Use of a plant extract from Salvia haenkei as an active agent in the treatment of heart diseases

Abstract

The present invention relates to the use of Salvia haenkei extract as an active agent in the treatment of heart diseases, which can provide a significant contribution in terms of slowing down, alleviating, reducing and / or preventing changes and damages in heart tissues. In addition, the present invention also relates to a pharmaceutical composition comprising Salvia haenkei extract and a suitable pharmaceutically acceptable excipient for use in the treatment of heart diseases.

Description

[0001] Description Field of the Invention

[0002] The present invention relates to the use of Salvia haenkei extract as an active agent in the treatment of heart diseases. The Salvia haenkei extract provides a significant contribution in slowing down, alleviating, reducing and / or preventing alterations and damages of heart tissues (both muscle tissue and connective tissue). In addition, the present invention also relates to a pharmaceutical composition comprising the Salvia haenkei extract and a suitable pharmaceutically acceptable excipient for use in the treatment of heart diseases.

[0003] Background

[0004] The term "heart disease" or "heart condition" without distinction refers to a variety of acute and / or chronic conditions that can affect one or more of the components of the heart. Heart diseases represent the most common chronic diseases and the associated sanitary cost. In addition, heart diseases are now the leading cause of death.

[0005] The most relevant heart conditions include cardiac fibrosis, arrhythmia, long QT syndrome (QTL), heart failure or decompensation, and cardiotoxicity. The heart diseases can be caused by a variety of factors, including hypertension, diabetes, myocardial infarction, chemotherapy, or heart transplantation.

[0006] In cardiac fibrosis, the myocardium, i.e., the heart tissue containing cardiomyocytes incorporated in the extracellular matrix (ECM) with collagen fibers, remodels with an increase in type I collagen deposition. Cardiac fibrosis can be: responsive to arterial hypertension and long-term overwork, usually without loss of cardiomyocytes; infiltrative as in metabolic disorders such as sphingolipidosis; and reparative after myocardial infarction related to damage of cardiomyocytes.

[0007] Cardiac fibroblasts activated in an unbalanced manner differentiate into myofibroblasts, which express growth factors, pro-inflammatory factors, and profibrotic factors, and secrete a large amount of metalloproteinases and other enzymes that degrade the extracellular matrix, promoting their own migration; in addition, the deposition of collagen and other proteins leads to the formation of scars, which over time make the interstitium harder (stiffer), impairing heart function.

[0008] The evaluation of cardiac fibrosis can be carried out by endomyocardial biopsy and magnetic resonance imaging or by measuring biomarkers of fibrosis such as serum fibronectin, TGF-β, and MMP (matrix metalloproteinases).

[0009] Long QT or long QT syndrome is a rare cardiac condition in which the recovery time of the heart's ventricles to prepare for the next contraction after one contraction is prolonged. This dysfunction is detectable as an alteration in specific parts of the electrocardiogram trace (QT interval and T wave). In many cases, long QT syndrome is associated with arrhythmias, which are life-threatening changes in the myocardial rhythm. Long QT syndrome can be congenital or may be secondary to drug ingestion or blood electrolyte alterations.

[0010] Cardiotoxicity is defined as toxicity affecting the heart during pharmacological treatment (usually oncological pharmacological treatment). The most well-known cardiac damage is left ventricular dysfunction, which can lead to chronic and progressive heart failure and sometimes even death. In addition to a complete clinical evaluation using an ECG, the two most commonly used diagnostic methods are echocardiography and the dosage of two cardiac biomarkers, troponin and natriuretic peptides. Traditionally, subclinical cardiotoxicity has been detected by using echocardiography or other imaging to evaluate a decrease in left ventricular ejection fraction (LVEF).

[0011] Thus, generally speaking, heart conditions will manifest themselves as alterations in the electrophysiological function of the heart, usually associated with arrhythmias and / or heart failure. Any condition that damages the heart or reduces its function, thereby altering the heart's ability to pump oxygenated blood throughout all blood vessels and organs, has serious consequences that can manifest throughout the body.

[0012] Currently available treatment strategies for heart diseases generally aim to slow down the heart rate, such as using beta-blocker drugs, so as to reduce the likelihood of arrhythmias occurring. Alternatively, in order to reduce cardiac fibrosis, therapies of uncertain efficacy are used, such as angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists, beta-blockers, endothelin antagonists, and finally statins. Alternatively, an implantable cardioverter defibrillator in the patient's body is the most commonly used non-drug therapy for patients at high risk of sudden cardiac death. This therapy is clearly invasive.

[0013] Therefore, the object of the present invention is to provide a new, effective and non-invasive treatment method for treating heart conditions that can act at the level of both myocardial and connective heart tissues. Summary of the Invention

[0015] The above object has been achieved by the use of an extract of Salvia haenkei as an active agent in the treatment of heart diseases as reported in claim 1.

[0016] In another aspect, the present invention relates to a pharmaceutical composition comprising an extract of Salvia haenkei and a suitable pharmaceutically acceptable excipient for use in the treatment of heart diseases.

[0017] For the purposes of the present invention, the heart diseases include: cardiac fibrosis, arrhythmia, long QT syndrome (QTL), heart failure, heart attack, and cardiotoxicity. Brief Description of the Drawings

[0019] The features and advantages of the present invention will become apparent from the following detailed description, working examples provided for illustrative purposes, and the accompanying drawings, in which:

[0020] - Figure 1 Shows: (A) the protocol of the in vitro experiment of Example 2 (SH = Salvia haenkei extract; MEA = multi-electrode array), (B) an exemplary ECG trace (showing the QT interval), and (C) the results of QT recordings of beating cardiomyocytes (CM) from untreated and SH-treated human subjects.

[0021] - Figure 2 Shows: (A) the protocol of the in vitro experiment of Example 3 (SH = Salvia haenkei extract; MEA = multi-electrode array, DOXO = doxorubicin), (B) an exemplary ECG trace (showing the QT interval), and (C) the results of QT recordings of beating CM treated with and without SH after DOXO exposure.

[0022] - Figure 3 Shows: (A) the protocol of the in vivo experiment of Example 4, (B) representative echocardiography images of each group tested, and (C) the quantification of the results.

[0023] - Figure 4 Shows: (A) the protocol of collagen synthesis and, according to Example 5, the evaluation of procollagen and secreted collagen-I formation in cardiac fibroblasts with and without SH at the basal level and after exposure to Tgfb-1 (western blot analysis and quantification results are shown in Figures B and C, respectively).

[0024] - Figure 5 Shows: (A) the staining of collagen-I fibers and collagen-II fibers in cardiac fibroblasts with and without SH at the basal level and after exposure to Tgfb-1 according to Example 5, and the quantification of the staining (B). Detailed Description of the Invention

[0026] Salvia haenkei is a shrub from Bolivia and Peru, commonly known as "prawn sage" due to the color and shape of its shrimp-like flowers. Morphologically, Salvia haenkei is characterized by lanceolate leaves with serrated edges that are over 12 cm in length. They are light green in color and have a wrinkled surface. The inflorescence is very long, over 20 cm, and is defined as a "raceme", i.e., the flowers are inserted along the same flower axis at the same length of pedicels at different heights on the central axis.

[0027] For the preparation of the extract, the aerial parts of the plant are usually used, i.e., the stems, leaves, flowers, or a mixture thereof. These parts can be used fresh or after drying under controlled conditions. In both cases, the individual parts or their mixture are brought into contact with a suitable extraction solvent by using conventional extraction methods such as maceration or percolation, or more sophisticated techniques such as extraction using ultrasound, microwaves, pressure, or supercritical fluids.

[0028] After separating the exhausted plant, the extract can be used as it is, or after replacing the extraction solvent with one or more solvents suitable for human use (such as glycerol or diols if not used in the extraction phase). Preferably, the extraction solvent is removed to give a dry extract. For the removal of the extraction solvent, the preferred techniques are evaporation under reduced pressure and low temperature, as well as atomization.

[0029] The extract can also undergo subsequent purification steps to remove potential contaminants (such as lipophilic pesticides), impurities (such as chlorophyll), or to increase the concentration of secondary metabolites.

[0030] The Salvia haenkei extract thus obtained contains a collection of terpenoids, especially diterpenoids and triterpenoids (Almanza, G. et al., (1997) Clerodane diterpenoids and an ursane triterpenoid from Salvia haenkei, Computer-assisted structural elucidation, Tetrahedron, 53(43), pp. 14719 - 14728), as well as gallic acid and its derivatives and chlorogenic acid and its derivatives. Some of these compounds are specific to this species of Salvia and distinguish it from other species in the same genus, reasonably contributing to the characteristic activity of its extract.

[0031] The dry extract can be admixed with suitable excipients, for example to make it smoother, less hygroscopic or to standardize the content of secondary metabolites. Among the excipients that can be used are, for example, silica, maltodextrin, microcrystalline cellulose.

[0032] In solvents suitable for the preparation of Salvia haenkei extracts, solvents of medium polarity are preferably chosen because they are able to effectively extract the secondary metabolites of the plant. Preferably, such extraction solvents have a dielectric constant of from 8 to 60.

[0033] Examples of extraction solvents that can be used are alcohols having up to 4 carbon atoms (including diols and triols), aldehydes, ketones, organic esters, chlorinated compounds, and mixtures thereof. When miscible, such solvents can also be used in admixture with water.

[0034] Preferred solvents include methanol, ethanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, acetone, ethyl acetate and mixtures thereof, either as such or in admixture with water.

[0035] In a preferred embodiment, the extraction solvent is an aqueous-alcoholic solution, even more preferably an alcoholic solution of 40%-80%. The alcohol is preferably methanol or ethanol.

[0036] Particularly preferred is the embodiment in which the extraction solvent is a 60%-80% ethanol solution.

[0037] Preferably, the preparation of the Salvia haenkei extract comprises the following steps:

[0038] 1. Collecting the aerial parts of Salvia haenkei,

[0039] 2. Extracting with a solvent,

[0040] 3. Separating the spent plants from the liquid extract, and

[0041] 4. Removing the solvent to give a dry extract.

[0042] The aerial parts in step 1. can be fresh or pre-dried. If the aerial parts are fresh and just harvested, the relatively large amount of water physiologically present in the plant should be taken into account.

[0043] Accordingly, the present invention relates to the use of Salvia haenkei extract as an active agent for use in the treatment of heart diseases.

[0044] For the purposes of the present invention, the term "treatment" is intended to include the administration of an extract of Salvia haenkei or a pharmaceutical composition comprising said extract to a subject suffering from a heart disease or at risk of developing a heart disease, for the purpose of improving the overall condition of the subject's heart tissue, and for the purpose of slowing down, alleviating, reducing and / or preventing any alteration in the function of the subject's heart tissue.

[0045] Preferably, the heart disease is: cardiac fibrosis, arrhythmia, long QT syndrome (QTL), heart failure, heart attack or cardiotoxicity.

[0046] These heart diseases can be caused by a variety of factors, including hypertension, diabetes, myocardial infarction, chemotherapy, hypercholesterolemia or heart transplantation. Optionally, the heart condition is drug-induced, such as for example chemotherapy drugs.

[0047] Preferably, the extract is administered to a subject in need thereof at a dose of 0.1 mg - 1500 mg per day.

[0048] In a preferred embodiment, the extract is administered via a systemic route at a dose of 1 mg - 5000 mg per day, more preferably by oral administration, and the effective dose varies according to the extent and severity of the disease to be treated.

[0049] Preferably, the daily dose of the drug is about 1500 mg, preferably from 0.1 mg to 1000 mg, preferably administered in a divided manner about once a day or 2 - 3 times a day.

[0050] In another aspect, the present invention relates to a pharmaceutical composition comprising an extract of Salvia haenkei and a pharmaceutically acceptable carrier for use in the treatment of heart diseases.

[0051] In another aspect, the present invention relates to a food supplement comprising an extract of Salvia haenkei for use in the treatment of heart diseases.

[0052] The pharmaceutical composition or food supplement may be administered via the oral route.

[0053] Preferably, the pharmaceutical composition or food supplement comprising an extract of Salvia haenkei is in liquid form, more preferably the extract of Salvia haenkei is at a concentration of 0.1 mg / ml - 500 mg / ml of the composition or supplement, more preferably at a concentration of 1 mg / ml - 100 mg / ml.

[0054] Preferably, the pharmaceutical composition or food supplement comprising the Salvia haenkei extract is in solid form, and more preferably the Salvia haenkei extract is at a concentration of 0.1 mg / mg - 500 mg / mg of the composition or supplement, and more preferably at a concentration of 1 mg / mg - 100 mg / mg.

[0055] Preferably, the pharmaceutical composition comprises the Salvia haenkei extract and at least one additional active agent selected from the group consisting of: cardiotonic agents, antiarrhythmic agents, vasodilators, antihypertensive agents, antiplatelet agents, and diuretics.

[0056] An article for oral administration of a pharmaceutical composition comprising the Salvia haenkei extract may be in the form of: tablets, capsules, lozenges, orally dispersible films, soft gelatin capsules, granules or powders, orally dispersible powders, liquid solutions, dressings, or suspensions. As is known in the art, tablets, capsules, and lozenges may also contain common excipients in addition to the active ingredient, such as extenders such as lactose, calcium phosphate, sorbitol, etc.; lubricants such as magnesium stearate, polyethylene glycol (PEG); binders such as polyvinylpyrrolidone, gelatin, sorbitol, gum arabic, flavoring agents, disintegrants, and dispersants.

[0057] Liquid pharmaceutical products, which are usually in the form of aqueous or oily solutions or suspensions, may contain conventional additives such as dispersants.

[0058] All of the pharmaceutical preparations described above can be prepared by methods known in the pharmaceutical art.

[0059] It should be understood that all aspects determined to be preferred and advantageous for the Salvia haenkei extract should be considered similarly preferred and advantageous for the pharmaceutical composition and its uses.

[0060] It should also be understood that all combinations of the preferred aspects of the Salvia haenkei extract of the present invention, as well as the pharmaceutical composition and its uses, as reported above are hereby considered to be disclosed.

[0061] The following are working examples of the present invention provided for non - limiting, illustrative purposes, demonstrating the efficacy of the Salvia haenkei extract in the treatment of heart diseases. Examples

[0062] Example 1.

[0063] Preparation of Salvia haenkei extract

[0064] Harvest 10 kg of the aerial parts of Salvia haenkei from field crops. The aerial parts are then subjected to a drying process in a ventilated dryer under controlled conditions.

[0065] In this way, 1.95 kg of dried plant is obtained, which is ground in a bladed mill to give dried and ground Salvia haenkei.

[0066] This is used as raw material for the subsequent solvent extraction carried out as described below:

[0067] 1. Introduce 100 g of dried and ground Salvia haenkei into a static percolator and cover it completely with 200 ml of a water and ethanol 30%-70% v / v mixture. Let it stand for 2 hours and recover the extraction solvent (170 ml) from the bottom of the percolator and set it aside (extract 1);

[0068] 2. The wet plant remaining in the percolator is covered with a fresh aliquot of 70% aqueous ethanol (170 ml), and it is left to stand for 2 hours. Recover the solvent (165 ml - extract 2);

[0069] 3. Repeat the extraction described in point 2 until the dry residue of the recovered extract is less than 5% of the total dry residue extracted up to that point. At this point, the extraction is considered complete and the exhausted wet plant is eliminated. Six extractions are required;

[0070] 4. Combine the extracts obtained from the individual extraction steps (from extract 1 to extract 6), filter, and concentrate in a rotary evaporator under reduced pressure at low temperature. Continue until a concentrated viscous solution (35 ml) is obtained;

[0071] 5. Transfer the concentrated extract to a steel tray and insert it into a cabinet dryer under vacuum with the heating set to 30 °C. After 12 hours, the solvent is completely removed (weight loss of the extract less than 10%, i.e., dry residue higher than 90%). 14.3 g of the complete dry extract is obtained. Drug: The dry extract ratio (DER) is 7:1 (extract 1A).

[0072] 6. Add 10 g of maltodextrin (DE 10) to the obtained dry extract to improve its consistency, and grind and sieve the mixture to obtain 23.7 g of ground dry extract.

[0073] Different natural dry extracts were prepared by applying the same procedure but different extraction solvents.

[0074] The table summarizes the results of various extractions:

[0075]

[0076]

[0077] In the following examples, an aqueous-alcoholic extract of Salvia haenkei of Example 1A was used, which is abbreviated as "SH".

[0078] For in vitro experiments, the extract was reconstituted with absolute ethanol to a stock concentration of 10 mg / ml (SH is insoluble in water at this concentration) and kept at -20 °C. Depending on the assay, working solutions were obtained by diluting the stock solution in water-based RPMI medium at concentrations of 10 μg / ml, 100 μg / ml, and 300 μg / ml.

[0079] For in vivo experiments, considering that the water intake of adult rats is about 30 ml / day - 40 ml / day, 1.25 g of the SH extract was diluted in 300 ml of water to achieve a daily dose of approximately 0.5 mg / Kg (extract / body weight).

[0080] Example 2

[0081] Effect of SH on electrophysiological properties of beating human iPS-CM (basal)

[0082] In vitro tests were performed on human specialized cardiac cell cardiomyocytes (CM) derived from iPSCs. Human iPSCs were generated by overexpressing four Yamanaka factors (Oct3 / 4, Sox2, Klf4, c-Myc) in adult somatic cells. Then, stable colonies of the stem cells were redifferentiated into CMs as described by Pianezzi, E. et al. ("Role of somatic cell sources in the maturation degree of human induced pluripotent stem cell-derived cardiomyocytes". Biochim Biophys Acta Mol Cell Res, 2019: page 118538).

[0083] Differentiated cells showed spontaneous beating activity at 10.0 ± 1.4 days post-infection. Fifteen days after the onset of spontaneous beating activity, beating iPSC-CMs were isolated from the culture wells by microdissection and gently plated onto a multi-electrode array plate (MEA) with a standard 60 electrodes. An MEA with high spatial (200 mm) resolution (60MEA-200 / 30iR-Ti; Multi Channel Systems, Reutlingen, Germany) was used. Extracellular field potentials (FPD) were recorded for spontaneous beating clusters of iPSC-CMs at 37 °C.

[0084] On day 1 of recording (approximately 25 days post-infection, DPI), Salvia haenkei (SH) extract was added to the culture medium at a maximum concentration of 10 μg / ml ( Figure 1 A). The field potential duration reflecting electrical systole was measured from the start of the sharp positive deflection to the peak of the secondary slow deflection ( Figure 1 B); for simplicity, this measurement was designated as "QT" and rate-corrected (QTc) by the Bazett formula.

[0085] As Figure 1 shown in C, SH did not cause any statistically significant change in the QT interval; thus, under basal conditions, the electrophysiological properties of CMs were not affected by the presence of 10 μg / ml of SH.

[0086] Example 3

[0087] Effect of SH on electrophysiological properties of beating human iPS-CM (with cardiotoxic treatment)

[0088] Sublethal concentrations (10 M - 7 M) of doxorubicin (DOXO) cancer treatment in cardiomyocytes (CMs) impairs the function of ion channels in CMs and results in cardiotoxicity with QT interval prolongation, which is the basis of arrhythmogenic cardiomyopathy in patients. A recently developed platform for simulating DOXO-induced electrophysiological dysfunction has been used here to test whether SH can rescue the harmful effects of DOXO on the QT interval. DOXO was added to the culture medium of beating CMs, and the medium was changed after 3 hours, and SH at a concentration of 10 μg / mL was added ( Figure 2 A).

[0089] Traces were recorded every 24 hours for 7 consecutive days starting from the start of DOXO treatment (day 0 - 3 h).

[0090] As expected, treatment with DOXO induced QT interval prolongation, while treatment with SH surprisingly reversed QT interval prolongation. SH also had a positive effect on stabilizing the heart rate ( Figure 2 C).

[0091] Example 4

[0092] Cardioprotective effect of SH in an animal model of cardiotoxicity

[0093] Adult female Sprague-Dawley rats (250 g - 300 g body weight) were injected with 6 intraperitoneal doses of DOXO (Sigma), which were delivered at regular intervals from day 1 to day 11 (cumulative dose = 15 mg / kg). During the treatment period, the rats had free access to water containing SH. The rats received a daily dose of approximately 0.5 mg / Kg of SH.

[0094] For echocardiography assessment, animals were anesthetized with 2.0% - 2.5% isoflurane at the indicated time points. Transthoracic echocardiography was performed using a Vevo 2100 high-resolution imaging system (VisualSonic) equipped with a 13 MHz - 24 MHz linear transducer. Analyses were performed on day 0 (baseline), day 12, and day 19. Two-dimensional short-axis M-mode echocardiography was performed. All M-mode trace measurements were averaged over at least three cardiac cycles. The researchers were blinded to the identity of the animals.

[0095] Representative echocardiography images of three different treatment groups at 12 days are shown in Figure 3 B. In DOXO-treated animals not receiving SH, the left ventricular end-systolic volume (LVESV) increased over time, followed by a significant decrease in the left ventricular ejection fraction (LVEF). At days 12 and 19, co-administration of SH significantly decreased LVESV while increasing LVEF. The left ventricular end-diastolic volume (LVEDV) also significantly decreased at day 19 ( Figure 3 C). These results indicate that SH prevents the harmful effects of DOXO-induced on overall cardiac function.

[0096] Advantageously, in untreated and unaffected animals, SH alone had no effect on cardiac function ( Figure 3 C).

[0097] Example 5

[0098] Effect of SH on reducing collagen-I secretion by fibroblasts

[0099] The biosynthesis of collagen and the secretion by cardiac-activated fibroblasts (myofibroblasts) are key phenomena regulating cardiac fibrosis, leading to maladaptive remodeling in heart failure. Collagen is synthesized as pre-pro-α-chain: after multiple post-translational modification steps, the pre-pro-α-chain self-assembles into a triple helix called procollagen. Procollagen is exported and converted extracellularly into collagen by cleavage of the propeptides ( Figure 4 A). By using an antibody directed against the C-peptide CPII, it is possible to distinguish intracellular procollagen from the extracellular isoform.

[0100] The ability of SH to reduce both procollagen formation and secreted collagen-I in cardiac fibroblasts was tested at the basal level and after activation of the myofibroblasts by exposure to Tgfb-1.

[0101] As shown by representative immunoblot analysis ( Figure 4 B, Figure 4 C), SH reduced collagen synthesis in cardiac fibroblasts at the basal level and after stimulation with Tgfb-1.

[0102] This effect was dose-dependent and superior to secreted collagen-I, thus indicating that SH reduces collagen deposition in the fibrotic heart.

[0103] Without being bound by theory, this effect is most likely the underlying cause of the cardioprotective effect observed in vivo (see Example 4).

[0104] The role of SH in reducing collagen deposition was further evaluated by "picrosirious red", which stains collagen-I and collagen-II fibers ( Figure 5 A).

[0105] As shown by representative images and quantification ( Figure 5 B), SH had no effect on fiber deposition under basal conditions, while it significantly reduced collagen secretion and fiber formation when fibroblasts were activated by Tgf-b stimulation.

Claims

1. Use of Salvia haenkei extract as an active agent in the preparation of a medicament for treating heart diseases, wherein the extraction is carried out in a solvent of ethanol:water = 70:30 v / v; wherein the heart diseases are at least one of the following: cardiac fibrosis, arrhythmia, long QT syndrome, and cardiotoxicity.

2. The use according to claim 1, wherein the heart disease is cardiac fibrosis.

3. The use according to claim 1, wherein the heart disease is cardiotoxicity.

4. The use according to any one of claims 1 - 3, wherein the extract is administered via the oral route.

5. Use of a pharmaceutical composition comprising Salvia haenkei extract and a pharmaceutically acceptable vehicle in the preparation of a medicament for treating heart diseases, wherein the extraction is carried out in a solvent of ethanol:water = 70:30 v / v; wherein the heart diseases are at least one of the following: cardiac fibrosis, arrhythmia, long QT syndrome, and cardiotoxicity.

6. The use according to claim 5, wherein the medicament is in the form of: tablets, capsules, lozenges, orally dispersible films, granules or powders, liquid solutions, dressings, or combinations thereof.

7. The use according to claim 5, wherein the medicament is in the form of: soft gelatin capsules, orally dispersible powders, or suspensions.

8. The use according to any one of claims 5 - 7, when the medicament is in liquid form, the Salvia haenkei extract is at a concentration of 0.1 mg / ml - 500 mg / ml of the medicament; or when the medicament is in solid form, the Salvia haenkei extract is at a concentration of 0.1 mg / mg - 500 mg / mg of the medicament.

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