FIELD OF THE INVENTION

This invention is directed to compositions and methods useful for the treatment and prevention of thrombosis.

REFERENCE TO GOVERNMENT GRANTS

The research in this patent application was supported in part by National Institute of Health Grant No. HL43183. The U.S. Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Almost half of all persons in the United States and Europe die of atherosclerosis. Two third of deaths caused by atherosclerosis are caused by thrombosis of one or more arteries. The remaining third are caused by thrombosis or hemorrhage of vessels in other organs of the body, especially the brain, causing strokes, but also in the kidneys, liver, gastrointestinal tract, limbs, etc.

One of the mechanisms that mediate thrombosis associated with atherosclerosis involves elevated levels of Lp(a) . This lipoprotein, which is a fraction of low density lipoproteins ( DL) and has an attached lipoprotein (a) , is synthesized in liver cells and has been identified as being a major determinant of intravascular thrombosis in atherosclerosis patients. Abnormally high levels of LDL and very low density lipoproteins (VLDL) are also known to be

associated with high cholesterol levels and increased risk of atherosclerosis. Therefore, it is highly desired to modulate cholesterol, VLDL, LDL as well as Lp(a) levels in order to prevent atherosclerosis and thrombosis. Deposition and calcification of deposited cholesterol may form atherosclerotic plaques. The atherosclerotic plaque can cause a local blood clot, or thrombus, which can occlude a blood vessel. Vessel wall injury is another cause of thrombus. The thrombus usually begins when a vessel wall is injured or when an atherosclerotic plaque has grown so large that it has broken through the intima, thus coming in contact with the flowing blood. Because the plaque or injury presents a rough surface to the blood, blot platelets begin to adhere to it and fibrin begins to be deposited. Blood cells then become entrapped and form a clot that grows until it"occludes the vessel. Antiplatelet drugs such as aspirin have had some success in preventing clot formation associated with atherosclerotic changes in the arterial circulation, e.g., coronary arteries. Anticoagulants such as warfarin and heparin are also used to inhibit clot formation, primarily in the venous circulation. However, these treatments are associated with increased risk of bleeding. New methods of interfering with abnormal clot formation are thus greatly desired. Normally, clots slowly break down in vivo . The plasma fibrinolytic system is responsible for this in vivo lysis of blood clots. Circulating plasminogen activators (PA) such as tissue plasminogen activator (tPA) and urokinase (UK) are essential for the normal activation of the fibrinolytic system. In fact, low levels of plasminogen activators have been linked to an increased risk of thromboembolic events in both venous and arterial systems. Thus, methods of increasing in vivo levels of plasminogen activators are greatly desired. Furthermore, methods of increasing the efficacy of plasminogen activators such as tPA and UK as well as streptokinase (SK) , an exogenous

plasminogen activator, is also greatly desired. Furthermore, it would be desirable to enhance the activity of plasminogen.

Hence, the treatment of thrombosis can be targeted at a number of levels. At one level, Lp(a) levels may be modulated. Cholesterol lowering agents such as nicotinic acid are known in the art to be effective to lower serum cholesterol in patients with abnormally high cholesterol levels. This effect is via inhibition of VLDL synthesis and is also associated with decreased LDL levels. In addition, nicotinic acid has also been shown to increase HDL levels and to lower Lp(a) levels. Unfortunately, nicotinic acid has an unpleasant side effect, flushing, and a potentially more serious side effect, liver damage. While a tolerance develops toward the former effect after a few days, nicotinic acid has not been widely accepted by patients and doctors. The liver toxicity of nicotinic acid, however, appears to be primarily a dose-related effect, which is difficult to deal with considering the high doses of nicotinic acid used for lowering serum cholesterol. Thus, it would be a significant improvement if a nicotinic acid analog was developed which would be a potent inhibitor of Lp(a) synthesis, and which would modulate blood serum cholesterol levels, but which would have neither the flushing effect nor the hepatotoxic effect of nicotinic acid. It would also be desirable to treat thrombosis at the clot formation level. Fibrin fibers provide the structural backbone of clots, which also contain platelets and other cells. Normally, fibrinogen is first converted to fibrin by thrombin, and subsequently, fibrin polymerizes through the formation of cross-links to form a fibrin fiber network. Thus, it may be desirable to modulate this conversion and stabilization by causing a pharmacological agent to bind to fibrinogen to affect the fibrin assembly process, leading to altered fibrin aggregation and assembly, and decreased Factor Xlll-induced cross-linking. Since clot lysability is inversely related to the extent of cross- linking, decreased cross-linking will lead to greater clot

lysability to a given concentration of plasmin, the physiological clot-lysing enzyme. Thus, by targeting this mechanism, clots may be more easily lysed and the need for traditional anticoagulants, such as heparin and warfarin, with their associated risks, superseded.

Increased plasmin production may also be targeted. Plasminogen activators activate the production of plasmin which is integral to fibrinolytic activity. The administration of plasminogen activators has been used for the treatment for thrombosis. Currently there are four plasminogen activators approved for clinical use in the United States, tPA, SK, UK and APSAC (anisoylated (lys)- plasminogen streptokinase activator complex) . There is, however, considerable interest in more efficacious plasminogen activators, such as plasminogen activators having increased fibrinolytic activity.

Stimulation of plasminogen activator synthesis in vivo would also be an effective method of preventing and treating thrombosis. While studies have shown that sulfonylureas, polyamines, and nicotine/cotinine induce PA synthesis in endothelial cells, there are currently no approved drugs useful as PA synthesis stimulators. Effective PA synthesis stimulators are thus greatly desired.

Enhancement of plasminogen is also an effective method of treating thrombosis and is, therefore, greatly desired.

SUMMARY OF THE INVENTION

Methods and compositions for preventing or treating thrombosis are provided-by the present invention.

In accordance with some embodiments of the present invention, a pharmaceutical composition useful for lowering blood Lp(a) having the formula

wherein R ₂ , ₄ , R ₅ and „ are independently selected from the group consisting of H, halogen, electron withdrawing groups and electron donating groups (hereinafter Formula I) in a pharmaceutically acceptable carrier, is provided by the present invention. Methods useful for preventing and treating thrombosis by administration of the composition of Formula I to a patient are also provided.

In other embodiments of the present invention, pharmaceutical compositions useful for stimulating PA synthesis are provided which have the formula

where j is H or halogen (hereinafter Formula II) in a pharmaceutically acceptable carrier. Methods of treating and preventing thrombosis by administering the compound of Formula II to a patient are also provided by the present invention.

In still other embodiments of the present invention, pharmaceutical compositions having the formula

where X is H or an electron withdrawing group;

Y is H or an electron donating group; and Z is H or an electron donating group (hereinafter Formula III) , in a pharmaceutically acceptable carrier are provided by the present invention. Compositions of Formula III may be administered to treat and prevent thrombosis by stimulating PA synthesis.

Methods of enhancing the efficacy of plasminogen activators and plasminogen are provided in accordance with some embodiments of the present invention. In still other embodiments of the present invention, acetylated plasminogen activators and/or acetylated plasminogen are provided. These acetylated compositions may be administered to patients to treat thrombosis in.accordance with the present invention.

Finally, non-steroidal anti-inflammatory drugs such as meclofenamic acid may be administered to prevent thrombosis by modulating fibrin clot assembly, cross-linking and lysability. Methods of treating and preventing thrombosis by administering such agents are also provided by the present invention. Thus, it is an object of. the present invention to provide compositions useful to treat or prevent thrombosis.

Further, in accordance with some embodiments of the present invention, methods of treating and preventing thrombosis are provided. These and other objects of the invention will become apparent through a reading of the detailed description and accompanying claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the treatment and prevention of thrombosis at a number of levels.

Thrombosis may be prevented by lowering the blood levels of Lp(a), which interferes with normal fibrinolysis. Thus, in

accordance with some embodiments of the present invention, nicotinic acid analogs having the formula

wherein R ₂ , R ₄ , R ₅ and g are independently selected from the group consisting of H, halogen, electron withdrawing groups and electron donating groups (hereinafter Formula I) in a pharmaceutically acceptable carrier are provided. By electron withdrawing group, any of the groups known to those skilled in the art to be effective as an electron withdrawing group are intended, such as, for example COOH, CONH ₂ , CN, N0 ₂ and S0 ₃ H. Electron donating group is intended to mean any of the moieties known to those skilled in the art to act as an electron donating group. Some exemplary electron donating groups include NH ₂ , O-lower alkyl, OH and lower alkyl. Lower alkyl, as used herein refers to C - C ₆ alkyl. Of course, any electron withdrawing or donating groups known to those skilled in the art which are in keeping with the basic tenets of the present invention are also encompassed by the present invention. It is also preferred, in some embodiments of the present invention, to acetylate compounds of the present invention. For example, compounds comprising OH groups may be acetylated. Compositions of Formula I may be synthesized by modification of methods used to synthesize nicotinic acid, such as were taught by Woodward et al., Ind. Eng. Chem . -3JJ.540 (1944) and McElvain, J. Am . Chem. Soc. j>2-2283 (1941), incorporated by reference herein in their entirety. The compounds of Formula I may be administered to prevent and treat thrombosis by lowering Lp(a) levels. While not wishing to be bound to any particular theory, it is believed that the administration of compositions of Formula I also contribute to lower

cholesterol buildup by lowering serum cholesterol, VLDL and LDL levels and by elevating HDL levels in blood serum. In accordance with further embodiments of the present invention, compositions which enhance fibrinolytic activity are provided. Compounds having the formula

H

where R _x is H or halogen (hereinafter Formula II) in a pharmaceutically acceptable carrier are provided. In some preferred embodiments of the present invention, R _x is H, F or Cl. Compositions of Formula II may be synthesized from the starting material 2-amino-phenylsulfonamide or derivatives thereof. Preferable derivatives include 2-amino (5-chloro)- phenylsulfonamide and 2-amino (5-fluoro)-phenylsulfona ide. Reacting starting material with chloroacetic acid in the presence of Na ₂ C0 ₃ provides compositions of the present invention such as 3-oxo-l,2,4-benzothiadiazine 1,1 dioxide and 7-chloro and 7-fluoro derivatives thereof. HC1 is a byproduct of this reaction. The resulting product resembles the antihypertensive drug, diazoxide, which is commercially available. However, the structure is modified to resemble the class of compounds sulfonylureas which induce synthesis of plasminogen activators in endothelial cells. By making such structural alterations, significant changes in the property of the molecule result. For example, sulfonylureas block K ⁺ channels, while diazoxide is known to be a K ⁺

channel opener. In other embodiments of the present invention compositions having the formula

where X is H or an electron withdrawing group; Y is H or an electron donating group; and Z is H or an electron donating group, (hereinafter Formula III) in a pharmaceutically acceptable carrier are provided. By electron withdrawing group, any of the groups known to those skilled in the art to be effective as an electron withdrawing group are intended, such as, for example, COOH, CONH ₂ , CN, N0 ₂ and S0 ₃ H. Electron donating group is intended to mean any of the moieties known to those skilled in the art to act as an electron donating group. Some exemplary electron donating groups include NH ₂ , O-lower alkyl, OH and lower alkyl. Lower alkyl, as used herein, refers to C, - C ₆ alkyl. Of course, any electron withdrawing or donating groups known to those skilled in the art which are in keeping with the basic tenets of the present invention are also encompassed by the present invention. It is also preferred in some embodiments of the present invention to acetylate compounds of the present invention. For example, compounds comprising OH groups may be acetylated. Such compositions of Formula III may be synthesized by modifications of the procedure described in Acheson et al. , J. Chem . Res . (S) , 222 (1979), J. Chem . Res . (M) 3901-3942 (1979) , incorporated herein by reference in its entirety. While not wishing to be bound to any particular theory, it is believed that compositions of Formula II and III stimulate the synthesis of plasminogen activators, thereby activating fibrinolysis or clot lysis. The present invention is also directed to modification of plasminogen activators to enhance their

fibrinolytic activity. Plasminogen activators such as tPA, SK, UK and APSAK are presently approved for clinical use in the United States. By acetylating these and other plasminogen activators, enhanced fibrinolytic activity results. Similarly, acetylation of plasminogen also enhances its fibrinolytic activity. Furthermore, it is possible, in some aspects of the present invention, to acetylate naturally occurring plasminogen activators and plasminogen, in vivo . Plasminogen activators may be acetylated by incubation with different acetyl, for example, acetylsalicylic acid.

In accordance with other embodiments of the present invention, the fibrin assembly and cross-linking process is modulated in order to eliminate abnormal clot formation. Non-steroidal anti-inflammatory drugs are provided which modulate the fibrin assembly and cross-linking process, thus making fibrin clots more readily lysable. For example, the non-steroidal anti-inflammatory drug meclofenamic acid has been found to inhibit α-polymerization of fibrin and, thus, promote clot lysability, via non-covalent binding to fibrinogen. Other preferred non-steroidal anti-inflammatory drugs include analogs of meclofenamic acid, mefenamic acid, and analogs thereof. Meclofenamic acid and mefenamic acid are commercially available from, for example, Warner-Lambert, Ann Arbor, MI. Analog, in the context of the present invention, is intended to encompass all chemical compositions which are derivatives of meclofenamic acid and mefenamic acid, but which retain similar physical and functional characteristic and which cause decrease or elimination of the incidence of thrombosis. All of the pharmaceutical compositions of the present invention are preferably administered internally, such as orally, intravenously, or intramuscularly in a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention can be administered alone or in combination with other anti-thrombotic agents that affect blood or fibrin clot formation and lysis. For example, combined administration of

acetylated plasminogen activator and acetylated plasminogen causes a synergistic effect. Furthermore, compositions of the present invention may be applied in conjunction with other antithrombotic agents to a patient predisposed to thrombosis or suffering from thrombosis as long-term antithrombotic therapy. Compositions of the present invention may be administered to a patient suffering from thrombosis in an amount effective to reduce vessel blockage caused by such thrombosis. Furthermore, compositions of the present invention may be administered to patients predisposed to thrombosis in amounts effective to reduce the incidence of thrombotic formation. Amounts to be administered will, of course, vary with the patient in accordance with factors well known to those skilled in the art, such as patient weight, age, sex and physical condition. The present invention is further described in the following examples which, while illustrative, should not be construed as limiting of the present invention.

EXAMPLES Example 1

Preparation of Plasma

Human blood was treated with 0.05 M Na citrate and plasma is separated at 2000 X g for 10 minutes.

Example 2 Preparation of Clot

20 μl of (0.2 M CaCl ₂ + 50 U/ml thrombin) was added to 200 μl of human plasma prepared in accordance with Example 1 in an Eppendorf tube.. A rounded wood was introduced into the tube to permit fibrin to stick around the wood. The tube was incubated undisturbed for 18 hours at room temperature. Thereafter, the clot was removed and rinsed with saline solution (0.15 M, 0.5 M Tris-Cl, pH 7.4 , 4°C) . The clot was squeezed gently against the tube wall of a glass tube filled with 5 ml cold saline solution until the clot was clear

(white) . Using filter paper, all residual liquid was removed from the clot.

Example 3

Dose Response Study Clots were formed in accordance with Example 2 using human serum which was preincubated with 10, 100, 300, 500 and 1000 μM meclofenamic acid at 37°C for 30 minutes. Samples were reduced and run on SDS polyacrylamide gel to separate the a, β , y, 7-7 dimer (y ₂ ) , and α-polymer (α _n ) . The bands were quantitated on a densitometer for 10, 100, 300, 500 and 1000 μM meclofenamic acid. The <x/β ratio was significantly increased at 500 μM and 1000 μM, and the ctjβ ratio was significantly decreased at 300 μM, 500 μM and 1000 μM. No significant changes were observed in the y/β or y ₂ /β ratios. Clot lysis studies using lysometry showed concentration-dependent increase in clot lysis rate.

Example 4

Time Course Study

Clots were formed in accordance with Example 2 using human serum which was preincubated with 10, 100, 300, 500 and 1000 μM meclofenamic acid at 37°C for 30 minutes. The clotting reaction was stopped at 5, 15,- _^ 30 minutes and 1, 2, 4, 5, 18 and 24 hours. Samples were reduced and run on SDS polyacrylamide gel. Using 0.5 mM meclofenamic acid, a significantly lower a _D /β ratio was detected as early as 15 minutes, reaching an asymptotic difference at about 2 hours.

Example 5 Binding Study

Purified fibrinogen (PF) was dialyzed against Tris- Cl pH 8-0 buffer overnight. 320 μl of the PF solution was equilibrated with 1, 3, 10 and 20 mM of ^I4 C-meclofenamic acid at 37°C for 1 hour. 250 μl of the sample was transferred into a Centrifree micropartition system (AMICON) centrifuge tube and spun at 1.800 X g for 1 hour. 50 μl of ¹⁴ C

meclofenamic acid (total) and 50 μl from the centrifuge tube (free) , were counted. The counts trapped in the Centrifree filter were also counted. The fraction of fibrinogen bound was calculated as follows: Total - Free

= fraction bound

Total The results are as follows:

¹⁴ C-meclofenamic acid % bound (mM)

1.0 5.6

3.0 16.0

10.0 14.1 30.0 14.7

Thus, this system revealed approximately 15% binding by meclofenamic acid to fibrinogen under the conditions used.

Example 6

Effect of Acetylation of Plasminogen Activators Fixed concentrations of two plasminogen activators, tPA (25 units/ml) and SK (5 units/ml) , were incubated individually with freshly prepared solutions of 0.25 μM, 0.25 mM and 25 mM acetylsalicylic acid as the acetylating agent. In separate experiments, plasminogen was incubated with the same concentrations of acetylsalicylic acid. The incubation was performed in 0.05 mM Tris buffer, pH 8.8, at 37°C for two hours in sealed 1.5 ml plastic Eppendorf tubes. Subsequently, the acetylated plasminogen activators, together with appropriate controls, were tested for plasminogen activator activity using a chromogenic assay modified from the methods of Lijnen et al., Thrombos Haemostas 51:108-109 (1984) , incorporated by reference herein, in its entirety. Briefly, each plasminogen activator was combined with plasminogen, poly-L-lysine (as fibrin substitute) , and a chromogenic substrate S-2251 in Tris buffer in individual wells of a 96 well Corning plastic plate. The substrate is susceptible to amidolysis by plasmin and a color change

results upon cleavage which can be quantitated as a change in optical density at 405 nm by a spectrophotometer. The results showed an increasing plasminogen activator activity with increasing acetylsalicylic acid concentration during the preincubation, indicating concordant relationship between acetylation of these proteins and their plasminogen activator activity; salicylic acid by itself had no effect on plasminogen activator activity. The percent increases in the activity of acetylated tPA, after the three acetylsalicylic acid concentrations were 51 ± 55, 81 ± 56, and 81 ± 56%, respectively. The percent increase in the activity of acetylated SK, after the three acetylsalicylic acid concentrations were 4 ± 1, 10 ± 3, and 17 ± 3%, respectively. The percent increases in the fibrinolytic activity of acetylated plasminogen, after the three acetylsalicylic acid concentration were 4 ± 2, 10 ± 3, and 17 ± 4%, respectively. In addition, there was significant synergism between the acetylated plasminogen activators and the acetylated plasminogen.

Example 7

^12S I-fibrin Plate Assay to Determine Plasminogen Activator Activity

Compounds of Formula II and III are tested for their effect in stimulating plasminogen activator activity by the fibrin plate method as described in Kuo et al. , J. Clin . Invest. JL 730-737 (1988) which is incorporated by reference herein in its entirety. Bovine aortic endothelial (BAE) cells—are incubated with test compounds, the supernatant medium harvested after 24 hours of incubation and assayed for PA activity.

Each well of a 96 well Coster tissue culture plate is coated with ¹² I-fibrin monomer (15,000 cp ) and unlabeled fibrin monomer (1.25 μg), and dried at 56°C for 3 hours prior to use. Purified tPA, used as the standard, and supernatant medium samples are first treated with 0.75% SDS and then with 7.5% Triton X-100. Aliquots of the resulting mixtures are

transferred to the ¹²⁵ I-fibrin-coated plate, after the addition to each well of an assay buffer containing 1 μg of purified human plasminogen. The assay buffer consists of 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA and 0.1% gelatin. Standard curves are constructed for each plate (l-

20 mU tPA/ well) . After a two hour incubation at 37°C in a water bath, fibrinolysis is determined on the basis of the released radioactivity and expressed as a percentage of total trypsin-induced releasable radioactivity. Non-plasminogen related fibrinolytic activity was around 5%. Test compounds increase fibrinolytic activity to levels higher than the fibrinolytic activity of the standard.

Example 8 Elisa Assay to Determine Lp(a)

Compounds of Formula I are tested for their effect in stimulating Lp(a) synthesis using a commercially available ELISA assay system. Cultured liver cells and hepatoma cells, as well as other cell types, are incubated with test compounds and the supernatant medium harvested after specific durations of incubation and assayed for Lp(a) levels.