ACTIVITY

TECHNICAL FIELD

This invention relates to a composition containing a mussel extract having anti¬ inflammatory activity. In particular, the invention relates to a composition containing an anti-inflammatory mussel extract where the anti-inflammatory activity is enhanced by the addition of choline or a choline derivative. The invention also relates to a method of treating inflammation, particularly inflammation caused by arthritis, using the mussel extract composition of the invention.

BACKGROUND

The anti-inflammatory properties of mussels have been known for some time (Halpern 2000, Sinclair et al 2000, Darlington and Stone 2001), especially in relation to relieving inflammation and pain associated with arthritis (McFarlane et al 1975, Croft 1979, Gibson et al 1980, Rainsford and Whitehouse 1980, Audeval and Bouchart 1986).

Studies have also shown that mussel preparations can produce a more general anti- inflammatory effect (Rainsford and Whitehouse 1980, Miller and Ormrod 1980, Couch et al 1982, Miller et al 1993).

The anti-inflammatory properties of extracts from mussels are also described in various published patent specifications.

US 4,455,298 describes pharmaceutical preparations that contain an extract from the New Zealand green-lipped mussel (Perna canaliculus) useful in preventing, alleviating or treating gastro-intestinal irritation or ulcer formation.

WO 00/53198 describes a method of inhibition of a lipoxygenase pathway by administering to a human or animal a lipid extract of Perna canaliculus or Mytilus edulis (blue mussel). Lipoxygenase pathways are known to play a role in inflammation.

WO 96/05164 describes an anti-inflammatory preparation comprising a purified active fraction isolated from a lipid extract of Perna canaliculus or Mytilus edulis, and

5,11,14,17-eicosatetraenoic acid which is postulated to be the major constituent of the active fraction.

US 6,346,278, WO 97/09992, and EP 0850068 describe the anti-inflammatory, and particularly anti-arthritic, treatment of a human or animal by the administration of a lipid extract of Perna canaliculus or Mytilus edulis. They also describe an ointment or lotion formulated for the transdermal administration of a mussel lipid extract in an oil, such as olive oil.

WO 85/05033 and US 4,801 ,453 describe a method of stabilising the activity of an extract of Perna canaliculus by the addition of organic aliphatic acids (e.g. tartaric acid), alkali metals, or alkaline earth metal salts.

Compositions that contain a mussel extract together with other substances are also known. For example, WO 00/56164 describes a pet food product for use in the maintenance of healthy joints and the alleviation of arthritic symptoms. The product comprises an active extract of Perna canaliculus and vitamin E in olive oil. Vitamin E is added as an anti-oxidant.

WO 02/24211 describes a method for modulating the immune response to an immunogen in a human or animal by administration of a lipid extract of Perna canaliculus or Mytilus edulis. Again, vitamin E is added as an anti-oxidant.

WO 01/01976, WO 01/05411 , US 20020018787, and WO 00/71140 describe the use of an extract of Perna canaliculus in conjunction with glucosamine and methylsulfonylmethane (WO 01/01976), with bark extract or shark cartilage (WO 01/05411), and with N,N-dimethylgiycine (US 20020018787 and WO 00/71140). The various substances added to the mussel extract are reported to enhance the anti¬ inflammatory activity of the extract.

There have been reports that phospholipids, especially in the oxidised form, induce an inflammatory response (Marathe et al 2000, Kadi et a/ 2002). This pro-inflammatory activity is believed to be, at least in part, due to the fragmentation of the phospholipids.

Surprisingly, the applicant has now shown that a phospholipid can enhance the anti¬ inflammatory activity of a mussel extract. In particular, the applicant has shown that when lecithin is added to a lipid extract from Perna canaliculus the anti-inflammatory activity of the lipid extract is enhanced.

The major component of lecithin is the phospholipid phosphatidylcholine. Lecithin is the predominant source of choline in the human diet. The applicant has also shown that choline enhances the anti-inflammatory activity of a lipid extract from Perna canaliculus.

It is therefore an object of the invention to provide a composition which comprises a mussel extract and choline or a choline derivative, which composition has an enhanced anti-inflammatory effect, or to at least provide a useful choice.

STATEMENTS OF INVENTION

In a first aspect of the invention there is provided a composition containing: (i) a mussel extract having anti-inflammatory activity; and (ii) choline or a choline derivative; where the anti-inflammatory activity of the mussel extract is enhanced by the addition of the choline or the choline derivative.

In a preferred embodiment of the invention, the mussel extract is an extract from the New Zealand green-lipped mussel (Perna canaliculus). Preferably, the extract is a lipid extract.

Preferably, the composition contains a mussel extract and choline.

A preferred choline derivative is phosphatidylcholine or lecithin.

Preferably, the composition also comprises an ingestible oil. A preferred ratio of mussel extract to ingestible oil is one part of mussel extract to nine parts of ingestible oil. The ingestible oil is preferably olive oil.

In another preferred embodiment, the composition also comprises vitamin E.

Preferably, the amount of choline in the composition is in the range of 1:10 to 1:30 by weight. Typically, the amount of choline in the composition will be in the range of 1 :20 to 1:25 by weight, for example 1:23 by weight.

A preferred ratio of the choline derivative in the composition is in the range of 1:100 to

1:300. The range of 1:150 to 1:200 is further preferred. An especially preferred ratio, where the choline derivative is lecithin, is 1:184 by weight.

In a second aspect of the invention there is provided a method of treating or preventing inflammation in a human or animal using a composition of the first aspect of the invention. Preferably, the inflammation is caused by arthritis.

The composition may preferably be administered in a ratio of 2 mg of composition per gram of body weight of the human or animal.

In a third aspect of the invention there is provided the use of a mussel extract and choline or a choline derivative in the manufacture of a medicament for the treatment or prevention of inflammation.

In a fourth aspect of the invention there is provided a process for preparing a composition of the first aspect of the invention including the step of adding choline or a choline derivative to a mussel extract.

Preferably, the process includes the steps of contacting meat from one or more mussels with a solvent, separating the mussel meat from the solvent, and at least partially removing the solvent to give the mussel extract.

In a preferred embodiment, the mussel meat is freeze dried and powdered prior to contact with the solvent.

In one embodiment of the invention, any suitable organic solvent may be used. Preferably, the solvent is ethanol, methanol, or dichloromethane, or a mixture of any such solvents. The preferred solvent is anhydrous ethanol. In an alternative embodiment of the invention, the solvent may be a supercritical fluid such as supercritical CO ₂ .

DETAILED DESCRIPTION

As used herein, the term "mussel extract" is intended to mean an extract obtained from the meat of any mussel variety including, but not limited to, the New Zealand greeπ- lipped mussel (Pema canaliculus) and the blue mussel {Mytilus edulis). The extract may be obtained by any solvent extraction technique including extraction with an organic solvent such as ethanol and including supercritical fluid extraction.

Choline is an essential nutrient that is widely distributed in foods, principally in the form of phosphatidylcholine, but also as free choline. It is also found in foods in the form of the phospholipid sphingomyelin. Choline is necessary for the structure and function of all cells and is crucial for sustaining life.

Choline plays many roles in the body. The three major metabolic functions of choline are as a precursor for phosphatidylcholine biosynthesis, as a precursor for acetylcholine biosynthesis and as a methyl donor. In addition to serving as a precursor for phosphatidylcholine, choline is the precursor of the phospholipid sphingomyelin. Phosphatidylcholine and sphingomyelin are structural components of biological membranes. These phospholipids also serve as precursors for the intracellular messengers ceramide and diacylglycerol. Choline is also the precursor of the signaling lipids, platelet-activating factor (PAF) and sphingosylphosphoryl-choline.

Choline is also known as 2-hydroxy-N,N,N-trirnethylethanarninum, (beta-hydroxyethyl) trimethylammonium or bilineurine. The major commercial salts for supplementation are choline chloride and choline bitartrate. The chemical structure of choline is:

As used herein, the term "choline derivative" means any compound derived from choline and any compound that incorporates choline as part of its chemical structure or any compound that can be metabolised to give choline. Choline derivatives contemplated for this invention include, but are not limited to, phosphatidylcholine, acetylcholine, and L-alpha-glycerylphosphorylchoIine.

In biological systems, choline is most frequently found as a component of the phospholipid phosphatidylcholine where it is bound through a phosphate to an acylated glycerol. Phosphatidylcholine is commonly present in most phospholipids and is the major component of commercially available lecithin. It is represented by the following chemical structure:

o

Λ R πnd R ¹ = fatty acids residues v R ⁱ

Or

-N+fCHjfc

The term lecithin itself has different meanings when used in chemistry and biochemistry than when used commercially. Chemically, lecithin is phosphatidylcholine. Commercially, it refers to a natural mixture of neutral and polar lipids. Phosphatidylcholine is present in commercial lecithin in concentrations of 20 to

90%. Most of the commercial lecithin products contain about 20% phosphatidylcholine.

Lecithins containing phosphatidylcholine are produced from vegetable, animal and microbial sources, but mainly from vegetable sources. Soybean, sunflower and rapeseed are the major plant sources of commercial lecithin. Soybean is the most common source. Eggs contain from 68 to 72% phosphatidylcholine, while soya contains from 20 to 22% phosphatidylcholine.

Recent research has shown that choline is important for cell membrane integrity, controlling fat and cholesterol buildup in the body, preventing fat from accumulating in the liver, and facilitating the movement of fats in cells. Choline also helps regulate the kidneys, liver, and gallbladder. It is important for nerve transmission, and is needed for normal brain functioning, particularly in infants.

Lecithin is used in dietary supplements to prevent arteriosclerosis, protect against cardiovascular disease, improve brain function, increase energy levels, repair damage from alcoholism, help in digestion of fats, aging, immune system disorders, AIDS, herpes, and chronic fatigue syndrome.

In efforts to determine the nature of the constituents responsible for the anti¬ inflammatory activity of mussels, fractions enriched with carbohydrate and fractions enriched with lipid have both been shown to be anti-inflammatory, although the lipid fraction has been shown to have the higher activity. The identification of the active constituents continues to elude investigators. It may be that there are multiple molecules responsible for the anti-inflammatory activity.

The applicants have shown that when choline is added to a lipid extract from Perna canaliculus, the anti-inflammatory activity of the extract is stimulated when assessed using a carrageenan-induced joint swelling model. A significant increase in anti¬ inflammatory activity is attained with a relatively small quantity of choline (1 part of choline to 23 parts of mussel extract).

Other bases that are involved in phospholipid structures, such as inositol of phosphatidylinositol, serine of phosphatidylserine, were shown to not have any effect on the anti-inflammatory activity of the mussel extract.

The applicants also found that lecithin increases the anti-inflammatory activity of an extract from Perna canaliculus. The source of the lecithin does not seem to matter, although lecithin from egg yolk appears to be more active.

It will be appreciated that choline from any source and any choline derivative from any source are anticipated to enhance the anti-inflammatory activity of a mussel extract.

The ratio of choline to mussel extract or the ratio of choline derivative to mussel extract does not appear to be critical. The invention relates to any such ratio that provides enhancement of anti-inflammatory activity of the mussel extract. The ratio for choline may typically be in the range of 1:10 to 1:30 by weight. The ratio for lecithin may typically be in the range of 1:100 to 1:300.

The addition of lecithin to an extract from Perna canaliculus was also found to enhance the anti-angiogenic activity of the extract. Angiogenesis (the formation of new blood vessels) is implicated in a number of inflammatory conditions.

It will be appreciated that the composition of the invention may be administered in any suitable manner including, but not limited to, oral administration, topical administration, and administration by injection intravenously, subcutaneously, intradermally or intraperitoneally. Suitable formulations of the composition of the invention include capsules, tablets, granules, powders, creams, ointments, and injectable solutions.

The invention is further described with reference to the following examples. It is to be appreciated the invention is not limited to the examples.

EXAMPLES

Example 1 - Extraction

Powdered Perna canaliculus was mixed and stirred with ethanol (preferably anhydrous ethanol) at ambient temperature for approximately 18 hours. The liquid phase was decanted and then filtered to remove any solid material. The volume of ethanol was reduced by distillation, but the ethanol was not removed completely. Both olive oil and vitamin E were added and then the remaining ethanol removed completely by distillation (preferably at a low temperature). Olive oil was added in the ratio of 1 part of mussel oil to 9 parts of olive oil. Further filtration to remove any remaining solid material gave the mussel-olive oil product used in the following examples.

Example 2 - In vivo assay of acute joint inflammation

The acute anti-inflammatory activities of compositions of the invention were assessed using the carrageenan model of acute inflammation in the hind foot-pads of rats (Ormrod and Miller 1991). Six Lewis rats (3 male, 3 female) were selected as a control group and six Lewis rats (3 male, 3 female) were selected as an experimental group.

The volume displacement of all hind feet was measured. For the experimental group, the experimental solution was injected intraperitoneally into each rat. For all 12 rats, 100 μl of 2.5% λ-carrageenan was injected into each hind foot-pad one hour later. After four hours, the volume displacement of each foot was measured. The percentage increase in volume of each foot was calculated.

Results (1) Choline

Choline added to the mussel-olive oil product at a ratio of 1 part to 230 by weight gave a 1.8 times increase in the anti-inflammatory effect when evaluated with the

carrageenan model of joint inflammation (Table 1). This increase is highly significant statistically.

Table 1 - Inhibition of Joint Inflammation - choline

(Carrageenan Model)

% Increase in Joint % Inhibition

Swelling ± SEM a. control (n=12) 92.47% ± 2.23 b. product (2mg/g body weight) 82.58% ± 2.63 10.70 c. product (2mg/g body weight) + choline at 1 mg/230mg of product 74.75% ± 2.93 19.16

a v b: p<0.001 ; a v c: p<0.0001 ; b v c: p<0.05;

(2) Inositol and Serine

When inositol or serine (other components of phospholipid) were investigated, no effect on the antagonistic action of mussel-olive oil product in the joint inflammation model at similar ratios of inositol or serine to mussel-olive oil product was found (Table 2).

Table 2 - Inhibition of Joint Inflammation - inositol and serine

(Carrageenan Model)

% Increase in Joint % Inhibition

Swelling ± SEM a. control (n=12) 86.38% + 3.74 b. product (2mg/g body weight) 80.63% ± 2.22 6.66 c. product (2mg/g body weight) + inositol at 1 mg/230mg of product 80.82.% ±2.61 6.44 d. product (2mg/g body weight) + serine at 1 mg/230mg of product 79.56% ±2.89 7.90

(3) Lecithin

When lecithin was added to the mussel-olive oil product at a ratio of 1 part to 1840 by weight, there was a 50% increase in the anti-inflammatory activity of the mussel-olive oil product (Table 3). This increased inhibition is even more statistically significant.

Table 3 - Inhibition of Joint Inflammation - lecithin

(Carrageenan Model)

% Increase in Joint % Inhibition

Swelling ± SEM a. control (n=12) 84.45% ± 4.57 b. product (2mg/g body weight) 72.55% ± 3.56 14.09 c. product (2mg/g body weight) + lecithin at 1mg/1840mg of product 66.74.% ±3.17 20.97

a v b: p<0.05; a v c: p<0.005; b v c: NS

Example 3 - In vitro assay of angiogenesis

The aorta was removed from a rat and cleaned of adhering fatty and connective tissues before being cut into rings of approximately 3 mm size (Nicosia and Ottinetti 1990, Brown et al 1996). Fibrinogen was layered in the bottoms of wells of multi-well culture plates and allowed to gel by thrombin action. A ring was then layered on the top of each gel and a further layer of fibrin placed on this. The fibrinogen was prepared in MCDB131 medium supplemented with antibiotics. The double layer of fibrin was then overlaid with MCDB131 containing the test materials. The gels were incubated at 37°C in an atmosphere of 3%CO2/97% air. The rings were examined using an inverted microscope and the growth of microvessels from their perimeters observed. Digital pictures were taken of these every 2 or 3 days and the extent of microvessel growth relative to the size of the ring determined using NIH Image software. From this the rate of growth of microvessels was determined for each well. Each test substance was assayed in triplicate and the mean growth rate calculated. The Student t-test was used for assessing statistical significance. A probability of less than 0.05 is regarded as significant.

Results A solution of mussel-olive oil product at 10 mg/ml antagonised angiogenesis when evaluated in the in vitro aortic ring model (Table 4). However, when egg white lecithin was added to the mussel-olive oil product at a concentration of 1 mg per 1840 mg of extract, the inhibitory activity was approximately doubled. Although the inhibition by the mussel-olive oil product was not statistically significant, the inhibition by the extract and lecithin was (p<0.0025).

Table 4

Inhibition of angiogenesis

(Aortic Ring Assay)

% Inhibition a. product (10mg/ml) 12.83 b. product (10mg/mI) + lecithin at 0.5 mg/ml of product 24.89

Although the invention has been described with reference to preferred embodiments thereof, it should be appreciated that variations and/or modifications may be made without departing from the scope of the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.

REFERENCES

Audeval and Bouchart (1986) Gazette Medicate, 93; 111.

Brown KJ etal (1996) Lab Invest, 75; 539. Couch et al (1982) The New Zealand Medical Journal , 95; 803.

Croft (1979) Relief From Arthritis, Thorsons Publishing Group, Rochester, Vt.

Darlington and Stone (2001) Brit J Nut., 85; 251.

Gibson ef al (1980) The Practitioner, 224; 955.

Halpern (2000) Allergie et Immunologie, 32; 272. Kadi et al (2002) Vascul. Pharmacol. , 38; 219.

Marathe ef al (2000) Free Radical Biol & Med., 28; 1762.

McFarlane et al (1975) The New Zealand Medical Journal, June; 569.

Miller and Ormrod (1980) The New Zealand Medical Journal, 92; 187.

Miller et al (1993) Agents Actions, 38; 139. Nicosia and Ottinetti (1990) LaJb Invest , 63; 115.

Ormrod and Miller (1991) Pharmaceut. Res., 8; 1270.

Rainsford and Whitehouse (1980) Arzneim.-Forsch. /Drug Res., 30 (ii); 2128.

Sinclair et al (2000) Allergie et Immunologie, 32; 261.

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INDUSTRIAL APPLICATION

The composition of the invention has enhanced anti-inflammatory activity compared to the mussel extract containing no choline or choline derivative. The composition is useful for treating inflammation. It is particularly useful for treating inflammation caused by arthritis.