KR20020061130A - Synergistic Compositions Comprising Ascorbate and Lysine for States Related to Extracellular Matrix Degeneration - Google Patents

Abstract

PURPOSE: Provided is a synergistic composition comprising ascorbate and lysine for prevention and treatment of diseases or pathological symptoms related to extracellular matrix degeneration. CONSTITUTION: The pharmaceutical composition characteristically contains ascorbate, at lest one kind of fibrinolytic enzyme inhibitor and pharmaceutically acceptable carrier in a sufficient amount for the treatment of collagenase related degenerative diseases. It optionally contains at least one kind of antioxidants.

Description

Synergistic Compositions Comprising Ascorbate and Lysine for States Related to Extracellular Matrix Degeneration}

The present invention relates to substances and compositions for the prevention and treatment of diseases or pathological conditions associated with the degeneration of the extracellular matrix, for example degenerative diseases, in particular atherosclerosis, cancer, infection or other inflammatory diseases.

Mammalian ovulation studies show that gonadotropin, prostaglandin and other substances stimulate the release of plasminogen activator (PA) from follicle cells in the ovary stroma. (Strickland, S., et al., (1976) J. Biol. Chem. 751: 5694-5702) (FIG. 1). PA then stimulates extracellular activation of plasminogen to plasmin. Plasmin is known to activate procollagenase with collagenase and thereby break down collagen.

Under pathological conditions, PA is secreted in a manner similar to follicle cells stimulated by hormones by cancer cells, macrophages and cells transformed by viruses (Unkeless, et al, (1974) J. Biol. Chem. 249 : 4295-4305) (FIG. 2). PA was found at high concentrations in metastatic lung tumors (Skriver, et al., (1984) J. Cell Biol. 99: 753-758). PA has also been known to be involved in various human tumors, as well as renal and bladder carcinomas (Corasanti, et al. (1980) J. Natl. Canc. Inst. 65: 345-351 and Ladehoff, A. (1962) Act. Path.Micro.Scand. 55: 273-280).

In the regulation of this mechanism, the function of lipid protein (a) (Lipoprotein (a); Lp (a)), which is a particle similar to low density lipid protein containing a unique glycoprotein called apoprotein (a), has been proposed. This particle has been suggested to be involved in wound healing and general cell repair (Brown, M. et. Al., (1987) Nature 330: 113-114). The cDNA sequence of apo (a) has significant homology with plasminogen with multiple repetitions of kringle 4, one kringle 5 and protease domain. Isomers of apo (a) vary in size from 300 to 800 kDa, and depend primarily on the number of genetically determined Kringle 4 structures (McLean, J. W., et al. (1987) Nature 300: 132-137). apo (a) has no plasmin-like protease activity (Eaton, DL, et al., (1987) Proc. Natl. Acad. Sci. USA 84: 3224-3228) and has been demonstrated to have serine protease activity (Salonen, E., et al. (1989) EMBO J. 8: 4035-4040).

Despite the lack of functional homology, the strong structural similarity to plasminogen makes it possible to understand the physiological and pathological roles of Lp (a). Like plasminogen, Lp (a) binds to lysine-sepharose, immobilized fibrin and fibrinogen, and plasminogen receptors of endothelial cells (Harpel, PC et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3847-3851; Gonzalez-Gronow, M. et al. (1989) Biochemistry 28: 2374-2377; Miles, L. et al. (1989) Nature 339: 301-302; Hajjar, KA, et al. (1989) Nature 339: 303-305). In addition, Lp (a) binds to other components of the arterial vessel wall, such as fibronectin and glycosaminoglycans. However, the exact nature of this binding is not well understood. Lp (a) increases plasma concentrations in cancer, atherosclerosis and other diseases. Conversely, the higher the incidence of these diseases, the lower the concentration of ascorbate (Knox, EA (1973) Lancet, ie 1465-1467; Wright, LC et al. (1989) Int. J. Cancer 43: 241-244). . Based on these and other observations, Lp (a) has been proposed as a substitute for ascorbate (Rath, M. & L. Pauling (1990) Proc. Natl. Acad. Sci. USA 87: 6204-6207) .

There is a need for therapeutic compositions that reduce the regression of extracellular matrices, particularly those resulting from plasmin-induced proteolysis and free radical-induced fibrin degradation. In particular, compositions that reduce regression and at the same time promote the synthesis of collagen, which is the first component of the extracellular matrix, prevent the growth of the disease.

The present invention provides a therapeutic composition comprising one or more fibrin degradation inhibitors in an amount sufficient to reduce plasmin- and free radical-mediated collagen production in a patient in need of treatment of a degenerative disease that destroys the extracellular matrix. Provided is a pharmaceutical composition for treating the disease. Another aspect of the invention relates to a composition comprising an activator suitable for enhancing collagen synthesis.

In another aspect of the invention there is provided a composition comprising ascorbate and optionally comprising at least one antioxidant. The term antioxidant, as used herein and in the claims, does not include ascorbate, which is itself a powerful antioxidant.

In addition, the present invention provides one or more fibrin degradation inhibitors, one collagen synthesis activator, ascrobate and optionally one or more antioxidants in an amount sufficient to reduce plasmin- and free radical-mediated collagen production. It provides a therapeutic composition comprising.

These and other aspects of the invention will be more readily understood in view of the following figures and detailed description of the invention.

1 is a flow chart of tissue regression induced by plasmin under physiological conditions that induce ovulation.

2 is a physiological flow chart in which the degeneration of the extracellular matrix proceeds as a result of proteolytic degradation induced by free radicals.

3 is a flow chart showing the physiological role of apo (a) in regulating proteolysis induced by plasmin and proteolysis induced by free radicals.

4 is a flow chart for the synergistic effect of ascorbate and fibrin degradation inhibitors in the treatment of proteolytic degradation induced by plasmin and free radicals.

The present invention relates to methods and compositions that slow or inhibit extracellular matrix degradation and thereby prove useful for the treatment of cancer, degenerative diseases, infections or other inflammatory diseases, or pathological conditions associated with extracellular matrix degradation. Many diseases appear to be caused or worsened as a result of extracellular matrix degradation. For example, cancer has usually been life-threatening after individual tumors have metastasized, ie, individual cells are released into the extracellular matrix and transported to other parts of the body for them to proliferate. Cancer as well as other degenerative diseases, such as advanced atherosclerosis, have a similar mechanism as the disease progresses. As used herein, the term “degenerative disease associated with collagenase” includes degenerative diseases, neoplastic diseases, infections or other inflammatory diseases, or pathological symptoms associated with extracellular matrix degradation. As shown in FIG. 2, two basic pathomechanisms are plasmin-induced proteolysis and free radical-induced proteolysis, which are synergistic in the progression of the disease. In particular, in the case of cancer treatment, the emphasis is on removing or killing the tumor, and little attention has been given to inhibiting the proliferation or figuring out the tumor blockage in addition to surgery. Such blockade may be achieved by substances that interfere with or block this pathogenic mechanism, such as fibrin degradation inhibitors. For example, tranexamic acid is uncertain but has been administered in an attempt to stop cancer proliferation (Marcus, G. (1984) Sem. Thromb. Hemost. 10: 61-70).

The present invention is based on the discovery that plasmin-induced proteolysis is a fundamental physiological feature of humans that can respond to controlling a number of different and irrelevant physiological processes. In particular, cancer cells and similar normal tissues in the body activate plasmin and thereby secrete PA, which leads to collagen degradation, whereby the degeneration of the intracellular matrix can be beneficial to secretory tissue. For example, follicle stroms in human ovaries must be regressed to release the egg into the reproductive tract. Follicle cells secrete PA, which activates plasminogen to produce plasmin. Plasmin promotes collagenase, resulting in the breakdown of the extracellular matrix proteins of the strom, resulting in the expansion of the follicles and ovulation of the eggs. Many cancer cells also produce large amounts of PA that are regulated within the same physiological pathway leading to the degeneration of collagen and extracellular matrix. In addition to these examples, degeneration has the deleterious effect of releasing individual cells in the tumor from the tumor and transporting them to distant parts of the body. This indicates that metastasis of all cancers is highly dependent on degradation of the extracellular matrix. As detailed in US Patent Publication No. 07 / 533,129, it was also found that certain substances compete with plasminogen and Lp (a) at binding sites on endothelial cells of the vessel wall. It has now been found that these substances, previously named Lp (a) binding inhibitors and herein termed fibrin degradation inhibitors, are antagonists to plasmin that converts procollagenase to collagenase. It has also been found that administration of such fibrin degradation inhibitors, especially in combination with ascorbate to enhance collagen synthesis, can prevent or at least withhold degradation of the extracellular matrix due to increased plasmin activation.

The term fibrin degradation inhibitor, as used herein and in the claims, includes all substances that act as antagonists of plasmin-induced proteolytic activity, in particular collagenase production induced by fibrin degradation. Some of these compounds are used clinically for the treatment of excessive fibrin degradation conditions at high doses.

In most cases, activated macrophages play an important role in the onset and spread of pathological symptoms. When activated, these cells secrete a variety of products, including enzymes such as procollagenases. In addition, as shown in Figure 2, it also secretes reactive oxygen intermediates such as peroxides, hydrogen peroxide and hydroxyl radicals, referred to herein as "free radicals" (Nathan, C. (1987) J. Clin. Invest. 79: 319 -326). In addition, other mechanisms of disease progression are mediated by peroxyl free radicals. Such oxidizing radicals can be produced by activated macrophages, in particular other cells under pathological symptoms. Similar to plasmin-induced proteolysis, free radicals induce extracellular matrix degradation, which promotes disease progression. Free radicals are known to induce proteolysis in a number of ways and play an important role in activating procollagenase to collagenase. Ascorbate is a powerful antioxidant, which is also an important therapeutic agent that inhibits proteolysis by free radicals. As shown in FIG. 4, the useful effect of ascorbate is that ascorbate, together with the fibrin degradation inhibitor, has a synergistic effect and inhibits free radical-induced proteolysis as well as plasmin-induced proteolysis. Ascorbate enhances the production of collagen (Murad, et al., 1981 Proc. Natl. Acad. Sci. USA 78: 2879-2882), and also promotes the production of lymphocytes that can be used to eradicate already generated cancer cells. Stimulate (Yonemoto et al., 1976 Proc. Amer. Assoc. Canc. Res. 17: 288).

Fibrin degradation inhibitors inhibit the dissolution of blood coagulants, resulting in a pro-coagulant effect. Because it is known that neoplasms are often associated with thromboembolic complications, these properties have somewhat limited or inhibited the use of such agents in the treatment of cancer and other diseases. Ascorbates are known to have properties that inhibit coagulation, for example by stimulating prostracycline and inhibiting thromboxane production. In addition, aspects of the present invention in which the fibrin degradation inhibitors bind ascorbate significantly increases the utility of the therapy by eliminating unwanted side effects of monotherapy. The present invention provides methods and compositions for both treatment and prevention of diseases associated with degradation of the extracellular matrix. Each such embodiment is described in turn below.

<General application>

The present invention provides a subject with an effective amount of at least one fibrin degradation inhibitor that inhibits the conversion of procollagenase to mediated collagenase mediated by plasmin and free radicals to collagen-associated degenerative diseases such as cancer and many Provided are methods and compositions for treating and preventing other diseases. In this method, disruption of the extracellular matrix that primarily forms collagen can be reduced or eliminated. Preferred fibrin degradation inhibitors are ε-aminocaproic acid (EACA), lysine, tranexamic acid (4-aminomethylcyclohexane carboxylic acid), p-aminomethylbenzoic acid (PAMBA), p-benzylamine sulfuric acid, α-N- Acetyl-lysine-methyl ester, cis / trans-4-aminomethylcyclohexane carboxylic acid- (1) (AMCHA), trans-4-aminomethylcyclohexane carboxylic acid (AMCA) and 4-aminomethyl-bicyclo -2,2,2-octane carboxylic acid (AMBOCA), including but not limited to. Effective amounts of fibrin degradation inhibitors or mixtures of one or more fibrin degradation inhibitors may also be used. In addition, by combining two or more fibrin degradation inhibitors, the toxicity may be reduced because the fibrin degradation material may have different catabolic pathways, potentially increasing the therapeutic effect. In addition, the methods and compositions of the present invention may include ascorbate in combination with a fibrin degradation inhibitor. As used herein, the term "ascorbate" includes any pharmaceutically acceptable salt of ascorbate, including sodium ascorbate as well as ascorbic acid itself. Other substances used in the treatment of cardiovascular diseases can also be administered simultaneously, including tocopherols, carotene, selenium, N-acetylcysteine, antioxidants such as probucol, and related substances such as vitamins, provitamins, and trace elements. . Ascorbate can be used alone because it stimulates collagen synthesis, but the doses shown in Table 1 for ascorbate and one or more fibrin breakdown inhibitors and antioxidants for the treatment of existing degenerative diseases (day weight (kg / kg body weight / day)). Table 1 shows that the compositions provide different concentration ranges of each component depending on whether the composition is administered orally or parenterally. The change in dosage reflects the change in severity of the disease. Therefore, if the subject wants to diagnose the progression stage of a specific degenerative disease, it can be seen that the dose at the high point of the above range can be used. However, where the prevention of collagen degradation is desired prior to the onset of serious symptoms, dosages at the low end of this range can be used.

Alternatively, the same pharmaceutical composition as described above can be used except for ascorbate. When ascorbate and fibrin degradation inhibitors are used in the same composition, they are simply mixed, or chemically using synthetic methods known in the art, such as compounds in the form of salts in which the ascorbate and inhibitor are covalently or ionically bonded. Can be combined. For example, ascorbate may be covalently linked to lysine, other amino acids, or ε-aminocaproic acid in ester bonds. Ascorbyl ε-aminocaproate or ascorbyl-aminomethylcyclohexane carboxylic acid are such examples. In this form, ascorbate residues can also be particularly effective in preventing unwanted lipid peroxidation.

Ascorbate and fibrin degradation inhibitors may be used alone or in combination with other agents used in chemotherapeutic therapies of cancer. The medicament includes, but is not limited to, a compound selected from the group consisting of antimicrobial derivatives, estrogen inhibitors, metabolic inhibitors, hormones, cytotoxic agents, nitrogen-mustard derivatives or steroids, and combinations thereof. For oral administration, other pharmaceutically acceptable inert carriers can be used. In addition, when administered orally, the active ingredient may be administered in the form of a tablet. Tablets may include binders such as tragacanth, corn starch or gelatin, disintegrants such as alginic acid and / or lubricants such as magnesium stearate. If liquid administration is desired, sugars and / or flavors may be used. When administration is parenterally injected in isotonic saline, phosphate buffers and the like can be used as pharmaceutically acceptable carriers.

In addition, the amino acid proline, proline salt or synthetic proline homologues can be used for the prevention and treatment of the pathological symptoms described in this patent.

In addition to its role as an inhibitor of LP (a) binding proline, it also requires a role as an amino acid component for protein synthesis. Collagen and other extracellular matrix proteins are particularly rich in lysine and proline residues, which make up about 25% of the total weight of collagen.

In contrast to lysine, proline can be synthesized in the body, while the rate of synthesis of this amino acid is partially optimized, especially for chronic diseases. In the pathology symptoms, when excessive collagen degradation lasts for months or the synthesis of collagen and other extracellular matrix molecules lasts for years, sufficient availability of proline determines the optimal production of new collagen and thereby It is an important factor to keep progress.

Therefore, it is essential that appropriate amounts of proline, proline salts and proline homologues are available to the body.

The availability of fibrin degrading inhibitors for cancer, angiohematoma and other such diseases depends heavily on the general health of the subject, particularly on excessive fibrin degradation conditions. Most fibrin degradation inhibitors (except lysine) are used clinically to treat the disease. As a result, monitoring of the subject's coagulation system and fibrin degradation system is recommended prior to and at the time of treatment. However, it should be noted that the hemostatic complications are not similar because the fibrin degradation inhibitors are also common protease inhibitors that have also been shown to inhibit coagulation (Aoki, N. et al., (1978) Blood 52: 1-12). Long-term administration of the fibrin degradation inhibitors requires formulations in the low range of the dosages of the fibrin degradation inhibitors shown in Table 1. As mentioned above, ascorbate is known to stimulate prostaglandin and prostacyclin synthesis and reduce thromboxane levels, thus exhibiting an inhibitory effect on aggregation. These features may be particularly desirable with the use of fibrin degradation inhibitors that counter strong coagulation side effects.

The ascorbate and fibrin degradation inhibitors described above can be administered separately. The therapeutic effect can be optimized by using a time release composition that makes the serum concentration of the agent relatively constant over time.

Component Dosages of the Compositions of the Invention Oral administration Parenteral administration Ascorbate: EACA tricanesamic acid p-aminomethyl benzoic acid lysine proline 5-500 mg / weight kg / day 25-2500 mg / kg1-1500 mg / weight kg / day Same 1-500 mg / weight kg / day Same 1-500 mg / weight kg / day equal 1-500 mg / weight Same kg / day 1-1500 mg / kg body weight / day Antioxidant: Tocopherol Carotene N-Acetyl Cysteine 0.1-500 1U / weight kg / day Same 0.1-10,000 1U / weight kg / day Same 0.1-5000 mg / weight kg / day Same

According to FIG. 3, another aspect of the present invention is the discovery that Lp (a) is a co-regulator of plasmin-induced proteolysis that is closely associated with tissue transformation and recovery. Due to its homology to plasminogen and plasmin, Lp (a) is a physiologically competitive inhibitor for plasmin-induced protein degradation. Apoproteins also have more than 100 disulfide bonds and therefore act as antioxidants like other protein-thiols.

All publications and patent applications cited herein are hereby incorporated by reference as if individually and individually indicated that each publication or patent application is incorporated by reference. It is now clear that the compositions and methods of treatment of the present invention for the prevention and treatment of pathological symptoms associated with extracellular matrix degradation represent a significant improvement over known methods, with certain preferred embodiments disclosed, illustrated and described above. It is understood that other embodiments are possible without departing from the invention described herein. Therefore, the present invention may be defined according to the claims as well as their equivalents.

Using the compositions of the present invention comprising ascorbate and fibrin degradation inhibitors, it is possible to reduce the degeneration of the extracellular matrix, in particular due to plasmin-induced proteolysis and free radical-induced fibrin degradation. In particular, it is possible to prevent the proliferation of various diseases by reducing the degeneration and at the same time promoting the synthesis of collagen, the first component of the extracellular matrix.

Claims (7)

A pharmaceutical composition comprising an amount of ascorbate and one or more fibrin degradation inhibitors, and a pharmaceutically acceptable carrier, sufficient to treat a degenerative disease associated with collagenase. The composition of claim 1 wherein the ascorbate is selected from the group consisting of pharmaceutically acceptable ascorbate salts, ascorbic acid and mixtures thereof. The fibrin degradation inhibitor according to claim 1, wherein the fibrin degradation inhibitor is lysine, lysine salt, or ε-aminocaproic acid, tranexamic acid, p-aminomethylbenzoic acid, p-benzylamine sulfuric acid, α-N-acetyl-lysine-methyl ester, Sheet / trans-4-aminomethylcyclohexane carboxylic acid- (1), trans-4-aminomethylcyclohexane carboxylic acid and 4-aminomethyl-bicyclo-2,2,2-octane carboxylic acid At least one component selected from the group consisting of compositions selected from synthetic homologs. The composition of claim 1, wherein the at least one component is selected from the group consisting of proline, or a pharmaceutically acceptable salt or synthetic proline homolog thereof. The composition of claim 1, further comprising at least one antioxidant. 6. The composition of claim 1, wherein the antioxidant is selected from the group consisting of tocopherol, carotene, selenium, N-acetylcysteine, probucol, and mixtures thereof. 7. The composition of any one of claims 1 to 6 comprising an active ingredient for the prevention and treatment of degenerative diseases associated with collagen, which is selected from vitamins, pro-vitamins and trace elements.