WO2021156145A1

WO2021156145A1 - Adonirubin and dha for heart health

Info

Publication number: WO2021156145A1
Application number: PCT/EP2021/052076
Authority: WO
Inventors: Igor Bendik; Angelika Sonja Johanna FRIEDEL; Pascale FUCHS BOSSERT; Heinrich HUG; Hubert Paul HUG; Bernd Mussler
Original assignee: Dsm Ip Assets B.V.
Priority date: 2020-02-03
Filing date: 2021-01-29
Publication date: 2021-08-12

Abstract

The present invention relates to the use of a combination of adonirubin and DHA for heart health. Together they lead to a decrease of MVD expression, whereas the single compounds did not show a significant effect in our cellular assay.

Description

ADONIRUBIN AND DHA FOR HEART HEALTH

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of adonirubin and the polyunsaturated fatty acid (PUFA) DHA for heart health. This combination reduces expression of diphosphomevalonate decarboxylase (mevalonate pyrophosphate decarboxylase, MVD) mRNA. MVD is an enzyme involved in cholesterol synthesis. Thus, the combination of adonirubin and DHA can be used to improve cardiovascular health by lowering cholesterol synthesis.

BACKGROUND OF THE INVENTION

Coronary heart disease (CHD) and hypercholesterolemia or high cholesterol are major health issues. The diphosphomevalonate decarboxylase (mevalonate pyrophosphate decarboxylase, MVD) protein is involved in the cholesterol biosynthesis pathway and has been identified as a potential enzyme to be targeted in the cholesterol synthesis pathway.

Adonirubin (CAS 4418-72-8, phoenicoxanthin, 3-hydroxy^^-carotene-4,4'-dione) is a carotenoid which is an intermediate in the pathway synthesizing astaxanthin from beta-carotene. It was originally identified in the petals of Adonis annua and later also found in astaxanthin producing bacteria such as Paracoccus carotinifaciens. It has been reported that astaxanthin can protect against glucocorticoid-induced cataract in chick embryos. Adonirubin has also been identified in quail retina that is used as non-primate small animal model for studying the roles of carotenoids in light protection.

Docosahexaenoic acid (DHA, CAS number 6217-54) has benefits in heart health and supplementation with DHA has been recommended for patients with elevated triglycerides and CHD.

There are reports that lower vitamin D levels correlate with a risk for CHD. In Australia, migrants from sufficient vitamin D zones prior to migration had reduced risks of CHD than Australian born residents. The prevalence of CHD, heart failure and peripheral artery disease increased significantly when the level of vitamin D decreased in the serum. The mechanisms of how vitamin D protects against CHD have not been described yet.

While statins and other drugs are routinely used to combat high cholesterol, they may have adverse side effects. It would be desirable to have an all-natural alternative to combat high cholesterol production by an individual.

DETAILED DESCRIPTION OF THE INVENTION

It has been found, in accordance with this invention that the combination of adonirubin and DHA can inhibit the expression of diphosphomevalonate decarboxylase (mevalonate pyrophosphate decarboxylase, MVD), and therefore inhibit cholesterol biosynthesis.

Thus, one embodiment of this invention is a method of inhibiting cholesterol biosynthesis is administering an effective amount of a composition comprising adonirubin and DHA to an individual in need thereof. A further embodiment is the use of a composition comprising adonirubin and DHA to inhibit cholesterol biosynthesis. A further embodiment is the use of a composition comprising adonirubin and DHA in the manufacture of a medicament which inhibits cholesterol biosynthesis.

Increased cholesterol synthesis is a factor in a number of conditions/diseases. These include: coronary heart disease, familial hypercholesterolemia, diabetes, and dementias such as Alzheimer's diseases and vascular dementias. Hypercholesterolemia may be the result of dietary factors, genetic factors, and diseases which affect cholesterol synthesis control.

As a high amount of cholesterol is involved in a number of cardiovascular diseases, a further embodiment is a method of preventing or lessening the risk of, or treating cardiovascular diseases in an individual comprising administering a composition comprising adonirubin and DHA to an individual in need thereof. A further embodiment is the use of a composition comprising adonirubin and DHA to prevent or treat cardiovascular diseases. A further embodiment is the use of a composition comprising adonirubin and DHA in the manufacture of a medicament, a nutritional supplement or a food or beverage which prevents or treats cardiovascular diseases, or maintains cardiovascular health. Particular cardiovascular diseases or conditions which are associated with high cholesterol include: atherosclerosis, heart diseases, heart attacks, stroke, stenosis, dyslipidemia, high blood pressure, hypertrophic cardiomyopathy, carotid atherosclerosis, macrovascular diseases, coronary artery disease, non-fatal myocardial infraction, myocardial infarction, peripheral vascular disease, venous thromboembolism and diabetes.

Thus, other embodiments of this invention include a method of preventing, lessening the risk, lessening the severity of, or treating a cardiovascular disease or condition, or maintaining cardiovascular health comprising administering a composition comprising adonirubin and DHA, wherein the cardiovascular disease is selected from the group consisting of: heart attacks, stroke, dyslipidemia, high blood pressure, diseases of the veins, chronic arterial occlusive diseases, congestive heart failure, myocardial infarction, diseases of the coronary arteries, and acute ischemic heart disease.

BRIEF DESCTIPTION OF THE FIGURES

FIGURE 1 shows the reduction of MVD synthesis. Here we compared treatment with adonirubin, DHA, and the combination compared to the control. Adonirubin is a precursor of astaxanthin. The structure is shown below:

We have shown that the combination of adonirubin and DHA reduces mRNA expression of MVD in HepG2 cells.

Definitions: "Preventing" as used herein includes delaying the onset of a condition or disease; lessening the severity of a symptom of a condition or disease, as well as stopping the condition or disease from occurring.

"DHA" includes various forms of DHA, such as a methyl ester, ethyl ester and other esters. It also includes physiological precursors, i.e. compounds such as triglycerides which are metabolized into DHA.

COMBINATIONS WITH OTHER ACTIVE INGREDIENTS

In some embodiments the composition comprises adonirubin, DHA and another active ingredient. Preferably the further active ingredient is Vitamin D or one of its metabolites.

Examples of further optional active ingredients include Vitamin E, water soluble tomato extract, resveratrol, Vitamin D, 25-hydroxy vitamin D3, hydroxytyrosol, resveratrol, polyunsaturated fatty acids (PUFAs), Vitamin A, astaxanthin and mixtures thereof.

DOSAGES

The adonirubin and DHA are preferably administered in the same formulation but may be administered separately.

A recommended daily dose of adonirubin would be up to about 50 mg/day; A preferred dosages are up to about 30 mg/day and from about 2 to 10 mg/day.

For DHA, the daily dosage amount is up to 4 g/day. The DHA may be used in a concentrated form (such as 90% or higher or even pure). Some preferred dosages include 500 mg/day, 1 gram/day and 2 g /day. The dosages may be administered as smaller doses to be consumed two or three times a day to ensure the correct amount is delivered in a more convenient way.

If desired, the daily intake can be divided into two or more dosages, such as twice a day tablets or in any other form. For non-human animals, the human dosages above can be adjusted to the animal's body weight.

FORMULATIONS The composition of the present invention is preferably in the form of a pharmaceutical, drug topical, inhaling form or orally applied and nutritional compositions, such as fortified food, fortified feed, or fortified beverages, or in form of fortified liquid food/feed (such as drinks, or shots), pills or capsules for humans or animals.

The dietary and pharmaceutical compositions according to the present invention may be in any galenic form that is suitable for administering to the animal body including the human body, especially in any form that is conventional for oral administration, e.g. in solid form, such as (additives/supplements for) food or feed, food or feed premix, fortified food or feed, tablets, pills, granules, dragees, capsules, and effervescent formulations such as powders and tablets, or in liquid form such as solutions, emulsions or suspensions as e.g. beverages, pastes and oily suspensions. The pastes may be encapsulated in hard or soft-shell capsules, whereby the capsules feature e.g. a matrix of (fish, swine, poultry, cow) gelatin, plant proteins or lignin sulfonate. Examples for other application forms are forms for transdermal, parenteral or injectable administration. The dietary and pharmaceutical compositions may be in the form of controlled (delayed) release formulations. It may also be topically applied, such as in a cream gel, foams, lotions, and ointments, and aerosols.

The dietary compositions according to the present invention may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents, gel forming agents, antioxidants and antimicrobials.

Examples of food are cereal bars, dairy products, such as yoghurts, and bakery items, such as cakes and cookies. Examples of fortified food are cereal bars, and bakery items, such as bread, bread rolls, bagels, cakes and cookies. Examples of dietary supplements are tablets, pills, granules, dragees, capsules and effervescent formulations, in the form of non-alcoholic drinks, such as soft drinks, fruit juices, lemonades, near-water drinks, enhanced water drinks, teas and milk-based drinks, in the form of liquid food, such as soups and dairy products (muesli drinks and yoghurts). Beverages encompass non-alcoholic and alcoholic drinks as well as liquid preparations to be added to drinking water and liquid food. Non-alcoholic drinks are e.g. soft drinks, sport drinks, fruit juices, vegetable juices (e.g. tomato juice), lemonades, teas and milk-based drinks. Liquid foods are e.g. soups and dairy products (e.g. muesli drinks).

EXAMPLE 1

Endogenous MDV Gene Down Regualtion

Methods:

Cells and treatments

The human hepato-carcinoma (HHC) cell line HepG2 was from the American Type Culture Collection (ATCC), Rockville, Maryland, USA.

Adonirubin (CAS 4418-72-8, phoenicoxanthin), rac-adonirubin, was used at a final concentration of 1 mM in medium for stimulation. The dilution was prepared in ethanol/tetrahydrofuran (EtOH/THF) at a ratio of 1:1 with a maximal concentration of 0.2 % of the solvent. Control cells were treated with EtOH/THF 1:1 at a final concentration of 0.2 %.

DHA was diluted to a concentration of 1 mM in to the cell culture medium.

HepG2 cells were stimulated with 1 mM Adonirubin, 20 mM DHA and in combination overnight. At least 5 independent replicates per treatment was measured. A mock stimulation was applied using vehicle as a control.

Total RNA extraction from HepG2 cells

After stimulation the HepG2 cells were harvested. The RNeasy Mini Kit from Qiagen, Hombrechtikon, Switzerland, was used to extract total RNA. The phenol-free total RNA extraction spin column procedure removed genomic DNA contamination. RNA concentration was measured using a Nanodrop spectrophotometer and quality was assured using an Agilent 2100 Bioanalyzer. Only RNA integrity number (RIN) values of more than 8.0 were considered as non-degraded and of good quality for Affymetrix GeneChip Array work flow.

Affymetrix GeneChip array work flow

The human Affymetrix GeneChip^® HTA 2.0 arrays were applied. The work flow was according standardized protocols by the supplier (Affymetrix Ltd., 3420 Central Expressway, Santa Clara, CA 95051, USA). The solutions of the GeneChip WT PLUS Reagent Kit were used applying the Affymetrix instructions. The yield of obtained labeled single strand cDNA was measured using a Nanodrop spectrophotometer, size of single strand cDNA and fragmentation was checked on the Agilent 2100 Bioanalyzer. After staining, hybridizing and washing, the hybridized Affymetrix Gene Chip arrays were scanned using the Affymetrix GeneChip^® Scanner 3000 (GCS 3000) series. The scanned data was pre-processed and cell intensity files (.CEL) were used to generate relative expression data of the expressed mRNAs using Partek software (Partek Genomics Suite software, version 7.18.0723, Partek Incorporated, 624 Trade Center Boulevard, St. Louis, MO 63005, USA). All arrays that passed the quality control after scanning were included into the statistical analysis. For statistics the 'analysis of variance' (ANOVA) was used and p-values were calculated. Significantly changed mRNA expression was consider for p< 0.001.

Results: HepG2 cells were either untreated or treated with 1 mM adonirubin, 20 mM DHA or with a combination of 1 mM adonirubin and 20 mM DHA. Expression of MVD mRNA (NCBI Reference Sequence: NM_002461) was measured using established GeneChip array hybridization technology as described above.

Compared to the untreated control MVD mRNA expression was changed only by a factor of 1.043 for 1 mM adonirubin and by a factor of -1.098 for 20 mM DHA, respectively. However, in both comparisons the p-value was too high to be considered statistically significant different. Using this method both treatments alone did not significantly differ from the control.

Only the combination of adonirubin and DNA resulted in a highly significant downregulation of MVD mRNA. Fold change was -1.2221 (p = 8.65 x 10⁶). See Figure 1.

Claims

Claims:

1. A composition comprising adonirubin and DHA.

2. A composition according to Claim 1 wherein the adonirubin and DHA are present in an effective amount to inhibit cholesterol biosynthesis.

3. A composition according to Claim 1 or Claim 2 which is for use in a person experiencing coronary heart disease, familial hypercholesterolemia, diabetes, and dementias such as Alzheimer's diseases and vascular dementias.

4. A composition according to any of Claims 1 to 3, which is for use in a person experiencing hypercholesteremia, heart attacks, stroke, dyslipidemia, high blood pressure, diseases of the veins, chronic arterial occlusive diseases, congestive heart failure, myocardial infarction, diseases of the coronary arteries, and acute ischemic heart disease.

5. A composition according to any of claims 1-4, further comprising another active ingredient selected from the group consisting of: include Vitamin E, water soluble tomato extract, resveratrol, Vitamin D, or one of its metabolites, 25-hydroxy vitamin D3, hydroxytyrosol, polyunsaturated fatty acids (PUFAs), Vitamin A and mixtures thereof.

6. Use of a composition comprising adonirubin and DHA for inhibiting cholesterol biosynthesis.

7. A method of inhibiting cholesterol biosynthesis is administering an effective amount of a composition comprising adonirubin and DHA to an individual in need thereof.

8. A pharmaceutical, nutraceutical, functional food, or food supplement comprising a composition according to any of claims 1-5.