ES2407089B1 - PENTACYCLIC TRITERPENS TO USE IN THE PREVENTION OR TREATMENT OF ACUTE OR UNDERWARD MIOCARDITIS - Google Patents

Abstract

Pentacyclic triterpenes for use in the prevention or treatment of acute or subacute myocarditis. # Use of a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory myocardial disease, preferably acute or subacute myocarditis, to prevent or treat a disease that occurs with the appearance of calcium salt deposits, to prevent or treat a disease that occurs with fibrotic accumulation. Preferably, said pentacyclic triterpene is oleanolic acid. # As well as a pharmaceutical or nutraceutical composition comprising a triterpene together with at least one pharmaceutically acceptable adjuvant and / or vehicle or, when the composition is nutraceutical, an adjuvant and / or nutraceutically acceptable carrier, to treat or prevent an inflammatory myocardial disease, preferably acute or subacute myocarditis, to prevent or treat a disease that occurs with the appearance of calcium salt deposits, to prevent or treat a disease that occurs with fibrotic accumulation. Preferably, said pentacyclic triterpene is oleanolic acid.

Description

PENTACICLIC TRITERPENS TO USE IN THE PREVENTION OR TREATMENT OF ACUTE OR UNDERWARD MIOCARDITIS

SECTOR OF THE TECHNIQUE

Use of a pentacyclic triterpene for the preparation of a therapeutic agent for the treatment of diseases of inflammatory origin of the myocardium and especially for protection against acute or subacute myocarditis and the alterations associated with them.

Likewise, the present invention also relates to the use of a pentacyclic triterpene for the preparation of a nutraceutical composition to prevent diseases of inflammatory origin of the myocardium and especially for the

protection against acute or subacute myocarditis and alterations associated with them.

STATE OF THE TECHNIQUE

Myocardial abnormalities - myocarditis - together with cardiomyopathies are the main cause of heart failure in patients under 40 years of age or young athletes (Eva Laraudogoitia and Ignacio Díez. Rev Esp Cardiol. Supl.

2006; 6:21 E-9E; Gupta S et al. Nat Clin Pract Cardiovasc Med. 2008; 5: 693-706).

Myocarditis is a non-ischemic inflammatory process of the heart muscle, and according to its evolution it can be acute, subacute or chronic. It should be noted that some patients recover spontaneously and completely and there are even subjects in whom the process goes unnoticed and resolves itself, without any sequela or symptom. The clinical presentation varies from unspecific systemic symptoms to sudden and unexpected death. About 10-20% of people with histological evidence of myocarditis develop the

chronic disease that leads to dilated cardiomyopathy (MeO) (Feldman AM et al. N. Engl J Med. 2000; 343: 1388-98), which is characterized by ventricular hypertrophy and causes heart failure leading to transplantation or death ( Theleman KP et al Am J Cardiol. 2001; 88: 1078-83).

The etiology and clinical presentation of myocarditis is varied and difficult to assess. Infectious agents, usually entrovirus, Coxsackie 83 (CVB3), or adenovirus, as well as toxins and drugs are the major inducers of the disease (Woodruff FJ. Am J Pathol. 1980; 101: 425-84; Burke M. Myocarditis.

10 Current Diagnostic Pathology. 1994; 1: 216-225).

The differential diagnosis between myocarditis in which cardiac damage is produced as a consequence of the activation of the immune system, and ischemia cardiomyopathy in which myocardial abnormalities are due to

15 An imbalance between myocardial oxygen supply and demand as a result of coronary pathologies has been possible with the new cardiac magnetic resonance imaging (MRI) techniques. Late gadolinium uptake (CTG) is a new technique used in CMR that detects areas of necrosis and myocardial fibrosis with high spatial resolution and reflects "true" changes

20 demonstrated by pathological anatomy, both in animal models and in diseases in humans, exactly. When necessary, the optimized pulse sequence is used to reflect differences in the relaxation time T2 spin-spin. Thus, while patients with acute infarction have early subendocardial segmental defects in perfusion with

25 RMC, with the corresponding CTG segmenting it subendocardial or transmural in a vascular distribution, those presenting myocarditis do not have early defects and focal or diffuse CTG not segmental or subendocardial (Sechtem U et al. Heart. 2007; 93: 1520-7).

The best animal model available to study myocarditis is experimental autoimmune myocarditis, a non-ischemic inflammatory disease of the heart muscle in rodents, which shares clinical, pathogenic and histopathological characteristics with human myocarditis. Experimental autoimmune myocarditis

5 (MAE) is a model of post-infectious myocarditis and cardiomyopathy that is induced in strains of mice susceptible to inoculation with CVB3 (Kawai Ch. Circulation. 1999; 99: 1091-1100; Esfandiarei M and McManus BM. Annu Rev Pathol Mech Ois. 2008; 3: 127-55.) Or by immunization with peptides derived from the myosin to cardiac heavy chain (MyHC-a) (Pummerer eh L, et al. J

10 Clin Invest. nineteen ninety six; 97: 2057-2062). Myocarditis induced by immunization with cardiac myosin or its peptide provides an experimental virus-free model. This model can be developed in the Balb / c strain and is characterized by the presence of giant cells in the lesions and infiltrate of eosinophils, maeralagos, C04 + T cells, with some C08 + T cells and 6 6220+ in the

15 myocardium In addition, it has been observed that Th2 cytokines, especially IL-4, as well as Th1, TNF "or IL-12, and Th17, IL-17 cytokines have a crucial role in the development of the disease. At later times, the Low inflammation and myocardial fibrosis becomes a characteristic of the disease.This phase is associated with systolic and diastolic dysfunction.In this experimental model, it

20 considers that the acute phase of the disease is 21 days, the subacute phase takes place between days 40 and 48, and the chronic phase between days 65 and 70.

The treatment of myocarditis is a great challenge, mainly due to the complex clinical characteristics of the disease, and it can be classified as symptomatic and specific. Symptomatic treatment is basically supportive and is applied to patients with heart failure of any grade. The specific and standardized treatment is much discussed and not yet defined. The response to treatment depends on the specific cause of the disease, the severity of irreversible tissue alterations at the onset of

treatment and the ability of the myocardium to compensate for these alterations. In the best case with the specific treatment, a rapid progression of the disease can be stopped, but no significant improvement in ventricular function has been achieved. Generally studies in models

5 murine experiments support the recommendations of the treatments, and several clinical trials have been carried out with the aim of defining clinical intervention guidelines.

Initial studies to evaluate the efficacy of drug treatment

10 non-specific anti-inflammatory or immunosuppressive drugs ruled out their widespread use, given that they did not show a significant beneficial effect or in some cases side effects appeared (Masan JW et al. N Engl J Med.

nineteen ninety five; 333: 269-275; Parrillo JE et al. N Engl J Med. 1989; 321: 1061-1068),

the importance of the moment of starting its application has even been discussed: 15 may be useful in late stages but are contraindicated in the acute phase (first two weeks of myocarditis) for increasing myocardial damage

(Rezkalla SH and Kloner RA. Am Heart J. 1989; 117: 706-8; Schultz J. et al, Mayo Clin Proc. 2009; 84: 1001-1009; O'Connell JB et al. Circulation. 1986; 73: 353-9; Kilboume ED. Et al. J Clin Inves !. 1956 Apr; 35 (4): 362-70.). It has been realized

20 other studies that include various immunomodulatory agents: immunosuppressants (Parrillo JE Circulation. 2001; 104: 4-6, Wojnicz R et al. Circulation 2001; 104: 39-45; Frustaci A et al., Circulation 2003; 107: 857- -863.), Immunoadsorption (Staudt A and Felix SB. Transfus Apher ScL 2007; 37: 187-90.), And interferon (Mirié M. et al. Eur Heart J. 1995; 16 Suppl 0: 150-2.) . Even so, the

The approach to the management and treatment of cardiomyopathies is a permanent subject of discussion since most of the different options have not been studied through extensive clinical trials and only recently has the etiology of the disease been considered as a variable to be taken into account. at the time of the treatment choice.

Therefore, it is a challenge to standardize the treatment of myocarditis, as well as to explore and identify new molecules that can stop the progression of histological and clinical deterioration, at a stage that has not yet occurred.

5 generalized and irreversible myocardial injury, or even that can improve cardiac function.

Thus, pharmacological research is focused on finding novel therapeutic agents, and in recent years the Vegetable Kingdom is proving to be an excellent source of resources for the discovery of these new compounds. In addition, multiple lines of research have been developed that aim to analyze the beneficial effects of the natural compounds present in plants, in order to relate the composition of the diet to the development / modulation of diseases of immuno / innamatory base and to define Possible nutritional therapeutic strategies. In this context, oleanolic acid is a pentacyclic triterpene from the group of oleananos present in numerous plants (Jager S, et al. Molecules 2009, 14: 2016-2031), some of them frequently used in traditional medicine in various countries. It is in the form of free acid or as saponin aglycone

20 triterpenoids (Liu J., J Ethnopharmacol 1995, 49: 57-68), highlighting its presence in the Olea europaea.

There are several publications since 1906 that recognize oleanolic acid as a permanent constituent of olive leaves and fruits. In the olive tree,

25 It is found mainly in leaves, green fruits, especially in the cuticle, and in the residual lye from olives dressing. It is also found in ripe olives, virgin olive oil and pomace oil.

There are numerous publications on the therapeutic properties of triterpenic compounds and in particular on oleanolic acid there are multiple studies that collect their biological and pharmacological activities. Between them

Highlights: hepatoprotective activity (Líu Y et al. Toxicology Letlers 1998, 95:77

5 85), anti-inflammatory (Sung HY et al. Nutr Res. 2010; 30: 831-9; Takada K, et al. Phytomedicine. 2010; 17: 1114-9; Mañez S, et al. Eur J Pharmacol. 1997, 334: 103-5), immunomodulatory (Chiang LC, et al. Planta Med. 2003; 69: 600-4), antitumor (Martin R. et al. Cancer Res. 2007; 67: 3741-51, Ju-Hong Feng , et al. The Open Natural Products Journal; 2009, 2: 48-52), neuroprotective (Martin R et

10 al. Biochem Pharmacol. 2010; 79: 198-208.), Vasodilator (Rodriguez Rodriguez R. et al. Br J Nutr. 2004; 92: 635-42), cardioprotective (Rodriguez Rodriguez Rodriguez, et al. Br J Nutr. 2009; 102: 1435-44), hypoglycemic (Sato H. et al. Biochem Biophys Res Commun. 2007; 362: 793-8), antidiabetic and lipid lowering (Gao O et al. Phytother Res. 2009; 23: 1257-62).

The present invention is related to the search for new treatments against inflammatory myocardial diseases, preferably myocarditis, and describes a new pharmacological application of oleanolic acid as an agent capable of significantly attenuating or preventing

20 clinical signs, inflammatory and immune system alterations (immuno-inflammatory alterations), as well as fibrotic lesions and dystrophic calcifications, associated with myocarditis.

DESCRIPTION OF THE INVENTION

Brief Description of the Invention

The object of the invention is the pharmacological application of a pentacyclic triterpene in the therapy of myocardial diseases such as for example

myocarditis The invention shows the ability of natural pentacyclic triterpenes present in the olive cuticle and olive leaf, as well as in oils where these fractions have an important presence (olive pomace oil), to attenuate in a way Remarkable clinical signs of myocarditis. This action is associated with a normalization of the heart size normalized by body weight, a decrease in the levels of cardiac damage markers, and a regulation of the immune and inflammatory response both at systemic and tissue level, characterized by a decrease in both pro-inflammatory cytokine levels as in the extravasation of immuno-inflammatory cells to the myocardium and by an increase in the levels of anti-inflammatory cytokines. It also manifests a protective effect against the formation of pathological fibrotic calcifications and accumulations. Therefore, it is an object of this invention to provide a pharmaceutical and / or nutraceutical composition with applications for treating the

15 myocarditis

Likewise, the present invention also relates to the use of a pentacyclic triterpene, to prevent or treat a disease that occurs with the appearance of deposits of calcium salts or with fibrotic accumulation.

In one of the embodiments of this invention, an acidic pentacyclic triterpene, preferably isolated from the olive, is described as oleanolic acid (3p-hydroxyolean-12-en-28-oic acid) represented by the chemical formula presented below:

HO # "

Acid or / Eanolic

as an effective component for the preparation of drugs or pharmaceutical compositions and nutraceutical compositions for treatment

or the prevention of myocardial diseases, preferably myocarditis.

Likewise, a pharmaceutical or nutraceutical composition useful for the treatment of an inflammatory myocardial disease, preferably myocarditis, is part of the invention to prevent or treat a disease that occurs with the appearance of calcium salt deposits, or to prevent or

10 treating a disease that occurs with fibrotic accumulation, which comprises an effective amount of a pentacyclic triterpene together with at least one adjuvant and / or acceptable pharmaceutical carrier.

Also within the scope of the invention is a pharmaceutical composition or

15 nutraceuticals in which the natural pentacyclic triterpene is oleanolic acid, as well as the use of the pharmaceutical composition in the treatment of a human being affected by an inflammatory myocardial disease, preferably myocarditis, which comprises the administration of said composition that reduces disease progression

Detailed description of the invention

A first object of the present invention relates to the use of a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory myocardial disease.

In this patent application, the term "pentacyclic triterpene" includes in addition to the free form of the compound, any one of its isomers, as well as any one of its pharmaceutically and biologically acceptable salts thereof (such as sodium salt, potassium salt, salt). calcium or magnesium salt, among others), solvates (complexes of variable stiochemistry with suitable solvents - in which it is reacted or those that precipitate or crystallize - include, but are not limited to, water, methanol, ethanol and acid acetic and similar) and chemical derivatives or prodrugs, suitable for inclusion in a pharmaceutical or nutraceutical composition

Likewise, "isomers" means both individual sórremos and mixtures thereof, in particular both enantiomers or individual diastereoisomers are understood as mixtures thereof. All of them can be obtained by conventional techniques such as separation from a mixture containing them.

On the other hand, the term "prodrug" as used herein includes any compound derived from a pentacyclic triterpene, for example, esters, including carboxylic acid esters, amino acid esters, phosphate esters, metal salt sulphonate esters, etc., carbamates, amides, etc., which, when administered to an individual, is capable of providing, directly or indirectly, said pentacyclic triterpene in said individual. Advantageously, said derivative is a compound that increases the bioavailability of pentacyclic triterpene when administered to an individual or that enhances the release thereof in a biological compartment. The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.

The pentacyclic triterpene used to make a pharmaceutical composition

or nutraceutical to prevent or treat an inflammatory myocardial disease as described in this patent application, it can be a natural pentacyclic triterpene, present in nature, or a synthetic pentacyclic triterpene, obtained by chemical derivation from a natural pentacyclic triterpene . The preparation of said synthetic pentacyclic triterpene can be carried out by conventional methods known to those skilled in the art.

As used in the present invention, the term "inflammatory myocardial disease" refers to a pathology that involves cardiac damage, fibrotic accumulations, has an inflammatory component and an uncontrolled immune response, and more specifically refers, by way of title.

10 illustrative and without limiting the scope of the invention, to myocarditis, dilated cardiomyopathy, rheumatic fever, cardiac pathology of Chagas disease and collagen diseases with heart injury.

In a preferred embodiment, the present invention relates to the use of

15 a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory myocardial disease selected from the group consisting of myocarditis, dilated cardiomyopathy, rheumatic fever, cardiac pathology of Chagas disease and collagen diseases with a lesion of heart.

In an even more preferred embodiment, the invention relates to the use of a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory myocardial disease that is myocarditis. Preferably, this disease is acute myocarditis or

25 subacute.

During the acute phase of the disease, an imbalance between proinflammatory and anti-inflammatory cytokines and an increase in adhesion molecule expression have been observed, with an intimate relationship between

severity of the clinical picture and their humoral levels (Matsumori A. Eur Heart J. 2002 (supplement 1) 142-145). The histopathological pattern is characterized by the mild infiltration of inflammatory cells and the expression of antigens of the major histocompatibility complex (HLA), with the consequent 5 myocyte necrosis and disorganization of its cytoskeleton. In the subacute phase there is an increase in cellular infiltration in the myocardium, especially by T cells. This phase is accompanied by the presence of circulating autoantibodies against myosin and other cardiac antigens, and can lead to heart failure and death associated with MeO. . Thus, a large variety of autoantibodies that react against cardiac autoantigens of the plasma membrane, cytoskeleton or internal structures have been identified in many patients with MeO (Nou! Sias M. et al. Eur Heart J. 2002 (suppl. 1 ) 154-162.). In the subacute form and particularly in the third stage of the disease, chronic myocarditis, interstitial fibrosis develops, replacing the fibers

15 dead muscles, and myofiber hypertrophy resulting in contractile dysfunction and ventricular dilation. Eventually, the presence of calcifications in the wall of the ventricles has also been observed in myocarditis (Oka K et al. Virchows Arch. 2005, 446: 259--264).

Additionally, the present invention also relates to the use of a pentacyclic triterpene as defined in this patent application, to prevent or treat a disease that occurs with the appearance of calcium salt deposits.

In a further aspect, the present invention also relates to the use of a pentacyclic triterpene as defined in this patent application, to prevent or treat a disease that occurs with fibrotic accumulation.

In another preferred embodiment, the present invention relates to the use of a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory myocardial disease, preferably acute or subacute myocarditis, a disease that occurs with

5 the appearance of deposits of calcium salts or a disease that occurs with fibrotic accumulation, characterized in that pentacyclic triterpene is a natural compound that is selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid, maslinic acid, uvaol, erythrodiol and any one of its isomers, salts, solvates or prodrugs. Preferably, triterpene

10 pentacyclic is oleanolic acid (3a-hydroxyolean-12-en-28-oic acid).

In another preferred embodiment, the present invention relates to the use of a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory myocardial disease,

15 Preferably acute or subacute myocarditis, a disease that occurs with the appearance of deposits of calcium salts or a disease that occurs with fibrotic accumulation, characterized in that pentacyclic triterpene is a natural compound belonging to the family of oleananos. Preferably, the pentacyclic triterpene is oleanolic acid or any one of its isomers,

20 salts, solvates or prodrug, it being especially preferred that the pentacyclic triterpene be oleanolic acid (3a-hydroxyolean-12-en-28-oic acid).

Oleanolic acid is found in nature as a free acid or as an aglycone of triterpenoid saponins (Liu J., J Ethnopharmacol1995, 25 49: 57-68) and has been isolated from different plant species, highlighting its

presence in Ofea europaea.

There are several publications since 1906 that recognize oleanolic acid as a permanent constituent of olive leaves and fruits. In the olive tree, it is found mainly in leaves, green fruits, especially in the cuticle, and in the residual lye coming from the dressing of the olives. It is also found in ripe olives, virgin olive oil and pomace oil.

Another object of the present invention is a pharmaceutical or nutraceutical composition comprising a pentacyclic triterpene together with at least one adjuvant and / or a pharmaceutically acceptable vehicle or, when the composition is nutraceutical, an adjuvant and / or a nutraceutically acceptable vehicle. , to prevent or treat an inflammatory myocardial disease, a

10 disease that occurs with the appearance of deposits of calcium salts or a disease that occurs with fibrotic accumulation.

The pentacyclic triterpene comprised in the pharmaceutical or nutraceutical composition of the invention has meaning defined above in this patent application.

In the sense used in this description, the term "pharmaceutically acceptable carrier and / or adjuvant" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the

20 preparation of pharmaceutical administration forms and includes adjuvants, solids or liquids, solvents, surfactants, etc.

In the sense used in this description, the expression "vehicle and / or nutraceutical acceptable adjuvant" refers to those substances, or

A combination of substances, known in the nutraceutical sector, used in the preparation of pharmaceutical administration forms and includes adjuvants, solids or liquids, solvents, surfactants, etc.

If desired, said pharmaceutical composition may further contain one or more therapeutic agents that, if necessary, enhance the therapeutic action of said pentacyclic triterpene compound or increase its spectrum of action. The additional therapeutic agent (s) are agents for treatment.

5 supporting the clinical symptoms of myocarditis. These therapeutic agents may be, for example, an antiarrhythmic drug, an inonotropic, an anticoagulant, an angiotensin-renin-aldosterone system blocker, a calcium-antagonist beta-adrenergic blocker or a diuretic. Non-limiting examples of these agents include captopril, enalapril, spironolactone,

10 furosemide, dobutamine, dopamine, losartan, calvedilol, bisoprolol or amiodarone.

Additionally, if desired, said nutraceutical composition may also contain one or more active ingredients that, if necessary, enhance the therapeutic action of said pentacyclic triterpene compound or that increase its

15 spectrum of action. These active ingredients can be for example active ingredients for the treatment of the pathogenic mechanisms of myocarditis. Non-limiting examples of these active ingredients may be nutritional supplements with antioxidant and / or anti-inflammatory properties and include carotenoids, flavonoids, polyphenols, fatty acids.

20 polyunsaturated omega-3, terpenoids, indoles, fibers, phallic acid, lipoic acid and lecithin.

The triterpene pentacyclic compound will be present in the pharmaceutical composition in a therapeutically effective amount, that is, in an amount appropriate to exert its therapeutic effect. In a particular embodiment, the pharmaceutical composition provided by this invention comprises between 0.01% and 99.99% by weight of a pentacyclic triterpene compound, in the form of a free compound, or one of its isomers, salts, solvates or prodrugs, and it can be presented in any pharmaceutical form of appropriate administration in

function of the route of administration chosen, for example, oral, parenteral, intraperitoneal or topical. A review of the different pharmaceutical forms of drug administration and their preparation procedures can be found, for example, in the Galician Pharmacy Treaty, C. Faulí ¡Trillo, 1st

5 edition, 1993, Luzán 5, SA de Ediciones.

Additionally, the pentacyclic triterpene compound will be present in the nutraceutical composition in an amount appropriate to exert its preventive effect. In a particular embodiment, the nutraceutical composition

10 provided by this invention comprises between 0.01% and 99.99% by weight of a pentacyclic triterpene compound, in the form of a free compound, or one of its isomers, salts, solvates or prodrugs, and can be presented in any nutraceutical form of proper administration such as liquid solutions, suspensions or solid forms such as tablets, capsules, powder, etc.

In a preferred embodiment, the pharmaceutical or nutraceutical composition as described in this patent application is characterized in that the triterpene pentacycle is a natural compound that is selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid, maslinic acid, uvaol ,

Erythrodiol and any one of its isomers, salts, solvates or prodrug. Preferably, the pentacyclic triterpene is oleanolic acid or any one of its isomers, salts, solvates or prodrug, it being especially preferred that the pentacyclic triterpene is oleanolic acid (3a-hydroxyolean-12-en28-oic acid).

In another preferred embodiment, the pharmaceutical or nutraceutical composition as described in this patent application, preferably when the pentacyclic triterpene is oleanolic acid, is characterized in that it can be used to prevent or treat acute or subacute myocarditis, since it reduces the

progression of said disease. Additionally, the pharmaceutical or nutraceutical composition as described in this patent application is characterized in that it can be used to prevent or treat a disease that occurs with the appearance of calcium salt deposits, or to prevent or treat a

5 disease that occurs with fibrotic accumulation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Inhibition of heart weight gain (normalized against

10 body weight) caused by the progression of the disease due to the effect of oleanolic acid, administered daily from the moment of the induction of the disease -OA 1 - until day 21 post-immunization. A. Male mice, and B. Female mice. C, healthy animals. MAE, sick animals. MAEoA1, sick animals treated with oleanolic acid (OA) from the moment of

15 induction of the disease.

Figure 2: Inhibition of increased heart weight (normalized against body weight) caused during the progression of the disease due to oleanolic acid, administered daily from the moment of induction of

20 the disease (A) or when myocarditis is established (from day 21 post-induction) (6) until day 21 and 65 post-immunization. Modulation of the serum levels of the BNP (C, O) peptide by the action of oleanolic acid, measured by commercial ELlSA kit. e, healthy animals. COA, healthy animals treated with OA. MAE, sick animals. MAEoA1, sick animals treated with OA

25 from the moment of disease induction. MAEoA2 • sick animals treated with OA when myocarditis is established (from day 21).

Figure 3: Inhibition of cell infiltration, fibrotic accumulations and calcification in the myocardium due to oleanolic acid. A.-Images of healthy mouse heart and mouse heart with MAE in which calcifications are observed (Red arrows). B.-Histological study of cardiac tissue on day 65 post-immunization analyzed by staining with Eosin & Hematosilin (E&H), Alizarin Red (RA) and Sirius Red (RS). MAE, sick animals. MAEoAl, sick animals treated with OA at the time of disease induction. MAEoA2, sick animals treated with OA with established myocarditis.

Figure 4: Modulation of pro-inflammatory protein expression in serum (A), and cardiac tissue (B) by oleanolic acid, measured by commercial ELlSA kit, on day 21 after disease induction. e, healthy animals. OA, healthy animals treated with OA. MAE, sick animals. MAEoA1, sick animals treated with OA from the moment of disease induction.

Figure 5: Modulation of the expression of anti-inflammatory proteins in cardiac tissue (A, B), and serum (C, O) by oleanolic acid, measured by commercial ELlSA kit. C, healthy animals. COA, healthy animals treated with OA. MAE, sick animals. MAEoA1, sick animals treated with OA from the moment of disease induction. MAEoA2, sick animals treated with OA when myocarditis is established (from day 21).

Figure 6: Modulation of serum cardiac anti-myosin antibodies by oleanolic acid, measured by ELlSA kit. e, healthy animals. COA1, healthy animals treated with OA. MAE, sick animals. MAEoA1, sick animals treated with OA from the moment of disease induction.

.

EXAMPLE OF REALIZATION

The invention describes in one of its embodiments how oleanolic acid is not only an agent capable of significantly attenuating clinical signs,

5 immuno-inflammatory, profibrotic and calcifications of experimental autoimmune myocarditis induced in Balb / c mice (animals 6-8 weeks of age), animal model of myocarditis, but significantly improves its evolution (see Example 1) :

* Delay in the start of the MAE through i.p. 50 mg / kg weight 10 oleanolic acid (OA1)

,., Improvement of the disease state MAE by administration Lp. 50 mglkg weight of oleanolic acid (OA2)

These actions are manifested through the following effects: i)

15 inhibition in the appearance of cardiac hypertrophy, ii) decrease in the levels of cardiac damage markers, iii) decrease in fibrotic accumulations and calcifications on the myocardium, iv) decrease in cell infiltration, v) decrease in the expression of proteins with relevance in proinflammatory processes, vi) increased protein expression with relevance in

20 anti-inflammatory processes and vii) decrease in circulating cardio-specific autoantibodies.

Therefore, the results suggest that the use of this natural triterpene has beneficial protective effects both in cardiac remodeling and in

25 the immuno-inflammatory and fibrotic component related to the evolution of the MAE, which may lead to an improvement of the clinical symptoms associated with myocarditis in the MAE model, as well as other cardiac muscle alterations.

The new pharmacological application of oleanolic acid as a therapeutic agent of inflammatory diseases of the myocardium and specifically myocarditis has been observed in an acute model of the disease, experimental autoimmune myocarditis induced in Balb / c mice (mice 6-8 weeks of age ).

Example 1.-Oleanolic acid improves the signs and symptoms of animals with MAE

In the tests, ten animals are used per group. MAE is induced in the manner described (Pummerer Cl, et al. J Clin Invest. 1996; 97: 2057-62) in Balb / c mice by administration of a peptide derived from the cardiac myosin-a heavy chain ( MyHC-061 4-629). Immunization is carried out with 200 g of the MyHC-C1s14-629 peptide in Freund's complete adjuvant, containing 5 mg / ml of Mycobacterium tuberculosis H37Ra. Mice are immunized by a subcutaneous injection of this emulsion on day O and 7. In addition, on day O, 500ng / 300 ~ 1 of Bordetella Perfussis toxin are also administered intraperitoneally. The administration of 50 mg / kg of oleanolic acid intraperitoneally is done once a day, beginning: 1.-the day of induction of the MAE (OA 1), and continuing until day 21 (acute phase) or 65 ( chronic phase) after the induction of the MAE, and 2.-after 21 days of induction of the MAE (OA2), when the acute or inflammatory phase has been reached, and continuing until day 65 after the induction of the MAE, when the chronic stage was reached. The clinical evolution of the animals (weight) is observed daily using a registration system and compared with a group of MAE animals to which the vehicle is administered, with untreated (healthy) control animals or with (healthy) control animals that receive the same daily dose of oleanolic acid. On days 21 or 65 the animals are sacrificed and the heart is weighed, normalizing the weight with the weight of the animal. In addition, a histopathological evaluation is performed to confirm the effect of improving the condition of the

disease.

The heart weight / animal weight index of each group is represented in 5 figures 1 and 2.

In Figure 1, it is shown that both female and male animals develop the clinical signs of the disease (increased heart size, normalized against body weight) after immunization with the peptide

10 derived from the heavy chain of cardiac myosin-a (MyHC-a614-S29) and that both respond favorably to semi-prophylactic treatment with 50 mg / kg of the natural triterpenic compound, oleanolic acid (OA 1), preventing development of hypertrophy. As indicated in Figure 2A, compared to the control group (healthy), the animals of the MAE groups show a

15 progressive increase in the size of the heart at the times studied: at 21 and 65 days after the induction of the disease. On the contrary, in none of the groups treated semi-prophylactically with 50 mg / kg of the natural triterpenic compound (OA1) is this increase in heart weight observed, compared to the untreated MAE group. In addition, when oleanolic acid is

20 administered once the disease has been established (50 mglkg OA2), the severity of the MAE is also significantly attenuated, since a significant reduction in heart size is observed on day 65 post-induction, compared to the size of the heart on day 21 in the untreated MAE group (Fig. 28). In both situations, the

25 increase in cardiac muscle, observing a prevention (Fig. 1 and 2A) or recovery effect (Fig. 28). These data are in line with the serum values of the "cerebral uretic nat" peptide (BNP) (Fig. 2 e and D). BNP, which occurs in the ventricle, is considered a marker of cardiac damage and the evolution of its serum values They allow an objective monitoring of the clinical evolution of the disease, which is useful as a prognostic marker and response to the established treatment.In Figure 2, it is observed that in serum of animals with SMA there is an increase in the levels of BNP that it is observed 21 days after induction and increases on day 65, in

5 comparison with healthy control mice. This increase is significantly reduced when mice are treated with oleanolic acid, both following the OA1 (semi-prophylactic) and OA2 (therapeutic) protocol.

10 In addition, as already mentioned, myocarditis is characterized by the presence of interstitial infiltration of immune-inflammatory cells and the appearance of their products, that is, of elevated levels of proinflammatory cytokines both in cardiac muscle and circulating in plasma. . Figure 3 A shows a heart corresponding to a healthy mouse and a heart

15 corresponding to a mouse with MAE for 65 days in which calcifications are observed (red arrows). A representative histological analysis, corresponding to cardiac tissue collected on day 65 after induction of myocarditis, is shown in Figure 3B. Tissues corresponding to MAE animals show the massive presence of immuno-inflammatory infiltrate (H&E), as well as

20 of calcification foci (RA) and fibratic accumulations (RS). In contrast, when the tissues belong to animals of the MAEoA1 or MAEoA2 protocol groups, a significant decrease in the immuno-inflammatory infiltrate is observed, compared to the MAE group. Also, in the tissues of MAEoA1 or MAEoA2 mice, no calcium deposits are observed

25 nor fibrotic accumulations.

The analysis of the expression of relevant proinflammatory proteins in the MAE, has led us to study the presence and modulation of the cytokines IL-17 and TNFa on day 21 after the induction of myocarditis (acute phase). In Figure 4 it is observed that both in serum and in the cardiac tissue of animals with MAE there is an increase in the expression of these proteins, compared to healthy control mice. This increase is significantly reduced when mice are treated with oleanolic acid, both following the

5 OA1 protocol as OA2.

Regarding the expression of anti-inflammatory proteins that could have a relevant function in the resolution of the MAE, it is represented in Figure 5. Figure 5 A and B shows that both serum and cardiac tissue of

10 MAE animals treated with oleanolic acid, following the OA1 protocol, there is a noticeable increase in the expression of IL-10, compared to untreated MAE mice. This increase is observed on day 21 post-induction and is maintained (at least) until day 65. In addition, we find that in healthy animals, controls, treated with oleanolic acid, an increase also occurs

15 significant levels of cytokine IL-10. Figure 5 shows that animals with active myocarditis treated with oleanolic acid following the therapeutic OA2 protocol experience an increase in plasma and tissue levels of IL-10 similar to that found in animals treated according to OA1 protocol, semi -prophylactic.

In relation to studies aimed at characterizing the autoimmune response, antibodies against the MyHC-a peptide, in Figure 6 it is shown that in the MAE group the mice have elevated levels of IgM, IgG, IgG1 at 21 days after induction of the disease. Instead, in the group to which it is administered

25 Oleanolic acid at the time of immunization immunoglobulin levels decrease dramatically. No significant differences were found between the different groups of animals for the IgG2a isotype.

Therefore the results suggest that this triterpene contained in the pomace oil could have protective effects in relation to the immunoinflammatory response and cardiac remodeling related to the evolution of the MAE, which could lead to an improvement of the clinical symptoms associated with the

5 myocarditis, in the MAE model, as well as other cardiac disorders. In addition, since this product is a natural substance that can be isolated from the olive, this compound could be used as a complementary contribution to the diet or in pharmaceutical preparations.

10 A PREFERRED EMBODIMENT OF THE INVENTION

Experimental animals

The animals have been kept in the animal faculty of the Faculty of Medicine of

15 the University of Valladolid, in cages with chip bed, feeding them with a diet of special feed for laboratory animals, with a variable consumption depending on weight and age of animal, water ad libitum, temperature

constant of 20-240 C and exposed to a light cycle of 12h / day (8.00 a.m. -8.00 p.m.) (Council of European Communities, 1986). The study protocols have

20 were approved by the Animal Research Ethics Committee of the Faculty of Medicine of the University of Valladolid.

Reagents used

25 The following chemicals used in the experiences were

provided by Sigma Chemieal Co. (SI. Louis, MO, USA): adjuvant

Complete of Freud, M. tuberculosis H37 RA, and B. pertussis toxin. The E LISA kit

for the detection of BNP it was RayBioteeh Ine (Noreross, GA, United States) and for IL-10, IL-17 and TNFa it was from the house eomerciallBL (Hamburg, Germany).

30 The natural oleanolic acid compound was supplied by Extrasynthese (Genay

Cedex, France). The peptide derived from the cardiac myosin-a heavy chain (MyHC-aS14_629) was synthesized by the laboratory of Dr. F. Barahona (CBM, Madrid).

5 The pentacyclic triterpene, oleanolic acid was initially dissolved in dimethylsulfoxide (DMSO) to prepare a stock solution of 10.2 M. Subsequent dilutions of triterpene were also made in DMSO. The final concentration of DMSO reached (less than 0.001%) did not significantly affect the results.

MAE induction

MAE is induced in female and male mice of the Balb / c lineage of 6-8 weeks following the protocol described by (Pummerer Ch L, et al. J Clin Invesl. 15 1996; 97: 2057-62). The animals receive on day O and 7 a subcutaneous dose of a mixture of 200 g of the peptide derived from the cardiac myosin-a heavy chain (MyHC-aS14-629), Freund's complete adjuvant and 5 mg / ml M. tuberculosis H37 RA. An i.p. of 500 ng / animal of B. pertussis toxin administered twice, with an interval of 48 hours. The animals are

20 examine daily to monitor weight loss and the onset of neurological symptoms.

Experience Development

25 The protective effect of oleanolic acid was revealed by Lp injection. 50 mg / kg daily weight of pentacyclic triterpene. This experience was carried out following two experimentation protocols: i) OA1 -The injection of 50 mg / kg of oleanolic acid begins on the day of immunization of the animals.

ii) OA2 - The injection of 50 mg / kg of oleanolic acid begins in the acute phase, when myocarditis is established (approximately on day 21 post-immunization).

Histological Analysis

Representative animals of the different groups were sacrificed at 65 days after induction of myocarditis. The collected hearts were weighed and then cut transversely into three pieces. After 24 h of fixation in 2% Paraformaldehyde-PBS, the hearts were embedded in paraffin, cut with a microtome in sections of 5 J.lm thick and stained with Eosin & Hematosilin to determine the infiltration of immunoinflammatory cells, Sirius Red to determine collagen and red accumulations

Alizarin to visualize the presence of calcifications. The histological examination was performed on a Nikon Eclipse 90i microscope (Nikon Instruments Inc) connected to a DXM1200C digital camera (Nikon Instruments Inc). Between 410 sections were examined per mouse.

Determination of BNP and cytokines in serum and cardiac tissue

Protein extracts from myocardium and serum samples were obtained from animals of the different experimental groups, sacrificed at 21 or 65 days after immunization. The tissue was homogenized in a solution: DA M Nael, 0.05% Tween 20, 0.5% BSA, 0.1 mM Phenyl methyl sulfonyl fiuoride, 0.1 mM benzetonium chloride, 10 mM EDTA and 20 KI aprotinin (100 mg of tissue / ml). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 oC and in the supernatants the concentration of IL-10, IL-17 and TNFa was determined by a commercial ELlSA kit. The BNP concentration was determined exclusively in serum samples. The cytokine concentration was determined by extrapolation of a standard curve and expressed as pg / mg of total protein.

Determination of antibody levels

The serum of the animals of the different groups was collected on day 21 post-immunization and the levels of the antibodies against the peptide derived from the cardiac myosin-to heavy chain (MyHC-061 4-629) were determined using the ELlSA technique . Thus, a 96-well plate was incubated with 0.5

10 ~ g / well of the MyHC-a61 + 629 peptide in PBS at 4 'C overnight.

The next day a 5% BSA block was performed and the wells were incubated in duplicate with the serum samples diluted 1: 60 in PBS with 10% STF for 2 hours at 20'C. Next, the plate was washed and

15 added polyclonal anti-mouse antibodies conjugated with HRP (IgM, IgG, IgG1 or IgG2a) for 90 min. After washing, the enzyme substrate (TM B) was added for 15 min and the reaction was stopped with 0.1 N Hel. The absorbance at 450 nm was read. The results are expressed as the average of the optical density at 450 nm.

Statistic analysis

Data are presented as mean ± SD. The results are analyzed using the ANOVA variance test followed by the post-hoc Bonferroni analysis for

25 multiple comparisons, using the Graph Pad Prism program, version 5 (GraphPad Software Inc, La Jolla, CA). The difference between experimental values has been considered significant since P <0.05.

Claims (7)

1.-Use of a pentacyclic triterpene in the preparation of a pharmaceutical or nutraceutical composition to prevent or treat an inflammatory disease of myocardium. 2. Use of a pentacyclic triterpene according to claim 1, characterized in that the inflammatory myocardial disease is acute or subacute myocarditis. 3. Use of a pentacyclic triterpene according to claim 1, to prevent or treat a disease that occurs with the appearance of calcium salt deposits. 4. Use of a pentacyclic triterpene according to claim 1, to prevent or treat a disease that occurs with fibrotic accumulation. 5. Use of a pentacyclic triterpene according to any one of claims 1 to 4, characterized in that said compound is a triterpene 20 natural pentacyclic selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid, maslinic acid, uvaol, erythrodíol and any one of its isomers, salts, solvates or prodrug. 6. Use of a pentacyclic triterpene according to claim 5, characterized in that the pentacyclic triterpene is oleanolic acid. 7. Pharmaceutical or nutraceutical composition comprising a pentacyclic triterpene together with at least one pharmaceutically acceptable adjuvant and / or vehicle or, when the composition is nutraceutical, an adjuvant and / or vehicle 2.

nutracéulicamenle acceptable, to treat or prevent an inflammatory myocardial disease.

5

8. Pharmaceutical or nutraceutical composition according to claim 7, characterized in that the inflammatory myocardial disease is acute or subacute myocarditis.

\OR

9. Pharmaceutical or nutraceutical composition according to claim 7, to treat a disease that occurs with the appearance of calcium salt deposits. 10. Pharmaceutical or nutraceutical composition according to claim 7, for treating a disease that occurs with fibrotic accumulation3.

fifteen

11. Pharmaceutical or nullification composition according to any one of claims 7 to 10, characterized in that the natural pentacycle triterpene selected from the group consisting of oleanic acid, uric acid, betulinic acid, maslinic acid, uvaol, erythrodiol and any one of its isomers , salts, solvates or prodrug.

twenty

12. Pharmaceutical or nullification composition according to the claim characterized in that the pentacyclic triterpene is oleanolic acid. eleven ,