ES2611667B1 - GALIC ACID FOR THE TREATMENT OF PATHOLOGIES OF THE HYPERACTIVATION OF THE RENINE ANGIOTENSIN ALDOSTERONE SYSTEM (SRAA) - Google Patents

Abstract

The present invention is the use of gallic acid or its pharmaceutically acceptable salts in the preparation of a medicament for the treatment of a pathology by hyperactivation of the Renina Angiotensin Aldosterone system, SRAA. In particular of a cardiovascular disease, a cerebrovascular disease, a renal disease or diabetes.

Description

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GALIC ACID FOR THE TREATMENT OF PATHOLOGIES OF THE HYPERACTIVATION OF THE RENINE ANGIOTENSIN ALDOSTERONE SYSTEM

(SRAA)

DESCRIPTION

TECHNICAL SECTOR

The invention is within the field of medicine and the pharmaceutical industry. It can also be used in dietary supplements in the diet or in the food industry.

BACKGROUND OF THE INVENTION

The medical use of gallic acid in the treatment of cancer and in immunodeficiencies taking advantage of its action as an antioxidant has been described in the art.

More widely it is known for its vasoconstrictor capacity inducing an elevation of blood pressure. Its specific use for blocking the Renin Angiotensin Aldosterone System (SRAA) and its usefulness in the treatment of related diseases has not been described.

The SRAA plays a fundamental role in the regulation of blood pressure, sodium balance and plasma volume. However, hyperactivation of SRAA through Angiotensin II (Ang II), its main effector, is also partly responsible for the increase in blood pressure and the development of lesions in target organs. These lesions are asymptomatic in a first phase, such as microalbuminuria or hypertrophy of the left ventricle, but can lead to serious clinical or even fatal repercussions in what has been called the cardiovascular and renal continuum. The cardiovascular and renal continuum begins with the development of risk factors, continues with asymptomatic organic lesion and ends with the evolution towards peripheral renal, cerebrovascular, cardiac and vascular disease. The SRAA is formed by a set of peptides and enzymes that lead to the synthesis of Ang II and aldosterone.

Cardiovascular, metabolic and renal disease are pathologies caused by hyperactivation of SRAA and are recognized and named as such in the

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technique (Giner-Galvaña V. et al. “Direct Renin Inhibition: from Physiology to Pharmacology and Clinical Implications.” Rev. Esp. Cardiol Suppl. 2011. 11 (D): 25-36; Ferrario, CM "Role of Angiotensin-II in Cardiovascular Disease: Therapeutic Implications of More Than a Century of Research." J. Renin Angiotensin Aldosterone Syst. 2006. 7: 3-14).

The activation of SRAA is located in the pathophysiological center of cardiovascular and metabolic disease as a whole (Giner-Galvaña V. et al. “Direct Renin Inhibition: from Physiology to Pharmacology and Clinical Implications.” Rev Esp. Cardiol. Suppl. 2011. 11 (D): 25-36). This fact is due to the fact that the actions of Ang II and aldosterone do not only focus on systemic hemodynamic variables such as saline reabsorption and vasoconstriction, but also have a local effect on inflammation, oxidative stress and cell proliferation.

In relation to cardiovascular disease, a clear association between hyperactivation of SARS and risk of cardiovascular events has been demonstrated, including cardiac hypertrophy, cardiac arrhythmias, endothelial dysfunction, atherosclerosis, thrombus formation and platelet aggregation. In addition, it has been shown that the alteration of any of the components of the SRAA represents an independent factor of suffering from myocardial infarction and of dying from a cardiovascular event. It has also been shown that the use of SRAA blockers has shown a positive impact on the morbidity and mortality of cardiovascular disease (Ferrario CM. “Role of Angiotensin II in Cardiovascular Disease: Therapeutic Implications of More Than a Century of Research”. J. Renin Angiotensin Aldosterone Syst. 2006. 7: 3-14).

As an aspect to consider, the HOPE (Heart Outcomes Prevention Evaluation) clinical trial demonstrates that the protective effect of RAAS blockade on cardiovascular morbidity is independent of the presence of arterial hypertension. This study demonstrates that the benefit caused by SRAA blockade was especially pronounced in diabetics, with a reduction in acute myocardial infarction, stroke or death of 25%, an effect that remained persistent after adjusting for the reduction in blood pressure.

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In relation to metabolic disease, hyperactivation of SRAA associated with diabetes has been demonstrated (Ribeiro-Oliveira A. et al. "The Renin-Angiotensin System and Diabetes: An Update." Vasc Health Risk Manag. 2008. 4 (4): 787-803) and to obesity (Sarzani R. et al. "Renin-Angiotensin System, Natriuretic Peptides, Obesity, Metabolic Syndrome, and Hypertension: an Integrated View in Humans." J Hypertens. 2008. 26 (5): 831- 43). And an important role of Ang II in metabolic syndrome has been described, mainly linked to its role in endothelial dysfunction, insulin resistance, inflammation and its proliferative effects (Ribeiro-Oliveira A. et al. "The Renin- Angiotensin System and Diabetes: An Update. ”Vasc Health Risk Manag. 2008. 4 (4): 787-803).

Another of the pathologies related to hyperactivation of ARS is kidney disease. In this regard, the use of SRAA blockers has also been shown to slow the progression of kidney disease in patients with type 2 diabetes and with kidney disease (Ferrario CM. "Role of-Angiotensin II in Cardiovascular Disease: Therapeutic Implications of More Than a Century of Research. ”J. Renin Angiotensin Aldosterone Syst. 2006. 7: 3-14).

In addition, hyperactivation of SRAA can cause many other pathologies related to the effects of Ang II as an inflammatory agent. It has been pointed out that the regulation of SRAA can preserve neuronal function and viability and combat different ocular disorders (Kurihara T. et al. "Renin-Angiotensin System Hyperactivation Can Induce Inflammation and Retinal Neural Dysfunction." Int. J. Inflamm. 2012 , Article ID 581695, 14 pages).

The active drugs on the blockade of this system are extremely useful against all pathologies related to hyperactivation of the SARS. In this regard, Ang II induces vasoconstriction, left ventricular hypertrophy and myocardial infarction. The molecule is also involved in the process of atherosclerosis as it causes vascular inflammation and endothelial dysfunction. In type 2 diabetes it causes an increase in tubular reabsorption of Na + and oxidative stress by acting on specific receptors (AT1) and through the hormone aldosterone, promoting cardiovascular and renal secretion of fibrogenic and inflammatory cytokines (Schmieder RE. Et al. "Renin-angiotensin System and Cardiovascular Risk”, Lancet. 2007. 369: 1208-1219).

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Ang II also regulates endothelial dysfunction, which is characterized by an imbalance between vasodilator and vasoconstrictor compounds synthesized by the endothelium. Vasodilators include nitric oxide (NO) and prostacyclines (PC), while endothelin-1 (ET-1) is the most important vasoconstrictor. Endothelial dysfunction causes a change in the vessels towards a proinflammatory and prothrombotic state and towards a decrease in dilation. This pathology is associated with vascular disease and is common in chronic heart failure, coronary artery disease, peripheral arterial disease, diabetes, chronic renal failure, hypertension and cerebrovascular disease (Bernatova I et al. "Endothelial Dysfunction in Spontaneously Hypertensive Rats: Focus on Methodological Aspects. ”2009. J. Hypertens. Suppl. 27 (6): S27-S31).

Brazil studies the antihypertensive properties in rats of a methanolic extract of papaya and its Inhibitory activity of Angiotensin Converting Enzyme (ACEI activity) in vitro (Brazil GA. Et al. “Antihypertensive Effect of Carica papaya Via a Reduction in ACE Activity and Improved Barorefex ”, Planta Med. 2014. 80 (17): 1580-1587). Said methanolic extract contains in homogeneous amounts ferulic acid, caffeic acid, gallic acid and the flavonoid quercetin, and showed ACEI activity in vitro. Angiotensin Converting Enzyme (RCT) is a key enzyme in the control of blood pressure and component of SRAA. On the other hand, it has been described that flavonoids and polyphenols have ACEI activity; In fact, quercetin has been identified as responsible for reducing blood pressure. The expert would find it suggested to relate the antihypertensive effect observed with quercetin and not with gallic acid since the document does not specifically associate gallic acid with this therapeutic effect. Therefore it should be considered that his teachings diverge in this regard from the present invention.

As an aspect to be taken into account, in the present application it is demonstrated that the blockage of the SRAA produced by gallic acid in vivo is not produced by an ACE inhibition.

Some patent documents relate the use of gallic acid with blood pressure. Specifically, the application US 20020068123 A1 describes a method for sweetening food, medicine or hygiene products with gallic acid.

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It takes advantage of the organoleptic properties of gallic acid and describes its medical uses, and among them that it is hypotensive (paragraph [0011]). However, this hypotensive ability is not demonstrated, nor is it really any other application related to it. The authors limit themselves to enumerating a series of various applications, among which there is also their ability to block the SRAA, and therefore should not be considered in the patentability discussion of the present invention.

On the other hand, the fact that a compound has a hypotensive effect is indicative of a drop in blood pressure under normal physiological conditions, which is not desirable. In the present invention it is demonstrated that gallic acid does not have a hypotensive but antihypertensive effect, this being understood as the lowering of blood pressure in conditions of hypertension as a disease.

US patent 6280776 B1 describes a plant extract rich in gallic acid capable of treating a subject's hypertension. The extract is extremely varied in its components. The document is not able to directly relate gallic acid to the therapeutic effect. According to the inventor, the extract obtained lowers the transaminases in such a way that one would expect a positive effect on many other symptoms of the patient, including blood pressure. However, this supposed decrease in blood pressure in the cirrhotic animals in which it is tested is not even reported. The fact is that a direct link between transaminases and blood pressure has not been described in the art, so the teachings of this publication should be considered speculative in this regard. The document also does not indicate the effect of gallic acid as a blocker of SARS.

WO 1996008483 A1 describes the medical properties and uses of sulphonated derivatives of gallic acid of its formula ID, which in turn includes methylated and sulfated derivatives. The medical uses it describes are all those symptoms mediated by endothelin, including endothelin-induced hypertension. The derivatives described have a very varied reactivity and different from the gallic acid from which they come, and do not suggest that the acid will exhibit the same activity. It is widely known that derivatives of dietary polyphenols have shown different bioactivity compared to free forms (Lodi F. et al. "Glucuronidated and Sulfated Metabolites of the Flavonoid Quercetin Prevent Endothelial Dysfunction but Lack Direct Vasorelaxant Effects in Rat Aorta." 2009.

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Atherosclerosis 2004 (1): 34-39). This document also does not describe or suggest the effect of gallic acid as a blocker of SARS.

Application US 3784695 A describes in mammals a hypotensive effect of esters of gallic acid, in particular of alkyl esters of chain between 1 and 9 carbon atoms, preferably butylgalate. Again, neither describes nor suggests the effect of gallic acid as a blocker of SARS. The document does not contemplate the case that the substituent is H, that is to say gallic acid itself. It is necessary to take into account that the effect of the esters in the organism is not extensible to the acid from which they come.

The bioavailability of the compound administered orally is conditioned by its digestion and its absorption in the digestive tract. Gastric digestion and intestinal absorption of free gallic acid with respect to its derivatives does not have to be coincidental. It is not only quantitative but also qualitative differences, since bioavailable compounds are different after the administration of free gallic acid or its derivatives. When a polyphenol is not absorbed in the small intestine, it will continue to the large intestine, where it will be extensively metabolized by the microbiota and can form completely different compounds from the initial one. Being free molecules, free gallic acid and its derivatives also differ in polarity and solubility, and these different properties will affect absorption in the small intestine, making the amount of compounds that reach the colon different in both cases. The new compounds formed in the colon may have more or less biological activity, but they will be different from the initial compounds. In Example 4 of the present application, it is demonstrated that after administration of free-form gallic acid, up to 23% more metabolites from the metabolism of the microbiota of the colon can be quantified in plasma. In this sense, the expert would not find in this document any indication that suggests thinking about the same reactivity between them.

Sutra studies the effect of some polyphenol compounds on model rats in Metabolic Syndrome, including gallic acid (Sutra T. et al. "Preventive Effects of Nutritional Doses of Polyphenolic Molecules on Cardiac Fibrosis Associated with Metabolic Syndrome: Involvement of Osteopontin and Oxidative Stress ”, J. Agric. Food Chem. 2008. 56: 11683-11687) .It does not indicate the effect of the acid

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Gallic as a blocker of SRAA, but it is responsible for the prevention of increased blood pressure. The authors identify themselves as the first capable of demonstrating this function in vivo, although it refers to another publication that also reports the prevention of hypertension in spontaneously hypertensive rats (SHR) with the administration of green tea, rich in gallic acid. The model used by Sutra is designed so that the animals present hypertension, and on that model the gallic acid would avoid a new ascent. However, the prevention of a rise in blood pressure is not directly related to an antihypertensive effect of the same compound. Thus, for example, the administration of a high concentration of polyphenols in a person who already has high blood pressure will not have the same effects as in a healthy person; although momentarily it could cause a decrease, the blood pressure will rise again at the moment when the patient stops taking said concentration of polyphenols.

Kang studies in hypertensive rats (SHR) the effect of an extract of Spirogyra sp. with a high content of gallic acid, although without defining said content (Kang N et al. "Gallic Acid Isolated from Spirogyra sp. Improvescardiovascular Disease Through a Vasorelaxantand Antihypertensive Effect". Environ.Toxicol. Pharm. 2015. 39: 764772). extract demonstrates antihypertensive activity in vivo and ACEI activity and vasorelaxing effect in vitro, however, in the present application it is demonstrated that the blockage of SRAA produced by gallic acid in vivo is not produced by an ACE inhibition, so that the expert would not find suggested the object of the present invention from the teachings of Kang.

The problem of the technique is posed as obtaining an alternative treatment to pathologies caused by hyperactivation of the SRAA system in a patient. The solution proposed by the present invention is the use of gallic acid for the treatment of said pathologies.

DESCRIPTION OF THE INVENTION

The present invention is the use of gallic acid or its pharmaceutically acceptable salts in the preparation of a medicament for the treatment of a hyperactive pathology of the Renina Angiotensin Aldosterone system, SRAA, in a subject,

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preferably human. An alternative way of expressing the invention is gallic acid or its pharmaceutically acceptable salts for use in the treatment of a hyperactive pathology of the Renina Angiotensin Aldosterone system, SRAA, in a subject, preferably human. Another alternative way of expressing the invention is a method of treating a pathology by hyperactivation of the Renina Angiotensin Aldosterone system, SRAA, which comprises the administration of gallic acid or its pharmaceutically acceptable salts in a subject in need thereof, preferably human.

A preferable aspect is that said pathology is selected from a cardiovascular disease, a cerebrovascular disease, a renal disease and diabetes, more preferably type 2 diabetes. The treatment of hypertension, atherosclerosis and the nephroprotective action of gallic acid are also within the scope. of the present invention.

Another highly preferable aspect of the invention is a pharmaceutical composition comprising gallic acid and pharmaceutically acceptable excipients. Another preferable aspect is a food supplement comprising gallic acid and food additives.

Gallic acid acts as a blocker of SRAA. In the present application, a blocker of the SRAA is understood as a compound that acts on any of the components of the SRAA blocking its action.

The results shown in the present application in Example 6 indicate the positive effect of gallic acid on endothelial function. This indicates that its effect is dependent on an increase in the concentration of NO as a vasodilator factor, and partially mediated by prostacyclin (PC). It has also been shown to produce a significant decrease in plasma levels of endothelin (ET-1) as a vasoconstrictor factor. All results demonstrate the clear vasoprotective role of gallic acid, with the implications derived from this fact for cardiovascular, renal and cerebrovascular disease.

On the other hand, from a technical point of view it is obvious that any of the metabolites that result from the absorption of gallic acid in the small intestines and

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coarse and its subsequent microbial metabolism, identified in plasma and aorta, could present the therapeutic effect of the present invention; in particular, 3-O-methyl gallic acid, gluic acid glucuronide, methyl acid sulfate, dimethyl gallic acid, trimethylgalic acid or, catechol, methylpyrogallol, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid or 3,4-dihydroxybenzoic acid.

BRIEF DESCRIPTION OF THE FIGURES

Unless otherwise indicated, all doses indicated in this description refer to the body weight of the animal.

Figures 1a and 1b show the concentration of Ang I and Ang II, respectively, in plasma of SHR rats at 6 hours after administration of 1 mL of water (control) or 6.64 mg / kg of gallic acid animal. Data represent the mean ± ESM for a minimum of 6 animals. ** P <0.01. * P <0.05.

Figure 2 shows the activity of the RCT in plasma collected at 6 h after administration of 1 mL of water (control) or 6.64 mg / kg of gallic acid animal intragastrically in SHR rats. Data represent the mean ± ESM for a minimum of 6 animals. Statistical differences between treatments not found.

Figure 3a shows the content of compounds derived from gallic acid absorbed in small intestine and colon measured in plasma (white bar) and aorta (grated bar) of SHR rats, 6 h after consumption of 6.64 mg / kg of animal Gallic acid. Data represent the mean ± ESM for a minimum of 6 animals.

Figure 3b shows the percentage of compounds derived from gallic acid absorbed in the small intestine and colon measured in plasma (1) or aorta (2) of SHR rats, 6 h after consumption of 6.64 mg / kg of Gallic acid animal. Of the total metabolites derived from gallic acid detected in plasma (0.25 ^ M) (1) 23% come from absorption in the colon (white) and 77% from absorption in the small intestine (dotted). Of the total metabolites derived from gallic acid detected in aorta (2) (4.79 nmol / g), 88% came from absorption in the colon (white) while 12% from absorption in the small intestine (dotted). Data represent the mean ± ESM for a minimum of 6 animals.

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Figure 4a shows the decrease in systolic blood pressure (SBP) obtained in SHR rats to which 1 mL of water is administered as a control (□, a), gallic acid in doses of 3.5 mg / kg of animal ( ▲, a), 6.64 mg / kg of animal (•, bc) and 14 mg / kg of animal (-, c) or 50 mg / kg of animal of Captopril (■, b), depending on the time spent after administration. Data represent the mean ± ESM for a minimum of 6 animals. Different lowercase letters next to the treatments indicate significant differences between treatments measured by two-way ANOVA (Post-hoc Tukey).

Figure 4b shows the decrease in diastolic blood pressure (PAD) obtained in SHR rats given 1 mL of water (control, □, a), gallic acid at doses of 3.5 mg / kg (▲, ac), 6.64 mg / kg of animal (•, bc) and 14 mg / kg (-, bc) or 50 mg / kg of Captopril animal (■, b), depending on the time spent after administration. Data represent the mean ± ESM for a minimum of 6 animals. Different lowercase letters next to the treatments indicate significant differences between treatments measured by two-way ANOVA (Post-hoc Tukey).

Figure 4c shows the decrease in SBP obtained in Wistar-Kyoto (WKY) normotensive rats that are the normotensive control of SHR rats to which 1 mL of water (control, ◊) or 6.64 mg / kg of animal of gallic acid (■), depending on the time spent after administration. Data represent the mean ± ESM for a minimum of 6 animals. No statistical differences were found between treatments.

Figure 4d shows the decrease in PAD obtained in WKY normotensive rats to which 1 mL of water (control, ◊) or 6.64 mg / kg of gallic acid (■) is administered, depending on the time spent after administration . Data represent the mean ± ESM for a minimum of 6 animals. No statistical differences were found between treatments.

Figure 5a shows the decrease in SBP obtained in SHR rats produced at 6 h after intragastric administration of 1 mL of water with intraperitoneal saline injection with (grid bar) or without (white bar) 30 mg / kg of animal of NG-nitro-L-arginine methyl ester (L-NAME) at 4 h after ingestion, or 6.64 mg / kg of gallic acid with intraperitoneal solution injection

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saline with (dotted bar) or without (grated bar) L-NAME at 4 h after its intake. Data represent the mean ± ESM for a minimum of 6 animals. Different lowercase letters indicate significant differences between treatments measured by two-way ANOVA (Post-hoc Tukey).

Figure 5b shows the decrease in diastolic blood pressure (PAD) obtained in SHR rats produced at 6 h after intragastric administration of 1 mL of water with intraperitoneal saline injection with (grid bar) or without (bar white) 30 mg / kg of NG-nitro-L-arginine methyl ester animal (L-NAME) at 4 h after its ingestion, or 6.64 mg / kg of gallic acid with intraperitoneal saline injection with (dotted bar) or without (grated bar) L-NAME at 4 h after ingestion. The data represent the mean ± ESM for a minimum of 6 animals. Different lowercase letters indicate significant differences between treatments measured by two-way ANOVA (Post-hoc Tukey).

Figure 6a shows the decrease in SBP obtained in SHR rats produced at 6 h after intragastric administration of 1 mL of water with intraperitoneal saline injection with (grid bar) or without (white bar) 5 mg / kg of indomethacin animal at 4 h after its intake, or 6.64 mg / kg of gallic acid animal with intraperitoneal saline injection with (dotted bar) or without (grated bar) 5 mg / kg of animal of indomethacin 4 h after its intake. Data represent the mean ± ESM for a minimum of 6 animals. Different lowercase letters indicate significant differences between treatments measured by two-way ANOVA (Post-hoc Tukey).

Figure 6b shows the decrease in SBP obtained in SHR rats produced at 6 h after intragastric administration of 1 mL of water with intraperitoneal saline injection with (grid bar) or without (white bar) 5 mg / kg of indomethacin animal at 4 h after its ingestion, or 6.64 mg / kg of gallic acid with intraperitoneal saline injection with (dotted bar) or without (grated bar) 5 mg / kg of indomethacin animal 4 h after your intake. Data represent the mean ± ESM for a minimum of 6 animals. Different lowercase letters indicate significant differences between treatments measured by two-way ANOVA (Post-hoc Tukey).

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Figure 7 shows the concentration of endothelin (ET-1) (pg / mL) in plasma collected at 6 h after administration of 1 mL of water (control) or 6.64 mg / kg of gallic acid intragastrically. . Data represent the mean ± ESM for a minimum of 6 animals. * P <0.05.

EXAMPLES

With the intention of showing the present invention in an illustrative manner but in no way limiting, the following examples are provided.

Example 1: Experimental designs

Experimental Design 1

Spontaneously hypertensive male (SHR) rats (n = 12) and Wistar-Kyoto (WKY) normotensive rats of 17-20 weeks of age and weight between 300 and 360 g (Charles River Laboratories, Spain) were used. The rats remained with a stable ambient temperature of 23 ° C and with 12-hour light-dark cycles, ingesting water and food freely available. At 17 weeks of life, the systolic and diastolic blood pressure measurements according to Example 5A began after the administration of water (control) or of different doses of gallic acid (3.5, 6.64 and 14 mg / kg of animal) . With WKY rats only the dose of gallic acid of 6.64 mg / kg of animal was tested. Once the determinations were completed, the SHR rats were allowed a rest period of 1 week to eliminate the body's gallic acid and restore blood pressure to initial values.

Experimental Design 2

Once the blood pressure of the SHR rats described in experimental design 1 was restored, 6 of them were given intra-gastric 1 mL of water (control) and another 6 rats, 6.64 mg / kg of gallic acid dissolved in water in a dose of 1 mL. At 6 h after administration the rats were sacrificed and the blood and aorta were collected. The plasma was obtained by centrifuging the blood at 2000 x g, for 15 min at 4 ° C, and preserved at -80 ° C until further analysis.

Experimental Design 3

To evaluate the effect of gallic acid on endothelial function (Example 6A and B), the animal model of SHR rats, which have endothelial dysfunction and

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in which therefore there is an imbalance between vasodilator and vasoconstrictor factors. To do this, male SHR rats (n = 12) between 17 and 20 weeks were used to which 1 mL of water (control) or 6.64 mg / kg of gallic acid animal was administered intragastrically. Four hours later they were injected intraperitoneally with NG-nitro-L-arginin methyl ester (L-NAME) (30mg / kg of animal) or indomethacin (5 mg / kg of animal), and 2 hours later the systolic and diastolic blood pressure. The time elapsed since the intake of gallic acid or water was 6 h. L-NAME is an inhibitor of the NO-producing endothelial enzyme (eNOS). Indomethacin is an inhibitor of cyclooxygenase, an enzyme that is involved in the production of prostacyclin. The method used to measure blood pressure was that described in Example 5A. The conditions of animal housing were as described in experimental design 1.

All the experimental designs used were approved by the ethical committee of the Rovira y Virgili University (Barcelona, Spain).

Example 2: Effect of gallic acid on the components of the Renin Angiotensin System (SRA) in plasma.

The components of the SRA, Ang I and Ang II were determined by ultra high pressure liquid chromatography (UHPLC) of the plasma of SHR rats given water (1 mL) or gallic acid (6.64 mg / kg of animal) collected at 6 h after intragastric administration (Experimental design 2). Previously the plasma samples were purified by precipitation. For this, 2.5 ^ L of the compound [Asn1, Val5] -Angiotensin II (internal standard, IS) dissolved in 0.1 M acetic acid was added to 100 ^ L of plasma, and the mixture was diluted 4 times with a solution of water: acetonitrile (1: 9, v: v). The resulting solution was mixed in a vortex and left at 4 ° C for 10 min. Subsequently, it was centrifuged at 15,000 rpm for 15 min at 4 ° C in a Mikro 200 R centrifuge (Hettich GmbH & Co.KG). An aliquot of the supernatant (200 ^ L) was collected and dried using a stream of nitrogen. The dried residue was re-dissolved in 50 ^ L of a solution of water: acetonitrile (1.1: 0.9 v: v) with 0.1% formic acid and transferred to a glass vial. The final concentration of the IS in the mixture was 25 ^ g / L.

The different SRA compounds were separated using a 1290 LC Series UHPLC system (Agilent Technologies) using a reverse phase C18 column

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Acquity BEH (300 A, 100 x 2.1 mm, 1.7 ^ m from Waters Corp.) The separation was performed under a gradient elution of 0.1% formic acid in water (phase A) and 0, 1% formic in acetonitrile (phase B) at a flow rate of 0.4 mL / min. The elution gradient was: 5% B (0-0.2 min), 5-40% B (0.2-2.5 min), 40% B (2.5-4.0 min), 40 -98% B (4.0-4.5 min), 98% B (4.5-7.5 min) and 98-5% B (7.5-8.0 min). The column was equilibrated for 2 min before each separation. The column temperature was 35 ° C and the injection volume was 2 ^ L.

This UHPLC system was coupled to an Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Technologies, CA, USA) equipped with an Agilent Jet Stream (AJS) ionization source. Electrospray ionization was performed in positive mode (ESI). ESI conditions were: capillary voltage, 3 k; injector voltage, 500 V; gas temperature, 350 ° C; flow rate, 12 L / min; nebulizer pressure, 20 psi; gas temperature ("sheath"), 350 ° C, gas flow ("sheath"), 12 L / min, lens voltage 110 and 60 V, and multiplier voltage 0 V. As collision and fogging gas nitrogen was used. The acquisition was made by monitoring multiple reactions (MRM). All data obtained were processed using the Agilent MassHunter Quantitative Analysis software (version B.06.00).

For the quantification of Ang I and Ang II, calibration curves were performed with human Ang I and Ang II human (Sigma-Aldrich, Madrid, Spain) dissolved in 0.1 M acetic acid and MilliQ water, respectively. Eight different concentrations were made in a solution of water: acetonitrile (1.1: 0.9, v: v) containing 0.1% formic acid. As shown in Figures 1a and 1b, the intake of gallic acid produced a significant decrease in the plasma levels of Ang I and Ang II blocking the RAS system, thus decreasing the content of Ang II in plasma.

Example 3: Measurement of the activity of angiotensin converting enzyme (RCT)

The plasma ACE activity of SHR rats given water (1 mL) or gallic acid (6.64 mg / kg of animal) collected at 6 h after intragastric administration (Experimental design 2) , was determined using the fluorimetric method described by (Miguel M. et al. "Antihypertensive, ACE-inhibitory and Vasodilator Properties of an Egg White Hydrolysate: Effect of a Simulated Intestinal

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Digestion ". Food Chem. 2007. 104 (1): 163-168). Briefly, plasma aliquots were incubated in triplicate at 37 ° C for 15 min with 40 of the assay buffer containing the ECA substrate (5 mM Hip-His-Leu, 0.1 M sodium tetraborate, 300 mM NaCl, pH 8.3; Sigma-Aldrich, Barcelona, Spain) The reaction was stopped with the addition of 0.35 M NaOH and the resulting volume was set in 96-well polystyrene plates (Thermo Scientific, MERCK) The concentration of the product produced (His-Leu) was then determined by fluorescence Aex = 350 nm and Aem = 520 nm at 37 ° C after 30 min incubation with 2 % O-phthaldialdehyde in methanol on a FLx800 fluorimeter (Bio-Tek-IZASA) It was quantified with a calibration curve with different concentrations of the enzyme ECA obtained from rabbit lung (Sigma-Aldrich) The activity of the ECA was expressed as mU RCT / mL of plasma As shown in Figure 2, no differences were observed in the activity of the enzyme produced by r the intake of gallic acid.

Example 4: Separation, identification and quantification of Gallic Acid and its metabolites in biological samples.

To carry out this study, plasma and aortas obtained from SHR rats that had been given water (1 mL) or gallic acid (6.64 mg / kg of animal) collected at 6 hours later were used. of its intragastric administration (Experimental design 2). For the analysis, a high efficiency liquid chromatography (HPLC) device was used in tandem with a triple quadrupole mass spectrometry (MS) that allowed quantification of gallic acid and its metabolic derivatives.

Purification and concentration of analytes in biological samples.

Prior to the chromatographic analysis of the flavanols and their metabolites in rat tissues, plasma and aorta samples were treated by liquid-solid extraction (LSE) in tandem and extraction in solid micro-phase (^ SPE). Briefly, the LSE procedure involved the addition of 50 ^ L of 1% ascorbic acid and 100 ^ L of 4% phosphoric acid to 60 mg of lyophilized dry tissue. All tissue samples were extracted 4 times with 400 ^ L of a 4% water / methanol / phosphoric acid solution (94.4 / 4.5 / 1.5 v / v / v), then subjecting the sample to ultrasound for 30 s with an ice water bath and then centrifuging for 15 min at 17150 xga at 4 ° C. The supernatants obtained from the LSE tissues were cleaned by ^ SPE using OASIS Plates HLB ^ -30 micron elution (Waters Corporation). Micro-cartridges

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they were sequentially conditioned with 250 ^ L of methanol and 250 ^ L of 0.2% acetic acid. 300 ^ L of 4% phosphoric acid and 50 ^ L of a solution of 1000 g / mL IS were added to 350 ^ L of the tissue extract, and the mixture was loaded onto the plate. The charged plates were washed with 200 ^ L of Milli-Q water and 200 ^ L of 0.2% acetic acid. The retained gallic acid and its metabolites were then eluted with 100 ^ L of a solution of acetone / water Milli-Q / acetic acid (70 / 29.5 / 0.5, v / v / v). The eluted solution was injected directly into the HPLC-MS / MS system. The volume of sample injected was 2.5 ^ L.

Quantification of compounds by HPLC-MS / MS

Chromatographic analysis by HPLC-MS / MS was performed using a 1200 LC Series HPLC coupled to an MS / MS 6410 (Agilent Technologies). Chromatographic separations were performed with a Zorbax SB-AQ column (150 mm x 2.1 mm ID, 3.5 microns in particle size, Agilent Technologies). The mobile phase consisted of 0.2% acetic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL / min. The elution gradient consisted of: 5-55% of B from 0 to 10 min; 55-80% of B from 10 to 12 min; 80% B isocratic from 12 to 15 min; and 80-5% from 15 to 16 min. A stabilization time (“post-run”) of 10 min was applied. The electrospray ionization conditions (ESI) were: drying gas temperature of 350 ° C and a flow rate of 12 L / min, 45 psi of gas pressure nebulizer and 4000 V of capillary voltage. The MS / MS operated in the negative mode, and the data acquisition of gallic acid and its metabolites was performed in MRM mode using MassHunter software (Agilent Technologies). The quantification of the different compounds was made from calibration lines of the analytes included in plasma.

As shown in Figures 3a and 3b, it turned out that in the aorta the metabolite content found was much higher than in plasma. However, their distribution in the two biological samples was different; Plasma metabolites prevailed in the small intestine (73 vs. 23%), while in the aorta cells, those in the large intestine (88 versus 12%). The metabolites found are shown in the following table I.

Table I

Plasma Aorta

 Compound

 mm SEM nmol / g SEM

 Gallic acid

 0.04 0.02 0.53 0.01

 3-O-methyl gallic acid

 0.09 0.03 0.05 0.00

 Gallic acid glucuronide

 0.00 0.00 0.00 0.00

 Gallic Acid Methylsulfate

 0.07 0.02 0.00 0.00

 Dimethyl gallic acid

 0.00 0.00 0.00 0.00

 Trimethylgalic acid

 0.00 0.00 0.00 0.00

 Catechol

 0.01 0.00 1.80 0.12

 Methyl pyrogallol

 0.01 0.00 0.00 0.00

 3- hydroxybenzoic acid

 0.03 0.01 1.90 0.06

 4-hydroxybenzoic acid

 0.00 0.00 0.32 0.06

 3,4-dihydroxybenzoic acid

 0.01 0.00 0.20 0.04

 TOTAL

 0.25 4.79

Example 5: Effect of gallic acid on cardiovascular diseases

5 5.A. Effect on hypertension: Measurement of the antihypertensive activity of gallic acid

in spontaneously hypertensive rats

The measurement of the blood pressure of the SHR rats (Experimental Design 1) was carried out with a modification of the tail cuff technique, originally described by Buñag (Buñag RD. Validation in Awake Rats of a Tail-Cuff 10 Method for Measuring Systolic Pressure. ”1973. J. Appl. Phys. 34 (2): 279-82). Usually, this technique allows obtaining only PAS measurements; However, the equipment used in this study (model Le50001 Letica) makes it possible to differentiate PAS and PAD from animals. Before placing the cuff on the tail of the rats, they were exposed to a temperature close to 37 ° C to facilitate dilation of the caudal artery. To ensure the reliability of the measurement, the animals were accustomed to the procedure 2 weeks before carrying out the test.

The administration of the gallic acid to be tested was carried out by intragastric tube in a period of time between 9 am and 10 am in the morning. The SHR 20 rats used for the study at that time had PAS and PAD values, respectively between 210 and 220 mm Hg, and between 180 and 190 mm Hg,

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respectively. Both measures were taken in the animals every 2 hours until 48 hours post-administration. Three different concentrations of gallic acid 3.5, 6.64 and 14 mg / kg body weight of the animal were tested.

As a negative control to establish the circadian variation of the PAS and the PAD, rats were used which were administered by intragastric tube 1 mL of water. As a positive control, rats were used which were administered by intragastric tube 50 mg / kg of Captopril, a prototype ACEI drug. This dose of Captopril was administered to each rat in a volume of 1 mL.

The results obtained were grouped and the mean ± the standard error of the mean (ESM) was obtained for a minimum of 6 homogeneous trials. Data from the three groups of animals were compared in a 2-way analysis of variance (ANOVA) using the Tukey test at each post-administration time with the SPSS statistical program (IBM SPSS Statistics software version 20.0) and the difference for values of p <0.05 was considered significant.

As shown in Figures 4a and 4b, the decrease in SBP and PAD were maximum at six hours after the administration of Captopril. On the contrary, the administration of gallic acid in a concentration of 3.5 mg / kg did not produce a significant decrease in SBP and PAD, except for 6 h, observing the same effect as that produced after the intake of 1 mL of water, where they remained stable in the post-administration times evaluated. However, after administration of 6.64 or 14 mg / kg of gallic acid, in both cases there was a decrease in SBP after 2 h of its administration, being maximum at 6 h for the two concentrations tested, observing PAS values similar to those obtained with captopril (over 40 mmHg). The antihypertensive effect of the dose of 6.64 mg / kg remained until 24 h after administration, as was captopril. PAD drops similar to those produced by captopril were also observed when 14 and 6.64 mg / kg of gallic acid were administered, being maximum at 4 or 6 h, respectively, although lower PAD values were observed after intake. of 6.64 mg / kg of gallic acid. The values of the PAS and the PAD observed 48 hours after the different administrations were similar to those that the animals had before them.

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5. B. Discarding the hypotensive effect of gallic acid in normotensive rats

In order to rule out the possible hypotensive effect of gallic acid in normotensive rats, the PAS and PAD of WKY rats (Experimental design 1), normotensive control of the SHR were recorded, to which 6.64 mg / kg had been administered (n = 6) The administration of gallic acid was performed intragastrically, dissolved in 1 mL of water. The method used to measure the blood pressure of the animals and the conditions of housing of the rats were the same as those used in Example 2. The PAS and PAD values of the WKY rats at the start of the study were between 124 and 135 mm Hg, and between 95 and 100 mm Hg, respectively.

As shown in Figures 4c and 4d, the hydrolyzate did not modify either the PAS or the PAD of the treated animals. These results allow us to rule out possible undesirable effects of the compound tested on the blood pressure of normotensive subjects.

Example 6: Effect of gallic acid on endothelial function

6. A. Effect of gallic acid mediated by the endothelial vasodilator NO

To evaluate the effect of gallic acid on endothelial function, the animal model of SHR rats was used, where as already indicated there is endothelial dysfunction (Bernatova I et al. "Endothelial Dysfunction in Spontaneously Hypertensive Rats: Focus on Methodological Aspects", 2009. J. Hypertens, Suppl. 27 (6), S27-31) Firstly, it was evaluated whether the effect of gallic acid was mediated by NO and for this purpose it was administered to L-NAME animals. This in vivo study corresponds to the experimental design 3.

As shown in Figures 5a and 5b, when the enzyme eNOS is inhibited, gallic acid does not produce a decrease in PAS and PAD, so its effect is completely suppressed. The effect of gallic acid is mediated by the endothelial vasodilator NO because if its producing enzyme (eNOS) is inhibited its effect disappears

6.B. Effect of gallic acid partially mediated by PC endothelial vasodilator Similar to the previous Example, another in vivo experiment was performed in which the indomethacin SHR animals were administered. Indomethacin is an inhibitor of cyclooxygenases, enzymes responsible for the production of prostacyclin, which is a

endothelium specific vasodilator. The study design corresponds to the experimental design 3.

Figures 6a and 6b show that administering this inhibitor partially reverses the decrease in PAS and PAD produced by gallic acid. It turns out that the effect of gallic acid is partially mediated by the PC endothelial vasodilator because if its producing enzyme, cyclooxygenase, is partially inhibited its effect.

On the other hand, Figure 7 shows how the administration of gallic acid produces a plasma decrease in the vasoconstrictor endothelin-1 (ET-1). A significantly lower ET-1 content is observed in the plasma of rats treated with gallic acid than in water. The concentration of ET-1 in plasma was carried out using the endotelin one Elisa kit (Enzo Life Science). The plasma used was that obtained from SHR rats according to experimental design 2.

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Claims (5)

ES 2 611 667 A1 1. Use of gallic acid or pharmaceutically acceptable salts thereof in the preparation of a medicament for the treatment of a pathology by 5 hyperactivation of the Renina Angiotensin Aldosterone system, SRAA, in a subject. 2. Use according to claim 1, characterized in that said pathology is selected from a cardiovascular disease, a cerebrovascular disease, a renal disease and diabetes. 3. Use according to claim 2, characterized in that said diabetes is diabetes 10 type 2. 4. Use according to any one of claims 1 to 3, characterized in that said subject is human. 5. Pharmaceutical composition comprising gallic acid and pharmaceutically acceptable excipients. 15 6. Food supplement comprising gallic acid and food additives. twenty