Derivatives of Lithothamnion sp. with potent anti-inflammatory activity

Abstract

A process for the production of an extract of a residue of Lithothamnion species wherein the residue of Lithothamnion has had siliceous materials and debris removed, been bleached, cleansed, and steri

Description

Field of the Invention This invention relates to the processing of Lithothamnion species, a calcified red macroalgae, in order to provide compounds or compositions with an anti-inflammatory functionality and to compositions produced by the method. Such compositions have the ability to inhibit the toll like receptor (TLR) inflammation cascade in macrophages. In addition, the compounds and compositions of the invention have an anti-inflammatory effect and in addition can act synergistically with a statin to reduce inflammation triggered by cholesterol crystals, a key event associated with inflammation in atherosclerotic cardiovascular disease. Background to the Invention CXI CXI Cardiovascular disease is a leading cause of mortality and morbidity worldwide. The management of the risk factors for atherosclerotic cardiovascular disease (CVD), of which elevated low density lipoprotein cholesterol (LDL-C) is one, is called primary prevention if it is administered to someone who has not previously experienced an atherosclerotic vascular event. For the purpose of risk reduction in patients without manifest CVD, only management of an elevated LDL-C has been shown to be of clinical benefit. Lipid-altering agents encompass several classes of drugs that include hydroxymethylglutaryl (HMG) CoA reductase inhibitors or statins, fibric acid derivatives, bile acid sequestrants, cholesterol absorption inhibitors, and nicotinic acid. These drugs differ with respect to their mechanism of action and to the degree and type of lipid lowering they can bring about. Thus, the indications for a particular drug are influenced by the underlying lipid abnormality. Lipid lowering, at least with statins, is beneficial for primary and secondary prevention of coronary heart disease in patients with dyslipidemias. The mechanisms of benefit seen with lipid lowering are incompletely understood. Regression of atherosclerosis occurs in only a minority of patients; furthermore, clinical benefits of lipid lowering are seen in as little as six months, before significant regression could occur. Thus, other factors must contribute; such as plaque stabilization, reversal of endothelial dysfunction, and decreased thrombogenicity. CXI CXI Statins are effective in reducing the risk of cardiovascular disease and have been recommended in clinical guidelines as frontline treatment for the prevention of cardiovascular disease. Various adverse events have been reported in clinical use, including muscle problems, liver dysfunction, renal insufficiency, diabetes, and eye conditions. Previous studies have shown that uptake and persistence with statin treatment is poor and, as a result, millions of patients could be missing out on life saving treatment. This underuse is partly because of concerns about potential adverse effects, and such concerns are particularly evident when statins are used for primary prevention in asymptomatic patients without a history of cardiovascular disease. In these individuals, who have a lower average risk of cardiovascular disease, the absolute benefits of statins are smaller than in a secondary prevention population with previous cardiovascular disease events, and therefore the benefit-to-harm balance of treatment might be less favourable. Nevertheless, recent guidelines have recommended wider use of statins for primary prevention, making a large population at low risk of cardiovascular disease eligible for treatment and exposed to the risks of adverse effects. Most previous systematic reviews of statins focused on efficacy or secondary prevention populations, making it difficult to determine the specific risks of adverse effects in patients without a history of cardiovascular disease. Reviews that examined harms in primary prevention have provided conflicting results, particularly for muscle problems, which were inconsistently defined and involved a wide range of muscle conditions with different severities. In the United States, if the liver enzyme, ALT level increases more than three times, it is recommended to change the drug or reduce the dose without stopping the statin immediately, and in Europe, it is recommended to check liver enzyme levels again after stopping the statin for 4 to 6 weeks. Formulations that allow reduced doses of statins for primary prevention will reduce side effects and improve patient compliance. However reduced doses will have less antiinflammatory activity to slow the progression of the inflammatory processes in atherosclerotic plaques increasing the potential for a stroke event. The inclusion of additional functional components with anti-inflammatory activity will reduce inflammation and improve the stability of the plaques. There is thus a need for components which can allow for a reduction in the dose of statin without losing efficacy. The harvesting and primary processing of calcified Lithothamnion macro-algae species for use in animal feed and human food is known in the art (e.g. PCT / IB1998 / 00142). The processing of the material for these applications involves sterilising, drying and milling to reduce particle size of the material. Its use has been described in different applications including dietary supplements, animal feed and water treatment. The primary uses of Lithothamnion sp. in human food and feed applications is as a rumen buffer and as a plant source of calcium and 72 other minerals. Additional health benefits have been reported in the literature. CXI CXI The starting material for processing is a residue of Corallinaceae, particularly Lithothamnium species, which are seaweeds found in cold and temperate seas. Corallinaceae are coralline algae, and a number of sub- species exist which are differentiated by morphological differences, but these differences vary depending on the local seabed and weather conditions. These algae lay down calcium carbonate in their cell walls which gives them a hard stony texture. The living Corallinaceae generally have a red color due to the presence of the pigment phycoerythrin in their structure, but when dead their color is white or yellowish. Corallinaceae occur naturally in cold and temperate seas and have been reported in Norway, Canada, Scotland, Ireland and France. There are two known Lithothamnion species, Lithothamnion tophiforme and Lithothamnion glaciale, which are both found growing in the North Atlantic Ocean. Once harvested the crude product consists primarily of mineral substances, particularly calcium carbonate and magnesium carbonate. This product is sometimes known as Maerl. The starting material is a dredged product in which the siliceous materials and debris are removed by intensive washing and the product is then bleached, cleansed and sterilized, particularly by the use of hydrogen peroxide, in accordance with the disclosure in WO 98 / 33508. Additional processing of calcified Lithothamnion sp. for providing or enhancing a specific biological functionality has not been previously disclosed. Processing Lithothamnion sp. to enhance the anti-inflammatory effect will have significant benefits in terms of robustness of the effect and the magnitude of the effect in a biological system. Object of the Invention It is an object of the present invention to provide methods of processing Lithothamnion sp. to provide compounds or compositions having beneficial biological properties or health benefits. An object of the present invention is to provide compositions with an antiinflammatory functionality. A further object is to provide compositions which have the ability to inhibit the toll like receptor (TLR) inflammation cascade in macrophages. In addition, a further object is to provide compositions which can act synergistically with a statin to reduce inflammation triggered by cholesterol crystals, a key event associated with inflammation in atherosclerotic cardiovascular disease. A further object is to provide methods of treating a variety of inflammatory diseases. LO CXI i— CM Summary of the Invention According to the present invention there is provided a process for the production of an extract of Lithothamnion species comprising treatment of Lithothamnion species from which the siliceous materials and debris has been removed and which has been bleached, cleansed and sterilized to produce a residue of Lithothamnion species comprising treatment of the residue with acid at pH of approximately 3.8 to 5.5, blending the acidified mixture at a temperature of about 18 to 22 degrees centigrade in the presence of an anti-foaming agent, maintaining the mixture at a pH of above 3.8 and then filtering the mixture through a sieve with pores in the range 3 to 4 mm to produce the extract. Preferably the pH is maintained in the range pH 4.0 to 5.0, more preferably at pH 4.5. The antifoaming agent may be composed of mono and diglycerides derived from Glycerol Monostearate, Glycerol monopalmitate, Glycerol monooleate, Glycerol monolaurate, Glycerol monoricinoleate. Suitably the anti-foaming agent may be the commercially available E 471 sold under the trade name Struktol (RTM) B420 by Schill and Seilacher. Preferably pressure is applied to assist passage of the material through the sieve. Preferably the acid is food grade acid selected from acetic, lactic, tartaric, sulphuric, hydrochloric, citric acid or malic acid or a mixture of these acids. In some embodiments the acid is a mixture of citric and malic acids. Preferably the filtration step is completed within about one hour of the start of filtration. Once the reaction is complete, as evidenced by substantially no CO2 being emitted, filter aid may be added to the reaction mixture before it is filtered through the sieve. The reaction mixture may be allowed to rest after addition of the filter aid. A level of <0.1% CO2 in the exhaust air of the reaction vessel in which the reaction is conducted is considered to indicate that the reaction is complete. The filter aid may be diatomaceous earth or perlite. CXI Suitably the process is carried out at a temperature at or below 22°C, to ensure that crystallization doesn't take place. CXI Suitably the filtered material is spray dried. The spray dried material may then be sieved through a filter with a pore size of about 400 to 600pm, more preferably about 500 pm. In another aspect, the invention provides a product whenever produced by a process as described above, for use in combination with a statin to reduce cholesterol levels in a subject. The product maybe a white or off white powder with a solubility in water of up to lg / 100 ml, which is odorless and tart to the taste and having a calcium content of at least 12 weight percent and magnesium content of at least 1% by weight, the product having an anti- inflammatory activity in a murine bone derived macrophage cell model primed for a pro- inflammatory response with LPS. The product may have a pH (1% aqueous solution) of about 4.0 min. The product may also have a particle size of about 500pm max. The product may have a total viable count of about 5,000cfu / g max. The product may further have anti- inflammatory activity on macrophages stimulated with LPS and cholesterol crystals. The product may also lower IL 1 beta levels in human PBMC under LPS and cholesterol stimulation, when compared to untreated human PBMC. The IL 1 beta levels may be lowered in the presence or absence of statins. In a still further aspect the present disclosure provides use of the product to reduce inflammation in a subject, in combination with a statin to reduce cholesterol levels in a subject. The invention thus provides a method of reducing the amount of statin required to reduce cholesterol in a subject by co-administration of the product together with a statin, which in turn can reduce the adverse effects of statin therapy. In another aspect the present disclosure provides a method of treatment of inflammatory diseases such as atherosclerosis, ulceralative colitis, osteoarthritis, Irritable Bowel Syndrome, Crohn’s disease, psoriasis, eczema, or epidermolysis bullosa. CXI CXI The invention also provides a pharmaceutical composition comprising the product of the process described above together with pharmaceutically acceptable carriers and excipients for use in combination with a statin to reduce cholesterol levels in a subject. The pharmaceutical composition may comprise a white or off white powder derived from Lithothamnion species by acid treatment, the powder having a solubility in water of up to lg / 100 ml, which is odorless and tart to the taste and having a calcium content of at least 12 weight percent and magnesium content of at least 1% by weight, the product having an anti- inflammatory activity in a murine bone derived macrophage cell model primed for a pro- inflammatory response with LPS. Brief Description of the Drawings Figure 1 is a flow diagram of a process for the preparation of Lithothamnion sp. biomass for the enhancement of anti-inflammatory activity. Figure 2 demonstrates the lack of cell toxicity of unprocessed Lithothamnion (UPL) and processed Lithothamnion (PL). There is no loss of murine macrophage cell viability in the presence of PL up to a concentration of 2mg / ml. This data indicates that there is no cytotoxic effects from the PL. Figure 3 demonstrates the anti-inflammatory effect derived from the process. The effect of anti-inflammatory activity is demonstrated in a murine bone derived macrophage cell model, primed for a pro-inflammatory response with LPS. Figure 4 shows the anti-inflammatory activity of PL on macrophages stimulated with LPS and cholesterol crystals. This test system provides a cell culture model for the inflammatory processes in an atherosclerotic plaque. Figure 5 shows IL 1 beta levels from Human PBMC under LPS and cholesterol stimulating conditions in the absence, presence or combined treatment with PL and statin. CXI CXI Detailed Description of the Drawings Example 1 : Extract Preparation One embodiment of the process of the invention is shown in Fig. l.The process for the production of the extract of Lithothamnion species involves treatment of Lithothamnion species from which the siliceous materials and debris has been removed and which has been bleached, cleansed and sterilized to produce a residue of Lithothamnion species. This process has been described in PCT / IB1998 / 00142. Generally speaking the Lithothamnion species is harvested, cleaned and subjected to intensive cleaning by for example, bleaching and sterilising in hydrogen peroxide for from 8 to 24 hours, further washing in water, drying in a sterile fluid bed and final milling under bacterially controlled conditions. This produces a Lithothamnion species residue. The residue is then treated with acid at pH of approximately 4.5, in a reaction vessel and the acidified mixture is blended at a temperature of about 19 to 20 degrees centigrade in the presence of an anti-foaming agent. The anti-foaming agent may be Struktol (RTM). The mixture is maintained at a pH of above 4 until substantially all CO2 has been released as determined by there being <0.1% CO2 in the reaction vessel exhaust. Diatomaceous earth is then added as a filter aid and the mixture reacted together for about 2 hours. It is then filtered through a sieve with a pore size of 3.4 mm under pressure of 5 to 7 bar. The filtered material is then spray dried and sieved through a filter with a pore size of about 500 pm The product of the process has the following properties:- General characteristics of the product Appearance White / Off White Powder Solubility Soluble up to Ig / lOOmls with vigorous mixing Odour Odourless Taste Acidic / tart Typical Analysis PL is derived from a natural marine source and as such is subject to seasonal variations. The values below are typical. Chemistry / Physical Calcium (ICP) Magnesium (ICP) 12% min. 1% min. LO Moisture (AOAC) 6% max. CM Lead (ICP) Ippm max. Arsenic (ICP) 1.5ppm max. 1“ Cadmium (ICP) Ippm max. CM Mercury (ICP pH (1% aqueous solution) Particle Size Bulk Density (Tapped) Microbiological Total viable count Yeast &Moulds E. Coli Coliforms Enterobacteriaceae Staphylococcus aureus Salmonella O.lppm max. 4.0 min. 500pm max. 0.2-0.7g / cm3 5,000cfu / g max. lOOcfu / g max. Absent in 1g Absent in 1g Absent in 1g Absent in 1g Absent in 25g Nutritional Profile 35g max per 100g Ash Protein Fat Carbohydrates Energy Value <0.5g <0.5g <0.5g 59 kcal / 247 kJ Example 2 : Assessment of Biological properties MATERIALS AND METHODS CXI CXI Macrophage cell culture The J774A.1 murine macrophage cell line was purchased from the American Tissue Culture Collection (ATCC, Rockville, United States). Cells were cultured as recommended by the ATCC in Dulbecco’s Modified Eagle’s medium (DMEM; 4.5 g / L D-glucose, Sigma-Aldrich Ltd., Wicklow, Ireland) supplemented with 10% v / v heat-inactivated foetal bovine serum (FBS) (SigmaAldrich Ltd., Wicklow, Ireland). Cells were maintained at 37°C in an incubator under a 5% CO2 and 95% relative humidity. Prior to the experiments, cells were detached from the flask using a scraper, centrifuged at 1,000 rpm for 3 min at room temperature (RT) and re-suspended in antibiotic-free growth media. Cells were counted using a hemocytometer and seeded at a density of 100,000 cells / cm2 in a 96-well plate for 24 h prior to incubation with LPS (lOng / ml) and PL (0.125, 0.25, 0.5, 1 and 2 mg / ml) or UPL (2, 4 and 8 mg / ml) for 6 h or 24 h. Control cells were also run in parallel and subjected to the same changes of medium. In vitro culture of human PBMCs Blood samples were obtained from healthy volunteers by venipuncture. Briefly, peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophils (PMN) were subsequently separated by dextran sedimentation (Dextran 500, 8% (w / v), density 1.113 ± 0.001 g / ml) followed by gradient centrifugation (dextran:blood ratio of 1:1). PBMC and PMN cell suspensions were washed twice in RPMI 1640, 8% human serum (HS) by centrifugation. Total leucocytes were adjusted to a final concentration of 2.5 * 106 cells / ml and cultured in RPMI 1640, 8% HS in 24-well tissue culture plates. Viability Assays Cell viability was measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Vybrant cell proliferation assay kit (Molecular Probes, V13154). Briefly, J774A.1 macrophage cells were cultured at a density of 100,000 cells / cm2 in a 96-well plate for 24 h. The cells were subsequently treated with different concentrations of PL (0.125-2 mg / ml) or UPL (2, 4 and 8 mg / ml) for 6 h and 24 h in the presence of LPS (lOng / ml) and incubated with MTT for another 4 h at 37°C and 5% CO2. The index of the cell viability was determined by measuring formazan production with an ELISA reader (Thermofisher Scientific, Varioskan LUX) at an absorbance of 548 nm. Cell viability was determined relative to the untreated control cells. CXI Determination of Nitric Oxide production J774A.1 cells were cultured in a 96-well plate at a density of 100,000 cells / cm2 at 37°C for 24 h. Then, the cells were treated with different concentrations of PL (0.125-2 mg / mL) for 6 h or 24 h in the presence of LPS (lOng / ml). Controls were untreated (NT) J774A. 1 cells. Afterwards, NO in the culture supernatants was measured by using a Greiss reagent [1% sulphanilamide in 5% phosphoric acid and 0.1% N-(l-naphthyl)- ethylenediamine dihydrochloride] kit for nitrite determination (Molecular Probes, G- 7921). The absorbance at 540 nm was measured, and the concentration of nitrite was calculated using a calibration standard curve constructed using sodium nitrite dissolved in DMEM. CXI Enzyme-Linked Immunoabsorbent Assay Cell culture supernatants were collected from J774A.1 murine macrophages, LPS-primed J774A.1 murine macrophages and primed J774 / V1 murine macrophages treated with PL (0.125-2mg / ml) after 6 h and 24 h. Levels of TNF~a and IL-6 were measured using murine TNF-a and murine IL-6 ELISA Kits (AssayGenie, Dublin, Ireland) following the manufacturer’s instructions and analyzed using a microplate reader (Thermofisher Scientific, Varioskan LUX) at 450nm. IL-ip levels in supernatants from LPS (10ng / ml)-primed human PBMCs and cholesterol (CC) stimulating conditions in the absence, presence or combined treatment with PL (0.5-2mg / ml) and statin (ST) (12.5-50 pM) were measured using a human IL-ip ELISA kit (AssayGenie, Dublin, Ireland) according to the manufacturer’s instructions. Statistical Analysis Statistical analysis was performed using GraphPad Prism version 8 with one-way ANOVAs with Tukey's post-test. All data show the mean and standard deviation (S.D.). All replicates are biological. CXI CXI The invention has surprisingly identified the potential of natural marine materials to provide novel bioactivities to improve human health. This invention allows the processing of calcified red algae from the Lithothamnion species in order to obtain a material which possess anti-inflammatory activity. This anti-inflammatory activity has been demonstrated by measuring a reduction in the amount of inflammatory cytokines being produced by macrophage cells in vitro when primed with the bacterial endotoxin, lipopolysaccharide (LPS) and other stimuli. The anti-inflammatory activity of the obtained material has also been demonstrated to act synergistically with antiinflammatory drugs including statins. It achieves this by reducing cytokine production caused by exposure of the immune system's macrophage cells, to pro-inflammatory stimuli that are relevant to the onset and progression of inflammation associated human diseases. The processed material is suitable for use in dietary supplements and pharmaceutical formulations for administration to human subjects for providing antiinflammatory effects. The words “comprises / comprising” and the words “having / including” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Claims

1. A process for the production of an extract of Lithothamnion species comprising treatment of Lithothamnion species from which the siliceous materials and debris has been removed and which has been bleached, cleansed and sterilized to produce a residue of Lithothamnion species comprising treatment of the residue with acid at pH of 3.8 to 5.5, blending the acidified mixture at a temperature of 18 to 22 degrees centigrade, in the presence of an anti-foaming agent, maintaining the mixture at a pH of above about pH 3.8 and then filtering the mixture through a sieve with pores in the range 3 to 4 mm to produce the extract.

2. A process as claimed in claim 1 wherein the pH is maintained at 4.0 to 5.0.

3. A process as claimed in claim 1 or 2 wherein pressure is applied to assistLO passage of the material through the sieve.CXI4. A process as claimed in any preceding claim wherein the acid is a food grade1 acid selected from acetic, lactic, tartaric, sulphuric, hydrochloric, citric acid or malic"1” acid or a mixture of these acids.CXI5. A process as claimed in any preceding claim wherein the filtration step is completed within about one hour of the start of filtration.

6. A process as claimed in any preceding claim wherein once the reaction is complete as evidenced by substantially no CO2 being emitted from the reaction mixture, a filter aid is added to the reaction mixture before it is filtered through the sieve.

7. A process as claimed in any preceding claim wherein the reaction mixture is allowed to rest after addition of the filter aid.

8. A process as claimed in any preceding claim wherein process is carried out at a temperature of at or below 22°C.

9. A process as claimed in any preceding claim wherein the filtered material is spray dried.

10. A process as claimed in claim 9 wherein the spray dried material is sieved through a filter with a pore size of about 500 pm.

11. A composition whenever produced by a process as claimed in any preceding claim, for use in combination with a statin to reduce cholesterol levels in a subject.

12. A composition derived from Lithothamnion species by treatment with acid, which is a white or off white powder with a solubility in water of up to lg / 100 ml, which is odorless and tart to the taste and having a calcium content of at least 12 weight percent and magnesium content of at least 1% by weight, the product having an antiinflammatory activity in a murine bone derived macrophage cell model primed for a pro- inflammatory response with LPS, for use in combination with a statin to reduce cholesterol levels in a subject.

13. The composition as claimed in any of claims 11 to 12 for use in a method of treatment of inflammatory diseases such as atherosclerosis, ulceralative colitis, osteoarthritis, Irritable Bowel Syndrome, Crohn’s disease, psoriasis, eczema, or epidermolysis bullosa.

14. The composition as claimed in any of claims 11 to 12 for use in a method of treating atherosclerotic cardiovascular disease.

15. A pharmaceutical composition comprising the composition for use as claimed in any of claims 11 to 12 together with pharmaceutically acceptable carriers and excipients.

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