Composition based on olive extract suitable for cardioprotection

Abstract

The present invention relates to compositions containing a mixture of three active substances, which are derived from dive extract and are hydroxytyrosol, oleuropein, and oleocanthal, said compositions being suitable for cardioprotection.

Description

[0001] Composition based on olive extract suitable for cardioprotection

[0002] Description of the Invention

[0003] The present invention relates to compositions containing a mixture of three active substances, which are derived from olive extract and are hydroxytyrosol, oleuropein, and oleocanthal, said compositions being suitable for cardioprotection.

[0004] Hydroxytyrosol can be extracted from olive leaves and fruit and is a molecule with antioxidant, anti-inflammatory, anti-atherogenic, and antithrombotic properties. In vitro studies show that, in addition to its antioxidant action, it improves endothelial dysfunction, lipid profile, and has anti-inflammatory properties. It can therefore be considered a cardioprotective agent [Bertelli M, Kiani AK, Paolacci S, Manara E, Kurti D, Dhuli K, Bushati V, Miertus J, Pangallo D, Baglivo M, Beccari T, Michelini S. Hydroxytyrosol: A natural compound with promising pharmacological activities. J Biotechnol. 2020; 309: 29-33],

[0005] Oleuropein is a phenolic compound of the olive, composed of three structural subunits: hydroxytyrosol, elenolic add, and a glucose molecule, Oleuropein has been reported to possess a range of biological actions, including anti-dyslipidemic, anti-obesity, antidiabetic, antioxidant, anti-atherogenic, antihypertensive, anti-inflammatory, and hepatoprotective activity [Ahamad J, Toufeeq I, Khan MA, Ameen MSM, Anwer ET, Uthirapathy S, Mir SR, Ahmad J. Oleuropein: A natural antioxidant molecule in the treatment of metabolic syndrome. Phytother Res. 2019; 33(12): 3112-3128], as well as cardioprotective activity [I. Andreadou, E.K. Iliodromitis, E. Mikros, M. Constantinou, A. Agalias, P. Magiatis, E. Kamber, A. L, Skaltsounis, A, Tsantili-Kakoulidou, D. Th. Kremastinos. The olive constituent oleuropein exhibits anti-ischemic, anti-oxidative and hypolipidemic effects in anesthetized rabbits. J Nutr, 136: 2213-2.219, 2006].

[0006] Oleocanthal is a minor component of olive oil with strong anti-inflammatory activity. Given that the pathogenesis of many chronic diseases involves inflammation and oxidative stress, oleocanthal is a promising agent for the prevention of these conditions. Anti-inflammatory, antioxidant, antimicrobial, anticancer, and neuroprotective effects of oleocanthai have been reported. Oleocanthai has been shown to suppress cancer cells of melanoma, breast, liver, and colon. Neurological studies have focused on oleocanthal's effects against Alzheimer's disease, Oleocanthai improved amyloid-beta protein clearance from neurons and reduced astrocyte inflammation [Pang KL, Chin KY. The Biological Activities of Oleocanthai from a Molecular Perspective. Nutrients. 2018; 10(5): 570].

[0007] Document W02020095236A1 refers to compositions where oleuropein is present at approximately 30%, hydroxytyrosol at least approximately 50%, tyrosol at least approximately 25%, oleocanthai at least approximately 2%, along with suitable pharmaceutical excipients. However, the compositions described in that document are suitable for anti-inflammatory activity* and not for cardioprotection.

[0008] The product Olivomed by the company Intermed S.A. contains hydroxytyrosol and exhibits a positive effect on reducing body mass index (W0202Q152131A1) and improving vascular and endothelial function in coronary patients [Association of hydroxytyrosol enriched olive oil with vascular function in chronic coronary disease. Ikonomidis I, Katogiannis K, Chania C, lakovis N, Tsoumani M, Christodoulou A, Brinia E, Pavlidis G, Thymis J, Tsilivarakis D, Kountouri A, Korakas E, Lambadiari V, Triposkiadis F, Skaltsounis L, Tseti I, Iliodromitis EK, Andreadou L Eur J Clin Invest. 2023 Mar 13:el3983].

[0009] However, to date, no compositions have been reported in the prior art that contain a specific mixture of the three active substances derived from olive extract— hydroxytyrosol, oleuropein, and oleocanthal— which compositions are suitable for cardioprotection as described in the present invention,

[0010] Given the considerable difficulty in reducing infarct size after myocardial ischemia / reperfusion, the discovery of a safe, preventive, supplementary option is currently an urgent clinical need for improving the quality of life of patients at high cardiovascular risk.

[0011] The present invention, taking into account all of the above, addresses and resolves the aforementioned problems of the prior art, and does so with unexpectedly improved results in terms of cardioprotection, such as reducing infarct size after myocardial ischemia / reperfusion and lowering fasting glucose levels.

[0012] More specifically. the present invention is defined by the foilowing definitions:

[0013] Definition 1. A composition containing a mixture of three active substances derived from olive extract, namely hydroxytyrosoi, oleuropein, and oleocanthal, wherein the oleocanthal is at least ¼ the weight of oleuropein, and the composition is suitable for cardioprotection.

[0014] Definition 2. A composition for use according to definition 1, wherein the hydroxytyrosoi :oleuropein:oieocanthal weight ratio is approximately 1: at least approximately 3: at least approximately 2, with a measurement accuracy of ± 0,2.

[0015] Definition 3. A composition for use according to any of definitions 1 to 2, in the form of a soft capsule with a mass of 500 to 1000 mg, preferably 750 mg, wherein the total mass of the three active components— hydroxytyrosoi, oleuropein, and oleocanthal— is from 10 to 20 mg, preferably 15 mg.

[0016] Definition 4. A composition for use according to any of definitions 1 to 3, suitable for administering hydroxytyrosoi at a dose of at most 5 to 10 mg / kg patient body weight, preferably at most 5,9 mg / kg patient body weight. Definition 5. A composition for use according to any of definitions 1 to 4, suitable for administering oleuropein at a dose of at most 10 to 30 mg / kg patient body weight, preferably at most 20,6 mg / kg patient body weight.

[0017] Definition 6. A composition for use according to any of definitions 1 to 5, suitable for administering oleocanthal at a dose of at most 5 to 20 mg / kg patient body weight, preferably at most 11,6 mg / kg patient body weight.

[0018] Definition 7. A composition according to any of definitions 1 to 6, suitable for administration of the mixture of hydroxytyrosol, oleuropein, and oleocanthal in a dose ratio of hydroxytyrosol :oleuropein:oleocanthal as 5,9:20,6:11,6 mg per at least 1 kg of patient body weight.

[0019] Definition 8. A composition for use according to any of definitions 1 to 7, formulated with pharmaceutically acceptable excipients into oral dosage forms, preferably soft capsules.

[0020] Definition 9. A dietary supplement comprising the composition according to any of definitions 1 to 8.

[0021] The compositions of the present invention may be formulated into oral dosage forms such as tablets, soft capsules, hard capsules, granules for oral suspension, oral solutions, or oral suspensions, preferably soft capsules. For humans, soft capsules are particularly advantageous because of the easy and comfortable administration as well as precision of the dosage and precision in targeting and liberation rate of the actives.

[0022] The olive extract may be from either olive fruit and / or olive leaves.

[0023] It is noted that the addition of elenolic add offers no substantial advantage to the triple combination of the present invention. It was unexpectedly found that the compositions of the present invention offer the advantage of reducing infarct size after myocardial ischemia / reperfusion.

[0024] Additionally, it was surprisingly found that the compositions of the present invention offer the advantage of reducing fasting glucose levels in circulation.

[0025] Moreover, it was surprisingly found that the compositions of the present invention containing the triple combination simultaneously reduce both the infarct size after myocardial ischemia / reperfusion and fasting glucose levels — an effect not achieved to the same extent when the active ingredients are administered individually, e.g., oleuropein alone or oleocanthal alone.

[0026] Finally, it was surprisingly observed that the more specific the subject matter of the above definitions, the more pronounced the advantages of the present invention.

[0027] The present invention is further described by the following indicative, non-limiting, examples 1 and 2 and Figures 1 and 2.

[0028] Example 1: In vivo evaluation of the administration of the mixture of the three active substances in reducing infarct size after myocardial ischemia / reperfusion.

[0029] Model of mice with metabolic syndrome was used. Mice were male wild-type (WT) C57BI6 aged 10-12 weeks. For the induction of metabolic syndrome, a Western Diet (TD 88137, Harlan-Teklad) was selected, rich in carbohydrates, fats, and cholesterol, which was administered to all animals throughout the protocol duration, i.e., for 14 weeks. The induction of metabolic syndrome was confirmed by specialized laboratory assessment including measurement of body weight, blood pressure, glucose levels, and lipid profile.

[0030] At the Sth week, the animals were randomized. Each mouse received daily oral administration of the composition of the present invention, which contains a mixture of the three active substances: oleuropein (20,6 mg / kg animal body weight), hydroxytyrosol (5,9 mg / kg animal body weight), and oleocanthal (11,6 mg / kg animal body weight). The control group received 5% DMSO.

[0031] At the end of the 14-week period, all animals underwent surgery to induce myocardial ischemia and reperfusion, and infarct size was determined after appropriate staining. Specifically, each mouse was anesthetized with a solution consisting of ketamine 100 mg / kg animal body weight, xylazine 20 mg / kg animal body weight, and atropine 0,6 mg / kg animal body weight, and placed supine on a heated platform. A tracheostomy was performed to secure artificial ventilation using a small animal ventilator. After stabilizing respiration, the mouse was repositioned laterally with the left side up, and a thoracotomy was performed to expose the myocardium. The pericardium was incised to better visualize the coronary vasculature, enabling identification and ligation of the left anterior descending (LAD) artery. Ligation of the artery induced ischemia for 30 minutes in a large part of the left: ventricle, which turned a lighter pink color. Subsequently, the ligation was released, and the myocardium was allowed to reperfuse for 120 minutes.

[0032] For infarct size measurement, the aorta was catheterized, and physioiogical saline was initially perfused to flush the myocardium, followed by injection of Evans Blue dye, which delineated the ischemic area of the heart. The myocardium was immediately frozen. It was then cut into slices, which were incubated at an elevated temperature with TTC (Triphenyltetrazolium Chloride) reagent to stain viable myocardial areas red and infarcted areas, i.e. areas were cell death occurred, white. The slices were then fixed in formalin solution (NSBF) for at least 24 hours, examined under a Zeiss 459300 stereoscope (Carl Zeiss Light Microscopy, Gottingen, Germany), and photographed with a Canon Powershot A620 camera (Canon, Tokyo, Japan). The images were processed using the ImageJ 1,51 software to determine the total area of each cross- section (All), the area at risk (AAR) that was ischemic but remained viable, and the infarcted area (Ischemic, I). The percentages AAR / AII % and I / R % were calculated. Using GraphPad Prism 7.00 software, the values were compiled, graphically displayed, statistically analyzed, and parameters such as mean and standard error of the mean (mean ± SEM) were calculated. Comparison of numerical experimental variables between groups was performed using one-way ANOVA and post-hoc Tukey test. The results showed that the composition of the present invention, containing the specific mixture of the three active substances, statistically significantly reduces infarct size (see Figure 1).

[0033] Example 2: In vivo evaluation of the administration of the mixture of the three active substances in reducing fasting glucose

[0034] Model of mice with metabolic syndrome was used. Mice were male wild-type (WT) C57BI6 aged 10-12 weeks. For the induction of metabolic syndrome, a Western Diet (ID 88137, Harlan-Teklad) was selected, rich in carbohydrates, fats, and cholesterol, which was administered to all animals throughout the protocol duration, i.e., for 14 weeks. The induction of metabolic syndrome was confirmed by specialized laboratory assessment including measurement of body weight, blood pressure, glucose levels, and lipid profile.

[0035] At the 8th week, the animals were randomized. Each mouse received daily oral administration of the composition of the present invention, which contains a mixture of the three active substances: oleuropein (20,6 mg / kg animal body weight), hydroxytyrosol (5,9 mg / kg animal body weight), and oleocanthal (11,6 mg / kg animal body weight). The control group received 5% DMSO.

[0036] Each mouse received daily oral administration of the composition of the present invention which contains oleuropein (20,6 mg / kg animal body weight), hydroxytyrosol (5,9 mg / kg animal body weight), and oleocanthal (11,6 mg / kg animal body weight). Fasting glucose was measured at the beginning of the protocol, at the Sth week, and at the 14th week. Fasting glucose was assessed using a suitable blood glucose meter with a single drop of blood obtained from the tail vein after 8 hours of fasting and was elevated in all animals at the Sth week due to the diet.

[0037] For the statistical analysis of fasting glucose, repeated measurements were taken at three different time points for each animal, analysis was performed using two-way ANOVA with Tukey's multiple comparisons test for statistical hypothesis testing.

[0038] The results showed that the composition of the present invention, containing the specific mixture of the three active substances, statistically significantly reduces fasting glucose at the 14th week (see Figure 2). Figure 1: In vivo evaluation of the administration of the mixture of the three active substances In reducing infarct size after myocardial ischemia / reperfusion. The combination statistically significantly reduces infarct size compared to the control group receiving solvent (5% DMSO), where the combination consists of hydroxytyrosol:oieuropein:oleocanthal 5,9:20,6:11,6 mg / Kg of mouse body weight. One-way ANOVA with Tukey's test was used for the comparison.

[0039] Figure 2: In vivo evaluation of the administration of the mixture of the three active substances in reducing fasting glucose. The combination statistically significantly reduces fasting glucose compared to the control group, where the combination consists of hydroxytyrosol:oieuropein:oleocanthal 5,9:20,6:11,6 mg / Kg of mouse body weight. Two-way ANOVA was used for the comparison.

[0040] Both examples demonstrated the benefits described for the specific mixture of the three active substances of the present invention.

[0041] From the two examples, it is established that the composition of the present invention, which contains the specific mixture of the three active substances, simultaneously reduces the infarct size after myocardial ischemia / reperfusion and lowers fasting glucose levels.

[0042] When dosing the composition in humans based on the corresponding per kilogram dosage used in mice, humans typically require proportionally tower quantities per kilogram of body weight compared to mice.

Claims

Claims1 A soft capsule comprising a mixture of three active substances derived from olive extract, namely hydroxytyrosol, oleuropein, and oleocanthal, wherein the oleocanthal is at least one-quarter (14) of the oleuropein by weight, and wherein said composition is suitable for cardioprotection, wherein the weight ratio hydroxytyrosol:oieuropein:oleocanthal is from 1 .: at least 3 : at least 2, with a measurement accuracy of ± 0.2, wherein the mass of the soft capsule is from 500 mg to 1000 mg, preferably 750 mg, and wherein the total mass of the three active ingredients hydroxytyrosol, oleuropein, and oleocanthal is from 10 mg to 20 mg, preferably 15 mg.

2. A soft, capsule according to claim 1, suitable such that hydroxytyrosol can be administered at a maximum dose of 5 to 10 mg / kg patient body weight, preferably at a maximum dose of 5,9 mg / kg patient body weight.

3. A soft capsule according to any of claims 1 to 2, suitable such that oleuropein can be administered at a maximum dose of 10 to 30 mg / kg patient body weight, preferably at a maximum dose of 20,6 mg / kg patient body weight.

4. A soft capsule according to any of claims 1 to 3, suitable such that oleocanthal can be administered at a maximum dose of 5 to 20 mg / kg patient body weight, preferably at a maximum dose of 11,6 mg / kg patient body weight.

5. A soft capsule according to any of claims 1 to 4, suitable such that the mixture of three active substances comprising hydroxytyrosol, oleuropein, and oleocanthal can preferably be administered at a dose of hydroxytyrosol:oieuropein:oleocanthal of 5,9:20,6:11,6 mg per at least 1 kg of patient body weight.

6. A dietary supplement comprising the soft capsule according to any of claims 1 to 5.