WO2022015141A1 - Pharmaceutical use of pirfenidone for reducing cardiac fibrosis in patients with cardiomyopathy and/or cardiac steatosis and/or covid-19 - Google Patents

Abstract

The present invention relates to the field of medicaments that reduce cardiac fibrosis and also the expression of troponin I, specifically to the pharmaceutical use containing 5-methyl-1-phenyl-2-(1H)-pyridone (Pirfenidone) as an active ingredient, alone or in combination with any other bioactive molecule, specifically for the management of patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19.

Description

PHARMACEUTICAL USE OF PIRFENIDONE FOR THE REDUCTION OF CARDIAC FIBROSIS IN PATIENTS WITH CARDIOMYOPATHY AND/OR CARDIAC STEATOSIS AND/OR

COVID-19 FIELD OF INVENTION

The present invention is related to the field of medicine, specifically to drugs that reduce cardiac fibrosis and also the expression of troponin I, specifically to the pharmaceutical use that contains 5-methyl-1-phenyl-2-(1H)-pyridone (Pirfenidone) as an active ingredient alone or in combination with any other bioactive molecule, specifically for the management of patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19.

BACKGROUND OF THE INVENTION

Cardiomyopathy, cardiac steatosis and/or cardiac fibrosis.

Chronic inflammation is a common molecular basis for several chronic diseases, such as atherosclerosis, autoimmune diseases, neurodegenerative diseases, cancer, among others. Accumulating evidence suggests that chronic inflammation also plays a crucial role in cardiac fibrosis, cardiac steatosis, and/or cardiomyopathy.

Obesity is a chronic, multifactorial and largely preventable disease that affects all types of socioeconomic groups, and is caused by an imbalance between dietary energy intake in relation to energy expenditure. Fatty acids are the main energy lipids used metabolically by cardiac tissue, accounting for approximately 70% of the heart's energy requirements under normal conditions. However, in pathological situations such as advanced hypertrophy and other cardiomyopathies, cardiac energy metabolism shifts from fatty acids to glucose for ATP (adenosine triphosphate) production. The high-fat/high-cholesterol (HFHC) diet, commonly known as the Western diet, induces obesity, which is associated with the metabolic syndrome, type 2 diabetes (DM2: diabetes mellitus 2). ), non-alcoholic steatohepatitis (NASH: Non-Alcoholic SteatoHepatitis) and cardiovascular diseases. Lipid accumulation in cardiac tissue leads to alterations in cardiac morphology and function due to glucose intolerance, low-grade inflammation, and impaired insulin sensitivity. The diet rich in fats (HFD: High Fat Diet) and in general the diet known as Western ("Western Diet") composed of fats and carbohydrates in high quantities induces myocardial oxidative stress, defective intracellular signaling, hypertrophy of cardiomyocytes, interstitial fibrosis and dysregulation of genes such as peroxisome proliferator-activated receptors (PPARs), including Pparo and Ppary. Therefore, these pathological changes, mainly interstitial fibrosis, can cause myocardial stiffness.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease. Most patients with non-alcoholic steatohepatitis (NASH) show insulin resistance, independent of body weight. Insulin resistance is considered a better predictor of liver damage in patients with the non-alcoholic fatty liver disease (NAFLD), more than the visceral adiposity or fibrosls scoring system. commonly used. Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic component of the metabolic syndrome and its characteristics are similar to obesity, inflammation, insulin resistance and type 2 diabetes. Therefore, it is important to treat patients with the disease. non-alcoholic fatty liver disease (NAFLD) as well as its associated diseases. The exact mechanism of the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is still unknown. One theory suggests that insulin resistance and excess fatty acids in the circulation lead to simple hepatic steatosis, where insulin resistance promotes progression to non-alcoholic steatohepatitis (NASH). Insulin resistance is thus the spearhead in the progression of non-alcoholic fatty liver disease (NAFLD), there is a strong association between non-alcoholic fatty liver disease (NAFLD) and insulin resistance, where 70 -80% of obese and diabetic patients have non-alcoholic fatty liver disease (NAFLD). Immune cells, macrophages/Kupffer cells, natural killer cells, and T cells contribute to the progression of nonalcoholic steatohepatitis (NASH); in particular the resident and recruited hepatic macrophages derived from bone marrow, which are the most abundant cells and largely responsible for the secretion of inflammatory mediators such as TNFalpha and IL-1beta, leading to systemic insulin resistance and steatohepatitis non-alcoholic (NASH) (Kltade H, et al. Nonalcoholic fatty liver dlsease and Insult resistance: new insights and potential new treatments. Nutriente. 2017; 9, 387). Obesity leads to the development of non-alcoholic fatty liver disease (NAFLD) through liver dysfunction caused by hepatic steatosis. In obese patients with concomitant type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), hyperinsulinemia and dyslipidemia are more severe than in patients without non-alcoholic fatty liver disease (NAFLD). Excess fatty acids, whose production is induced by lipogenesis and fatty acid synthesis, as well as fatty acid oxidation, circulating in peripheral tissues, including the liver and fat tissue, where they accumulate, result in resistance to insulin. Adiponectin secreted by fatty tissue regulates fatty acid oxidation and inhibits lipid accumulation in both adipose tissue and liver. It also maintains glucose homeostasis throughout the body, including hepatic insulin sensitivity. Studies have shown that serum adiponectin levels are lower in patients with non-alcoholic fatty liver disease (NAFLD) than those without. A decrease in adiponectin in the development of nonalcoholic fatty liver disease (NAFLD) or type 2 diabetes affects fatty acid metabolism and promotes a state of chronic inflammation in the liver (Kitade H, et al. Nonalcoholíc fatty liver disease and insulin resistance: new insights and potential new treatments. Nutrients. 2017; 9, 387.

Cardiac fibrosis. Cardiac fibrosis is a common pathophysiology of most myocardial diseases, and is associated with systolic and diastolic dysfunction, arrhythmogenesis, and adverse outcomes. Cardiac fibrosis is characterized by excessive accumulation of extracellular matrix in the myocardium. Because the adult mammalian myocardium has negligible regenerative capacity, the most intense fibrotic remodeling of the ventricle is found in diseases associated with acute myocardial death.

For example, after acute myocardial infarction, the sudden loss of a large number of cardiomyocytes triggers an inflammatory reaction, ultimately leading to the replacement of the dead myocardium with a collagen-based scar. Several other pathophysiologic conditions induce more insidious interstitial and perivascular collagen deposition in the absence of complete infarction. Aging is associated with progressive fibrosis that may contribute to the development of diastolic heart failure in elderly patients. Excess pressure, induced by hypertension or aortic stenosis, results in extensive cardiac fibrosis that is initially associated with increased stiffness and diastolic dysfunction; the persistence of the pressure increase can eventually lead to ventricular dilatation and combined diastolic and systolic heart failure. Volume overload due to valvular regurgitant lesions can also lead to cardiac fibrosis, characterized by disproportionately large amounts of noncollagenous matrix.

Hypertrophic cardiomyopathy and postviral dilated cardiomyopathy are also often associated with the development of significant cardiac fibrosis. In addition, a variety of toxic injuries (such as alcohol consumption or anthracycline use) and metabolic disorders (such as diabetes and obesity) induce progressive fibrotic changes in the myocardium in both patients. humans and in experimental models. The pathophysiological mechanisms that lead to fibrotic remodeling of the ventricle are different in patients with various heart diseases; but the cellular effectors of fibrotic remodeling are common and the signaling pathways involved are similar.

Regardless of the pathophysiological mechanisms responsible for the development of the fibrotic response, cardiomyocyte death is usually the initial event responsible for the activation of fibrogenic signals in the myocardium. In other cases, noxious stimuli (such as pressure overload or myocardial inflammation) can activate profibrotic pathways in the absence of cell death. Various cell types are involved in fibrotic remodeling of the heart, either by directly producing matrix proteins (fibroblasts) or indirectly by secreting fibrogenic mediators (macrophages, mast cells, lymphocytes, cardiomyocytes, and vascular cells). The relative contribution of the various cell types often depends on the underlying cause of the fibrosis; however, in all conditions associated with cardiac fibrosis, the transdifferentiation of fibroblasts into secretory and contractile cells, termed myofibroblasts, is the key cellular event driving the fibrotic response.

Myofibroblasts are phenotypically modulated fibroblasts that accumulate at sites of injury and combine ultrastructural and phenotypic characteristics of smooth muscle cells. They have an extensive endoplasmic reticulum, a characteristic of synthetically active fibroblasts. Alpha-smooth muscle actin (α-SMA) expression identifies differentiated myofibroblasts in injured tissues, but is not a requirement for the myofibroblastic phenotype since, in early stages, from reparative or fibrotic responses, myofibroblasts may lack expression of α-SMA; but they exhibit stress fibers composed of cytoplasmic actins, these cells are called proto-myofibroblasts.

The abundance of fibroblasts in normal myocardium and the marked induction of mediators that promote myofibroblast transdifferentiation after cardiac injury (such as fibronectin, TGF-β1 (Transforming growth factor beta-1), and ED-A (chlorpheniramine/phenylephrine) ) suggest that the activation of resident cardiac fibroblasts may represent the most important source of myofibroblasts in the fibrotic heart. A growing body of evidence suggests that circulating bone marrow-derived fibrocytes may represent an additional source of myofibroblasts in cardiac injury.

Fibrillar collagen accumulation in the cardiac interstitium is the hallmark of cardiac fibrosis. Type I and type III collagen synthesis is markedly increased in the fibrotic heart regardless of the etiology of the fibrosis. Also, the deposition of nonfibrillar collagens (such as collagen VI) may play an important role in the activation of fibroblasts.

Several other cell types, including cardiomyocytes, endothelial cells, pericytes, macrophages, lymphocytes, and mast cells, may contribute to the fibrotic process by producing proteases that are involved in matrix metabolism, secreting fibrogenic mediators, and proteins. matricellular cells, or by exerting contact-dependent actions on fibroblasts. The mechanisms of fibrogenic signal induction depend on the type of primary myocardial injury. Activation of neurohumoral pathways stimulates fibroblasts both directly and through effects on immune cell populations. Cytokines and growth factors, such as tumor necrosis factor-α, interieukin IL-1, IL-10, chemokines, members of the transforming growth factor-β family, IL-11, and platelet-derived growth factors they are secreted into the cardiac interstitium and play distinct roles in activating specific aspects of the fibrotic response. Secreted fibrogenic mediators and matricellular proteins bind to cell surface receptors on fibroblasts, such as receptors for cytokines, integrins. syndecans and CD44 (CD44 antigen is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration), and transduce intracellular signaling cascades that regulate genes involved in the synthesis, processing, and metabolism of the extracellular matrix. Endogenous pathways involved in the downregulation of fibrosis are critical for cardiac repair and may protect the myocardium from excessive fibrogenic responses. Due to the reparative nature of many forms of cardiac fibrosis, targeting fibrotic remodeling after myocardial injury poses great challenges. Although regression of fibrosis has been documented in several heart conditions, the mechanisms responsible for the reversal of fibrotic disease remain unknown. Removal of collagen and other matrix proteins from the fibrotic heart probably requires the activation of proteases. It is unknown whether specific subpopulations of antifibrotic macrophages and lymphocytes are involved in the resolution of fibrotic lesions. Furthermore, the functional characteristics and molecular profile associated with a cardiac fibroblast regression phenotype have not been investigated.

Fibrosis and cardiomyopathy.

Extensive evidence has documented the presence of myocardial fibrosis in patients with diabetes. Cardiac MRI often identifies myocardial scarring (replacement fibrosis) in diabetics without a history of myocardial infarction. These findings probably reflect silent coronary events. Numerous histopathological studies have shown that cardiac fibrosis in diabetic patients occurs independently of coronary atherosclerosis or hypertension. Myocardial fibrosis in diabetics is often accompanied by cardiomyocyte hypertrophy and evidence of microvascular abnormalities, such as thickening of the capillary basement membrane. Diabetes-associated interstitial fibrosis is associated with the accumulation of type I and III collagen, involves both the left and right ventricles, and has been described in type 1 and type 2 diabetes. Manifestations of cardiomyopathy range from microscopic alterations in cardiac myocytes to fulminant heart failure with inadequate tissue perfusion, fluid accumulation, and cardiac rhythm dysfunction. Cardiomyopathy can be classified into primary (genetic, mixed, or acquired) and secondary, resulting in varied phenotypes including hypertrophic, dilated, and restrictive categories. Hypertrophic cardiomyopathy is the most common primary cardiomyopathy and can cause exertional dyspnea, presyncope, atypical chest pain, heart failure, and death. sudden cardiac Dilated cardiomyopathy can be genetic or acquired and usually presents with classic symptoms of heart failure with reduced ejection fraction. Restrictive cardiomyopathy is much less common and is often associated with systemic diseases such as Type 2 Diabetes Mellitus. Acquired variants of cardiomyopathy include peripartum and stress-induced cardiomyopathy, as well as rare variants such as arrhythmogenic right ventricular dysplasia and left ventricular non-compaction. Diagnosis of cardiomyopathy includes electrocardiography and echocardiography tests, in addition to medical history and physical examination. Treatment of symptomatic heart failure should follow current American College of Cardiology/American Heart Association guidelines, which include drug therapy with beta-blockers, an angiotensin-converting enzyme (ACE) inhibitor. angiotensin converting enzyme). an angiotensin receptor blocker (ARB: Angiotensin Receptor Blocker), diuretics, or an angiotensin-neprilysin receptor inhibitor. Patients with more severe symptoms may be evaluated for placement of an implantable cardioverter defibrillator and heart transplantation in refractory cases.

Mechanisms contributing to the development of diabetic cardiomyopathy.

The exposure of the heart to the hyperglycemia of the diabetic environment, together with an increase in fatty acids and cytokines; are the mechanisms studied for the development of cardiomyopathy associated with DM2. Hyperglycemia increases enzymatic O-Glucosin acetylation of cardiomyocyte proteins and is deleterious. Increased formation of Advanced Nonenzymatic Glycation Endproducts (AGEs) also has deleterious effects. A diabetic autonomic neuropathy is present and linked to hyperglycemia. Exposure to increased levels of lipids, including fatty acids and triglycerides, increases the accumulation of fat vacuoles in cardiomyocytes that mediate cardiac lipotoxicity. Decreased insulin signaling is a hallmark of DM1 and DM2 (diabetes mellitus type 1 and 2) producing alterations in other signaling cascades, including decreased AMPK (Adenine Monophosphate Activated Protein Kinase) signaling: protein kinase activated by adenine monophosphate, an enzyme composed of three subunits, one subunit-a-catalytic and two non-catalytic subunits, β and Y) and increased signaling of protein kinase C (PKC: Protein Kinase C: Protein kinase C) and proteins mitogen-activated kinases (MAPKs: Mitogen-Activated Protein Kinases: Mitogen-activated protein kinases).

CQVID-19.

Coronaviruses belong to the Coronaviridae family of the Nidovirales order. The name of Corona is given by the shape of "crown" that is generated on the external surface of the virus in the form of points. Coronaviruses have a diameter between 65 and 125 nm and contain a single chain of RNA (RiboNucleic Acid: ribonucleic acid ) as nucleic material, with a length of 26 to 32 kb. The new coronavirus was named SARS-CoV-2 and the disease COVID-19. -CoV-2 belongs to the β group of coronaviruses (Shereen 2020, et al. COVID-19 infection: Origin transmission, and characteristics of human coronaviruses. J Adv Res. 2020; 24:91-98). All coronaviruses contain specific genes downstream of open reading frame 1 (orfl) that encode viral replication, nucleocapsid, and protein S formation proteins. The receptor-binding domain (RBD: Receptor-Binding Domain: domain receptor binding) of the S protein binds freely between viruses, therefore, the virus can infect multiple hosts. SARS-CoV-2, in addition to protein S, expresses other polyproteins, nucleoproteins, and membrane proteins, as well as RNA polymerase, protease 3 Upo chymotrypsin, papin-type protease, helicase, glycoprotein, and accessory proteins. The structural proteins of SARS-CoV-2 are encoded by 4 structural genes, including spike (S), envelope (e), membrane (m), and nucleocapsid (n) (Shereen 2020, et al. COVID -19 Infection: Origin transmission, and characteristícs of human coronaviruses (Infection by COVID-19: transmission of origin and characteristics of human coronaviruses. J Adv Res. 2020; 24:91-98),

Common symptoms of COVID-19 with other illnesses are fever, fatigue, dry cough, myalgias, and dyspnea. Additionally, some patients have headache, dizziness, abdominal pain, diarrhea, nausea and vomiting. The disease can lead to progressive respiratory failure due to alveolar damage and trigger death (Xu H et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa). oral mucosal epithelial cells.) Int J Oral Sci 2020, Feb 24;12(1):8). Reports have suggested that patients with severe COVID-19 have histories of hypertension, chronic kidney disease, cardiovascular disease or diabetes mellitus than those with mild disease (South AM et al. Controversies of renin-angiotensin system inhibition during the COVID-19 pandemic). Nat Rev Nephrol. 2020. doi: 10.1038/s41581-020-0279-4).

Previous studies with SARS-CoV have shown an increase in the expression of genes associated with inflammation and a reduction in interferon 1β (Smits SL et al. Exacerbated innate host response to SARS-CoV ín aged non-human primates). host to SARS-CoV in elderly non-human primates) PLoS pathogens, 2010 Feb 5;6(2);e 1000756), as well as an excessive production of type 2 cytokines that leads to a deficiency in the control of replication viral infection and a more prolonged proinflammatory response. This exacerbated response of the immune system causes irreversible damage to the lung tissue with sequelae after recovery. Likewise, the alteration of certain clinical values may indicate disease progression and prognosis: lymphopenia, leukocytosis, elevated alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), high sensitivity of cardiac troponin I, creatine kinase, D-dimer, ferritin elevated serum levels, IL-6, prothrombin time, creatinine, and procalcitonin (Zhou F et al. Ciinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study) mortality of adult hospitalized patients with COViD-19 in Wuhan, China: a retrospective cohort study). Lancet. 2020 Mar 28;395(10229):1054-1062). The main typical pulmonary manifestations shown in computed tomography of patients with COVID-19 are ground-glass opacities (GGO: Ground-Glass Opacification), consolidation, reticular pattern and disordered cobblestone pattern. Atypical manifestations of computed tomography include airway changes, pleural changes, fibrosis, nodules, etc. All of the above could be associated with the progression and prognosis of COVID-19 (Ye Z et al. Chest CT manifestations of new coronavirus disease 2019 (COVID-19): a pictorial review (manifestations of the new coronavirus disease 2019 (COVID- 19): a pictorial review. Eur Radiol 2020, Mar 19, doi: 10.1007/s00330-020-06801-0). The presence of interstitial fibrosis has been observed in cadaveric biopsies of patients infected with SAR$«CoV-2 (Luo, W et al. Clinical pathology of critical patient with novel coronavirus pneumonia (COVID-19) novel coronavirus pneumonia (COVID-19). Preprints.org. Pathology &

Pathobioiogy/Patología y Patobioiogia, 202002.0407), likewise, some patients have presented bronchial deformation due to fibrotic damage, suggesting the formation of lesions before the onset of symptoms (Li Y et al. Coronavirus Disease 2019 (COVID-19): Role of chest CT in diagnosis and management.American Journal of Roentgenology 2020 Mar 4:1-7; Li Y et al.Coronavirus Disease 2019 (COVID-19): Role of chest CT in diagnosis and management.American Journal of Roentgenology/ American Journal of Roentgenology 2020 Mar 4:1-7). Viral infections increase the risk of pulmonary fibrosis (Shertg G et al. Viral Infection Increases the Risk of Idiopathic Pulmonary Fibrosis: A Meta-Analysis). Chest. 2019 Nov 12. pii: 50012-3692(19)34200-X), so this condition can be a severe complication after recovery for COVID-19 patients.

Troponin.

Troponin I (Tnl), together with troponin T (TnT) and troponin C (TnC), is one of the 3 subunits that form the troponin complex of the thin filaments of skeletal muscle. Tnl is the inhibitory subunit; it blocks actin-myosin interactions and thus mediates striated muscle relaxation. The Tnl subfamily contains three genes: Tnl-skeletal-fast-twitch, Tnl-skeletai-slow-twitch, and Tnl-heartiac. This gene encodes the Tn i -cardiac protein and is expressed exclusively in cardiac muscle tissues. Mutations in this gene cause Familial Hypertrophic Cardiomyopathy 7 (HCM7: Familial Hypertrophic Cardiomyopathy 7) and Familial Restrictive Cardiomyopathy (RCM). : Restrictive Cardiomyopathy).

The possible cardiovascular consequences and manifestations are not adequately established in COVID-19 disease, which is why Tag Guo et al. carried out a retrospective study that included 187 patients with a confirmed diagnosis of COVID-19, of which 144 (77%) were discharged home and 43 (23%) died. The mean age was 58.50 years; 66 (35.3%) had a history of cardiovascular disease (CVD: Cardiovascular Diseases) and 52 (27.8%) presented acute myocardial damage during admission, observed by elevated plasma levels of troponin (TnT). Overall, mortality was higher in patients with elevated TnT levels compared to those with normal TnT (Troponin T) levels (31 (59.6%) vs. 12 (8.9% p < 0.001 j). Mortality was 7.62% in patients without CVD (Cardio Vascular Diseases) and normal levels of TnT; 13.33% in the subgroup with CVD and normal levels of TnT; 37.50% in patients without CVD and elevated levels of TnT and 69.44% in those with CVD and elevated levels of TnT Patients with CVD more frequently presented elevated TnT compared with those without CVD (36 [54.5%]) vs. 16 (13.2%) ). A linear correlation was shown between levels of TnT and C-reactive protein (β = 0.530; p < .001) and NT-proBNP (β = 0.613; p < .001). The use of intravenous glucocorticoids and mechanical ventilation was prescribed more frequently in patients with elevated levels of TnT compared to TnT in the normal range. During hospital admission, patients with elevated TnT presented more frequently malignant arrhythmias (including ventricular tachycardia and ventricular fibrillation), renal failure, and acute respiratory distress syndrome.

Pirfenidone.

Rirfenidone (PFD), 5-methyl-1-phenyr-2-(1H)-pindone, was initially developed as an anthelmintic and antipyretic agent. PFD is a small molecule with a molecular weight of 185.23 Da, its chemical formula is C12H11NO with high solubility in alcohol and chloroform, the molecule is capable of diffusing between cell membranes without the need for a receptor (Macias- Barragan J et al. The multifaceted role of pirfenidone and its novel targets. new goals. Fibrogenesis Tissue Repair) 2010; 3:16). PFD has been tested in various cellular and animal models of inflammation and fibrosis, where it has been shown to have anti-inflammatory effects, anti-oxidative stress and anti-proliferative properties. PFD is known to regulate key cytokines and growth factors in the fibrotic process. It inhibits various inflammatory mediators, has an antioxidant effect, and restores the immune response. The compound per se is a known compound, as well as its pharmacological effects described, for example, in Japanese KOKAI Application Nos. 87677/1974 and 1284338/1976, as an anti-inflammatory agent including its analgesic and antipyretic effects. US Patent Numbers U 53,839,346 issued Oct. 1, 1974; US3,974,281, issued Aug. 10, 1976; and US4,052,509, granted on October 4, 1977, where they describe the method of obtaining the molecule, as well as its use as an anti-inflammatory agent. Beneficial effects have been observed in PFD treatment of fibrotic diseases, including renal, hepatic, and pulmonary fibrosis, and multiple sclerosis, conditions that share abnormal collagen deposition. The anti-fibrotic activity of PFD is described in the Mexican patent 11X182,266 granted on July 29, 1996. In fibrosis, the positive balance for collagen synthesis is influenced by the production of TGF-β and other growth factors , which may be downregulated by PFD (Macias-Barragan J et al. The multifaceted role of pirfenidone and its novel targets. Fíbrogenesis Tissue Repair 2010; 3: 16).

On the other hand, Komíya et al. evaluated the therapeutic effect of PFD in a murine model of NASH (Non-Aicoholic SteatoHepatitis: steatohepatitis no alcohol) associated with obesity, insulin resistance and dyslipidemia. They showed that PFD markedly attenuates hepatic fibrosis in the experimental model, with a parallel reduction in hepatocyte apoptosis. They observed that PFD inhibits TN Faifa-generated tissue damage and the fibrogenic response. Sandoval et al. found that the administration of pirfenidone in a model of non-alcoholic steatohepatitis decreases the weight of the animal, cholesterol, lipoproteins and very low density triglycerides, and at the liver level it decreases hepatic macrosteatosis, inflammation, ballooning of hepatocytes, fibrosis, epididymal fat and adiposity. PFD restored insulin, glucagon, adiponectin, and resistin levels along with improved insulin resistance.

The invention US20080025986A1 of January 31, 2008 describes as an invention, methods of treating disorders mediated by TNFalpha, the methods are based on the administration of pirfenidone or analogs and a secondary therapeutic agent for the reduction of the synthesis of TNFalpha or the reduction of the binding of TNFalpha to its receptor. The invention provides methods for the treatment of NASH where the general method requires the administration of an individual amount of pirfenidone. It also provides the treatment method for type 2 diabetes with the combination of pirfenidone and insulin.

Background to the invention object of this document, the invention WO2018088886A1 of May 17, 2018 claims the use of a pharmaceutical composition in the form of extended release tablets containing pirfenidone for the treatment of NAFLO/NASH and advanced liver fibrosis, reducing serum cholesterol and triglyceride levels, as well as the content of accumulated fat in liver tissue, in the form of macrosteatosis and microsteatosis.

OBJECT OF THE INVENTION

The object of the invention is to make available the pharmaceutical use of 5-methyl-1-phenyl-2-(1H)-pyridone (Pirfenidone) as an active ingredient alone or in combination with any anti-angiogenic compound, nutraceuticals or any bioactive molecule. that modify the expression of Troponin I, specifically for the management of patients with cardiomyopathy and/or cardiac steatosis and/or cardiac fibrosis and/or COVID-19,

The technical details of the developed invention are described below. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the dual HFHC diet leading to obesity, fat accumulation and NASH, a) Experimental design showing weeks of diet and Pirfenidone treatment, b) Macroscopic comparison of body weight, terminal dissection of the abdominal cavity of visceral adipose tissue showing liver and heart size (scale bar: 5 mm), c) HFHC diet liver showing microvesicular and macrovesicular steatosis, scale bar: 150 pm (40X magnification); some neutrophilic inflammatory foci are indicated by black arrows, scale bar: 100 pm (20X magnification); perivenular fibrosis and ballooning hepatocytes are indicated by arrows; scale bar: 100 pm (20X magnification). Pericellular fibrosis with scale bar: 150 pm (40X magnification). Images illustrate representative mice per group (n = 5). PV: portal vein; 60: bile duct; HA: hepatic artery; NOT: normal diet.

Figure 2 shows the average daily caloric intake in groups of mice before and during PFD treatment. a) caloric intake measured prior to PFD treatment, b) caloric intake measured during PFD treatment, Data are expressed as means ± SEM (Standard error of the mean); n - 5 / group. For group comparisons in a), Welch's t-test was applied; in panel b), Kruskal-Wallis test followed by Mann-Whitney U post-hoc test. *** P < 0.001 vs. ND.

Figure 3 shows how PFD prevents HFHC diet-induced accumulation of cardiac lipids. Lipid (ad) accumulation in frozen heart sections was stained with oily red (ORO). Scale bar: 100 pm, 20X magnification (a); 200 pm, increase 12X (b, 12 weeks) and 150 pm, 40x magnification (b, 16 weeks), d, e Histograms skilled llpidos content from 4 ^to 16 weeks to week and 16th, respectively. Data are expressed as means ± SEM. For group comparisons (n = 3/group in d and n = 5/group in e), one-way ANOVA (Analysis of Variance) followed by Tukey's post hoc analysis. *P<0.05, **P<0.01 and ***P<0.001 vs. 4 weeks or ND; ## P < 0.01 vs, 8 weeks; #P < 0.05 vs. HFHC and ttP <0.01 vs. 12 weeks.

Figure 4 shows representative H&E stained cardiac histology features of HFHC diet fed mice. a) Micrographs of cardiomyocyte cross-sectional area, depicting cardiomyocyte size of groups of mice at 16 weeks (three cell membranes per photomicrograph are outlined in black), scale bar: 100 pm, 100X magnification. b) Histogram showing the cross sectional area of cardiomyocytes at 16 weeks (n = 5 / group), c), d) cardiac tissue reveals small inflammatory foci neutrofllicos (black arrows) from 4 · week to 16 and 16 ^a week, respectively; scale bar: 150 pm 40X magnification. Each point and error bar indicate the mean ± SEM. Statistical testing was performed using the Kruskal-Wallis test followed by a Mann-Whitney U test for individual comparison of means. *** P < 0.001 vs. ND; #P < 0.05 vs. HFHC. Figure 5 shows the histological analysis of the effects of PFD on HFHC-induced cardiac fibrosis. a), c), e) Representative photomicrographs of transverse sections of cardiac tissue showing the deposition of the endomysial and perimysial collagen network, stained by Masson's trichrome and picrosirium. Interstitial fibrosis and perivascular fibrosis are indicated by arrows (a, c, e); collagen fibers appeared at 8 weeks a. b), d) Representation of histograms of collagen deposition of Masson's trichrome staining (n = 3 / group in b and n = 5 / group in d). f, g Ventricular sections of mice were immunostained with α-SMA, signal mainly around vessels: arrows (n = 5/group). Scale bar: 150 pm, 40X magnification. Data are expressed as means ± SEM. For group comparisons (n = 5/group), one-way ANOVA followed by Tukey's post hoc analysis. **P<0.01 and ***P<0.001 vs. eighth week or ND; ### P < 0.001 vs. 12th week or HFHC. Figure 6 shows the effects of HFHC and PFD diet on the levels of mRNA gene expression in cardiac tissue. RT-qPCR for hypertrophy and fibrosis genes such as Desmtn, Tflfpl, Timpl, Col I, Col III (a); and for TNF-o, Nrf2, Sod1, Acox1, Srebp1 and Pgc1a (b) using ventricutar tissue. Data are expressed in bar plots as means ± SEM, Rare group comparisons (n = 5/group), one-way ANOVA followed by Tukey's post hoc analysis. *P<0.05, °P<0.01 and ***P<0.001 vs. NA; #P<0.05 and ###P<0.001 vs. HFHC.

Figure 7 shows the evaluation of inflammation, oxidative stress, myocardial damage and genes related to lipid metabolism. Western blotting image and quantification of NF-kB, Nrf2, troponin-l (a, b); and Pparo, Ppary, Acox1, Cpt1A, Lxra, and Srebp1 (c, d) using cardiac protein extracts from ventricular tissue. Band intensities were measured and are shown in histograms. Data are expressed as means ± SEM. For group comparisons (n = 5/group), one-way ANOVA followed by Tukey's post hoc analysis. ^M P < 0.01 and *** P < 0.001 vs. NA; #P < 0.05, ## P < 0.01 and ### P < 0.001 vs. HFHC.

DETAILED DESCRIPTION OF THE INVENTION

The characteristic details of the pharmaceutical use of prifenidone for the reduction of cardiac fibrosis in patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19 are clearly shown in the following description and in the accompanying illustrative drawings.

EXPERIMENT.

Animal model.

Male mice of the C57BL/6J strain of 4 weeks of age weighing 20-25 g before the experiment were fed with standard diet and water. purified ad libitum. After one week of acclimatization, the animals were randomly assigned into two groups; normal diet (ND, n = 5) and high fat/carbohydrate diet (HFHC, n = 10). ND was composed of 18% kcal from fatty foods. The HFHC diet consisted of 60% kcal from fatty foods and drinking water with 42 g/L carbohydrates (55% fructose and 45% sucrose). At the end of the eighth week, five HFHC mice were treated with approximately 350 mg/kg/day of PFD mixed with food (HFHC + PFD, n − 5), the dose chosen as reported in a mouse model of NASH, Another group of animals was put on an HFHC diet (n = 12) and sacrificed in groups (n = 3) at 4, 8, 12 and 16 weeks to control the development of cardiac steatosis and fibrosis. The results can be seen in figures 1 and 2.

Weight, fasting glucose and insulin sensitivity. Food intake was systematically measured three times a week between 9 and 10 am, energy intake from food and drink was calculated. Body weight and 4-hour fasting glucose were obtained each week during the study. Glucose levels were measured with a tail vein blood glucose meter. At the end of treatment, 48 h before sacrifice, mice were fasted for 4 h for insulin tolerance testing after intraperitoneal injection of short-acting recombinant human insulin at a standardized dose of 0.025 U. /mouse. Glucose levels were measured at 0, 30, 60, and 90 minutes after insulin administration. Finally, the area under the curve (AUC: Area Under Curve) was calculated.

The results show that the animals fed the diet high in fat increased their serum glucose levels compared to ND control animals. The HF group treated with PFD showed an increase in insulin sensitivity as measured by the Insulin Tolerance Test (ITT) which is shown by the decrease in the value of the area under the curve. The results are shown in figure 3.

Regarding weight, the animals treated with PFD significantly decreased their body weight, as well as the fat of the epididymis, demonstrating a lipolytic effect. immunoblot.

The frozen ventricular tissue was homogenized in RIPA buffer (RIPA: Radio immuno Precipitation Assay); 40 pg of total cardiac protein per lane was resolved on a 12% SDS-PAGE gel and transferred to PVDF (PVDF: Polyvinyldifluoride) membrane (Bio-Rad Laboratories). Membranes were incubated with 3% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 1 hour. Membranes were immunoblotted with primary antibodies specific for NFkB, Nrf2, Troponin I, beta-actin, PPAR-alpha, PPAR-gamma, Acox1, Cpt1A, LXR-alpha, and SREBP1. Primary antibodies were used at 1:1000 dilution and incubated overnight at 4°C. Subsequently, peroxidase-conjugated secondary antibody (Roche) was used at a 1:12000 dilution for 1 hour at room temperature. Protein bands were detected using the BM Chemiluminescence Western Blot Kit (8M: Methyl Bromide) (Roche Applied Science), and density analyzed via ChemiDoc XRS+ with Image Lab software (Bio-Rad Laboratories). . All western blots were performed on triplicate samples. The β-actin was used as a constitutive protein. The results can be seen in figure 4.

Dissection of the heart and liver.

Mice were anesthetized with tiletamine/zolazepam (15 mg/kg/pe, Zoletil® 50, Virbac), hearts and livers rapidly isolated. Previously, the heart of the mice was perfused through the left ventricle using a syringe with 5 ml of cold PBS (PBS: Phosphate Buffered Saline or Phosphate Buffered Saline). The atria were removed and the ventricles were cut into four sections; one was fixed in 4% paraformaldehyde (0.1 M PBS, pH 7.4) and embedded in paraffin; another was immersed in Tissue-Tek® OCT (OCT; Optical Coherence Tomography). The other two portions of the ventricles were used to extract protein and total RNA (ribonucleic acid), respectively. The results can be seen in Figure 5.

Immunohistochemistry. After deparaffinization and rehydration, the ventricular tissues were treated with _{3% H 2} O ₂ in methane! absolute for 20 minutes. Sections were incubated overnight at 4°C with rabbit anti-α-SMA monoclonal antibody. It is then detected with avidin-conjugated secondary antibodies and visualized with a Vectastain kit, PK-8800 (Vector Laboratories) and diaminobenzidine (Sigma-Aldrich). Sections were lightly counterstained with hematoxylin, before dehydration and mounting. The recovery of the antigen was carried out in a water bath set at 99 ºC for 25 minutes. in Tris-EDTA buffer (Tris-EDTA is responsible for protecting nucleic acids by inactivating DNAses or RNases), pH 9.0. For quantification, 10 random fields of ventricular areas were evaluated. α-SMA staining intensity was determined using ImageJ (ImageJ is a public domain digital image processing program programmed in Java developed at the National Institutes of Health: National Institutes of Health).

RNA isolation and RT-qPCR. Total RNA from ventricular tissue was isolated with Trizol reagent (Invitrogen) according to the manufacturer's instructions, and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). 2 pg of total RNA was reverse transcribed using 690 ng random primers, 0.72 mM deoxynucleotide triphosphate (dNTP) mix, 1x first-strand buffer, 3.6 mM dithiothreitol (DTT), 5 U RNase inhibitor, and 260 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) in a reaction volume of 50 pL, following the manufacturer's instructions. Each qPCR was carried out using 1 μL of cDNA, 1x Universal PCR Master Mix, and 1x specific Taqman primer/probe. The qPCR reactions (qPCR: quantitative Polymerase Chain Reaction or quantitative polymerase chain reaction) were performed on the LightCycler 96 instrument (Roche Molecular Systems). All data were run in triplicate, normalized according to Gapdh (Gapdh: glyceraldehyde-3-phosphate dehydrogenase) and 18S levels (18S ribosomal RNA (abbreviated 18S rRNA) is a part of ribosomal RNA, the S in 18S represents Svedberg units), and were analyzed using the 2-ΔΔCt method. The results can be seen in figures 6 and 7. Resulting pharmaceutical use.

From the analyzes carried out, it appears that:

• Troponin I overexpression is associated with cardiomyopathy and/or cardiac steatosis and/or cardiac fibrosis and/or COVID-19.

• Pirfenidone can be used for the reduction and/or modulation of cardiac fibrosis and the expression of cardiac troponin 1 in any pharmaceutical formulation that includes but is not restricted to Pirfenidone rapid release or standard pirfenidone, prolonged or extended release pirfenidone, slow-release pirfenidone, pirfenidone in liposomes of any lipid composition, aerosol pirfenidone, microemulsion pirfenidone, among others; pirfenidone alone or administered with an anti-angiogenic compound or other active ingredients or pirfenidone administered or formulated with other bioactive molecules or active ingredients.

• The pharmaceutical use whose active ingredient is Pirfenidone may contain other active ingredients, nutraceuticals or any bioactive molecule that modifies the expression of Troponin 1.

• The other inhibitors of gene expression of Troponin I can be inhibitors llb/llla (inhibitors of glycoprotein llb/llla: abciximab, eptifibatide and tirofiban) and bioactive molecules encoded by PRKCD genes (Protein Kinase C Delta: Protein kinase C Delta, the protein encoded by this gene is a member of the family of protein kinases C, which are specific serine and threonine protein kinases), PRKCA, PRKCB and PRKCG (Protein Kinase C Alpha, Beta and Gamma: Protein kinase C Alpha , Beta, Gamma, protein kinase C, is a family of specific protein kinases (alpha, beta and gamma) of serine and threonine that can be activated by calcium and the second messenger, diaciglyceroi), PPP2CA (This gene encodes the phosphatase 2A catalytic subunit. Protein phosphatase 2A is one of the four main Ser/Thr phosphatases, and is involved in the negative control of cell growth and division) and PAK3 (P21 (RAC1) Activated Kinase 3, The protein encoded by this gene is a serine-threonine kinase and forms an activated complex with GAS bound to RAS (P21), CDC2 and RAC1) or any other molecule that inhibits or modifies the expression of Troponin I.

• The pharmaceutical use of Pirfenidone and/or any other active ingredient and/or any other molecule that modulates the expression of troponin I acts in cardiomyopathy and/or cardiac sciatosis and/or cardiac fibrosis and/or COVID-19, as an adjuvant.

• The total daily dose of pirfenidone can be 800 mg, 1200 mg or 2400 mg.

• The formulation can be part of the treatment of patients with cardiomyopathy and/or cardiac steatosis and/or cardiac fibrosis and/or COVID-19, as an adjuvant.

• Troponin I overexpression can be of any other aetiology, including type II diabetes, non-alcoholic steatohepatitis, alcoholic steatohepatitis, metabolic syndrome, etc.

• The pharmaceutical composition can be presented in various pharmaceutical forms such as granules, tablets, capsules, drops, tablets, emulsion, microemulsion, suspension, syrup, bulk powder mixture or others.

Due to the above, the pharmaceutical use based on pirfenidone for the reduction of cardiac fibrosis in patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19 is as follows:

1. Pirfenidone that can be used for the reduction and/or modulation of cardiac fibrosis and the expression of cardiac troponin I in any pharmaceutical formulation that includes but is not restricted to rapid release plrfenidone or standard pirfenidone, prolonged or extended release pirfenidone , slow-release pirfenidone, pirfenidone in liposomes of any lipid composition, pirfenidone in aerosol, pirfenidone in microemulsion, among others; Pirfenidone alone or administered with an anti-angiogenic compound or other active ingredients or Pirfenidone administered or formulated with other bioactive molecules or active ingredients, a. The pharmaceutical use whose active ingredient is Pirfenidone may contain other active ingredients, nutraceuticals or any bioactive molecule that modifies the expression of Troponin I, where overexpression of Troponin I may be of any other etiology, including type II diabetes, non-alcoholic steatohepatitis, alcoholic steatohepatitis , metabolic syndrome, etc., b. The other inhibitors of Troponin I gene expression can be the llb/llla inhibitors (inhibitors of the glycoprotein llb/llla; abciximab, eptifibatide and tirofiban) and the bioactive molecules encoded by the PRKCD genes (the protein encoded by this gene is a member of the protein kinase C family, which are specific serine and threonine protein kinases), PRKCA, PRKCB and PRKCG (protein kinase C, is a family of specific protein kinases (alpha, beta and gamma) of serine and threonine that can be activated by calcium and the second messenger, diacylglycerol), PPP2CA (this gene encodes the phosphatase 2A catalytic subunit, protein phosphatase 2A is one of the four main Ser/Thr phosphatases, and is involved in negative control of cell growth and division) and PAK3 (P21 (RAC1) Activated Kinase 3, the protein encoded by this gene is a serine-threonine kinase and forms an activated complex with GAS bound to RAS (P21), CDC2 and RAC1 ) or any other molecule that inhibits or modifies the expression of Troponin I, c. The total daily dose of pirfenidone can be 800 mg, 1200 mg or 2400 mg.

In turn, the pharmaceutical formulation can be presented as follows;

1. The pharmaceutical composition can be presented in various pharmaceutical forms such as granules, tablets, capsules, dragees, tablets, emulsion, microemulsion, suspension, syrup, bulk powder mixture or others.

Regarding the treatment of the pharmaceutical formulation:

1. The pharmaceutical composition can be part of the treatment of patients with cardiomyopathy and/or cardiac steatosis and/or cardiac fibrosis and/or COVID-19, as an adjuvant.

The above description of the disclosed definitions is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these definitions and/or implementations will be readily apparent to those skilled in the art, and the principles generic terms defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be given the widest scope consistent with the following claims and the principles and novel features described herein.

Claims

CLAIMS Having sufficiently described my invention, I consider as a novelty and therefore I claim as my exclusive property, what is contained in the following clauses:

1. A composition for pharmaceutical use for the reduction of cardiac fibrosis in patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19 characterized by comprising a. Pirfenidone which can be used for the reduction and/or modulation of cardiac fibrosis and the expression of cardiac troponin I in any pharmaceutical formulation including but not restricted to rapid release pirfenidone or standard pirfenidone, prolonged or extended release pirfenidone, pirfenidone slow release, pirfenidone in liposomes of any lipid composition, pirfenidone in aerosol, pirfenidone in microemulsion, among others; pirfenidone alone or administered with an anti-angiogenic compound or other active ingredients or pirfenidone administered or formulated with other bioactive molecules or active ingredients, b. This composition for pharmaceutical use whose active ingredient is Pirfenidone may contain other active ingredients, nutraceuticals or any bioactive molecule that modifies the expression of Troponin I, where overexpression of Troponin I may be of any other etiology, including type II diabetes, non-alcoholic steatohepatitis , alcoholic steatohepatitis, metabolic syndrome, etc., c. The other inhibitors of Troponin I gene expression can be the Ilb/IIIa inhibitors (Hb/llla glycoprotein inhibitors: abciximab, eptifibatide and tirofiban) and the bioactive molecules encoded by the PRKCD genes (the protein encoded by this gene is a member of the protein kinase C family, which are serine and threonine-specific protein kinases), PRKCA, PRKCB, and PRKCG (protein kinase C, is a family of serine and threonine-specific (alpha, beta, and gamma) protein kinases that can be activated by calcium and the second messenger, diacylglycerol), PPP2CA (this gene encodes the catalytic subunit phosphates» 2A, protein phosphatase 2A is one of the four main Ser/Thr phosphatases, and is involved in the negative control of the growth and cell division) and PAK3 (P21 (RAC1) Activated Kinase 3, the protein encoded by this gene is a serine-threonine kinase and forms an activated complex with GAS bound to RAS (P21), CDC2 and RACt) or any other molecule that inhibits or modifies the expression of Troponin I, d. The total daily dose of pirfenidone can be 800 mg, 1200 mg or 2400 mg,

2. The composition for pharmaceutical use to reduce cardiac fibrosis in patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19 according to claim 1, where said composition for pharmaceutical use can be presented in pharmaceutical forms such as: to. Granules, tablets, capsules, dragees, tablets, emulsion, micro emulsion, suspension, syrup, bulk powder mixture or others.

3. The composition for pharmaceutical use to reduce cardiac fibrosis in patients with cardiomyopathy and/or cardiac steatosis and/or COVID-19 according to claim 1, can be part of patients with: a. Cardiomyopathy and/or cardiac steatosis and/or cardiac fibrosis and/or COVID-19, as an adjuvant in treatment.