UA72215C2 - Pharmaceutical composition containing inhibitor of collagenase fibrinolysis and ascorbate - Google Patents

Abstract

A composition for the prevention and treatment of degenerative diseases, associated with collagen, comprising at least one inhibitor of collagen fibrinolysis and ascorbat in therapeutically acceptable dose and pharmaceutically acceptable carrier, at that the inhibitor of collagen fibrinolysis is not lysine or its salt.

Description

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, including but not limited to such diseases as degenerative diseases, especially arteriosclerosis, infections and other inflammatory diseases. In studies of ovulation in mammals, it appears that gonadotropins, prostaglandins, and some other substances stimulate release from follicular cells injected into the ovarian stroma. (5(sKiapa, 5., eiai., (1976) 9. Vioi. Speth. 751:5694-5702) (Fig. 1). Then PA stimulates the extracellular activation of plasminogen to plasmin, and plasmin, as is known, activates procollagenase to collagenase, which then breaks down into collagen.

Under pathological conditions, PA is also secreted by cancer cells, macrophages, and cells transformed by viruses in a manner similar to ovarian cells that have been hormonally stimulated (pke!ev5, et al. (1974) U. Vioi. Speth. 249:4295-4305) ( Fig. 2). PA has been found in high concentrations in metastatic lung tumors (5 Kiimeg, et al. (1984) 9. Sei! Visi. 99: 753-758). PA has also been found in association with various human tumors, such as carcinoma of the kidney and bladder (Sogazapii, et al. (1980) ..

Mai! Sape Ipvia 65:345-351 And Adenoi, A. (1962) Act. Raiip. Misgo. Zsapa 55:273-280).

A special role in the regulation of this mechanism has been proposed for the fraction of low-density lipoprotein, which carries a unique glycoprotein called apoprotein (a) (apo (a)). It was suggested that this fraction participates in wound healing and general cellular regeneration (Vhom/p., M., ei ai., (1987) 330:113-114).

The DNA sequence of aro(a) shows striking homology to plasminogen, with multiple repeats of Kipdie 4, one Kipdie 5, and a protease domain. Aro (a) isoforms vary in the range from 300 to 800 KOa and differ mainly in their genetically determined Kiipdie number 4 of the structure (Me! eap, ULMU., and others. (1987) 300:132 - 137). While aro (a) does not have any plasmin-like protease activity (Eayup, O.I... and others., (1987) Rgos.

May). Asad 5 USA 84:3224-3228), serine protease activity was demonstrated (ZaiIopep, E., et al. (1989)

EMVO). 8:4035 - 4040).

Despite its lack of functional homology, the strong structural similarity to plasminogen is crucial in understanding the physiological and pathological role of Gr(a). Like plasminogen, it has been shown that Gr(a) forms a lysine-sepharose bond, immobilizes fibrin and fibrinogen, and the plasminogen receptor on endothelial cells (Na!grei, R.S. and others. (1989) Rgos. Mai!. Asad, USA 8613847 - 3851;

Suoplaieg - Sgopomu, M. and others. (1989) Biochemistry 28:2374 - 2377 Mie5, I. and others. (1989) 339,301 - 302 Naiag, KA, et al. (1989) 339:303-305). In addition, Gr(a) showed the possibility of forming a connection with other components of the arterial wall, such as fibronectin and glycosaminoglycans. The exact nature of these connections, however, is poorly understood. Gr (a) plasma levels are increased in cancer, atherosclerosis, and other diseases. Inversely, low levels of ascorbate were associated with a high risk of these diseases. (Kpoh, E.A. (1973)

Iapsei 1465 - 1467; MUpdunE, H.S. other. (1989) Yipi. uU Sapseg 43:241-244). Based on this and other observations, it was assumed that Gr (a) is a substitute for ascorbate. (Rai, M. and G. Racipd (1990) Rgos. Mai)!. Asad

All together. USA 87:6204 - 6207).

Thus, there is a need for a therapeutic composition to reduce the degradation of the extracellular matrix, especially caused by plasmin-induced and free radical-induced proteolysis and, accordingly, fibrinolysis. Especially valuable would be a composition that would simultaneously reduce degradation and increase the synthesis of collagen, the primary component of the extracellular matrix, and thus help prevent the progression of diseases. The pharmaceutical composition is used for the treatment of degenerative diseases originating from degeneration of the extracellular matrix, and is administered in the form of a composition containing at least one fibrinolysis inhibitor in an amount sufficient to inhibit plasmin and free radical mediators of collagenase production in a subject in need thereof treatment. Another aspect of the invention is a composition including an activator suitable for increasing collagen synthesis.

Another aspect of the invention provides a composition comprising ascorbate and any one or more antioxidants. The term "antioxidant" throughout the description and formula is intended to exclude ascorbate, which is itself a powerful antioxidant. Also provided as an aspect of the present invention is a therapeutic composition comprising at least one inhibitor of fibrinolysis, one activator of collagen synthesis, ascorbate, and optionally one or more antioxidants, in amounts sufficient to reduce levels of plasmin and free radical mediators of collagenase production.

These and other aspects of the invention will become more apparent upon consideration of the following figures and detailed description of embodiments of the invention.

Brief Description of Figures

Fig. 1 - flow chart of plasmin-induced tissue degradation in physiological conditions preceding ovulation;

Fig2 is a flow diagram of the physiology of the genesis of the degeneration of the extracellular matrix as a result of free-radical-induced proteolysis;

Fig. 3 is a flow diagram showing the physiological role of aro (a) in the regulation of plasmin and free radical-induced proteolysis; and

Fig. 4 is a flow diagram of the synergistic effect of ascorbate and fibrinolysis inhibitors in the therapy of plasmin and free-radical-induced proteolysis.

The present invention relates to methods and compositions that slow down or inhibit extracellular matrix degradation, thereby being useful in the treatment of cancer, degenerative diseases, infectious diseases or other inflammatory diseases or pathological conditions associated with the degradation of the extracellular matrix A number of diseases occur or are exacerbated as a result of degradation of the extracellular matrix. Cancer, for example, becomes life-threatening after an individual tumor metastasizes, that is, after individual cells become free of the extracellular matrix and travel to other parts of the body where they can proliferate. Compared to cancer, other degenerative diseases, like progressive atherosclerosis, have similar mechanisms by which the diseases develop. As used herein, the term "collagenase-associated degenerative diseases" (collagenoses) include degenerative diseases, neoplastic diseases, infections or other inflammatory diseases or pathological conditions associated with the degradation of the extracellular matrix. As shown in Figure 2, two main pathomechanisms are plasmin-induced and free-radical-induced proteolysis, which may act synergistically in reactive disease progression.Especially in the case of cancer, treatment has focused on the removal or destruction of tumors, and little attention has been paid to inhibition of proliferation or ways to detect inhibition of tumor growth than, surgical treatment . This containment can be achieved by substances that interfere with or block these pathways, such as fibrinolysis inhibitors. In several cases, tranexamic acid has been administered in an attempt to stop the proliferation of cancer, with inconclusive results (Magsiv, Sh. (1984) Zet. Tpgotbr. Netozvi. 10:61 - 70).

This invention is based in part on the discovery that plasmin-induced proteolysis is a fundamental physiological feature in humans, responsible for the regulation of many different, and apparently unrelated, physiological processes. In particular, cancer cells and normal tissues similarly within the organ secrete RA, which activates plasmin and, as a result, collagen degradation, where the degeneration of the extracellular matrix has a positive effect on the secreting tissue. For example, the stroma of a human ovarian follicle must degenerate in order to release an egg into the reproductive tract. Follicular cells secrete RA, which activates plasminogen into plasmin. Plasmin activates collagenase, which then proteolysis of the extracellular matrix of the ovary, expanding, rupturing, and releasing the egg. Many cancer cells produce large amounts of RA, which then works within the same physiological pathway, ultimately leading to collagen and extracellular matrix degeneration. In addition, degeneration has the deleterious effect of allowing individual cells within the tumor to free themselves to be carried to distant parts of the body. Metastasis in all cancers appears to depend largely on degradation of the extracellular matrix. As described in more detail in the US patent application Me07/533,129, it was also found that some substances compete with plasminogen and p(a) for binding to sites on the endothelial cells of the blood vessel walls. Now it was discovered that these substances previously named

And p (a)-binding inhibitors, and here called fibrinolysis inhibitors, are antagonistic to plasmin in its conversion from procollagenase to collagenase. Thus, it is disclosed that the administration of these fibrinolysis inhibitors, especially in combination with ascorbate, which promotes collagen synthesis, can prevent or at least slow down the degradation of the extracellular matrix due to increased plasmin activation.

The term "fibrinolysis inhibitor" throughout the description and formula includes all substances that act as an antagonist for plasmin-induced proteolytic activity, especially fibrinolysis induced by collagenase production.

Some of these compounds, in high doses, are in clinical use for the treatment of hyperfibrinolytic conditions. Activated macrophages play an important role in the initiation and spread of the most pathological conditions. When activated, these cells secrete various products, including enzymes such as procollagenase. In addition, as shown in Figure 2, they secrete reactive oxygen intermediates such as superoxide, hydrogen peroxide, and hydroxyl radical, hereinafter called "free radicals" Mainap, S. (1987). 9.

Siip. Ipmevi 79:319-326. Thus, another mechanism of reactive progression of the disease is the mediated effect of peroxide radicals. These radicals can be generated by activated macrophages and other cells, especially under the influence of pathological conditions. Similar plasmin-induced proteolysis, free radicals, stimulate the degradation of the extracellular matrix and, thus, contribute to the progression of the disease. Free radicals are known to stimulate proteolysis in several ways, an important one being the activation of procollagenase to collagenase. Ascorbate is a powerful antioxidant and thus an important therapeutic agent for limiting free radical proteolysis. As shown in Figure 4, the beneficial effects of ascorbate suggest that ascorbate in combination with fibrinolysis inhibitors has a synergistic effect and inhibits plasmin-induced as well as free-radical-induced proteolysis. Ascorbate promotes collagen production (Mygady, and others. 1981 Rgos. May). Asad 5 USA 78:2879-2882), and also stimulates the production of lymphocytes, which can be used in the fight against already formed cancer cells (Uopetoio, eia!. 1976

Rgos. Ateg. Av5os Sape Nev. 17:288).

Fibrinolysis inhibitors inhibit the dissolution of blood clots, thus having a procoagulation effect. These properties have so far limited or prevented the use of these agents in the treatment of cancer and other diseases, because it is known that neoplasms are often associated with thromboembolic complications.

Ascorbate is known to have anticoagulant properties by stimulating, for example, prostacyclin and inhibiting the formation of thromboxane. Thus, an aspect of the invention where a fibrinolysis inhibitor is combined with ascorbate would greatly increase the utility of this therapy, eliminating the undesirable side effects of monotherapy. The present invention provides methods and compositions for the treatment and prevention of diseases associated with the degradation of the extracellular matrix. Each of these embodiments is discussed in turn below.

The present invention provides a method and composition for the treatment and prevention of collagen-associated degenerative diseases such as cancer and many others by administering to a subject an effective amount of at least one fibrinolysis inhibitor that inhibits plasmin and free-radical mediated conversion of procollagenase to collagenase. Thus, the destruction of the extracellular matrix, which is mainly formed by collagen, is reduced or eliminated.

Preferred fibrinolysis inhibitors include, but are not limited to, aminocaproic acid (AMCC), lysine, tranexamic acid (4-aminomethylcyclohexane-carboxylic acid), p-paraaminomethylbenzoic acid (pAMBEC), p-benzylamine-sulfuric acid, o-M-acetyl -lysine-methyl ether, cis/trans-4-aminomethylcyclohexane-carboxylic acid (AMCGCC), trans-4-aminomethylcyclohexane carboxylic acid (AMCGCC), and 4-aminomethylbicyclo-2,2,2-octane carboxylic acid (AMCGCC). An effective amount of a fibrinolysis inhibitor or a mixture of one or more fibrinolysis inhibitors may also be used.

Thus, the combination of two or more fibrinolysis inhibitors can potentially increase the therapeutic effect while reducing toxicity, because fibrinolytics can have different pathways of catabolism. In addition, the presented method and composition of fibrinolysis inhibitors can be combined with ascorbate. As used herein, the term "ascorbate" includes any pharmaceutically acceptable salt of ascorbate, including sodium ascorbate, as well as ascorbic acid directly. Other substances,

used for diseases can also be introduced in parallel, including antioxidants, such as tocopherol, carotene, selenium, M-acetylcysteine, probucol and similar substances: vitamins; provitamins; and trace elements. Although ascorbate can be used alone due to its stimulating effect on collagen synthesis, the combination of ascorbate with at least one of the fibrinolysis inhibitors and an antioxidant in doses (kilogram of body weight/day) as shown in Table 1 is preferred for the treatment of pre-morbid conditions of degenerative diseases It should be noted that Table 1 shows different concentration ranges for each ingredient, depending on whether the composition is to be administered orally or parenterally. The difference in dosages is reflective of the type and severity of the disease. So this will be done if the subject has an established diagnosis in the advanced stage of a specific degenerative disease, dosages in the higher range can also be used. However, if it is desirable to prevent collagen degradation before the onset of serious symptoms, dosages in the lower end of this range can be used. Alternatively, a pharmaceutical composition identical to that described but allowing ascorbate can be used. In those cases where ascorbate and a fibrinolysis inhibitor are used in the same composition, they may simply be mixed together or may be chemically linked using art-known synthetic methods in which the ascorbate and inhibitor are covalently linked. bound, or form ionically bound salts. For example, ascorbate can be covalently linked to lysine, other amino acids, or ε-aminocaproic acid linked by an ether bond.

Ascorbyl ε-aminocaproate or ascorbyl-aminomethylcyclohexane-carboxylic acid are such examples. In this form, ascorbate may also be particularly effective in preventing the formation of unwanted lipid peroxides.

Ascorbate and fibrinolysis inhibitors can be used alone or in any combination with other agents used in cancer chemotherapy. These agents include, but are not limited to: compounds from the groups of antibiotic derivatives, antiestrogens, antimetabolites, hormones, cytostatics or steroids, and combinations thereof. In the case of oral administration, pharmaceutically acceptable, otherwise inert, carriers may be used. Thus, in the case of oral administration, the active component can be prescribed in tablet dosage form. The tablet may contain binding agents such as tragacanth, corn starch or gelatin; disintegrators such as alginic acid, and/or lubricants - magnesium stearate. If it is desirable to prescribe it in the form of a liquid dosage form, use corrigents of taste and smell.

If the administration is by parenteral injection in an isotonic solution, phosphate buffer solution or the like can be used as a pharmaceutically acceptable carrier. In addition, proline, its salts or synthetic analogues of proline can be used in the prevention and treatment of the pathological conditions described in this patent. In addition to its role as an inhibitor of IR(a), proline is also required as an amino acid component for protein synthesis. Collagen and other extracellular matrix proteins are particularly rich in lysine and proline, accounting for approximately 2595% of the total collagen mass.

While proline, unlike lysine, can be synthesized in the body, the rate of synthesis of this amino acid is suboptimal, especially in the case of chronic diseases. In these pathological conditions, when excessive collagen degradation lasts more than months or even years, the synthesis of collagen and other extracellular matrix molecules depends on the availability of proline and becomes a critical factor determining the optimal production of new collagen and, thus, restraining the progression of diseases. Therefore, it is necessary that an adequate amount of proline, its salt and analogues be sufficient in the body.

The desirability of using fibrinolysis inhibitors in the treatment of cancer, angiohematoma and other diseases will depend to some extent on the general state of health of the subject, especially in relation to hyperfibrinolytic conditions. Most fibrinolysis inhibitors (except lysine) are clinically used to treat such conditions. As a result, monitoring of coagulation and fibrinolytic systems is recommended before and during treatment. It should be noted that hemostatic complications are unlikely because these fibrinolysis inhibitors are general protease inhibitors that are also indicated as coagulants. Aoki, M. and others. (1978) Viosa 52:1-12.

Long-term administration of fibrinolysis inhibitors requires prescriptions in which fibrinolysis inhibitor dosages are in the lower dosage ranges given in Table 1. As mentioned above, ascorbate is known to stimulate prostaglandin and prostacyclin synthesis and reduce thromboxane levels, thus exerting an antiaggregation effect. These properties may be particularly desirable in combination with the use of fibrinolysis inhibitors to counteract the potential coagulation side effect.

Ascorbate and the fibrinolysis inhibitors described above can be administered separately. Further optimization of the therapeutic effect can be obtained by using a controlled release formulation to achieve constant serum concentrations of the agent over time.

Table 1

Dosage of components in the compositions of this invention oral administration parenteral administration

Ascorbate: 5-500mg/kg/weight/day 25-2500mg/kg

Amkk 1-1500mg/kg/weight/day

Tranexamova 1-5B0Omg/kg/weight/day acid

Pambk 1-1500mg/kg/weight/day

Lysine

Proline 1-1500mg/kg/weight/day

Antioxidants:

Tocopherol 0.1-500 IU/kg/weight/day

Carotene 0.1-10,000 IU/kg/weight/day

Mm-acetylcysteine 01.-5000mg/kg/weight/day

As for Fig. 3, another aspect of the invention is the discovery that Ir(a) is a co-regulator of plasmin-induced proteolysis, critically involved in tissue transformation and repair. Because of its homology to plasminogen and plasmin, it is a physiological competitive inhibitor of plasmin-induced proteolysis. In addition, apoprotein (a) has more than 100 disulfide bonds and therefore functions as an antioxidant like other protein-thiols.

All publications and patent applications cited in this specification are incorporated by reference. It will now be apparent that the compositions and therapeutic methods of the present invention for the prevention and treatment of pathological conditions associated with degeneration of the extracellular matrix have shown improvement over known methods, and it should be understood that although some preferred embodiments have been disclosed, illustrated and described above, other implementation options are possible if they do not go beyond the scope of the invention disclosed herein. Therefore, the scope of legal protection of this invention is determined by the following formula.

Ppvamine-induced proteolysis during ovulation

Foriculconi

Hormones of children

Dcutivator t

I tt PrakaNogM

Prokalalenaudtttnv GG

Kaleganett Degradation of tet Srvupyatsiya shyaksnniha.

Fig. 1

The mechanism of the development of khronoba:

Pplasma and vypnoradical-induced degradation of the cellular matrix urni takt,

Я Aktapovanny macrofats! Bucks cell

S Miresna-transdyurmysevna katzyna ra

Free dktivatoy relative pi plozmyvnhenu

Um, right

And and and. p

H GEDAVMIN. Pzefeelelennya Pidvaminogen iespatna satie KopagennVOZ

Shin LIN

Fig, 2

Functions of Apoprotein (a) and mechanisms of disease regression

IY and Ayetivevanikhy macrotskhag and gakose kpitina. c Virus-tranformed cell song ra AH), KK

Vilni Shsh-o Akhtuvator of the radical / u

Zh Grizmin Shzhnynia Plvaminotek and and and

Y pProakopaganama nt deputy ek Koijen

Fig. WITH

Therapeutic synergism of oxacarbate and protein inhibitor of disease regression

And the dilated macrophage

Cancer wedge! and !

I Viral-traneform cell y / r; Ascorbate Kh

Vtem: I Actinator plasminogen radicals in ra ry rai

Plvamin lashyujyama DlazMnOgaN x and i

With and and

It's about procopagenazatttti

And Niain and braided Kopag AKC are used by Kopag AMCEK

Fig. 4

Claims (7)

1. A pharmaceutical composition for the treatment of collagenase-related degenerative diseases, comprising at least one collagenase fibrinolysis inhibitor and ascorbate in therapeutically effective amounts, and a pharmaceutically acceptable carrier, provided that the collagenase fibrinolysis inhibitor is not lysine or a pharmaceutically acceptable salt thereof . 2. The composition according to claim 1, characterized in that said ascorbate is selected from the group consisting of pharmaceutically acceptable salts of ascorbate, ascorbic acid and mixtures thereof. 3. The composition according to claim 1, which is characterized by the fact that the said collagenase fibrinolysis inhibitor is selected from the group consisting of 5-aminocaproic acid, tranexamic acid, p-aminomethylbenzoic acid, p-benzylaminesulfuric acid, 5-M-acetyl-lysine-methyl ester, cis/trans-4-aminomethylcyclohexanecarboxylic acid-(I), trans-4-aminomethylcyclohexanecarboxylic acid and 4-aminomethylbicyclo-2,2,2-octanecarboxylic acid. 4. The composition according to any one of claims 1-3, which is characterized by the fact that it additionally contains at least one compound selected from proline, its pharmaceutically acceptable salt or a synthetic analog. 5. The composition according to any one of claims 1-4, which is characterized in that it additionally contains at least one antioxidant. 6. The composition according to claim 5, which is characterized in that said antioxidant is selected from the group consisting of tocopherol, carotene, selenium, M-acetylcysteine and probucol. 7. The composition according to any one of claims 1-6, which is characterized by the fact that it additionally contains at least one active agent selected from the group consisting of vitamins, provitamins and trace elements used in the treatment of collagenase-related diseases.