., l991 Nucleic Acids Res l9, 747-750 (synthesis of 3 ' exonuclease-resistant 0l; Srnl r 1 eotideg cnnt ~ i n; n ,r, 3 ~ terminal 3 0 ~h l~grh t~roami date modi f i cat ions ); incorporat ed herein by ref erence) .
The sequences of the 5~ fl~nking region of the redox-sensitive gene can also be used in triple helix (triplex) gene therapy. Ol;,~nl1rleotides 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 WO 95130415 2 1 8 9 3 3 6 r~

triple nucleic acid helices which block tran6cription of the gene (see, e.g., 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 calclll Ation~ and empirical iindings have been reported which provide guidance for the design of oligonucleotides for use in oligonucleotide-directed triple helix formation to inhibit gene expres~ion. For example, oligonucleotides should generally be greater than 14 nucleotides in length to ensure target sequence specificity (see, e.g., Maher et al., (1989);
Grigoriev et al., (1992) ) . Also, many cells avidly take up oligonucleotides that are less than 50 nucleotides in length (see e.g., Orson et al., ~1991); Holt et al., 1988 Mol. Cell. Bisl. 8, 963-973 ; Wickstrom et al ., 1988 Proc . Natl . Acad. Sci .
USA 85, 1028-1032) . To reduce susceptibility to intrAc~ l1 Ar degradation, for example by 3 ' exonucleases, a f ree amine can be introduced to a 3 ' terminal hydroxyl group of oligonucleotides without loss of sequence binding specif icity (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 intercAlatin~ agent, such as an acridine derivative, is covalently attached to a 5' terminal phosphate (e.g., via a pentamethylene bridge);
again without loss of sequence spe~~i f; city (Maher ~-et al., (1989); Grigo~iev et al., (1992) .
-WO95/30415 ~1 ~9336 Methods to produce or ~ynthesize oligo-nucleotides are well known in the art. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see 5 e . g ., Sambrook et al , Chapters 5, 6 ) to purely synthetic methods, for example, by the cyanoethyl rhn~ph~ramidite method using a Milligen or Beckman System lPlus DNA synthesizer (see also, Ikuta et al., in Ar~n, Rev~ Biochem. 1984 53, 323-356 10 (phosphotriester and phosphite-triester methods~;
Narang et al., in Methods Enzvmol., 65, 610-620 (1980) (phosphotriester method) . Accordingly, DNA
se~uences of the 5' flanking region of the redox-sensitive gene described herein can be used to 15 design and construct oligonucleotides including a DNA sequence consisting essentially of at least 15 consecutive nucleotides, with or without base modifications or intercalating agent derivatives, for use in forming triple helices specifically 20 within the 5~ fl~nk;ng region of a redox-sensitive gene in order to inhibit expression of the gene.
In some cases it may be advantageous to insert F-nh~n~PrS or multiple copies of the regulatory sequences into an expression system to facilitate 25 screening of methods and reagents for manipulation of expression.
V. Models and Screens Screens for disorders mediated by VCAM-l or a redox-sensitive gene are also provided that include 30 the quantification of surrogate markers of the disease. In one embodiment, the level of ~y;rl;7ed 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 35 "oxidative environment" of the host and the host~ 5 ~ W095/304l~ 21 B~336 P~
susceptibility to VC~M-1 or redox-sensitive ge~e mediated disease.
In another ' ' - , the level of circulating or cell-surface VCAM-1 or other d~ Liate marker 5 and the effect on that level of administration of an d~L~J~Liate antioxidant is quantified.
In yet another assay, the sensitization of a host~ 8 vascular endothelial cells to polyunsaturated fatty acids or their ~Y; ~i 7Pd 10 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 VQM-1 or other surrogate marker to a population norm.
In another ~ ; t, in vivo models of atherosclerosi5 or other heart or infl: tory diseases that are mediated by VQM-1 can be provided by administering to a host animal an excessive amount of PUFA or nYi~li 7Pd 20 polyunsaturated fatty acid to induce disease.
These animals can be used in rl; ni c~l research to further the understanding of these disorders.
In yet another embodiment of the invention, compounds can be assessed for their ability to 25 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, 30 for example, a human or an animal such as a mouse, to a high level of PUFA or ox-PUFA and then detPrm;nin~ the therapeutic efficacy of a test compound based on its ability to decrease circulating or cell surface VCAM-l r~nt~Pntration 35 Alternatively, an i~ vitro screen can be used that is based on the ability o the test compound to prevent the oxidation of a PUFA, or the interaction WO 95/30415 ~ 1 8 9 3 3 ~ r~

of a PUFA or ox-PUFA with a protein target in the presence of an ~ ;7;n~ sub~tance such as a metal, for example, copper, or an enzyme such as a peroxida~e, lipoxyge~ase, cyclooxygenase, or 5 cytochrome P450.
In another ~mhD~;r ~, vascular endothelial cells are exposed to TNF-~I or other VCAM-l ;nr~llr ;n~ material for an appropriate time and then broken by any appropriate means, for example by 10 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. Phi~rr--~u~;c~-l Con~po~itions Humans, e~uine, canine, bovine and other animals, and in particular, mammals, suf f ering f rom 20 cardiovascular disorders, and other ;nfli t~ry 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 r~n~nt~ation of, or 25 prevention of the f ormation of an oxidized polyunsaturated fatty acids, including but not limited to ~nr; ~; 7~d linoleic (Cl8 ~9~12), linolenic (Cl8 ~69~1~), arArh;~nn;c (C~O ~5~8-11-1~) and ~;cn~i~trienoic (C~0 1~8~ ) acids; other ~-~;r~i t;on 30 signal; or other active compound, or a pharm~ Puti- ~l 1 y 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, WO 95/30415 2 1 8 9 3 3 6 . ~

parenterally, intravenously, intradermally, subcutaneously, or topically.
As used herein, the term pharmaceutically acceptable ealts or complexes refers to salts or 5 complexes that retain the desired biological activity of the above-i~n~; fied 1~ _ ~n~R and exhibit minimal undesired toxicological effects.
Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for 10 example, hydrochloric acid, h~dLuLLl c acid, sulfuric acid, ~hn5phnric 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, nArhthAl enesulfonic acid, nArhrhAl enedisulfonic acid, and polygalacturonic acid; (b) 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,N-dibenzylethylene-diamine, D-gll1rn8Amln~, illm, tetraethylammonium, or ethyl~nP~;Amin~-; or (c) 25 comb;nA~lnnR of (a) and ~b); e.g., a zinc tannate salt or the like.
The active ~-m-ro~ln~l or a mixture of the compounds are administered in any appropriate manner, including but not limited to orally and 30 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 f rom once every other day to several times a day .
Preferred daily dosages are between approximately 1 35 and 3000 mg/patient/day, more preferably between approximately 5 and 500 mg/patient/day, and even 2~893 W0 95130415 3 ~ r more preferably, between approximately 25 and 500 mg/patient/day .
The active ingredient should be administered to achieve peak plasma concentrations of the active 5 compound of about 0.1 to 100 ~LM, preferably about l-lO~LM. This may be achieved, for example, by the intravenous injection of a solution or fnrr-llAtinn of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the 10 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 arterial angioplasty. As an example, 2-5 mL of a 15 physiologically acceptable solution that nnntAinq approximately 1 to 500 IlM of the compound or mixture of compounds is administered at 1-5 a Isph~res 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 30 once every other day to three times daily.
Longer term tr~A q can be employed to prevent the development of advanced lesions in high-risk patients. A long term treatment can extend f or years with dosages ranging f rom 0 . 5 to 35 500 mg/kg body weight administered at intervals ranging from once every other day to three times dai ly .

21~933 Wo 95130415 6 The active compo~nds can also be administered in the period immediately prior to and following coronary angioplasty as a means to reduce or eliminate the abnormal prolif erative and 5 ;nfli tory response that currently leads to clinically significant re-stenosis.
The active, _ 'q can be administered in conjunction with other medications used in the treatment of cardiovascular disease, including lO lipid lowering agents such as probucol and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents such as CO~ n; calcium channel blockers such as varapamil, diltiazem, and nifedipine; angiotensin 15 converting enzyme (ACE) inhibitors such as captopril and enalopril, and 5-blockers such as propanalol, t,-rhut~lol, and labetalol. The compounds can also be administered in combination with nonsteroidal ~nt;;nfl. tories such as 2 0 ibupro f en, indome thacin, f enop ro f en, me f enami c acid, flufenamic acid, sulindac. The compound can 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 a3 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 30 understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the profPq~i nn;~l judgment of the person administering or supervising the administration of the 35 compositions, and that the -nnc~ntration ranges set f orth herein are exemplary only and are not intended to limit the scope or practice of the .

~9336 claimed 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 compo6itions 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; nr~ as part of the composition .
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 tr~rJPrAn~h or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starchi a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or qArrhArin; or a flavoring agent such 2~ 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 li~uid carrier such as a fatty oil.
In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unlt, 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 of an elixir, suspension, syrup, wafer, chewing gum or the like.
A syrup may contain, in addition to the active 2 1 ~q336 o 95/30415 r~

compounds, sucrose as a sw~ot~n i nr~ agent and certain preservatives, dyes and colorings and f lavors .
The active compound or pharmaceutically 5 acceptable derivatives or salts thereof can- also be administered with other active material5 that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, Ant;;nfl tories, or 10 antiviral _ ln~lc Solutions or susp~n.cirn.C used for parenteral, intradermal, 5llhr~ltAn~ous~ or topical appl irAtinn can include the following r~ nPntR: 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; rh~lAt;nr~ agents 20 such as ethyl~n~ iAmin~tetraacetic acid; buffers such as acetates, citrates or ~h~sphAt~q and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, ~li qp~qAhle syringes or 25 multiple dose vials made of glass or plastic.
Suitable vehicles or carriers f or topical application are known, and include lotions, suspensions, o;n' tq, creams, gels, tinctures, sprays, powders, pastes, slow-release transdermal 30 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.
35 Examples of thickening agents include petrolatum, beeswax, xanthan gum, or polyethylene glycol, h ^ctAntq such as sorbitol, emollients such as WO 95/30415 ~ I '8 9 3 ~i 6 . ~

mineral oil, lanolin and its derivatives, or s~ualene. A number of solutions and o; tR are commercially available.
Natural or artif icial f lavorings or sweeteners 5 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 ti~R1 ~n~d for ~rPl ;~t;-~n to oral mucosal surfaces.
The active ~ wul-ds can be prepared with carriers that protect the compound against rapid release, such as a controlled release for~ ti~n, including implants and microe~capsulated delivery systems. siodegradable, biocompatible polymers can 15 be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation af such fOr~ t;~nR are patented or generally known to those skilled in the 2 O art .
If administered intravenously, preferred carriers are physiological saline or phosphate buf f ered sal ine ~ PBS ) .
The active r ,__ ~1 can also be administered 25 through a transdermal patch. Methods for preparing transdermal patches are known to those skilled in the art. For example, see Brown, L., and Langer, R., Transdermal Delivery of Drugs , Annual Review of Medicine, 39:221-229 (1988), incorporated herein by 3 O ref erence .
In another embodiment, the active , ullds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled ~elease formulation, including 35 i ,l~nts and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, Wo95/3041~ 2 J 8~33~

polyglycolic acid, collage~, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained 5 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 10 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 , iate lipid (s) (such as stearoyl rhr,sFh~tidyl 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 crnt~;nrr. An a~ueous solution of 2 0 the active compound or its , orhr~cphate, diphosphate, and/or tr;rhr,cph~te derivatives are then illtroduced into the rrnt~;nr~r. The rrnt~inr~r is then swirled by hand to free lipid material from the sides of the crnt~;n~r 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 30 invention. Such modifications and variations are ; nt~n~l~-d to come within the scope of the ~rp~n~ d

Claims

We claim.
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 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 (C18 .DELTA.9,12), linolenic (C18 .DELTA.6,9,12), arachidonic (C20 .DELTA.5,8,11,14) and eicosatrienoic (C20 .DELTA.8,11,14) 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 (e.g., IL-1?) , chemoattractants that promote the migration of inflammatory cells to a point of injury (e.g., MCP-1), growth factors (e.g., IL-6 and the thrombin receptor), and adhesion molecules (e.g., 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 of 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.
15. 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 to screen compounds for their ability 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, C1-6 alkoxy-C1-10 alkyl, C1-6 alkylthio-C1-10 alkyl, NR2R3, -(CHOH)nCH2OH, wherein n is 0, 1, 2, 3, 4, 5, or 6, -(CH,)nCO2R1, including alkylacetyl, alkylpropionyl, and alkylbutyryl, and hydroxy (C1-6) 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.
20. 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, nahthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid.

21. 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 NR4R5R6R7, wherein R4, R5, R6, and R7 are independently hydrogen, C1-6 alkyl, hydroxy(C1-6) alkyl, aryl, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, 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.
25. The method of claim 17, wherein B is NR2R3, wherein R2 and R3 are selected from the group consisting of alkyl; - (CHOH)n(CH2)nOH, wherein n is 0, 1, 2, 3, 4, 5, or 6; - (CH2)nCO2R1, - (CH2)nCO2R4;
hydroxy (C1-6) alkyl-; alkenyl; alkyl (CO2H), alkenyl (CO2H), alkynyl (CO2H), or aryl, or R2 and R3 can together constitute a bridge of the formula - (CH2) m-, wherein m is 3, 4, 5, or 6, and wherein R4 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 alkylheterocyclic 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 pharmaceutically-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, nifedipine, captopril, enalopril, propanalol, terbutalol, labetalol, ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, sulindac, and a corticosteriod.
30. The method of claim 17, wherein the dithiocarbamate is an amino acid derivative of the structure AO2C-R9-NR10-C(S)SA, wherein R9 is B or the internal regidue of an amino acid and R10 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(.alpha.-hydroxy acids), polyanhydrides, polysaccharides, and polycaprolactone.
34. The method of claim 1, wherein B-C(S)S- is pyrrolidine -N - carbodithioate.
35. 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.
40. The method of claim 17, wherein the dithiocarbamate is administered in a dosage of between 0.5 and 500 mg/kg body weight.

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 .beta.-blocker, a nonsteroidal antiinflammatory, and a corticosteroid.
43. A method for the suppression of VCAM-1 expression in human cells comprising administering 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 VCAM-1 comprising administering an effective amount of the dithiocarbamate described in claim 17.
45. A method for the treatment of a human endothelial disorder that is mediated by VCAM-1 comprising administering an effective amount of the dithiocarbamate described in claim 17.
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.

49. A pharmaceutical composition comprising an effective amount to treat cardiovacular disease of a compound disclosed in any of claims 17-34.
50. 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.