Dithiocarbamates for the treatment of atherosclerosis and other cardiovascular and inflamatory diseases

Technical field

This invention is in the field of methods and compositions for treating atherosclerosis and other cardiovascular and inflammatory diseases.

BACKGROUND OF THE INVENTION

Leukocyte adhesion to the endothelium (cell lining) is a significant and timely symptom in many diverse inflammatory diseases including atherosclerosis, autoimmune diseases, and bacterial and viral infections. This process is partially mediated by induced expression of cell lining surface adhesion molecules such as ICAM-1 (intracellular adhesion molecule-1), VCAM-1 (vascular adhesion molecule -1) and ELAM-1 (endothelial leukocyte adhesion molecule-1). These adhesion molecules bind to immune cells which elicit and spread an anti-inflammatory response. In particular, one of the adhesion molecules, VCAM-1, plays an important role in the binding of monocytes. Multiple signals induce the expression of cell surface adhesion molecules.

Atherosclerosis is a chronic inflammatory disease of the arterial intima characterized by foci of accumulation of leukocytes, smooth muscle cells, lipids, and extracellular matrix. The central feature and one of the earliest observable symptoms in the pathogenesis of atherosclerotic plaques is the adhesion of mononuclear leukocytes to separate segments of the arterial cell lining through VCAM-1 proteins on the surface of the vascular cell lining. Upon attachment to mononuclear leukocytes, the macrophage-loaded lipids or foam cells are transformed into a cell lining. Atherosclerosis begins as a high focal lesion in the vascular wall, usually in an area where normal, laminar blood flow divides, as it divides when blood vessels flow. These regions of low shear stress are characterized by abnormal local accumulation of oxidatively modified low density lipoprotein (ox-LDL).

Early symptoms in atherosclerosis pathogenesis have been suggested to be mediated by low-density lipoproteins that converge with oxygen-reactive substances to oxidatively modified low-density lipoprotein (Steinberg et al., Beyond Cholesterol: Modifications of low density lipoprotein that increase its atherogenecity, N. Eng J. Med 320: 915-924 (1989), Parthasarathy et al., Probucol inhibits oxidative modification of low density lipoprotein J. Clin Invest. 77 (2), 641-664 (1986). It is not clear what mechanism LDL is oxidized either intracellularly or extracellularly.

Current therapy for cardiovascular diseases and especially atherosclerosis does not treat the cause of the disease but instead treats the disease symptoms or less risk factors associated with the disease. Pharmaceutical compositions that are prescribed for these conditions include lipid lowering agents such as probucol and nicotinic acid, aspirin (which prevents platelet adhesion), antithrombotic agents such as cumadine, calcium channel blockers such as varapamil, diltiazem and i nifedipine, angiotensin converting enzyme (ACE) inhibitors such as captopril and enalopril, and B-blockers such as propanalol, terbutanol and labetalol. Because therapeutic agents are not selective, they can adversely affect a variety of different organs and have significant side effects. There are currently no formulations that are directed at inhibiting the binding of monocytes to cell surface adhesion molecules such as VCAM-1.

Cardiovascular disease is reported to be the leading cause of death in the United States in the current period and 90% of cardiovascular diseases are now diagnosed as atherosclerosis. There is therefore a strong need to recognize new methods and to obtain new pharmaceutical compositions for their treatment.

Dithiocarbamates are transit metal chelators that are clinically used in heavy metal poisoning (RC Baselt et al., Comparisons of antidotal efficacy of sodium diethyldithiocarbamate, D-penicillamine and triethylenetetramine, and acute toxicity of nickel carbonyl in rats, Res. Commun. Pathol. Pharmacol., 18 (4), 677-688 (1977), T. Menne and K. Kaaber, Treatment of pompholyx due to nickel allergy with chelating agents, Contact Dermatitis 4 (5), 289-290 (1978),

FW Sunderman, Clinical response to therapeutic agents in poisoning from Mercury Vapor, Ann. Clin. Lab. Sci. 8 (4), 259-69 (1978), F. w. Sunderman, Efficacy of sodium diethyldithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning, Ann. Clin. Lab. Sci. 9 (1), 1-10, (1979), GR Gale et al.,

Diethyldithiocarbamate in the treatment of acute cadmium poisoing, Ann. Clin. Lab. Sci. 11 (6), 476-483 (1981), MM Jones and MG Cherian, The search for chelate antagonists for chronic cadmium intoxication, Toxicology 62 (1), 1-25 (1990),

SG Jones et al., A comparison of diethyldithiocarbamate and EDTA as antidotes for acute cadmium intoxication Res. Commun. Chem. Pathol. Pharmacol. 38. (2), 271-278, (1982), A. Pages et al., Dithiocarbaraates in heavy metal poisoning: complexes of N, N-di- (1-hydroxyethyl) dithiocarbamate with Zn (II), Cd (II) 1, Hg (II), CH₃Hg (II);₆H₅Hg (II), J. Inorg. Biochem. 25 (1), 35-42 (1985), SK Tandon et al., The lead-chelating effects of substituted dithiocarbamates, Biomed. Environ. Sci. 3 (3), 299-305 (1990).

Dithiocarbamates are also used in support of cisplatin chemotherapy to prevent renal toxicity (MP Hacker et al., Effect of disulfiram (tetraethylthiuram disulfide) and diethyldithicarbamate in mice, Cancer Res. 42 (11), 4490-4499 (1982), Bodenner Selected protection against cis-diamminedichloroplatin (II) - induced toxicity in kidney, gut, and bone marrow by diethyldithiocarbamate, Canc., Res., 46, 2751-2755 (1986). The transition metal chelates should have the effect of blocking the formation of hydroxy groups by the intracellular route, by the Haber-Weiss-Fenton reaction (M. Saran et al., Radical reactions in vivo - an overview, Radiat. Environ. Biophys. 29 (4), 249-262). (1990).

The dicarbamate currently used in the treatment of alcohol dependence is disulfiram, which is a dimer of diethyldithiocarbamate. Disulfiram inhibits liver aldehyde dehydrogenase (K. Inoue et al., Effect of disulfiram and its reduced metabolite, diethyldithiocarbamate on aldehyde dehydrogenase of human erythrocytes, Life Sci. 30 (5), 419-424 (1982).

Dithiocarbamates have been reported to inhibit HIV virus replication and also enhance maturation of specific T cell subpopulations. This has led to clinical trials of diethyldithiocarbamate in AIDS patients (E. Reisinger et al., Inhibition of HIV Progression by Dithiocarb), Lancet 335. 679 (1990).

SUMMARY OF THE INVENTION

The present invention therefore relates to a method of treating atherosclerosis and other cardiovascular and inflammatory diseases.

Another object of the invention relates to pharmaceutical compositions for the treatment of atherosclerosis and other cardiovascular and inflammatory diseases.

Yet another object of the present invention is to provide a means for blocking the ability of cells to express gene products that are responsible for adhering leukocytes to these cell activation.

relates to methods involved in being known to be cells, and to

A summary of the present invention is described below.

Dithiocarboxylates, and in particular dithiocarbamates, have been found to block the induced expression of the VCAM-1 cell surface liner molecule and thus are useful in the treatment of atherosclerosis, postangioplastic restenosis, coronary arterial diseases, angina and other cardiovascular diseases as well as non-cardiovascular diseases. mediated by VCAM-1.

Importantly, certain dithiocarbamates, such as sodium pyrrolidine-N-dithiocarbamate (PDTC), do not significantly block the induced expression of other cell-surface adhesion molecules, such as ICAM-1 or ELAM-1, and therefore do not have an adverse effect on inflammatory responses, which does not mediate VCAM-1. Pyrrolidine-N-dithiocarbamate sodium was also found to exhibit no preferential toxicity to proliferating or abnormally dividing smooth muscle cells. Another dithiocarbamate, sodium N-methyl-N-carboxymethyl-N-carbodithioate, also inhibits VCAM-1 expression without significantly affecting ICAM-1, but does not inhibit preferential toxicity for abnormally dividing vascular smooth muscle cells. The ability of other active dithiocarbamates to selectively inhibit VCAM-1 (without inhibiting the expression of ELAM-11 or icam-1) and exhibit preferential toxicity to abnormally dividing smooth muscle cells is assessed according to the following methods.

Dithiocarbamates which are useful in the treatment of atherosclerosis and other cardiovascular and inflammatory diseases include, but are not limited to, compounds of the general formulas:

R¹SC (S) NR²R³ or R²R³N (S) CS-SC (S) NR²R³ In which

R¹ means a hydrogen atom, a pharmaceutically acceptable cation including but not limited to sodium and potassium, and a group of formula NR⁴R⁵R⁶R⁷where R⁴, R⁵, R⁶ and R⁷ are, independently of one another, a hydrogen atom, a straight, branched or cyclic alkyl group having 1 to 6 carbon atoms, a hydroxyalkyl group having 1 to 6 carbon atoms on which one or more hydroxy groups are present on any of the carbon atoms, or an aryl group;

R^Ä and R^J independently of one another means a straight, branched or cyclic alkyl group having 1 to 10 carbon atoms, a group of the formula - (CHOH)_n(CH₂)_nOH where n is 0, 1, 2, 3, 4, 5 or 6,

- (CH₂)_nWHAT₂R¹, - (CH₂)_nWHAT₂R⁴ or a (C 1 -C 6) hydroxyalkyl group; or

R² and R³ they form a bridge together '(CH₂)_mwherein m is 3 to 6 and

R⁴ means an alkyl, aryl, alkaryl or aralkyl group, including acetyl, propionyl and butyryl groups.

The active dithiocarboxylates and in particular the dithiocarbamates included within the scope of the present invention may be used to treat acute and chronic inflammatory diseases mediated by VCAM-1, including but not limited to rheumatism, osteoarthritis, asthma and dermatitis, and may be beneficial in the treatment of scattering. sclerosis.

The compounds are suitable for both primary and supportive medical treatment of cardiovascular diseases. The compounds may be used in adjunctive therapy in combination with formulations that are administered to reduce the risk of disease by reducing LDL and serum cholesterol. The method represents a significant advantage in the treatment of cardiovascular diseases, which continues with concomitant therapy simply by preventing the spread of the disease, and if used appropriately, this provides the opportunity to medically treat atherosclerosis by preventing new injury from ongoing disease and the damage that has already occurred is brought back. The compounds may be administered for the treatment of small vessel diseases in which treatment cannot be performed surgically, or angioplasty or other vascular diseases in which surgery cannot be selected for treatment. The compounds may also be used to stabilize patients prior to revascularization therapy.

The active compound or mixture of compounds is administered by any suitable route, including, but not limited to, oral and intravenous administration. The general dosage range will range from 0.5 to 500 mg / kg body weight, with a dosage schedule from one to two times daily. The duration of dosing will range from a single dose given only one to twice a day to a dose administered over two to six months.

The compounds may be administered directly into the vascular wall using balloon perfusion catheters in or towards the coronary region or other arterial angioplasty region. By way of example, administration of 2 to 5 ml of a physiologically acceptable solution containing about 1 to 500 µm of compound. The compound mixture is administered at one to five times atmospheric pressure. For the next six months, during the period of maximum risk of restenosis, the active compounds are administered by other appropriate routes and according to the schedule of doses.

A relatively short period of treatment with the active compounds is used to cause diminished damage caused by coronary arterial disease that cannot be treated either angioplastically or surgically. An example of a short treatment period is two to six months, at a dosage range of 0.5 to 500 mg / kg body weight, administered in the range of one to three times a day, and this example is not to be construed as limiting possible treatments.

A longer treatment period can be used to prevent progressive disease in high-risk patients. The long treatment period may last for years, with doses ranging from 0.5 to 500 mg / kg body weight, administered at intervals of once every day to three times a day.

The active compounds may be administered in the period immediately before and after coronary angioplasty as a measure to reduce or eliminate abnormal proliferation and an inflammatory response that simultaneously results in clinically significant restenosis.

The active compounds may be administered concomitantly with other drugs for the treatment of cardiovascular diseases, including lipid lowering agents such as probucol and nicotinic acid, platelet aggregation inhibitors such as aspirin, antithrombotic agents such as couadin, calcium channel blockers such as varzemam and nifedipine, angiotensin converting enzyme (ACE) inhibitors such as captopril and enalopril, and β-blockers such as propanalol, terbutalol and labetalol. The compounds may also be administered in combination with non-steroidal anti-inflammatory agents such as ibuprofen, indomethacin, phenoprofen, mefanamic acid, flufenamic acid or sulindac, or with corticosteroids.

Overview of the drawings

Fig. 1 is an illustration of an autoradiogram of mRNA obtained as described below, hybridized to either³²Β-labeled human VCAM-1 specific cDNA (rectangle A), E-selectin (ELAM-1) specific cDNA (rectangle B), or ICAM-1 specific cDNA (rectangle C). After pre-treatment for 30 minutes with 50 µl sodium pyrrolidinedithiocarbamate (PDTC), HUVE (human umbilical vein) cells are exposed to 10 U / ml IL-1b in the continuous presence of 50 µl pyrrolidine-N-dithiocarbamate sodium. A parallel control assay is performed in the same manner except that sodium pyrrolidine-N-dithiocarbamate is absent. At the indicated time intervals, the entire RNA is isolated and the 20 µg material is denatured with a 1.0% formaldehyde-containing aragose gel for electrophoresis and transferred to nitrocellulose, hybridized as described above and visualized by autoradiography. Lane 1-0 hours, lanes 2, 4, 6, 8 - OL-1 alone for 2, 4, 8 and 24 hours, lanes 3, 5, 7, 9 - IL-1 with PDTC for 2, 4, 8 and 24 hours.

Fig. 2 is an illustration of a radiogram of mRNA obtained as described below, hybridized to either³²Β-labeled human VCAM-1 specific cDNA (rectangle A), E-selectin (ELAM-1) specific cDNA (rectangle B), or ICAM-1 specific cDNA (rectangle C). HUVE cells are pretreated with the indicated concentrations of pyrrolidine-N-dithiocarbamate sodium, then exposed to IL-1b in the presence of pyrrolidine-N-dithiocarbamate sodium for 4 hours and examined for accumulation of VCAM-1 on mRNA by Northern blot hybridization analysis. Lane 1 control, lane 2 - IL-1 (10 U / ml), lane 3 IL-1b + sodium pyrrolidine-N-dithiocarbamate (0.05 pmol), lane 4 - IL-1 LB + sodium pyrrolidine-N -dithiocarbamate (0.5 μοί), lane

- IL-1b + sodium pyrrolidine-N-dithiocarbamate (5,0 μοί), lane

- IL-1b + sodium pyrrolidine-N-dithiocarbamate (50,0 μοί), lane

- IL-1b + sodium pyrrolidine-N-dithiocarbamate (100 μοί)

Fig. 3 is an illustration of an autoradiogram of mRNA obtained as described below, hybridized to either³²Β-labeled human VCAM-1 specific cDNA (rectangle A), E-selectin (ELAM-1) specific cDNA (rectangle B), or ICAM-1 specific cDNA (rectangle C). HUVE cells are pretreated as described in connection with FIG. 1 with 50 μιηοΐ sodium pyrrolidine N-dithiocarbamate, are exposed for 4 hours to the formulations below and examined for accumulation of VCAM-1 (rectangle A) and ICAM-1 (rectangle B) mRDA. Lane 1 - TNFα (100 U / ml), Lane 2 - TNFα + Sodium pyrrolidine-N-dithiocarbamate, Lane 3 - lipopolysaccharide (LPS) (100 ng / ml), Lane 4 - LPS + Sodium pyrrolidine-N-dithiocarbamate lane 5 - poly (I: C) (100 mg / ml), lane 6 - poly (I: C) + sodium pyrrolidine-N-dithiocarbamate.

Fig. 4 is a graph of relative surface expression of VCAM-1 and ICAM-1 cells in the presence (dark band) or absence (light band) of sodium pyrrolidine-N-dithiocarbamate and in the presence of a number of stimulation inducing types. Confluent HUVE cells are pretreated or not pretreated (CTL only) for 30 minutes with 50 μπιοί sodium pyrrolidine-N-dithiocarbamate and then exposed to the above preparations in the presence or absence (CTL only) of pyrrolidine-N sodium for the indicated time period. -ditiokarbamátu. Surface cell expression is determined by primary binding with VCAM-1 specific (84H10) monoclonal antibodies of the mice, followed by secondary binding with anti-mouse IgG of horseradish peroxidase. The amount is determined by determining the calorimetric conversion of TMB at 450 nm. Fig. 4 shows that multiple regulator signals induce VCAM-1, but not ICAM-1, by the general route of dithiocarbamate sensitivity in human vascular cell lining.

Fig. 5 is a graph of the relative expression of surface VCAM-1 cells (optical density, OD, 595 nmol) in TNFα-activated human umbilical vein lining against concentrations of various antioxidants. (PDTC is sodium pyrrolidine-N-dithiocarbamate, DETC is sodium N, N-diethyl-N-carbodithioate, also referred to as sodium diethyldithiocarbamate, NAC is N-acetylcysteine, and DF is desferoximine).

Fig. 6 is a graph of relative expression of surface VCAM-1 cells (optical density 595 nmol) in human umbilical vein lining, activated by TNFα, in the presence of a specific amount of antioxidant. (PDTC is sodium N-pyrrolidinedithiocarbamate, DiDTC is sodium N, N-diethyl-N-carbodithioate, SarDTC is sodium N-methyl-N-carboxymethyl-N-carbodithioate, IDADTC is trisodium N, N-di (carboxymethyl) -N- carbodithioate, MGDTC is sodium N-methyl-D-glucamine-N-carbodithioate, MeOBGDTC is 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 and NAC is N-acetylcysteine).

Fig. 7 is a graph of the percentage of HUVE cell binding Molt-4 cells, either unstimulated or stimulated with 100 U / ml TNFα for 6 hours in the presence or absence of sodium pyrrolidine-N-dithiocarbamate.

Fig. 8 illustrates the chemical structure of the following dithiocarbamates:

sodium N-pyrrolidine-N-carbodithioate, sodium N-methyl-N-carboxymethyl-N-carbodithioate, trisodium N, N-di (carboxymethyl) -N-carbodithioate, sodium N-methyl-D-glucamine-N-carbodithioate, sodium N, N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate), sodium N, N-diisopropyl-N-carbodithioate and sodium N- (4-methoxybenzyl) -D-glucamine-N-carbodithioate,

The invention is described in detail below.

The term alkyl, when used herein, and unless otherwise specified, refers to a saturated straight, branched, or cyclic hydrocarbon group having 1 to 10 carbon atoms, and includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl individually. , cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,

3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl.

The term alkenyl, as used herein, and unless otherwise specified, refers to a straight, branched, or cyclic hydrocarbon group of 2 to 10 carbon atoms containing at least one double bond.

The term alkynyl, when used herein and unless otherwise specified, refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms containing at least one triple bond.

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

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

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

The term aryl, when used herein and unless otherwise specified, refers to a phenyl group or a substituted phenyl group in which the phenyl ring contains at least one of the following substituents: hydroxy, carboxy, or a pharmaceutically acceptable salt thereof,₂an (alkyl) group, an alkoxy group, an alkyl group, or a glucamine residue.

The term alkoxy, when used herein, and unless otherwise specified, refers to a moiety having the structure -O-alkyl.

The term halogen as used herein includes fluorine, chlorine, bromine and iodine.

The term hydroxyalkyl as used herein refers to an alkyl group of 1 to 6 carbon atoms in which at least one of the hydrogen atoms attached to any of the carbon atoms is replaced by a hydroxy group.

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

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

I. Active Compounds

Dithiocarboxylates have been found to be useful in the treatment of atherosclerosis and other cardiovascular and inflammatory diseases. Dithiocarboxylates, including dithiocarbamates, can be used to block the ability of cells, including the cell lining, to express VCAM-1, which is a gene product known to be responsible for the adhesion of leukocytes to cells. The fact that dithiocarboxylates, including dithiocarbamates, inhibit VCAM-1 gene expression significantly supports the importance of oxidation as an initial signal in altered vascular-inflammatory cell interactions. The specific molecular mechanism by which carboxylates act to inhibit VCAM-1 gene expression is unknown.

At least one of the compounds, sodium pyrrolidinedithiocarbamate (PDTC), inhibits VCAM-1 gene expression at a concentration of less than 1.0 μιηοΐ. Thus, this compound exhibits preferential toxicity to proliferating or abnormally dividing vascular smooth muscle cells. Sodium pyrrolidine-N-dithiocarbamate was found not to block significantly the expression of ELAM-1 or ICAM-1, and therefore treatment with this compound has no adverse effects on the inflammatory response mediated by ELAM-1 or ICAM-1. This generally avoids the suppression of immunity. This can avoid systemic complications from the general suppression of adhesion molecules in a number of other cell types known to express them.

Dithiocarboxylates are compounds of the general structural formula

A-SC (S) -B, which are members of a general group of compounds known as thiol antioxidants, and which are otherwise referred to in relation to carbodithiols or carbodithiolates. It appears that part of formula SC (S) is essential for the therapeutic effect and that A and B may be any groups that do not adversely affect the efficacy or toxicity of the compound. A and B may be selected by one of skill in the art to impart desirable characteristics to a compound, including size, charge, toxicity and degree of stability (including stability in an acidic environment such as in the stomach or in a basic environment such as the intestinal tract). selection

A and B will also have an important effect on tissue distribution on the pharmacokinetic properties of the compound. In general, for the treatment of cardiovascular diseases, it is desirable that the compound accumulates and is placed in a layer of proximal artery containing a vascular cell lining. The compounds are preferably excreted via renal excretions.

Examples A and B include, but are not limited to, alkyl, alkenyl, alkynyl, alkaryl, araryl, haloalkyl, haloalkenyl, haloalkynyl, aryl, and alkaryl, hydrogen, C 1 -C 6 alkoxyalkyl, and 1. up to 10 carbon atoms in the alkyl moiety, an alkylthioalkyl group having 1 to 6 carbon atoms in the alkylthio moiety and 1 to 10 carbon atoms in the alkyl moiety, substituted (C 1 -C 10) alkyl group (wherein the substituent is independently hydroxy, carbonyl, or a carboxylic acid radical which is located on any of all 10 carbon atoms), NR²R³- CHOH_nCH₂OH where n is 0, 1, 2, 3, 4, 5 or 6, - (CH₂)_n^C02^rlIncluding acetyl, propionyl and butyryl, a (C 1 -C 4) hydroxyalkyl group (wherein at least one hydroxyl group is located on any of the carbon atoms) and A may be a pharmaceutically acceptable cation including sodium, potassium or a group of formula NR⁴R⁵R⁶R⁷to which the calculation is not limited.

In another embodiment, a dimer of formula may be administered

BC (S) S-SC (S) -B.

The dithiocarboxylates should be selected for use in the treatment of atherosclerosis and other cardiovascular and inflammatory diseases so as to have intrinsic lipophilicity for placement in the affected area. The compound does not reach the low variable region, such as the fat deposition site. In the preferred treatment of cardiovascular diseases, the pharmacokinetic properties of the compound should not be significantly impaired by heart failure due to cardiac perfusion or deficiency of the tannins.

The dithiocarboxylates must be physiologically acceptable. In general, compounds with a therapeutic index of at least 2 and preferably at least 5 or 10 are acceptable. The therapeutic index is defined as EC₅₀/ IC₅₀where EC symbol_5Q means the concentration of compound that inhibits VCAM-1 expression by 50% and the IC symbol₅₀ refers to the concentration of compound that is toxic to 50% of the target cells. Cell toxicity can be measured by direct cell counting, trypan blue exclusion or studies of varying metabolic activity, as well as incorporation³H-thymidine, as known to those skilled in the art. The therapeutic index of sodium pyrrolidine-N-dithiocarbamate in tissue culture is above 100 as measured by cell toxicity divided by the ability to inhibit VCAM-1 expression, upon activation of TNFα, in HUVE (human umbilical vein) cells. Initial studies on rapidly dividing human HT-18 glioma cells show that there is no toxicity at concentrations 100-fold higher than the therapeutic concentration. Disulfiram, an orally administered form of diethyldithiocarbamate, used in the treatment of alcohol dependence generally does not cause great clinical toxicity, as suitably administered.

There are several dithiocarbamates that are known to be toxic to genes. These compounds do not fall within the scope of this invention, which is limited to the administration of physiologically acceptable substances. An example of a gene toxic dithiocarbamate is zinc dimethyldithiocarbamate, known as a fungicidal agent. Furthermore, the anticholinesterase properties of certain dithiocarbamates may lead to neurotoxic damage (D. Miller, Neurotoxicity of the pesticidal carbamates, Neurobehav. Toxicol. Teratol. 4 (6), 779-787 (1982).

The term dithiocarboxylate as used herein includes, but is not limited to, dithiocarbamates of the general formula

R¹SC (S) NR²R³ or R²R³N (S) CS-SC (S) NR²R³ in which the thion atom, including but not limited to, is hydrogen or pharmaceutically acceptable cassette and potassium, without being on or a group of formula NR⁴R⁵R⁶R⁷where R⁴, R⁵, R⁶ and R⁷ are, independently of one another, a hydrogen atom, a straight, branched or cyclic alkyl group having 1 to 6 carbon atoms, a hydroxyalkyl group having 1 to 6 carbon atoms on which one or more hydroxy groups are present on any of the carbon atoms, or an aryl group an * 1

R and R^J independently represent a straight, branched or cyclic alkyl group having 1 to 10 carbon atoms, a group of the formula - (CHOH)_n(CH2)_nOH where n is 0, 1, 2, 3, 4, 5 or 6, ^-(CH2) nCO2R¹, - (CH2)_nWHAT₂R⁴ or a (C 1 -C 6) hydroxyalkyl group; or

R and R together form a bridge, such as - (CH₂wherein m is an integer from 3 to 6 and

R⁴ means an alkyl, aryl, alkaryl or aralkyl group, including acetyl, propionyl and butyryl groups.

Specific examples of suitable dithiocarbamates illustrated in FIG. 8 are sodium pyrrolidine-N-carbodithioate, sodium N-methyl-N-carboxymethyl-N-carbodithioate, trisodium N, N-di (carboxymethyl) -N-carbodithioate, sodium N-methyl-D-glucamine-N-carbodithioate, sodium N, N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate) and sodium N, N-diisopropyl-N-carbodithioate.

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

At the molecular level, sodium pyrrolidine-N-dithiocarbamate also shows that this compound inhibits the activation of the transcriptional regulator factor · Nf-κB in response to certain cytokinone and non-cytokinone stimulation (R. Schreck et al., Reactive oxygen intermediates and apparently used messengerts in the activation of the nF-kappa B transcription factor and HIV-1, EMBO J. 10 (8), 2247-2258 (1991),

R. Schreck et al., Dithiocarbamates as potent inhibitors of nuclear factor B activation in intact cells, J. Exp. Med. 175, 1181-1194 (1992)). However, by shifting the mobility of the gel from HUVE cell extracts with various increasing effects similar to kB, it was found that the cell lining activates the gene expression of VCAM-1 apparently by a new transcriptional regulatory factor, which is not Nf-kB. This suggests that sodium pyrrolidine-N-dithiocarbamate can regulate gene expression of the cellular lining by acting on a novel transcriptional regulator protein. VCAM-1 expression has also been shown to be induced by a number of factors in cultured Kaposi's sarcoma cells, which may be important in their pathogenesis. Pyrrolidine-N-dithiocarbamate sodium blocks VCAM-1 expression in Kaposi's sarcoma cells that are activated by TNF, IL-1 and poly (I: C).

Smooth muscle cells have also been found to produce a soluble form of VCAM-1 that can be secreted by cells and which can act as a natural antihistamine.

II. Biological activity

The ability of dithiocarboxylates to inhibit VCAM-1 expression can be measured in a variety of ways, including the methods detailed below in Examples 1-7. Illustratively, Examples 1 to 3, 6, and 7 describe an assessment of the biological effect of sodium pyrrolidine-N-carbodithioate (also referred to as as PDTC). These examples are not intended to limit the scope of the invention, which specifically includes the use of any of the compounds described above for the treatment of atherosclerosis and other types of inflammation and cardiovascular disease mediated by VCAM-1. Sodium pyrrolidine-N-dithiocarbamate can be readily replaced by any of the compounds described above and evaluated similarly.

Examples 4 and 5 provide comparative values ​​regarding the ability of a series of dithiocarbamates to inhibit VCAM-1 gene expression. The examples below illustrate that the claimed dithiocarbamates specifically block the ability of VCAM-1 to undergo expression by a vascular cell liner in response to a variety of signals known to be active in atherosclerosis and in response to inflammation.

Experimental procedures

Cell culture

HUVE cells isolated from human umbilical vein are cannulated, rinsed with Hank's solution to remove blood, and then incubated with 1% collagenase at 37 ° C for minutes. After collagenase removal, cells are cultured in M199 medium supplemented with 20% fetal calf serum (HyClone), µg / ml heparin (ESI Pharmaceutical, Cherry Hill, New Jersey, USA), 50 µg / ml cell lining growth supplement (Collaborative Research Incorporated, Bedford, Massachusetts, USA), 25 mmol Hepes buffer, 2 mmol L-glutamine, 100 µg / ml penicillin and 100 µg / ml streptomycin, and grown on tissue culture plates coated with 0.1% gelatin at 37 ° C. Cells are passaged at confluence by 1: 4 digestion. Cells are used in the first 8 passages.

Incubation with cytokines and other reagents

HUVE confluent cells are washed with phosphate buffered saline and then added to fresh environment.

Sodium pyrrolidine-N-dithiocarbamate is added at the indicated concentrations after prior treatment 30 prior to the addition of cytokines. Cytokines and other inducers are directly added to the medium at the times and concentrations indicated in each experiment. Human recombinant IL-1b is a genetic poison from the Upjohn Company (Kalamazoo, MI, USA). TNFα is obtained from Bohringer Ingelheim (Germany). Bacterial lipopolysaccharide (LPS), polyinosinic acid, polycitidic acid (Poly I: C) and pyrrolidine dithiocarbamate (sodium pyrrolidine-N-dithiocarbamate) are provided by Sigma Chemicals (St. Louis, Montana, USA). All other reagents are reagent grade.

RNA isolation

Total cellular RNA is isolated by simple extraction using an acidic mixture of guanidium thiocyanate, phenol and chloroform. The cells are rinsed with phosphate buffered saline and then lysed with 2 ml of guanidium isothiocyanate. The solution was acidified with 0.2 ml of sodium acetate (pH 4.0) and then extracted with 2.0 ml of phenol and 0.4 ml of a 24: 1 mixture of chloroform and isoamyl alcohol. The RNA was subjected to two ethanol preparations before, as used to analyze Nothern stains.

Staining analysis Northern blots of total cellular RNA are size fractionated using a 1% formaldehyde-containing agarose gel in the presence of 1 μg / ml ethidium bromide. RNA is transferred to a nitrocellulose filter and covalently bound by ultraviolet radiation using a Stratlinker UV Crosslinker (Stratagene, La Jolla, California, USA).

Hybridization was performed at 42 ° C for 18 hours in 5x SSC (1 x = 150 mmol sodium chloride, 15 mmol sodium citrate), 1% sodium dodecyl sulfate, 5x Denhardt's solution, 50% formaldehyde, 10% dextran sulfate and 100 µg / ml DNA salmon spermatozoa, denatured by cut. About 1 x 10⁶ to x ΙΟ⁶ μιη / ml labeled test sample (with a specific activity of less than 10⁸ cpm / µg DNA) was used for hybridization. Hybridization was then performed and the filtrates were washed with a final sharpness of 0.2 x SSC at 55 ° C. The nitrocellulose is stripped using boiling water prior to rehybridization with another test sample. The audiograph is determined at amplifying shielding at -70 ° C.

Test samples

DNA samples labeled ³²P is prepared using a random oligonucleotide primer method. The ICAM-1 assay is an Eco R1 fragment from human cDNA. The ELAM-1 assay is a 1.85 kb Hind III fragment from human cDNA. The VCAM-1 assay is a Hind ΙΙΙ-Xho I fragment from human cDNA consisting of nucleotides 132-1814.

Immunosorbent-coupled enzyme assay (ELISA) <

> HUVE cells are plated on 96-well tissue culture plates for 48 to 72 hours for assay. Primary antibodies in M199 medium with 5% fetal calf serum are added to each well and incubated at 37 ° C for 1 hour. Cells are then washed and incubated for 1 hour with peroxidase-conjugated anti-mouse IgG goat (Bio Rad) at a 1: 500 dilution in M199 with 5% fetal calf serum. The cells are then washed and the binding of the antibody is determined by the addition of 100 μΐ of a solution containing 10 mg / ml 3,3,5,5'-tetramethylbenzidine (Sigma) containing 0.003% hydrogen peroxide. The reaction is stopped by the addition of 25 μΐ of 8-normal sulfuric acid. Plates are read on an ELISA reader (Bio Rad) at an OD OD of 420 nmol, after blind reading for lines stained only with the second degree of antibody. Data represent the mean of triplicate measurements.

antibody

The monoclonal antibody (MAb) 4B9, suitable for the determination of vascular adhesion molecule-1 (VCAM-1) cells, is a generous donation from Dr. John Harlan of the University of Washington, USA. MAb E9A1F1, suitable for the determination of the cell lining adhesion molecule (ELAM-1), is a generous donation from Dr. Swerlicka from Emory University. Hybridomas producing MAb 84H10 that detect intramolecular adhesion molecules-1 (ICAM-1) grow in a conventional manner in the laboratory of the inventors. The antibodies are used as supernatant tissue culture.

Examples of the invention

Example 1

Pyrrolidine-N-dithiocarbamate sodium blocks IL-1b-mediated induction of VCAM-1 HUVE cells but not ICAM-1 or ELAM-1, mRNA accumulation

To determine whether the oxidative state of a cellular lining can accelerate basal or induced expression of cellular adhesion molecular genes, a human vascular cellular lining is exposed to an inducing cytokine, IL-1b (10 U / ml) in the presence or absence of a metal chelating thiol antioxidant and pyrrolidate pyrrolidine-N-dithiocarbamate, 50 μπιοί) for up to 24 hours. As shown in FIG. 1, IL-1b alone (lanes 2, 4, 6 and 8) induces at the expected rate and with transient induction of VCAM-1 (rectangle A), E-selectin (ELAM-1, rectangle B) and ICAM-1 (rectangle C) mRNA accumulation, with all peaks attained in 4 hours. However, in the presence of sodium pyrrolidine-N-dithiocarbamate, IL-1b-mediated induction of VCAM-1 mRNA accumulation is significantly inhibited above 90% (Rectangle A, lanes 3, 5, 7 and 9). In contrast, although IL-1b-mediated ELAM-1 induction is poorly inhibited at 2 and 24 hours (compare lanes 2 and 3 and 8 and 9 on rectangle B), sodium pyrrolidine-N-dithiocarbamate does not inhibit induction at 4 and 8 hours (lanes 5 and 7 on rectangle B). IL-1b-mediated induction from ICAM-1 mRNA accumulation is not adversely affected (Rectangle B, lanes 3, 5, 7 and 9). In fact, a slight increase in IL-1b induction from ICAM-1 mRNA accumulation (approximately 30%) is observed (compare lanes 4 and 5 on rectangle B). An equivalent amount of nitrocellulose transferred RNA per lane was confirmed by ethidium bromide staining and visual observation.

I

Dose-response value analyzes are performed to determine whether pyrrolidine-N-dithiocarbamate sodium inhibits the induction of VCAM-1 gene expression by IL-1b at a dose-dependent dose. As shown in FIG. 2, sodium pyrrolidine-N-dithiocarbamate inhibits IL-1b-mediated induction of VCAM-1 gene expression with a steep dose-response curve (Fig. 2, rectangle

A), with a calculated EC 50 of approximately 10 μπιοί, while the sodium pyrrolidine-N-dithiocarbamate does not inhibit IL-1b-mediated induction of ELAM-1 expression at these concentrations (Figure 2, rectangle B). IL-1b-mediated induction of ICAM-1 mRNA accumulation is increased by pyrrolidine-N-dithiocarbamate sodium at concentrations greater than 0.5 μπιοί (Figure 2, compare lane 2 and lanes 4 to 7 on rectangle C).

These data demonstrate that IL-1b uses dithiocarboxylate, and in particular the sensitive grade with dithiocarbamate, as part of its signaling mechanism for inducing VCAM-1 gene expression. These data also show that this sensitive degree * with dithiocarbamate does not play a significant role in inducing the expression of the ELAM-1 or ICAM-1 gene, which is mediated by IL-1b.

Example 2

Pyrrolidine-N-dithiocarbamate sodium blocks the induction of VCAM-1 HUVE cells by mRNA accumulation by multiple stimulation

To determine whether other well-described activators of VCAM-1 gene expression also use a sensitive grade with pyrrolidine-N-dithiocarbamate sodium, three separate classes of activators are tested: inducing agent mediated by another classical receptor (tumor necrosis factor, TNFα), non-mediating agent (lipopolysaccharide, LPS) and a novel inducing agent recently described (double stranded RNA, poly (I: C)). In all three cases, pyrrolidine-N-dithiocarbamate sodium significantly inhibited the induction of VCAM-1 mRNA accumulation in HUVE cells after 4 hours (Fig. 3, rectangle A). Although TNFα-mediated ELAM-1 gene expression is to some extent suppressed (Figure 3, lane 1 and rectangle B) from ELAM-1, LPS-mediated mRNA accumulation and poly (I: C) are free of adverse effects (Figure 3, lanes) 3 to 6, rectangle B). The induction of ICAM-1 mRNA accumulation is without adverse effect (Fig. 3, rectangle C). These values ​​indicate that structurally different inducing agents, acting in different ways, are involved in a general regulatory step specifically for inducing VCAM-1 gene expression.

Example 3

Pyrrolidine-N-dithiocarbamate sodium blocks HUVE cell surface expression of VCAM-1 induced by multiple stimulation

To determine whether, like mRNA, the induction of surface protein expression of VCAM-1 cell lining would also be inhibited by pyrrolidine-N-dithiocarbamate sodium, monoclonal antibodies were used in an ELISA assay to quantitate cell surface induction of VCAM-1 and ICAM-1 in cultured HUVE cells. As shown in FIG. 4, a larger number of sets of activating agents in the absence of sodium pyrrolidine-N-dithiocarbamate (-PDTC) induces a rapid and short-term accumulation of VCAM-1 (upper left rectangle) on the cell surface, reaching a peak at 6 hours. In the presence of sodium pyrrolidine-N-dithiocarbamate (+ PDTC, upper right rectangle), the induction of surface expression of VCAM-1 cells by all tested preparations is significantly inhibited (80-90%). Conversely, induced cell surface expression of ICAM-1 is without adversely affected under identical conditions (lower left and right rectangles).

These data indicate that, like mRNA accumulation, VCAM-1 cell surface expression is selectively inhibited by dithiocarbamates, and that multiple sets of activating agents use a similar sensitive dithiocarbamate mechanism to induce VCAM-1 gene expression.

Example 4

Relative efficacy of antioxidants in blocking TNFα induction of VCAM-1

To determine whether structurally similar or different antioxidants could inhibit VCAM-1 gene expression and with what potency, HUVE cells are exposed to TNFα for 6 hours in the presence or absence of 5 different antioxidants at different concentrations. As shown in FIG. 5, both diethyldithiocarbamate (DETC) and N-acetylcysteine ​​(NAC) inhibit VCAM-1 expression at concentrations of 5 and 30 pmol. In contrast, sodium pyrrolidine-N-dithiocarbamate and 50 μιηοΐ. The chelating agent (PDTC) is effective among iron metal, desferroximine, and has no effect at the concentrations tested.

Example 5

Pyrrolidine-N-dithiocarbamate sodium inhibits TNFα induction of VCAM-1 / VLA-4 mediated adhesion

The ability of various antioxidants to inhibit TNFα induction of VCAM-1 in HUVE cells is assessed as described in the Example.

4th Fig. 6 is a graph of relative surface expression of VCAM-1 cells (optical density, OD, 595 nmol) in TNFα activated HUVE cells versus given concentrations

PDTC (sodium N-pyrrolidinedithiocarbamate),

DiDTC (sodium N, N-diethyl-N-carbodithioate),

SarDTC (sodium N-methyl-N-carboxymethyl-N-carbodithioate), IDADTC (trisodium N, N-di (carboxymethyl) -N-carbodithioate), MGDTC (sodium N-methyl-D-glucamine-N-carbodithioate),

MeOBGDTC (sodium N- (4-methoxybenzyl) -D-glucamine-N-carbodithioate), DEDTC (sodium N, N-diethyl-N-carbodithioate),

Di-PDTC (sodium N, N-diisopropyl-N-carbodithioate) and NAC (N-acetylcysteine).

The least active compounds are sodium N- (4-methoxybenzyl) -D-glucamine N-carbodithioate and N-acetylcysteine.

Example 6

Pyrrolidine-N-dithiocarbamate sodium inhibits TNFα induction of VCAM-1 / VLA-4 mediated adhesion

To determine whether the pyrrolidine-N-dithiocarbamate sodium caused by the inhibition of VCAM-1 regulation is associated with functional consequences, binding of Molt-4 cells to HUVE cells is either unstimulated or stimulated with 100 U / ml TNFα for 6 hours in the presence or absence of sodium. pyrrolidine N-dithiocarbamate. Molt-4 cells have previously been shown to be bound to activated HUVE cells via a VCAM-1 dependent mechanism. As shown in FIG. 6, the percentage of HUVE-binding Molt-4 cells decreases as pyrrolidine-N-dithiocarbamate sodium is present in the environment.

Example 7

Sodium pyrrolidine-N-dithiocarbamate inhibits monocyte binding to thoracic aorta of rabbits fed with cholesterol

The experiment is performed to determine whether the thiol antioxidant sodium pyrrolidine-N-dithiocarbamate would be effective in blocking the first monocyte-binding atherosclerosis component in an experimental animal model. One adult New Zealand white rabbit, weighing 3.5 kg, receives an intravenous injection of pyrrolidine N-dithiocarbamate sodium (20 mg / kg, at a concentration of 20 mg / ml in PBS) once daily for 5 days. Injections are given by a temporary cannula into a vein in the peripheral region of the ear that is kept accessible by the flow of heparinized saline solution. The sodium pyrrolidine-N-dithiocarbamate solution is mixed fresh daily or every other day (stored under protection from light at 4 ° C) and filtered (0.45 mm filter pores) immediately before use. After the first injection, when the cannula is placed, the active substance is administered to the conscious rabbit without any apparent signs of agitation or other signs of the disease. On the second day injection, the rabbit receives food containing 1% by weight of cholesterol and this is continued for the remainder of the experiment. On the fifth day, the animal is mercifully killed and the thoracic aorta is excised and fixed. After appropriate preparation, the sample is imaged on a lower ISI DS-130 scanning electronic microscope equipped with a LaB emitter. Using a double-shaded image and a transparent grid, CRT shades, with 64 adjacent fields, are determined to cover an area of ​​approximately 1.3 mm at 620X magnification². Leukocytes (WBC) and erythrocytes (RBC) in which adhesion occurred were counted and recorded in each field.

The arc sample data is as follows: 5 WBC and -25 RBC per

1.3 mm². This level of WBC adhesion is similar to that of control animals fed normal food (approximately 7 cases per area and thoracic from 2 negative control experiments are observed in the arc). Positive control rabbits, fed 1% cholesterol for 4 days but without antioxidant administration, exhibited approximately five-fold increased adhesion corresponding to 38 WBC / 1.3 mm². A considerable amount of predominantly dead cell-size tissue was observed adhered to each arc sample. It is unclear whether this material originated artificially from the preparation or whether it was present in vivo and, if so, whether this is related to the administration of sodium pyrrolidine-N-dithiocarbamate. This study suggests that infusion of sodium pyrrolidine-N-dithiocarbamate can effectively block the initial adhesion of monocytes to the lining of the aorta.

III. Pharmaceutical compositions

Iaidia and other living creatures, particularly mammals suffering from cardiovascular diseases and other inflammatory conditions mediated by VCAM-1, may be treated by administering to the patient an effective amount of at least one of the compounds as defined above, or a pharmaceutically acceptable derivative or salt thereof, in a pharmaceutically acceptable carrier. substance or diluent. The active materials can be administered by any suitable route, for example, orally, parenterally, intravenously, intradermally or subcutaneously.

As used herein, the term pharmaceutically acceptable salts or complexes refers to salts or complexes that retain the desired biological activity of the compounds described above and have minimal undesirable toxicological effects. Non-limiting examples of such salts are

(a) acid addition salts formed from inorganic acids (eg hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like) and salts formed from organic acids (eg acetic acid, oxalic acid, tartaric acid, succinic acid; malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid),

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

c) combinations of (a) and (b), for example, zinc tannic acid salt and the like.

The active compound is contained in a pharmaceutically acceptable carrier or diluent sufficient to deliver a therapeutically effective amount to the patient without causing a variety of toxic effects in the treated patient. A preferred dose of the active compound for all of the above conditions is in the range of about 0.5 to 500 mg / kg, preferably 1 to 100 mg / kg per day. The effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be released. If the derivative itself exhibits activity, the active ingredient can be determined as above, using the weight of the derivative or by other means known to one skilled in the art.

The compound is usually administered in any suitable dosage unit form, including a unit containing from 1 to 3000 mg of active ingredient, suitably 5 to 500 mg of active ingredient per dosage unit form, but the possibilities are not limited thereto. An oral dose of from 25 to 250 mg is usually suitable.

The active ingredient should be administered after peak plasma concentrations of the active ingredient of about 0.1 to 100 μιηοΐ, preferably about 1 to 10 μπιοί have been reached. This may be achieved, for example, by intravenous injection of a solution or composition of the active ingredient, optionally in saline. or an aqueous medium or by administration of a bite containing the active ingredient.

The concentration of the active compound in the drug composition will depend on the absorption, distribution, inactivation, and rate of excretion of the drug substance, as well as other circumstances known to those skilled in the art. It should be noted that the dose value will also vary depending on the severity of the condition to be alleviated. Furthermore, it is to be understood that for any individual patient, the specific dosage regimen may be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the formulation, and that the concentration range is given by way of example only and is not meant to limit implementation of the claimed means. The active ingredient may be administered at once or divided into a number of smaller doses to be administered at varying intervals of time.

Oral compositions will generally include an internal diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be brought into intimate contact with excipients and used in the form of tablets, trieths or capsules. Pharmaceutically acceptable binders and / or excipients may be included as part of the composition.

Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of a similar nature:

binders such as microbiological cellulose, gum, tragacanth or gelatin, excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch, lubricants such as magnesium stearate or Sterotes, glidants such as is a colloidal silica, a sweetening agent such as sucrose or saccharin, or a flavoring agent such as menthol, methyl salicylate or an orange flavoring.

If the unit dose is a capsule, it may contain liquid carriers such as fatty oils in addition to those of the above type. In addition, the dosage unit forms may contain various other materials which modify the physical form of the unit dosage, for example, containing coatings of sugar, shellac or edible preparations.

The active compound or a pharmaceutically acceptable salt or derivative thereof may be administered as a component of an elixir, suspension, syrup, wafer, chewing gum and the like. A syrup may contain, in addition to the active compounds, sugar as a sweetening agent and certain preservatives, coloring and flavoring agents.

The active compound or pharmaceutically acceptable derivatives or salts thereof may also be administered with other active ingredients which do not impair the desired effect, such as antibiotics, antifungal agents, anti-inflammatory agents, or antiviral compounds. The active compounds may be administered with lipid lowering agents such as probucol or nicotinic acid, platelet aggregation inhibitors such as aspirin, antithrombotic agents with agents such as couadin, calcium channel blockers such as varapamil, diltiazem and nifedipine, angiotensin census, angiotensin inhibitors such as captopril and analopril, and β-blockers such as propanalol, terbutalol and labetalol. The compounds may also be administered in combination with non-steroidal anti-inflammatory agents such as ibuprofen, indomethacin, aspirin, phenoprofen, mefanamic acid, flufenamic acid and sulindac. The compound may also be administered with corticosteroids.

Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical administration may include the following components:

sterile diluent such as water for injection, saline, hardened oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediamine buffers such as acetates, citrates or phosphates and agents for the adjustment of toxicity such as sodium chloride or dextrose.

Parenteral formulations may be incorporated into ampoules, ready-to-use syringes or multiple dose vials made of glass or plastic.

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

The active compound can also be administered via a transdermal patch. Methods for making transdermal patches are known to those skilled in the art, see, for example, L. Brown and R. Lanter, Transdermal delivery of drugs, Ann. Rev. Med. 39, 221-229 (1988).

In another embodiment, the active compounds will be formulated with carriers that will protect the compound against rapid elimination from the body as a controlled release formulation, including implants from microencapsulated release systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for making such compositions will be known to those skilled in the art. These materials can also be obtained commercially from Alza Corporation and New Pharmaceuticals, Inc.

Liposomal suspensions may also be pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art. See, for example, U.S. Pat. 4,522,811. For example, liposome formulations may be prepared by dissolving a suitable lipid or lipids (such as stearoylphosphatidylethanolamine, steraroylphosphatidylcholine, arachadoylphosphatidylcholine and cholesterol) in an inorganic solvent, which then evaporates leaving a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate and / or triphosphate derivative is then introduced into the reservoir. The contents of the container are then rapidly swirled by hand to release free lipid material from the sides of the container and disperse the lipid aggregates to form a liposomal suspension.

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

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