WO2022043287A1

WO2022043287A1 - Adonirubin and dha to prevent chronic inflammation

Info

Publication number: WO2022043287A1
Application number: PCT/EP2021/073317
Authority: WO
Inventors: Igor Bendik; Angelika Sonja Johanna FRIEDEL; Pascale FUCHS BOSSERT; Bernd Mussler; Hubert Paul HUG
Original assignee: Dsm Ip Assets B.V.
Priority date: 2020-08-25
Filing date: 2021-08-24
Publication date: 2022-03-03

Abstract

The present invention relates to the use of adonirubin in combination with DHA to prevent inflammation via upregulation of the CARDS gene.

Description

ADONIRUBIN AND DHA TO PREVENT CHRONIC INFLAMMATION

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of adonirubin, a precursor of astaxanthin, in combination with the co-3 fatty acid DHA in preventing chronic inflammation via upregulation of CARDS mRNA. Thus the combination is useful in treating and preventing various inflammatory diseases.

BACKGROUND OF THE INVENTION

Caspase recruitment domains or caspase activation and recruitment domains (CARDs) are motifs found in proteins involved in processes relating to inflammation and apoptosis via CARD-CARD interactions.

Caspase recruitment domain family member 8 (CARD8) is involved in pathways leading to activation of caspases and it interacts with proteins, such as cryopyrin or NLRP3 and nucleotide-binding oligomerization domain-containing protein 2 (NOD2), to inhibit inflammation.

CARD8 inhibits inflammasomes. Inflammasomes are cytoplasmic complexes of various proteins that activate inflammatory responses. They are prominent in cells of the innate immune system, e. g. macrophages. An increase of expression of wildtype CARD8 (but not some mutant CARD8s) should be beneficial in chronic inflammations, e. g. Crohn's disease

Adonirubin (CAS 4418-72-8, phoenicoxanthin, 3-hydroxy-p,p-carotene-4,4'-dione) is an intermediate form in the pathway from p-carotene to astaxanthin. It was originally identified in the petals of Adonis annua and later also found in astaxanthin producing bacteria such as Paracoccus carotinifaciens. Due to its antioxidant activity adonirubin is already marketed for human nutrition as AdoniCare™ available from ENEOS Corp, Japan.

Docosahexaenoic acid (DHA, CAS number 6217-54) and other co-3 fatty acids have antiinflammatory effects and balance the excess of inflammatory co-6 fatty acids. DETAILED DESCRIPTION OF THE INVENTION

For the first time it has been found that DHA in combination with adonirubin, a precursor of astaxanthin and DHA, as shown below, upregulates CARD8 mRNA expression. Neither DHA nor adonirubin alone led to a significant upregulation of CARD8 mRNA expression.

Thus we have found that the combination of adonirubin and DHA exerts an anti-inflammatory effect and is useful to treat, prevent, or ameliorate the symptoms of diseases or conditions associated with a downregulation of CARD8 mRNA expression. These diseases include:

• inflammatory diseases affecting the lungs, such as diseases related to air pollution and chronic obstructive pulmonary disease (COPD)

• inflammatory diseases affecting the intestines, such as inflammatory bowel disease (IBD), Crohn's Disease, and irritable bowel syndrome (IBD)

• inflammatory diseases affecting the liver, such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NALFD);

• and other chronic inflammations such as aphthous stomatitis, pharyngitis cervical adenitis (PFAPA syndrome, and rheumatoid arthritis.

Since CARD8 inhibits inflammation via interacting with the inflammasome we have found an anti-inflammatory action of the combination of DHA with adonirubin. This applies especially for chronic inflammations, e. g.Crohn's disease, mediated by the innate immune system.

While not wishing to be bound by theory, we propose that some of these anti-inflammatory effects are increased by using a combination of DHA with adonirubin via upregulation of expression of CARD8.

Thus one embodiment of this invention is a method of treating or lessening the severity of a symptom of a disease or adverse condition selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation comprising: administering an effective amount of a composition comprising DHA and adonirubin to a person in need thereof. Another embodiment is a method of delaying the onset of a disease or condition selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation comprising: administering an effective amount of a composition comprising DHA and adonirubin to a person at risk of the disease or condition. Another embodiment of this invention is the use of a composition comprising DHA and adonirubin to treat, prevent, or lessen the symptoms of a disease or condition selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation.

Another embodiment is the use of a composition comprising DHA and adonirubin in the manufacture of a medicament to treat, prevent, or lessen the symptoms of a disease or condition selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation.

Another embodiment is an agent for treating or preventing inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation comprising the combination of DHA and adonirubin as an active ingredient.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1: Caspase recruitment domain family member 8 (CARD8) expression. HepG2 cells were treated overnight with vehicle control, 1 pM adonirubin, 1 pM DHA and 1 pM adonirubin/1 pM DHA combination. Gene expression of CARD8 was measured using the Affymetrix GeneChip Array methodology. Different probe set of the human CARD8 gene are present on the HTA 2.0 GeneChip array (NCBI accession numbers: NM_001184900 and NR033678). The gene expression is shown in box-plots, the expression units are transformed to Log2. P-values (P) are indicated in each panel. The biological replicates are indicated by n.

Adonirubin is a precursor of astaxanthin. The structure is shown below:

The structure of DHA is given below:

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.

"Adonirubin" includes rac-adonirubin and all stereoisomers.

A further embodiment is a nutritional supplement comprising the combination of DHA and adonirubin. Another embodiment is a pharmaceutical composition comprising the combination of DHA and adonirubin. Another embodiment is a medical food comprising the combination of DHA and adonirubin. Another embodiment is a composition comprising the combination of DHA and adonirubin for addressing the nutritional needs of a patient suffering from a disease selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation.

COMBINATIONS WITH OTHER ACTIVE INGREDIENTS

In some embodiments the composition comprises adonirubin, DHA and another active ingredient. 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, other carotenoids and mixtures thereof.

Alternatively, the combination of DHA and adonirubin may be the only active ingredients present in the composition, i.e. the composition may also comprise other ingredients which are required for formulation, but do not directly act as a nutraceutical nor pharmaceutical. 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.

In some embodiments the molar ratio of adonirubin to DHA is from 1:100. Additional embodiments have molar ratios of from 1 to 50; 1 to 20, and 1:1.

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/shel I 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 CARD8 Gene Up Regulation

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 pM 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 pM in to the cell culture medium.

HepG2 cells were stimulated with 1 pM Adonirubin, 20 pM 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 pM adonirubin, 20 pM DHA or with a combination of 1 pM adonirubin and 20 pM DHA. Expression of CARD8 mRNA (NCBI Reference Sequences: NM_001184900 or NR_033678) was measured using established GeneChip array hybridization technology as described above. As shown in Figure 1 the fold change expression of CARD8 mRNA with the combination, 1 pM adonirubin and 20 pM DHA, is significantly higher as compared to the treatments with the compounds alone, which is slightly evaluated. The fold change expression is in both cases, with the CARD NM_001184900 or the CARD8 NR_033678 probe, more than additive. Therefore, we observe synergistic effects with the combinations. Also the p-values obtained with the treatments of the combinations are very low and therefore, the values are highly significant (Figure 1).

Claims

9 Claims:

1. Use of a composition comprising DHA and adonirubin to treat, prevent, or lessen the symptoms of a disease or condition selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation.

2. Use according to claim 1 wherein the disease or condition is selected from the group consisting of: diseases related to air pollution, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), Crohn's Disease, irritable bowel syndrome (IBD), nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NALFD); aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA syndrome), and rheumatoid arthritis.

3. A pharmaceutical, nutraceutical or food comprising a composition comprising docosahexaenoic acid (DHA) and adonirubin.

4. A composition according to Claim 3 characterized in the composition is for addressing the nutritional needs of a patient suffering from a disease selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation.

5. A method of treating or lessening the severity of a symptom of a disease or adverse condition selected from the group consisting of: inflammatory diseases affecting the lungs, inflammatory diseases affecting the intestines, inflammatory diseases affecting the liver, and chronic inflammation comprising: administering an effective amount of a composition comprising DHA and adonirubin to a person in need thereof.