HK40016400A - Lipid combinations - Google Patents

Description

Lipid combinations

FIELD

The present disclosure generally relates to combinations of marine lipids. In particular, the present disclosure relates to a combination of lipids obtained from green-lipped mussel (Perna canaliculus) and krill, compositions and preparations comprising the combination and use of the combination and composition in therapy. The present disclosure also relates to processes for making krill oil and uses of krill oil in combinations and compositions.

Background

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Inflammation is a necessary physiologically adaptive response to injury and infection, without which humans and animals cannot survive. Its function is to eliminate the original cause of injury, remove uncomfortable factors (inflections), and initiate repair of tissue structure and function. The early acute phase of inflammation is typically characterized by heat, pain, redness and swelling. A common outcome of acute inflammation is recovery and repair of injury, however, an unbalanced acute inflammatory response and a long-term chronic inflammatory response may be detrimental in conditions such as sepsis.

Importantly, chronic inflammation has now been implicated in the pathology of a number of diseases affecting all tissues and organs, including osteoarthritis, rheumatoid arthritis, cardiovascular disease, cerebrovascular disease, respiratory disease, autoimmune disease, and sarcopenia (sarcopenia); in fact, chronic inflammation has been implicated in the process of aging itself.

In response to the inflammatory process, a range of drugs have been developed, the most effective being glucocorticoid steroid drugs which are capable of inhibiting excessive inflammation. However, steroid drug therapy is limited in broad clinical use, and is generally limited to only short-term use, due to its significant side effects. A second class of inflammatory drugs, known as nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and the like, was developed-see table 1. It is recognized that aspirin has anti-inflammatory activity and is part of this second class of anti-inflammatory drugs. These drugs are safer than steroid drugs and can be used for more chronic inflammatory conditions, such as osteoarthritis.

Table 1: list of non-steroidal anti-inflammatory drugs (NSAIDs) commonly used

Prostaglandins play a key role in the inflammatory response, with a significant increase in the presence of prostaglandins in inflamed tissues, resulting in pain and heat by raising the temperature and dilating the blood vessels, which causes redness and swelling of the sites where prostaglandins are released. NSAIDs are competitive site inhibitors of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), and thereby reduce prostaglandin synthesis. NSAIDs help to relieve the discomfort of fever and reduce inflammation and associated pain by reducing the production of prostaglandins. NSAIDs are commonly used to treat acute and chronic conditions characterized by pain and inflammation, such as osteoarthritis, rheumatoid arthritis, headache and migraine, and fever. However, NSAIDs also present considerable problems surrounding safety and may exhibit gastrointestinal, renal and cardiovascular toxicity. For example, aspirin can cause gastric bleeding within a few days of use, and in 7 months of 2015, the FDA reiterates previous warnings for cardiac hazards with common NSAID analgesics, excluding aspirin. These analgesics include ibuprofen (Advil, Motrin) and naproxen (Aleve) as well as the prescription-only NSAIDs.

Thus, there is a need for additional alternative anti-inflammatory treatments.

SUMMARY

It has now been found that certain combinations of mussel lipids and krill oil can advantageously provide additive or synergistic inhibition against the production or release of one or more pro-inflammatory mediators involved in the inflammatory process. In some embodiments, the combinations of the present disclosure may therefore provide novel therapeutic treatments for disorders characterized by inflammation or having an inflammatory component. In some embodiments, the combinations of the present disclosure can provide new therapeutic treatments for pain, such as pain associated with inflammation.

Thus, in one aspect, a combination comprising mussel lipid and krill oil is provided. The combination of mussel lipid and krill oil may be suitable for separate or simultaneous administration to a subject. In some embodiments, the combination is a composition comprising mussel lipid and krill oil.

In another aspect, a combination consisting of or consisting essentially of mussel lipid and krill oil is provided. The combination of mussel lipid and krill oil may be suitable for separate or simultaneous administration to a subject. In some embodiments, the combination is a composition consisting of or consisting essentially of mussel lipid and krill oil.

In another aspect, a combination comprising mussel lipid and krill oil is provided for use in therapy. In some embodiments, a combination comprising mussel lipid and krill oil is provided for use in treating inflammation in a subject. Also provided is a combination comprising mussel lipid and krill oil for use in treating pain in a subject. The combination of mussel lipid and krill oil may be suitable for separate or simultaneous administration to a subject. In some embodiments, the combination is a composition comprising mussel lipid and krill oil.

In another aspect, the present disclosure provides a method of treating inflammation in a subject in need thereof, comprising administering to the subject a combination comprising mussel lipid and krill oil. The present disclosure also provides a method of treating pain in a subject in need thereof comprising administering to the subject a combination comprising mussel lipid and krill oil. The combination of mussel lipid and krill oil may be suitable for separate or simultaneous administration to a subject. In some embodiments, the combination is a composition comprising mussel lipid and krill oil.

In another aspect, the present disclosure provides the use of mussel lipid and krill oil in the manufacture of a combination medicament for the treatment of inflammation. The present disclosure also provides the use of mussel lipid and krill oil in the manufacture of a combination medicament for the treatment of pain. The medicaments may be adapted for separate administration or simultaneous administration. In some embodiments, the combination is a composition comprising mussel lipid and krill oil.

In another aspect, the present disclosure provides a combination for treating inflammation, the combination comprising mussel lipid and krill oil. The present disclosure also provides a combination for treating pain, the combination comprising mussel lipid and krill oil. Mussel lipid and krill oil may be suitable for separate or simultaneous administration to a subject. In some embodiments, the composition is a composition comprising mussel lipid and krill oil.

In some embodiments, the mussel lipid is in the form of a dried mussel powder. In other embodiments, the mussel lipid is in the form of a lipid extract obtained from mussels ("mussel lipid extract"). In yet further embodiments, the mussel lipid may be in the form of a combination or composition of dried mussel powder and mussel lipid extract.

In some embodiments, the krill oil has a phospholipid content in the range of about 40-99% w/w, and in further embodiments, has a phospholipid content in the range of about 50-99% w/w, such as in the range of about 60-80% w/w.

In some embodiments, the combinations of the present disclosure may be useful in treating one or more disorders in a subject, wherein the disorder has an inflammatory component, and whereby inhibition of one or more pro-inflammatory molecules is therapeutically beneficial. In some embodiments, the combination may be suitable for treating one or more chronic disorders.

In some embodiments, e.g., for use in treating chronic inflammation, the combination may eliminate, avoid, or otherwise reduce the extent, severity, or duration of one or more side effects associated with the commonly available NSAIDs.

In another aspect, a process for producing krill oil having a phospholipid content of about 50% or more, such as about 60% or more, is provided, the process comprising the steps of:

(a) mixing krill biomass feed material with CO₂Contacting with a mixture of ethanol to extract krill oil; and

(b) mixing said krill oil with CO₂Contacting to extract at least a proportion of the non-polar lipid component such that the oil has a phospholipid content of at least 50% w/w.

In some embodiments, the krill oil obtained from this process has a phospholipid content of about 60% w/w or greater, for example having a phospholipid content of at least about 65% w/w or 70% w/w or 75% w/w or 80% w/w or 85% w/w or 90% w/w.

The present disclosure also relates to a process for enriching the phospholipid content of krill oil.

Thus, in another aspect, a process for increasing the phospholipid content of krill oil having a phospholipid content of less than 50% w/w to about 50% w/w or more is provided, the process comprising contacting krill oil having a phospholipid content of less than 50% w/w with CO₂A step of contacting to selectively remove the non-polar lipid component.

In some embodiments, the primary krill oil has a phospholipid content of less than about 50% w/w, such as less than about 40% w/w or less than about 30% w/w or 20% w/w. In some embodiments, the enriched oil so obtained has a phospholipid content of at least about 55% w/w, or at least about 60% w/w, or at least about 65% w/w, or at least about 70% w/w, or at least about 75% w/w, or at least about 80% w/w, or at least about 85% w/w, or at least about 90% w/w.

Another embodiment provides krill oil having a phospholipid content of about 50% w/w or greater (e.g., > 60% w/w) obtained by the process of the present disclosure.

Still further embodiments provide krill oil having a phospholipid content of about 50% w/w or 60% w/w or more for use in the combinations and compositions described herein.

Drawings

Figure 1A graphically depicts NO inhibition in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, olive oil and N- (3- (aminomethyl) benzyl) acetamidine (1400W).

Figure 1B graphically depicts NO release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, olive oil, and N- (3- (aminomethyl) benzyl) acetamidine (1400W).

Figure 2A graphically depicts TNF α inhibition in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, olive oil, and dexamethasone.

Fig. 2B graphically depicts TNF α release (%) in Lipopolysaccharide (LPS) and interferon γ (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, and olive oil.

Figure 3A graphically depicts IL-6 inhibition in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, olive oil and dexamethasone.

Figure 3B graphically depicts IL-6 release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, and olive oil.

Fig. 4A graphically depicts PGE2 inhibition in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, olive oil, and diclofenac.

Fig. 4B graphically depicts PGE2 release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil, each of mussel lipid extract and krill oil alone, and olive oil.

Fig. 5A graphically depicts NO release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY90-LY 50).

Fig. 5B graphically depicts NO release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY50-LY 10).

Fig. 6A graphically depicts NO release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY75-LY 60).

Fig. 6B graphically depicts NO release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY60-LY 45).

Figure 7 graphically depicts an equivalent response graph (isobologram) for synergistic NO inhibition by combinations of various concentrations of mussel lipid extract and krill oil (LY90-LY 10).

Figure 8A graphically depicts TNF α release (%) in Lipopolysaccharide (LPS) and interferon γ (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY90-LY 60).

Figure 8B graphically depicts TNF α release (%) in Lipopolysaccharide (LPS) and interferon γ (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY60-LY 30).

Figure 8C graphically depicts TNF α release (%) in Lipopolysaccharide (LPS) and interferon γ (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY40-LY 10).

Figure 9A graphically depicts TNF α release (%) in Lipopolysaccharide (LPS) and interferon γ (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY70-LY 55).

Fig. 9B graphically depicts TNF α release (%) in Lipopolysaccharide (LPS) and interferon γ (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY50-LY 35).

Figure 10 graphically depicts an equivalent plot of synergistic TNF α inhibition by combinations of various concentrations of mussel lipid extract and krill oil (LY90-LY 10).

Fig. 11A graphically depicts IL-6 release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY90-LY 50).

Figure 11B graphically depicts IL-6 release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY50-LY 10).

Fig. 12A graphically depicts IL-6 release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY70-LY 50).

Fig. 12B graphically depicts IL-6 release (%) in Lipopolysaccharide (LPS) and interferon gamma (IF γ) stimulated RAW264.7 cells for various concentrations of combinations of mussel lipid extract and krill oil (LY50-LY 30).

Figure 13 graphically depicts the equivalent response plots for synergistic IL-6 inhibition by various concentrations of combinations of mussel lipid extract and krill oil (LY90-LY 10).

Description of the invention

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the appended claims, unless the context requires otherwise, the word "consisting essentially of" and variations such as "consisting essentially of" will be understood to indicate that the recited elements are essential, i.e., essential, elements of the invention. The phrase allows for the presence of other unrecited elements that do not materially affect the characteristics of the invention, but excludes other unspecified elements that would affect the basic and novel characteristics of the invention as defined.

The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise.

The term "invention" includes all disclosures, aspects, embodiments, and examples as described herein.

As used herein, "about" refers to an amount, value, or parameter that can vary by as much as 10%, 5%, or 2% -1% of the stated amount, value, or parameter, and includes at least tolerances accepted in the art. When used in relation to stated integer values, "about" may include a variation of one integer on either side of the stated value, e.g., "50%" may include 49% and 51%. When preceding a recited range of values, it is intended to apply to both the upper and lower limits of that range.

Features described below may be applied independently to any aspect or embodiment unless the context indicates otherwise.

As used herein, "mussel lipid" refers to the lipid component extracted or obtained from the New Zealand Green Lip (NZGL) (or green shell) mussel (new zealand green-lipped mussel). Mussel lipids may comprise one or more of polyunsaturated long chain fatty acids (PUFAs) such as ALA, ETA, EPA and DHA, sterols, sterol esters, triglycerides, the non-polar lipids carotenoids and other components of (NZGL) mussel meat. Mussel lipid may be in the form of a dried mussel powder, or in the form of a lipid fraction extracted from mussel meat ("mussel lipid extract"). It is also contemplated that "mussel lipid" includes mixtures of mussel powder and mussel lipid extract, for example mussel lipid may be supplemented by the addition of mussel powder, or vice versa. In some embodiments, the mussel lipid is an isolated lipid fraction.

Mussel lipid powder may be prepared from fresh (raw), frozen or heat-treated NZGL mussel meat by any suitable drying means (e.g. freeze-drying, flash-drying or vacuum-drying) and comminuting means. In addition to fatty acids (including ALA, ETA, EPA, and DHA), mussel powder obtained by drying mussel meat will contain other potentially beneficial components including minerals, amino acids, peptides, proteins, and glycosaminoglycans (e.g. chondroitin-4-sulfate and chondroitin-6-sulfate). Processes for preparing mussel powder are known in the art.

Mussel lipid extracts may be obtained from fresh (raw), frozen, heat-treated or dried (e.g. freeze-dried, flash-dried or vacuum drum-dried) NZGL mussel meat (e.g. mussel meat in powdered, spray-dried or comminuted form) by any suitable method such as solvent extraction (e.g. acetone or ethanol-see e.g. WO2005073354a1, the contents of which are incorporated by introduction), enzymatic treatment (see e.g. WO2006128244, the contents of which are incorporated by introduction) or supercritical fluid extraction. In some embodiments, the mussel lipid extract is advantageously prepared by using supercritical CO₂Extracted from dried (e.g. freeze-dried) mussel meat (optionally stabilised against oxidation). An exemplary method for obtaining mussel lipid extract is described in WO97/09992 a1, the contents of which are incorporated by reference. Other methods will be known in the art.

In a preferred embodiment, the process is conducted under conditions, such as cold working, such that beneficial components, such as fatty acids, are not substantially destroyed and are largely retained.

An exemplary mussel lipid extract obtained according to the process described in WO97/09992 a1 is also known as mussel lipid extract(Pharmalink International Limited, hong Kong).With added vitamin E (0.15% w/w, asAdded as a preservative to combat oxidation) and include a combination of free fatty acids, triglycerides, sterol esters, non-polar lipids and carotenoids (Sinclair, a.j. et al, 2000) and are a source of long chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as other long chain fatty acids such as 5,9,12, 15-stearidonic acid, 5,9,12, 16-nonadecatetraenoic acid, 7,11,14, 17-eicosatetraenoic acid and 5,9,12,15, 18-heneicosapentaenoic acid.(formulated with olive oil into encapsulated oral dosage forms) toAnd Omega(for human consumption) andnominal sales (for dogs and cats).

In some embodiments, the mussel lipid extract for use in the combination of the present disclosure is formulated with vitamin E (e.g., added in an amount of about 0.2% w/w, or 0.15% w/w, or 0.1% w/w, or 0.05% w/w, or about 0.03% w/w or about 0.01% w/w). In some embodiments, the mussel is lipidated withIs used, i.e. a mussel lipid extract containing 0.15% w/w vitamin E. In some embodiments, mussel lipid optionally containing vitamin E is further formulated with a carrier oil such as olive oil. Although in some embodiments, the mussel lipid is a mussel lipid extract and contains added vitamin E, the addition of vitamin E is optional and, thus, in some embodiments, the mussel lipid extract is used neat, i.e. does not contain any other additional ingredients such as vitamin E.

Mussel lipids in various forms are also available from commercial suppliers.

Krill oil may be prepared from any suitable krill species, including Antarctic krill (Euphausia superba) (Antarctic krill), Pacific krill (euphorbia pacifica) (Pacific krill), Northern krill (magacytiphanes norvegica) (Northern krill)), brilliant krill (Euphausia crystaurophila) (ice krill)), cold krill (euphorbia frigida), long-bodied krill (Euphausia longirostris), Euphausia triandra (euphorbia triacantha), and Euphausia vachelli (Euphausia vallini). In some preferred embodiments, the krill oil is obtained from antarctic krill.

Marine lipids contain fatty acids, particularly omega-3 fatty acids, such as EPA and DHA, in free form and in triglyceride form. Similarly, krill oil is also rich in omega-3 fatty acids, however, krill oil contains a large amount of phospholipids with the fatty acids attached to the phosphate head groups via the glycerol moiety. It is this phospholipid-bound form of fatty acids that is more efficiently taken up into the cell membrane than the triglyceride form and is thus more readily bioavailable. Typical phospholipids found in krill oil may include: phosphatidylcholine, alkylacylphosphatidylcholine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine, lysoalkylacylphosphatidylcholine, phosphatidylethanolamine, alkylacylphosphatidylethanolamine, cardiolipin + N-acylphosphatidylethanolamine, lysophosphatidylethanolamine, and lysoalkylacylphosphatidylethanolamine. Krill oil also contains a considerable amount of astaxanthin, an antioxidant, which is also responsible for its red color.

In some embodiments, the krill oil contains at least about 1% w/w, 5% w/w, 10% w/w, or at least about 20% w/w phospholipids. In further embodiments, the oil contains at least about 25% w/w, or at least about 30% w/w, or at least about 35% w/w, or at least about 40% w/w, or at least about 45% w/w, or at least about 50% w/w, or at least about 55% w/w, or at least about 60% w/w, or at least about 65% w/w, or at least about 70% w/w, or at least about 75% w/w, or at least about 80% w/w, or at least about 85% w/w phospholipid, or at least about 90% w/w phospholipid95% w/w phospholipid, or at least about 97% w/w phospholipid, or at least about 98% w/w phospholipid, or at least about 99% w/w phospholipid. In some embodiments, the krill oil has a phospholipid content in the range of about 40-99% w/w. In some further embodiments, the krill oil has a phospholipid content in the range of about 60-99% w/w, for example in the range of about 65-90% w/w. As referred to herein, "enriched" krill oil refers to krill oil having a phospholipid content of at least about 60% w/w. Phospholipid content can be determined by any suitable means in the art, for example³¹P NMR analysis.

Processes for preparing krill oil, including phospholipid-rich krill oil, are known in the art. Typically, fresh, frozen and/or heat treated krill (e.g., antarctic krill or pacific krill) biomass may use a solvent (e.g., an alcohol such as ethanol, a ketone such as acetone, or dimethoxyethane) and/or a supercritical fluid (e.g., CO)₂) To extract. Some non-limiting exemplary processes for making krill oil are described in U.S. patent No. 9,028,877, U.S. patent No. 9,375,453, U.S. patent No. 6,800,299, U.S. patent No. 8,828,447, U.S. patent No. 9,150,815, U.S. patent No. 8,383,845, WO2007/123424, WO2011/050474, WO2015/104401, and WO2015/121378, the contents of which are incorporated herein by reference. Additional methods are also described herein. Krill oil may also be purchased from commercial suppliers.

In some advantageous embodiments, the krill oil has a water content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w or 1% w/w, or 0.5% w/w or less. In some embodiments, the krill oil has a residual extraction solvent content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5% w/w or less. In further embodiments, the krill oil has a water content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5% w/w or less, and a residual extraction solvent content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5% w/w or less. The water and solvent may be removed by any suitable means, such as mild heating of very short duration (e.g. 30 minutes, or 1 hour, or 2 hours, or 3 hours, at a temperature of about or less than about 60 ℃, or 50 ℃, or 40 ℃, and preferably such that the integrity of the ingredients is not substantially compromised), nitrogen flow, or lyophilization (freeze drying).

The combination is directed against one or more inflammatory mediators such as Nitric Oxide (NO), cytokines such as interleukins (e.g. IL-6), prostaglandins (e.g. PG-E)₂) And the inhibitory activity of TNF α can be useful in treating one or more disorders or symptoms in a subject, whereby inhibition of one or more of such molecules is therapeutically beneficial. In particular, the combinations of the present disclosure may be useful in treating excessive acute or chronic inflammation and/or symptoms associated therewith, such as one or more of pain, heat, redness and swelling. In some embodiments, the combination may be useful in treating inflammation of a disorder in which the pathology includes an inflammatory component and/or pain associated with such a disorder. Some non-limiting examples of disorders that include aspects of inflammation include atherosclerosis, allergies, asthma, autoimmune diseases (e.g., celiac disease, psoriasis, rheumatoid arthritis, psoriatic arthritis), fibromyalgia, gout, migraine, osteoarthritis, ulcerative colitis, cancer, cognitive impairment including alzheimer's disease (impaired diagnosis), type 2 diabetes, Delayed Onset Muscle Soreness (DOMS), crohn's disease, and ankylosing spondylitis. In some embodiments, the combinations of the present disclosure may be useful in treating joint pain or improving joint mobility associated with osteoarthritis or rheumatoid arthritis. In some embodiments, the combination of the present disclosure is used in the treatment of PGE₂Inhibition of (a) may be beneficial in disorders such as rheumatoid arthritis, migraine and pain (pain may be nociceptive (somatic or visceral) pain and/or neuropathic pain).

It is to be understood that the combinations described herein may be adapted for separate administration or simultaneous administration. Where suitable for simultaneous administration, the combination may be provided and/or administered as an intimate composition (intragranular) or mixture comprising both the mussel lipid extract and the krill oil, or as discrete dosage forms of each of the combined components. Where the mussel lipid extract and krill oil are each provided and/or administered separately, they may be administered simultaneously, one after the other or each at a different time.

In further embodiments, mussel lipid and krill oil may be formulated, optionally in combination with one or more pharmaceutically acceptable carriers and/or additives, or separately. Some examples of suitable carriers are edible oils such as olive oil, castor oil, linseed oil, grape seed oil, fish oils (e.g., tuna oil), rapeseed oil, vegetable oils, sunflower oil, chia oil (chia oil), soybean oil, sesame oil, algal oil, and mixtures thereof. One or more optional additives, such as antioxidants, vitamins (e.g., fat soluble vitamins (A, D, E and K) or water soluble vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C), dietary minerals, amino acids, odor and taste masking agents, emulsifiers, pharmaceutically acceptable alcohols (e.g., ethanol, glycerol, propylene glycol, and polyethylene glycols) or other viscosity modifiers, surfactants (e.g., polysorbates), suspending agents, lactose, dextrose, sucrose, mannitol, sorbitol, glucose, lubricants, binders, starches, absorption promoters, preservatives, and the like may also be included. Such as astaxanthin and its esters, fatty acids in the form of free acids, acid esters, triglycerides or phospholipids (e.g. EPA, DHA), sterols, sterol esters, amino acids, peptides and proteins and glycosaminoglycans (e.g. chondroitin sulphate). Other anti-inflammatory foods such as fully ground forms or extracts thereof, e.g., turmeric (curcumin), ginger, garlic, clove, and the like, may also be optionally incorporated.

The formulated combination may be prepared according to methods known in the art. Such methods include the step of bringing the mussel lipid extract and/or krill oil into close association with a carrier, optionally together with one or more additive ingredients. It will be understood that any carrier or additive will be pharmaceutically acceptable.

Thus, in some embodiments, mussel lipid and krill oil are formulated alone or together with a carrier oil such as olive oil. In some embodiments, the carrier oil constitutes from about 10% w/w to about 90% w/w, for example about 20% w/w to about 80% w/w of the combination or composition. In further embodiments, the carrier oil comprises about 25% w/w, or about 30% w/w, or about 35% w/w, or about 40% w/w, or about 45% w/w, or about 50% w/w, or about 55% w/w, or about 60% w/w, or about 65% w/w, or about 70% w/w, or about 75% w/w of the combination or composition. In some embodiments, the weight ratio of carrier oil to the combined amount of mussel lipid and krill oil is about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or about 1:1.5, or about 1:2, or about 1:2.5, or about 1: 3.

Although any form of administration is contemplated herein, such as oral, parenteral, topical, transdermal or subcutaneous administration, advantageously, in some embodiments, the combinations of the present disclosure may be provided and/or administered in an oral dosage form. In some embodiments, the combination may be present in bulk form (bulk form), e.g., as a liquid, syrup, paste, semi-solid wax, dispersion, suspension, emulsion (e.g., water-in-oil or oil-in-water), pulverized powder, or microencapsulated powder, from which individual doses may be measured. The measurement and/or administration may be by any means, such as a spoon or spoon, syringe, dropper, or measuring cup. The measured dose may be administered directly to the subject or mixed, poured or dusted on food or beverage.

In other embodiments, the combination is advantageously present in a unit oral dosage form, i.e. a fixed dosage form. Some examples of suitable unit oral doses include individually packaged ampoules, tubes, filled syringes, pouches, chewables (chews), and capsules, including hard gel capsules and soft gel capsules.

One example of a suitable unit oral dosage form is a capsule in the form of a hard or soft shell. The shell may comprise one or more of gelatin, pullulan, hypromellose, PVA copolymers, carrageenan or other saccharide components such as starch or cellulose, or mixtures thereof, and may also comprise colorants, opacifiers, plasticizers (e.g., sorbitol, xylose, maltitol, and glycerol), and the like. Methods for encapsulating marine oils and lipids such as mussel oil and krill oil are known in the art. See, e.g., WO2015/121378, the contents of which are incorporated herein by reference. In some embodiments, where the krill oil is separately encapsulated, the krill oil may be encapsulated in the absence of optional additional agents such as viscosity modifiers, that is, the capsule fill consists essentially of krill oil.

In some advantageous embodiments, the combination of mussel lipid and krill oil of the present disclosure is present in the form of a softgel capsule, for example a softgel capsule comprising both mussel lipid and krill oil, or a softgel capsule alone, wherein the mussel lipid and krill oil are separately encapsulated, optionally together with suitable carriers and/or additives. Suitable softgel capsules may be prepared from gelatin (or alternatively, sugar sources such as pullulan and hypromellose), optionally together with one or more plasticizers such as sorbitol and glycerol (glycerin), and additives such as colorants and opacifying agents. In one example, the soft gel capsule shell can comprise gelatin and one or both of sorbitol and glycerol.

Microencapsulation is a process by which tiny droplets or particles are surrounded by a coating wall or embedded in a matrix to form a powder, wherein the coating or matrix forms a functional barrier that avoids or reduces the propensity for chemical reactions such as oxidation. In addition, it may provide a potential taste or odor masking effect. Thus, in some embodiments, mussel lipids and/or krill oil may be microencapsulated, either individually or together, to form a powder. Commonly used microencapsulation methods include emulsification, spray drying, freeze drying, in-line electrospray, extrusion, coacervation, supercritical fluid techniques and in-situ polymerization. Coating materials include natural and synthetic polymers, carbohydrates (e.g., starch, glucose), proteins (e.g., casein, gelatin), and mixtures thereof. (see, e.g., Bakry, A.M. et al, Comprehensive Reviews in Food Science and Food safety, 15, 143, 2016, and references cited therein, WO2014/170464 and WO2014/169315, the contents of which are incorporated herein by reference). Mussel lipid and/or krill oil may be microencapsulated together with one or more carriers or additives as described above. The microencapsulated mussel lipid and/or krill oil powder may be further encapsulated, for example in a hard shell capsule unit dosage form. The powdered microencapsulated lipid or oil may optionally be combined with one or more carriers or additives.

In some embodiments, the combinations of the present disclosure may be taken with food or beverages, for example, by dusting, stirring, mixing into or onto food or beverages the combination of mussel lipid extract and krill oil, or by other means of applying or incorporating the combination of mussel lipid extract and krill oil into or onto food or beverages. Thus, the combination may be provided in a form for incorporation into or onto a beverage or foodstuff. In some embodiments, the combination may also be formulated in the preparation of foods and beverages to provide functional foods.

In some embodiments, mussel lipid and krill oil are formulated alone or with a carrier oil such as olive oil and optionally with an antioxidant (e.g. vitamin E).

Subjects to be treated by the combinations of the present disclosure include mammalian subjects, such as humans, primates, felines, canines, bovines, equines (equines), porcines (porcines), lagomorphs (leporines), ovines (ovines), and caprines (caprines), and include livestock animals (e.g., cows, horses, sheep, pigs, and goats), companion animals (e.g., dogs, cats, rabbits, guinea pigs), and captive wild animals. Laboratory animals such as rabbits, mice, rats, guinea pigs, and hamsters are also contemplated as they may provide a convenient testing system.

Any of the dosage forms described above may be applied for human or veterinary use as appropriate.

A therapeutically effective amount is intended to include an amount of the combination that is jointly effective, when administered according to a desired dosing regimen, to at least partially achieve a desired therapeutic effect, including one or more of: reducing, eliminating, or reducing the duration, severity, and/or frequency of inflammation, and/or one or more symptoms of inflammation (e.g., heat, pain, swelling, redness) of the particular disorder or condition being treated; preventing or delaying the occurrence of the particular disorder or condition being treated; inhibiting the progression of the particular disorder or condition being treated; or (partially or completely) stop or reverse the occurrence or progression of the particular disorder or condition being treated.

Suitable dosages and dosing regimens may be determined by the attending physician or veterinarian and may depend on the particular condition/symptom being treated, the severity of the condition, and the general age, health and weight of the subject. Suitable daily dosages of mussel lipid and/or krill oil may independently range from about 10mg to about 10g, for example about 10mg, 20mg, 30mg, 40mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g, 2.1g, 2.2g, 2.3g, 2.4g, 2.5g, 2.6g, 2.7g, 2.8g, 2.9g, 3.0g, 3.2g, 3.5g, 3.7g, 4.5g, 0.5g, 0g, 5.5g, 0g, 0.5g, 0g, 5.5g, 0.5g, 0g, 5.5g, 0g, 0.5g, 0g, 5g, 0g, 5.8 g. In some further embodiments, the daily dose of the combination may range from about 20mg to about 15g, e.g., about 20mg, 30mg, 40mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g, 2.1g, 2.2g, 2.3g, 2.4g, 2.5g, 2.6g, 2.7g, 2.8g, 2.9g, 3.0g, 3.2g, 3.5g, 3.7g, 4.0g, 5.5g, 0.5g, 0.6 g, 5g, 0.7 g, 2.8g, 2.9g, 3.0g, 5g, 5.5g, 0g, 0.5g, 0g, 13.7 g, 12.7 g, 12.9 g, 5.9 g, 5g, 0g, 0.9 g, 5g, 0.9 g, 13.9 g, or 14.9 g.

In some embodiments, a single unit dose (e.g., soft gel capsule) may comprise a combination of about 10mg, 20mg, 25mg, 30mg, 40mg, 50mg, 60mg, 70mg, 75mg, 80mg, 90mg, 100mg, 110mg, 120mg, 125mg, 130mg, 140mg, 150mg, 160mg, 165mg, 170mg, 175mg, 180mg, 190mg, 200mg, 210mg, 220mg, 225mg, 230mg, 240mg, 250mg, 260mg, 265mg, 270mg, 275mg, 280mg, 290mg, 300mg, 310mg, 320mg, 325mg, 330mg, 340mg, 350mg, 360mg, 365mg, 370mg, 375mg, 380mg, 390mg, 400mg, 410mg, 420mg, 425mg, 430mg, 440mg, 450mg, 460mg, 465mg, 470mg, 475mg, 480mg, 490mg, or about 500mg, optionally formulated with a carrier oil (e.g., olive oil). In some further embodiments thereof, the combination further comprises vitamin E. In some further embodiments thereof, the combination comprises mussel lipids (e.g., as PCSO-524) in an amount in the range of about 10% w/w to about 90% w/w of the total amount of mussel lipids and krill oil, and the krill oil is included in an amount in the range of about 90% w/w to about 10% w/w of the total amount of mussel lipids and krill oil, i.e., a weight ratio of mussel lipids to krill oil of from about 10:90 to 90:10, e.g., a weight ratio of mussel lipids to krill oil of about 15:85, or about 20:80, or about 25:75, or about 30:70, or about 35:65, or about 40:60, or about 45:55, or about 50:50, or about 55:45, or about 60:40, or about 65:35, or about 70:30, or about 75:25, or about 80:20, or about 85: 15.

The dose may conveniently be administered once daily, or the daily dose may be divided and administered multiple times per day (e.g. two, three or four times). In some embodiments, a combination of the present disclosure may be administered once a week, twice a week, three times a week, or more times a week, e.g., on alternate days. In some embodiments, treatment may be continuous or chronic, e.g., over a period of at least 6-12 months or at least 2-3 years, or ongoing.

The combination of the present disclosure, e.g., in any one daily dose, may comprise mussel lipids in an amount ranging from about 1% w/w to about 99% w/w of the total amount of mussel lipids and krill oil, and krill oil in an amount ranging from about 99% w/w to about 1% w/w of the total amount of mussel lipids and krill oil, i.e., a weight ratio of mussel lipids to krill oil of from about 1:99 to 99: 1. In some embodiments, the combination comprises mussel lipids in an amount in the range of about 5% w/w to about 95% w/w of the total amount of total mussel lipids and krill oil, and krill oil in an amount in the range of about 95% w/w to about 5% w/w of total mussel lipids and krill oil, i.e., a weight ratio of mussel lipids to krill oil of from about 5:95 to 95: 5. In some embodiments, the combination comprises mussel lipids in an amount in the range of about 10% w/w to about 90% w/w of the total amount of total mussel lipids and krill oil, and krill oil in an amount in the range of about 90% w/w to about 10% w/w of total mussel lipids and krill oil, i.e., a weight ratio of mussel lipids to krill oil of from about 10:90 to 90: 10. In still further embodiments, the combination comprises mussel lipid and krill oil in a weight ratio of about 15:85, or about 20:80, or about 25:75, or about 30:70, or about 35:65, or about 40:60, or about 45:55, or about 50:50, or about 55:45, or about 60:40, or about 65:35, or about 70:30, or about 75:25, or about 80:20, or about 85: 15.

While the combinations of the present disclosure may be administered as the sole anti-inflammatory treatment for any one or more disorders, they may also be administered in combination with an administration regimen of one or more NSAIDs such as celecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, telolorac, mefenamic acid (mefenamic), meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, and tolmetin. In some embodiments, the combinations of the present disclosure may eliminate or reduce potential side effects associated with NSAIDs, for example, by eliminating or substantially eliminating the need for additional NSAID-based therapy, or by reducing the dose and/or frequency of dosing of NSAIDs required to achieve a beneficial therapeutic effect.

As discussed above, in one or more embodiments, the krill oil used in the combinations disclosed herein may advantageously have a phospholipid content of at least about 50% w/w or higher, preferably at least about 60% w/w or higher. Many prior art processes for extracting high phospholipid (e.g., greater than about 50% w/w or 60% w/w) krill oil from krill meal (krill meal) use CO₂And CO₂A combination of/ethanol. However, it is recognized that the higher the phospholipid content of krill oil, the more viscous the krill oil, with krill oil having a content of about 60% or greater typically existing as a viscous paste at ambient temperature. This presents manufacturing challenges, particularly in industrial or commercial scale oil production, as higher temperatures are required to evaporate the solvent used in the extraction process from the viscous material, where heat related damage to the oil is more likely to occur. Furthermore, increased pressure is then required to transfer the oil from the extraction tank to the packaging tank.

An exemplary prior art process is described in WO 2007123424. This document describes a two-step process whereby the feed material is first fed with pure CO₂Extraction to extract only neutral lipids (i.e. non-polar triglycerides), leaving a material enriched in phospholipids by means of removal of the non-polar triglycerides. The phospholipid-enriched material is then treated with CO₂+ gtoreq10% ethanol co-solvent to extract polar phospholipids and the remaining non-polar triglycerides together from krill feed biomass. This process inefficiently uses plant capacity on a commercial scale because during both stages of the process, the majority of the volume in the high pressure extractor is filled with non-extractable protein, carbohydrate and ash components in the feed biomass. This is also fundamentally a less efficient batch operation (as compared to a more efficient continuous operation), which may adversely affect process costs. Effectively, the enrichment for delivering the final polar phospholipid content into the finished oil is achieved in a first step directly on an industrial scale bulk heterogeneous solid raw material. Accurate final oil enrichment requires accurate prior knowledge of the polarity of the solid feedLipid content and non-polar lipid content, and how well each will be extracted later. In practice, uncertainty on a commercial scale can translate into expensive over-enrichment, which then requires final blending back to the required specifications. This again adversely affects the economics of the process.

US9,735,453, US9,078,905, US9,028,877, US9,320,765 and US9,072,752 describe the use of CO₂Or CO₂Extracting krill with about 5% ethanol to extract neutral lipids (nonpolar triglycerides), and then extracting with CO₂V 20% ethanol extracts krill oil with high amounts of phospholipids, astaxanthin esters and/or omega-3 fatty acids from the bulk heterogeneous solid material. These processes share the disadvantages described above.

The present disclosure now describes a process that, in some embodiments, may reduce, minimize or eliminate one or more of the disadvantages discussed above when producing krill oil comprising high or enriched phospholipids according to prior art methods, particularly on an industrial or commercial scale (e.g., when producing batches of oil on the order of at least about 50kg, 100kg, 200kg, 300kg or 500kg and greater). Accordingly, the present disclosure also provides a process for preparing krill oil having a phospholipid content at a level of at least about 50% w/w or higher, preferably to at least about 60% w/w or higher; and a process for enriching an oil of lower phospholipid content (less than about 50% w/w) to a level of at least about 50% w/w or higher, preferably to at least about 60% w/w or higher.

In some embodiments, the present disclosure provides a 2-step process for producing krill oil having a phospholipid content of at least about 50% w/w, wherein the first step comprises extracting a first krill oil having a phospholipid content of less than about 50% w/w from krill biomass, and then removing at least a proportion of non-polar lipid components (e.g., triglycerides) from the first krill oil to obtain a second krill oil enriched in phospholipids (i.e., having a higher phospholipid content) compared to the first krill oil.

In contrast to the prior art processes discussed above, some embodiments of the process are biological from krillNon-selective extraction of oil from the mass feed material begins. Extraction of both polar lipids (e.g., phospholipids) and non-polar lipids (e.g., triglycerides) from solid feed powders may use CO₂And ethanol (e.g., azeotropic ethanol-a water-ethanol mixture comprising about 95% ethanol).

In some preferred embodiments, it can be used in CO₂At least about 15% w/w or about 20% w/w ethanol, for example in the range of about 17-22% w/w. In other embodiments, it may be used in CO₂At a mass ratio of at least about 25% w/w ethanol, such as in CO₂At least about 26% w/w ethanol, or at least about 27% w/w ethanol, or at least about 28% w/w ethanol, or at least about 29% w/w ethanol, or at least about 30% w/w ethanol is used.

In some embodiments, including any one of the other embodiments discussed in this paragraph, the extraction temperature is at or below about 60 ℃, such as at or below about 55 ℃, or at or below about 50 ℃, or at or below about 45 ℃, or at or below 40 ℃, or at or below 35 ℃, or at or below 30 ℃, to advantageously reduce or minimize the risk of product degradation. The extraction pressure may be set to ensure that for a selected temperature and CO₂Supercritical, subcritical and/or near-critical conditions of ratio to ethanol. In some embodiments, the pressure is in the range of about 200-. In some embodiments, the pressure value or pressure range results in sufficient solvent density to ensure that extraction of the non-polar lipid does not become a rate limitation of the process. In some embodiments, the extraction pressure conditions may be adjusted throughout the extraction process to move between supercritical, subcritical or near-critical conditions. In some embodiments, subcritical conditions and/or near-critical conditions are used, the conditions for which will depend on CO₂The binary mixing ratio (mix ratio) with ethanol (or the ternary mixing ratio in the case of azeotropic ethanol which also contains water) varies. Lifting deviceThe extraction time may be determined by a person skilled in the art and may depend inter alia on the extraction conditions and the desired economic optimization. In some embodiments, the extraction time is generally in the range of about 1 to 15 hours, such as about 2 to 10 hours, for example about 2 to 5 hours, or about 3 to 6 hours, or about 4 to 5 hours. This in turn may depend on, among other things, the amount and particle size of the biomass feed material. Larger particles will allow the use of higher solvent flow rates while still maintaining static biomass and uniform solvent contact. However, larger particles also exhibit increased diffusion requirements for the solvent to reach the center of the particle, where more solvent is needed. Thus, in some embodiments, the particle size of the feed material is about 1-5mm, for example about 2-3 mm.

In some further embodiments, the extraction pressure is about 300 bar and the extraction temperature is about 60 ℃.

The oil separation can be carried out at a lower temperature (e.g., about 25-35 ℃) and pressure (e.g., about 25-60 bar).

The resulting extracted oil comprises both polar lipids (e.g., phospholipids) and non-polar lipids (e.g., triglycerides) and may have a phospholipid content of less than about 50% w/w, or less than about 45% w/w, or less than about 40% w/w, or less than about 35% w/w, or less than about 30% w/w, or less than about 25% w/w, or less than about 20% w/w, or less than about 10% w/w.

In some advantageous embodiments, the water and ethanol present in the oil may then be removed using any suitable method, such as evaporation under vacuum (optionally with gentle heating, e.g., about 65 ℃ or less), nitrogen flow, or lyophilization. In some embodiments, the oil is subjected to evaporation under vacuum, optionally with mild heating. Further, this is followed by a short residence time (e.g., 1-3 seconds) at a higher temperature (e.g., about 70 ℃, about 75 ℃, or about 80 ℃) to remove the water and ethanol co-solvent from the low viscosity polar lipid and non-polar lipid mixture, with a high proportion of non-polar lipids providing low viscosity. In some preferred embodiments, the temperature advantageously does not exceed about 60 ℃ throughout the mild heating under vacuum in order to avoid or minimize degradation of the components of the extracted oil. In further embodiments, the temperature advantageously does not exceed about 55 ℃, or about 50 ℃, or about 45 ℃, or about 40 ℃, or about 35 ℃, or about 30 ℃, or about 25 ℃ throughout the mild heating under vacuum.

In some embodiments, the residual volatile content (water and ethanol) after evaporation is about or less than about 3% w/w, as this can minimize the possibility that residual ethanol and water adversely affect the separation of lipids in the subsequent enrichment step. In further embodiments, the residual volatile content is about or less than about 2.5% w/w, or about or less than about 2.0% w/w, or about or less than about 1.5% w/w, or about or less than about 1.0% w/w, or less than about 0.5% w/w, or less than about 0.3% w/w, or less than about 0.1% w/w.

At this stage, after evaporation, the oil is still fluid and can be easily analyzed for example for phospholipid content and/or omega-3 fatty acid content due to the presence of the non-polar lipid component. This is important because accurate analysis is required in order to calculate the desired enrichment and thus the phospholipid content of the final oil achieved in the subsequent steps. In particular, where a final high phospholipid content is desired, over-enrichment (i.e. further removal of non-polar lipids), even to a small extent, may lead to processing problems due to excessive viscosity. The evaporated oil may be thoroughly mixed to ensure homogeneity, optionally after transfer to an intermediate product tank. Optionally, the oil may be gently heated (e.g., at a temperature of less than or about 60 ℃ or about 55 ℃, or about 50 ℃ or about 45 ℃, or about 40 ℃, or about 35 ℃, or about 30 ℃, or about 25 ℃) to help maintain a fluid and homogeneous material for analysis. In some embodiments, CO is passed through the first step as compared to the first step bulk solid biomass used in the prior art processes discussed above₂The lower viscosity properties of the unenriched oil obtained by the/EtOH extraction may advantageously allow for more accurate compositional analysis, as bulk homogeneity may be more easily achieved. In some embodiments, this may advantageously avoid, minimize, or otherwise reduce selective extraction of phospholipids at subsequent non-polar lipidsMay otherwise produce an undesirably sticky or immovable solid product.

The second step of the process involves the extraction, preferably or selectively, of non-polar lipids (triglycerides) from the oil obtained in the first step. If the non-enriched oil obtained by the first extraction step has been transferred to the intermediate tank, the non-enriched oil is returned to the extraction facility. Subjecting the oil further to CO₂Extraction, in some preferred embodiments under supercritical conditions, for example at about 300 bar or greater than about 300 bar and at about 60 ℃, in order to selectively extract the non-polar lipids (triglycerides). In some embodiments, the extraction may begin at a lower pressure and then incrementally increase to a desired level (e.g., about 300 bar). The non-polar lipids (triglycerides) may be gradually extracted, thereby enriching the remaining raffinate, until the desired compositional goal is achieved, such as a phospholipid content of at least about 50% w/w, or at least about 55% w/w, or at least about 60% w/w, or at least about 65% w/w, or at least about 70% w/w, or at least about 75% w/w, or at least about 80% w/w, or at least about 85% w/w, or at least about 90% w/w, or at least about 95% w/w, or at least about 97% w/w, or at least about 98% w/w, or at least about 99% w/w phospholipids.

In some embodiments, after the desired amount of non-polar lipids has been extracted to achieve the desired level of phospholipid enrichment, partially depressurizing the extraction vessel allows the residual pressure to assist in draining the now-enriched (and more viscous) raffinate as needed. By draining from the still partially pressurized extractor, the draining of the enriched high viscosity oil can be tolerated to a greater extent. In this way, extremely viscous materials can be transferred for forward blending and formulation.

In one or more embodiments, the process may allow for semi-continuous processing, where individual extraction vessels are replaced while continuously rotating, but one at a time, while other extraction vessels continue to operate. In this way, stops for replacing multiple extractor batches may be avoided.

In other embodiments, the second step described herein may be used to enrich the phospholipid content of any krill oil having a phospholipid content of less than about 50% w/w, so as to obtain krill oil having a phospholipid content of at least about 50% w/w.

In some embodiments, the starting krill oil has a phospholipid content of about 45% w/w or less, or about 40% w/w or less, or about 35% w/w or less, or about 30% w/w or less, or about 25% w/w or less, or about 20% w/w or less, or about 10% w/w or less. In some embodiments, the final enriched oil may have a phospholipid content of at least about 55% w/w phospholipids, or at least about 60% w/w phospholipids, or at least about 65% w/w phospholipids, or at least about 70% w/w phospholipids, or at least about 75% w/w phospholipids, or at least about 80% w/w phospholipids, or at least about 85% w/w phospholipids, or at least about 90% w/w phospholipids, or at least about 95% w/w phospholipids, or at least about 97% w/w phospholipids, or at least about 98% w/w phospholipids, or at least about 99% w/w phospholipids.

In some embodiments, the enriched krill oil has a final water content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5% w/w or less. In some embodiments, the krill oil has a residual extraction solvent content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5w/w or less. In further embodiments, the krill oil has a water content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5w/w or less and a residual extraction solvent content of about 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1% w/w, or 0.5w/w or less. In still further embodiments, the enriched krill oil has a residual solvent (water and ethanol) content of 5% w/w or less, or about 4% w/w, or 3% w/w, or 2% w/w, or 1.5% w/w, or 1% w/w, or 0.5w/w, or 0.3% w/w, or 0.1% w/w, or less.

In still other embodiments, the final enriched krill oil has a phospholipid content of at least about 60% w/w phospholipids, and a residual solvent content of about 3% w/w or less.

Other embodiments as described herein relating to krill oil may also be suitable as appropriate for the krill oil produced by the process of the present disclosure.

The following examples are provided for the purpose of illustrating some embodiments of the present disclosure and are not intended to limit the generality of the foregoing description.

Examples

Example 1Preparation of-62% w/w phospholipid krill oil

1. Krill oil extraction from krill meal

With CO at a temperature of 60 ℃ and a pressure of 300 bar₂Ethanol extraction of krill meal (feed ratio of ethanol to krill meal of about 3.0-3.5: 1w/w), wherein the ethanol mass fraction is in the range of about 17-22% w/w. ethanol/CO₂The duration of the phase extraction is between 10 hours and 15 hours. Extracted oil/CO₂the/EtOH mixture was separated at a pressure of 45 bar and 25 ℃.

Several batches of oil obtained by this process were blended to provide krill oil containing both polar and non-polar components and having a phospholipid content of about 42% (see table 1-1 below).

Tables 1 to 1: PL in krill oil samples³¹P NMR analysis

2. Selectively extracting triglycerides from the krill oil obtained from step 1.

5.9kg of feed krill oil (having the composition set out in tables 1-1 above) was directly loaded into a single 10.7L extraction vessel (155mm diameter). The feed material is CO at a temperature of 60 ℃ and a pressure of 300 bar₂And (4) extracting.

The extract, which mainly comprises triglycerides, is recovered and is significantly less viscous than the incoming krill oil loaded into the extraction vessel.

In the extraction ofAfter reaching 97% of the theoretical amount of material that can be extracted, the depressurization process of the extraction vessel is started. The facility depressurizes from 300 bar to 100 bar at a constant ramp rate over a period of 15 minutes, wherein the CO₂The cycle continues, albeit at 50% of the extraction flow. During this time, the measured temperature at the outlet of the extractor was reduced from the 60 ℃ operating temperature to 50 ℃. The extractor was then further depressurized from 100 bar to 75 bar over an additional period of 15 minutes without the pump running. Thereafter, the contents of the separation vessel are emptied.

Emptying the enriched krill oil content of the extraction vessel from the bottom of the vessel well below the raffinate surface level while the vessel is still at process temperature and 75 bar pressure, thereby avoiding high pressure CO being discharged with the raffinate oil₂Is lost. The recovery of the enriched oil takes about one hour and during this period the pressure of the vessel is reduced from 75 bar to 54 bar, since the remaining CO in the extraction vessel₂Expanded to occupy the space previously occupied by the raffinate oil.

After the discharge of the enriched oil has ended, it was observed that some enriched krill oil remained on the distributor and bottom surfaces of the vessel in the extraction vessel, which was estimated to be less than 2% of the total mass of the enriched oil. On a commercial scale, any such remaining oil is recycled to subsequent krill oil batches for use in the extractor.

The enriched oil was heated in an oven at 55 ℃ for 1 hour prior to astaxanthin and phospholipid analysis. This allows the sample to be sufficiently fluid for agitation to achieve a homogeneous sample for analysis.

Tables 1-2 summarize the quality of the feed, extracted oil and enriched oil as well as the Phospholipid (PL) and astaxanthin (Asta) contents. Very little phospholipids (<1g/100g extract) and astaxanthin (<2mg/100g extract) were co-extracted. Overall, the mass of the extract obtained from the enrichment process was 98% of the theoretical extract required for enrichment to 62% phospholipids.

Tables 1 to 2: summary of Phospholipid (PL) and astaxanthin (Asta) contents

Tables 1-3 and tables 1-4 summarize the compositional content of the enriched oil.

Tables 1 to 3: PL in krill oil samples³¹P NMR analysis

Phospholipid (PL)		Wt% of total PL	g/100g sample
				Phosphatidylcholine	PC	72.9	45.2
Alkyl acyl phosphatidyl choline	AAPC	10.0	6.2
				Phosphatidylinositol	PI	1.3	0.8
Phosphatidylserine	PS	0.7	0.4
				Lysophosphatidylcholine	LPC	7.6	4.7
Lysoalkylacylphosphatidylcholine	LAAPC	0.6	0.4
				Phosphatidylethanolamine	PE	1.1	0.7
Alkylacylphosphatidylethanolamine	AAPE	1.3	0.8
				Cardiolipin + N-acylphosphatidylethanolamine	CL/NAPE	3.8	2.4
Lysophosphatidylethanolamine	LPE	0.5	0.3
				Lysoalkylacylphosphatidylethanolamine	LAAPE	0.2	0.1
Total PL content			62.0

Tables 1 to 4: GC analysis of fatty acids

Example 2-

Mussel lipid extract is prepared according to WO97/09992 and prepared asThe form of (Pharmalink International Limited, hong Kong) was used. Krill oil was prepared by the process of example 1 with the compositional content as set out in tables 1-3 and tables 1-4 above.

Sample preparation

Fresh samples were prepared daily. The samples were mixed by inversion prior to sampling. The sample was weighed into a 1.5mL centrifuge tube and made up to 100mg/mL with ethanol to prepare a stock solution (stock). Stock mixtures were prepared by weighing out the oil in the correct ratio and then making up to concentration with ethanol. Serial dilutions of the stock solutions in ethanol (serialdilution) were prepared. Serial dilutions were then diluted in cell culture medium (1/100) before addition to cells with final 1/10 dilutions (in triplicate). This resulted in a 0.1% ethanol concentration for all doses and controls. Krill oil contains about 62% w/w phospholipids. Mussel lipid extract of musselThe form of (1) is used.

The abbreviations used in presenting the results are set forth in Table 2-1 below:

table 2-1: abbreviations

And (3) determination:

anti-inflammatory activity was determined in Lipopolysaccharide (LPS) and interferon gamma (IFN γ) stimulated murine macrophage RAW264.7 cells, which were cultured in standard cell culture medium and incubated with LPS and IFN γ in the presence or absence of different test compounds/extracts and positive controls. Including NO, PGE₂And LTB₄The production of inflammatory mediators of the cytokines TNF α and IL-6 was measured by established methods using commercial kits. Using relevant internal controls, each sample was tested with at least 3 concentrations (using 3 replicates, the maximum concentration was 100 μ g/ml) (n-9) (table 2-2). The cytotoxicity of each sample was also determined by MTT assay. No cytotoxicity was detected for any of the concentrations tested.

The measurement parameters for each measurement are summarized in Table 2-2. Briefly, for anti-inflammatory assays, cultured RAW264.7 cells were counted and plated in 96-well plates (0.8 × 10)⁵Cells/well) and incubated for the specified plating time. The medium was then aspirated and replaced with fresh medium, followed by addition of the test compound. Compounds were incubated for 1h before addition of the stimulus (stimulant). The plates were then incubated for between 4-18h and the supernatants were analyzed for the medium of interest, and the remaining cell viability was determined by MTT.

Positive controls were selected based on their broad use in similar assays, including N- (3- (aminomethyl) benzyl) acetamidine (1400W), a slow, tight binding inhibitor of Inducible Nitric Oxide Synthase (iNOS) (Garvey, e.p. et al, JBiol Chem,1997,21:272(8): 4959-63); and dexamethasone, a commonly used cytokine inhibitor. Diclofenac is a common non-steroidal anti-inflammatory agent and is known as an inhibitor of Cyclooxygenase (COX) enzyme that produces PGE 2.

Tables 2 to 2: measuring parameters

Results

1. Nitric oxide assay

NO is a free radical metabolite that has been shown to have many physiological functions, both as a signaling molecule and as a toxic agent in inflammation (Coleman, 2001). NO is derived from the oxidation of L-arginine by three types of Nitric Oxide Synthases (NOs); three types of nitric oxide synthases are the constitutive forms originally described in murine macrophages, neuronal and endothelial NOS, and the inducible form iNOS (Nathan & Xie, 1994; Stuehr & Marletta, 1985). The inducible form is continuously activated upon expression and is therefore regulated at the transcriptional level by NF-. kappa.B stimulated by inflammatory molecules such as LPS and IFN γ. Production of NO by iNOS experiences a lag time of several hours, after which NO is produced at much higher (nM) sustained levels (Nathan & Xie, 1994). Inducible forms of NOS are likely to be involved in inflammation and are more easily assessed in vitro due to the higher levels of NO produced.

NO is an unusual signaling molecule. Since there is NO specific cell surface receptor for NO, NO enters the cell indiscriminately, with the effect depending on cell type and NO concentration, thus producing a wide range of physiological responses. NO causes increased vascular permeability, vasodilation and free radical production, which causes tissue damage and elimination of pathogens (Guzik, korbout, & Adamek-Guzik, 2003). These physiological changes are associated with inflammation with increased blood flow, allowing more immune cells to enter the affected tissue, thereby destroying the pathogen.

The results of the NO inhibition assay are depicted in fig. 1A and 1B and tables 2-3.

Tables 2 to 3: NO inhibited IC₅₀

LY

Krill

90％LY

75％LY

Olive oil

1400W

IC50

～107

～286

44.8

40.9

Is inactive

1.6

2. Tumor necrosis factor-alpha

TNF α is a cell signaling protein (cytokine) primarily involved in the acute phase inflammatory response. Macrophages are the major source of TNF α, although TNF α can be released by many other cell types such as CD4+ lymphocytes, Natural Killer (NK) cells, neutrophils, mast cells, eosinophils, and neurons. TNF α is produced by the activation of MAPK and NF- κ B. It acts to increase its own production and the production of other inflammatory cytokines such as interleukin-1 beta (IL-1 beta). TNF α induces fever, apoptotic cell death, cachexia, inflammation, and inhibits tumorigenesis and viral replication. TNF α is implicated in a number of disease states including sepsis, traumatic injury, ischemia, asthma, burns, irritable bowel syndrome, alzheimer's disease, cancer, major depressive disorder, arthritis and multiple sclerosis (Cairns, Panacek, Harken, & Banerjee, 2000; Dowlati et al, 2010; Swardfager et al, 2010).

The results of the TNF α inhibition assay are depicted in fig. 2A and 2B and tables 2-4.

Tables 2 to 4: IC for TNF alpha inhibition₅₀

LY

Krill

90％LY

75％LY

Olive oil

Dexa

IC50

～238.7

～103.3

89

57

DNF*

3.328e-005

Mathematical model without fitting dose response curves

3. Interleukin-6

Similar to TNF α, IL-6 is thought to be a pro-inflammatory cytokine. IL-6 is secreted by T cells and macrophages that stimulate an immune response. IL-6 causes increased production of neutrophils in the bone marrow. It supports the growth of B cells and antagonizes T cell differentiation into regulatory T cells. It is able to cross the blood brain barrier and initiate PGE in the hypothalamus₂Thereby changing the body's temperature set point (Banks, Kastin,&Gutierrez,1994)。

the results of the IL-6 inhibition assay are depicted in FIG. 3A and FIG. 3B and tables 2-5.

Tables 2 to 5: IL-6 inhibited IC₅₀

LY

Krill

90％LY

75％LY

Olive oil

Dexa

IC50

22.4

13.4

11.5

10.4

～106.8

1.19

4. Prostaglandin E₂

Prostaglandin E2 (PGE)₂) Is one of the lipid mediators produced from Arachidonic Acid (AA) by the action of the enzyme Cyclooxygenase (COX) and is associated with the induction of fever, pain sensation and inflammation. Aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandins, including PGE₂) The biosynthesis of (a) produces antipyretic, analgesic and anti-inflammatory effects (Kawahara, k. et al 2015 and Kawabata a, 2011).

The results are depicted in fig. 4A and 4B and tables 2-6.

Tables 2 to 6: PG-E₂Inhibited IC₅₀

LY

Krill

90％LY

75％Ly

50％LY

Diclofenac acid

Olive oil

Is inactive

Is inactive

～112

～118

55.3

0.12

DNF*

Mathematical model without fitting dose response curvesSummary of the results

In this assay system, mussel lipid extract and krill oil were demonstrated to inhibit NO, TNF α and IL-6 alone, but not PGE2 at the concentrations tested.

The combination of mussel lipid extract and krill oil is more effective in inhibiting NO, TNF α and IL-6 than either mussel lipid extract or krill oil alone. In PGE₂Neither mussel lipid extract nor krill oil exhibited inhibitory activity in the assay alone, but in the combination.

Example 3: synergistic effect

Mussel lipid extract is prepared according to WO97/09992 and prepared asThe form of (1) is used. Krill oil was prepared by the process of example 1 with compositional content as set out in tables 1-3 and tables 1-4 above.

Samples, formulations and combinations

Before sampling the experiment, theAnd stock samples of krill oil high in phospholipids were mixed by inversion. The samples were weighed into 15mL centrifuge tubes and made up to 100mg/mL with ethanol to prepare stock solutions. The mixture is prepared by mixing the diluted oils in the correct ratio. Serial dilutions were prepared in ethanol. Then the series isDilutions were diluted in cell culture media (1/100) before addition to cells with final 1/10 dilutions (in triplicate). This resulted in a 0.1% ethanol concentration for all doses and controls. These doses are prepared fresh daily. Table 3-1 shows the abbreviations used for each sample.

Table 3-1: sample name abbreviation

Measurement of

Anti-inflammatory activity was determined in Lipopolysaccharide (LPS) and interferon gamma (IFN γ) stimulated murine macrophage RAW264.7 cells, RAW264.7 cells cultured in standard cell culture medium (DMEM, fetal bovine serum 5%) and incubated in the presence or absence of different test compounds/extracts and controls. Production of inflammatory mediators including NO, the cytokines TNF α and IL-6 was measured by established methods using a commercial ELISA kit (suppliers listed in tables 3-2). Using relevant internal controls, each sample was tested with at least 6 concentrations (using 3 replicates, the maximum concentration was 100 μ g/ml) (n-9) (shown in table 3-2). The cytotoxicity of each sample tested was determined by MTT assay. No cytotoxicity was detected for any of the concentrations tested.

The assay parameters used for each assay are summarized in Table 3-2. Briefly, for the NO assay, TNF α assay and IL-6 assay, cultured RAW264.7 cells were counted and plated in 96-well plates (0.8 × 10)⁵Cells/well) and incubation continued for 48 h. The medium was then aspirated and replaced with fresh medium, followed by addition of the test compound. Compounds were incubated for 1h before addition of the stimulus. The plates were then incubated for 18h and the supernatants were analyzed for the medium of interest, and the remaining cell viability was determined by MTT.

Positive controls were selected based on their broad use in similar assays, including N- (3- (aminomethyl) benzyl) acetamidine (1400W), a slow, tight-binding inhibitor of Inducible Nitric Oxide Synthase (iNOS) (Garvey et al, 1997); and dexamethasone, a commonly used cytokine inhibitor.

Tables 3-2: anti-inflammatory assay and positive control

Synergy computing

The synergy was expressed as a Combination Index (Combination Index). Synergy Combination Index (CI) and equivalent stress IC50 weights were calculated using the Compsyn software. The dose response curve generated in Graphpad Prism was transformed into 10 points representing the curve. These 10 points are then input into the Compsyn program, which generates a curve that fits the data points. This approach is preferred to closely replicate the complete dose response curve in the synergy program. The Compsyn fit curve was then used for synergy calculations. The pattern of synergy within the range tested can be observed with an equivalent plot plotting the relative contribution to activity of each component at IC 50. There is a straight line drawn between the two drugs being blended (Biavatti, 2009). Values below the line indicate synergy, values above the line are considered additive, and values above the line are antagonistic.

Results

1. Nitric oxide assay

The combination is tested to determine whether the combination exhibits synergistic inhibition of the inflammatory signaling molecule NO. The combinations were first tested in 10% increments. The most active combination observed was LY 60. An additional 5% increment around LY60 was then tested.

Dose response curves for the tested combinations LY90-LY10 are depicted in fig. 5A, fig. 5B, fig. 6A and fig. 6B. IC50 values and combination indices for NO inhibition are presented in tables 3-3 and 3-4. A combination index of less than 1 indicates synergy. The iso-plot for the 10% increments is depicted in fig. 7.

Tables 3 to 3: IC for NO inhibition of combination LY90-LY10₅₀Value of

Tables 3 to 4: IC for NO inhibition of combinations LY75 to LY45₅₀(CI) and combination index

TNF alpha assay

The combination was tested to determine if the combination exhibited synergistic inhibition of the inflammatory cytokine TNF α. The combinations were first tested in 10% increments. The most active combination observed was LY 50. An additional 5% increment around LY50 was then tested.

Dose response curves for the tested combinations LY90-LY10 are depicted in fig. 8A, fig. 8B, fig. 8C, fig. 9A and fig. 9B. IC50 values and combination indices for TNF α inhibition are presented in tables 3-5 and tables 3-6. A combination index of less than 1 indicates synergy. The iso-plot for the 10% increments is depicted in fig. 10.

Tables 3 to 5: IC for TNF alpha inhibition₅₀Value of

No possible estimate

Tables 3 to 6: IC for LY70-LY35₅₀And CI (n is 9)

IL-6 assay

The combination was tested to determine if the combination exhibited synergistic inhibition of the inflammatory cytokine IL-6. The combinations were first tested in 10% increments. The most active combination observed was LY 60. An additional 5% increment around LY60 was then tested.

Dose response curves for the tested combinations LY90-LY10 are depicted in fig. 11A, fig. 11B, fig. 12A and fig. 12B. IC50 values and combination indices for IL-6 inhibition are presented in tables 3-7 and tables 3-8. A combination index of less than 1 indicates synergy. The iso-plot for the 10% increments is depicted in fig. 13.

Tables 3 to 7: IC for IL-6 inhibition₅₀(n＝3)

Tables 3 to 8: IC for IL-6 inhibition₅₀And combined index

Summary of the results

Mussel lipid extract and krill oil used in combination in this assay system were shown to meet the mathematical criteria for synergy in terms of inhibition of NO, TNF α and IL-6.

Example 4: study of patients

A combination of mussel lipid extract (in the form of PCSO-542) and krill oil (61% PL) in capsule form at a ratio of PCSO-542 to krill oil of 75:25 is administered to patients suffering from various pain/inflammation conditions. The composition of the capsules is presented in table 4-1.

Table 4-1: composition of 150mg oil blend capsule

Comprises 0.15% w/w vitamin E

(i.e., about 0.056 mg/capsule)

Within a single dose, two doses or three doses, the dose will generally range from 2-8 capsules per day. Prior to starting treatment with this combination, patients have typically taken one or more NSAIDs including paracetamol or ibuprofen to manage pain. The results are depicted in Table 4-2.

Tables 4-2: summary of patient results

Reference to the literature

Biavatti，M.W.(2009).Synergy：an old wisdom，a new paradigm for pharmacotherapy.Brazilian Journal of Pharmaceutical Sciences，45(3)，371-378.

Cairns，C.B.，Panacek，E.A.，Harken，A.H.，&Banerjee，A.(2000).Bench to bedside：Tumor necrosis factor-alpha：From inflammation toresuscitation.Academic Emergency Medicine，7(8)，930-941.

Coleman，J.W.(2001).Nitric oxide in immunity and inflammation.Int Immunopharmacol，1(8)，1397-1406.

Dowlati，Y.，Herrmann，N.，Swardfager，W.，Liu，H.，Sham，L.，Reim，E.K.，&Lanctot，K.L.(2010).A Meta-Analysis of Cytokines in MajorDepression.Biological Psychiatry，67(5)，446-457.doi：10.1016/j.biopsych.2009.09.033

Garvey，E.P.，Oplinger，J.A.，Furfine，E.S.，Kiff，R.J.，Laszlo，F.，Whittle，B.J.R.，&Knowles，R.G.(1997).1400W is a slow，tight binding，and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo.Journal ofBiological Chemistry，272(8)，4959-4963.

Guzik，T.J.，Korbut，R.，&Adamek-Guzik，T.(2003).Nitric oxide and superoxide in inflammation and immune regulation.J Physiol Pharmacol，54(4)，469-487.

Kawabata，A.，(2011).Prostaglandin E₂ and Pain-An Update.Biol Pharm Bull，34(8)，1170-1173

Kawahara，K.(2015).Prostaglandin E₂-induced inflammation：Relevance of prostaglandin E receptors.Biochim Biophys Acta，1851(4)，414-421

Nathan，C.，&Xie，Q.W.(1994).Nitric oxide synthases：roles，tolls，and controls.Cell，78(6)，915-918.

Sinclair，A.J.，Murphy，K.J.and Li，D.(2000)Marine lipids overview″news insights and lipid composition of Lyprinol″.Allerg Immunol(Paris)32(7)，261-271

Stuehr，D.J.，&Marletta，M.A.(1985).Mammalian nitrate biosynthesis：mouse macrophages produce nitrite and nitrate in response to Escherichia colilipopolysaccharide.Proc Natl Acad Sci USA，82(22)，7738-7742.

Swardfager，W.，Lanctot，K.，Rothenburg，L.，Wong，A.，Cappell，J.，&Herrmann，N.(2010).A Meta-Analysis of Cytokines in Alzheimer′s Disease.BiologicalPsychiatry，68(10)，930-941.

Claims (58)

1. A combination comprising mussel lipid and krill oil, wherein the combination is suitable for administration separately or sequentially.

2. The combination of claim 1, in the form of a composition comprising mussel lipid and krill oil.

3. The combination according to claim 1 or 2, wherein the krill oil has a phospholipid content of at least about 40% w/w.

4. The combination according to claim 3, wherein the krill oil has a phospholipid content of at least about 60% w/w.

5. The combination according to any one of claims 1-4, wherein the krill oil has a water content of about 5% w/w or less.

6. The combination according to claim 5, wherein the krill oil has a water content of about 3% w/w or less.

7. The combination according to claim 5, wherein the krill oil has a water content of about 1% w/w or less.

8. The combination according to any one of claims 1-7, wherein the krill oil has an extraction solvent content of about 5% w/w or less.

9. The combination according to claim 8, wherein the krill oil has an extraction solvent content of about 3% w/w or less.

10. The combination according to claim 8, wherein the krill oil has an extraction solvent content of about 1% w/w or less.

11. The combination of any one of claims 1-10, wherein the mussel lipid is in the form of a mussel powder.

12. The combination of any one of claims 1-10, wherein the mussel lipid is in the form of a mussel lipid extract, optionally containing vitamin E.

13. The combination of any one of claims 1-12, wherein the weight ratio of mussel lipid to krill oil is in the range of 1:99 to 99: 1.

14. The combination of claim 13, wherein the weight ratio of mussel lipid to krill oil is about 5:95, or about 10:90, or about 15:85, or about 20:80, or about 25:75, or about 30:70, or about 35:65, or about 40:60, or about 45:55, or about 50:50, or about 55:45, or about 60:40, or about 65:35, or about 70:30, or about 75:25, or about 80:20, or about 85:15, or about 90:10, or about 95: 5.

15. The combination according to any one of claims 1-14, which is in an oral unit dosage form.

16. The combination of claim 15, wherein the oral unit dosage form is a soft gel capsule.

17. The combination of claim 15 or 16, wherein the oral unit dosage form comprises from about 10mg to about 10g of mussel lipid.

18. The combination of claim 17, wherein the oral unit dosage form comprises a lipid in an amount of about 10mg, 20mg, 30mg, 40mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g, 2.1g, 2.2g, 2.3g, 2.4g, 2.5g, 2.6g, 2.7g, 2.8g, 2.9g, 3.0g, 3.2g, 3.5g, 3.7g, 4.0g, 4.5g, 5.0g, 5.5g, 6.7 g, 0.9 g, 5.9 g, 5.0g, 5.5g, 0g, 5.5.5 g, 0g, 5.8 g, 5.9 g, 5g, 5.0g, 5.8 g, 5.

19. The combination according to any one of claims 15-18, wherein the oral unit dosage form comprises from about 10mg to about 10g krill oil.

20. The combination according to claim 19, wherein the oral unit dosage form comprises about 10mg, 20mg, 30mg, 40mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g, 2.1g, 2.2g, 2.3g, 2.4g, 2.5g, 2.6g, 2.7g, 2.8g, 2.9g, 3.0g, 3.2g, 3.5g, 3.7g, 4.0g, 4.5g, 5.0g, 6.7 g, 0.9 g, 5.9 g, 5.0g, 5g, 0.9.9 g, 0g, 5.0g, 5g, 8g or about 5g krill.

21. The combination according to any one of claims 15-20, wherein the oral unit dosage form comprises about 10-500mg of the combination.

22. The combination of claim 21, wherein the oral unit dosage form comprises about 50-300mg of the combination.

23. The combination according to any one of claims 1-22, further comprising one or more pharmaceutically acceptable carriers and/or additives.

24. The combination of any one of claims 1-22, consisting of or consisting essentially of mussel lipid and krill oil.

25. A composition comprising mussel lipid and krill oil.

26. The composition according to claim 25, wherein the krill oil has a phospholipid content of at least about 50% w/w.

27. The composition of claim 25 or 26, wherein the mussel lipid is in the form of a mussel lipid extract, optionally containing vitamin E.

28. The composition of any one of claims 25 to 27, wherein the weight ratio of mussel lipid to krill oil is about 5:95, or about 10:90, or about 15:85, or about 20:80, or about 25:75, or about 30:70, or about 35:65, or about 40:60, or about 45:55, or about 50:50, or about 55:45, or about 60:40, or about 65:35, or about 70:30, or about 75:25, or about 80:20, or about 85:15, or about 90:10, or about 95: 5.

29. The composition of any one of claims 25-28, further comprising a carrier oil.

30. The composition of claim 29, wherein the carrier oil comprises from about 10% w/w to about 90% w/w of the total composition.

31. The composition of claim 29, wherein the weight ratio of carrier oil to the combined amount of mussel lipid and krill oil is from about 3:1 to about 1: 3.

32. The composition of any one of claims 25-31, in unit dosage form.

33. The composition of claim 32, wherein the composition is encapsulated in a soft gel capsule.

34. The composition of claim 32 or 33, comprising about 10-500mg of mussel lipid and krill oil combined.

35. The composition of claim 34, comprising about 50-300mg of mussel lipid and krill oil combined.

36. The combination according to any one of claims 1-24 or the composition according to any one of claims 25-35, for use in treating inflammation in a subject.

37. A method of treating inflammation in a subject in need thereof, the method comprising administering to the subject a combination according to any one of claims 1-24 or a composition according to any one of claims 25-35.

38. Use of mussel lipid and krill oil in the manufacture of a combination medicament for the treatment of inflammation.

39. The use of claim 38, wherein the medicaments are suitable for separate administration or simultaneous administration.

40. The use of claim 39, wherein the medicament is in the form of a composition of mussel lipid and krill oil.

41. A combination for use in the treatment of inflammation, the combination comprising mussel lipid and krill oil.

42. The agent of claim 41, adapted for separate administration or simultaneous administration.

43. The combination according to any one of claims 1-24 or the composition according to any one of claims 25-35, for use in the treatment of pain in a subject.

44. A method of treating pain in a subject in need thereof, the method comprising administering to the subject the combination according to any one of claims 1-24 or the composition according to any one of claims 25-35.

45. Use of mussel lipid and krill oil for the manufacture of a combination medicament for the treatment of pain.

46. The use according to claim 45, wherein the medicaments are suitable for separate administration or simultaneous administration.

47. The use of claim 46, wherein the medicament is in the form of a composition of mussel lipid and krill oil.

48. A combination for use in the treatment of inflammation, the combination comprising mussel lipid and krill oil.

49. The agent of claim 48, adapted for separate administration or simultaneous administration.

50. A process for preparing krill oil having a phospholipid content of about 50% or greater, comprising the steps of:

(a) mixing krill biomass feed material with CO₂And BContacting the mixture of alcohols to extract krill oil; and

(b) mixing said krill oil with CO₂Contacting to extract at least a proportion of the non-polar lipid component such that the oil has a phospholipid content of at least 50% w/w.

51. The process of claim 50, wherein the krill biomass feed material is contacted with CO₂From about 15% w/w to about 30% w/w ethanol.

52. The process of claim 50 or 51, wherein step (a) is carried out at a temperature of about 60 ° or less.

53. The process of any one of claims 50-52, wherein step (a) is carried out at a pressure of about 300 bar or greater than about 300 bar.

54. The process of any one of claims 50-53, wherein step (b) is performed at a temperature of about 60 ° or less.

55. The process of any one of claims 50-54, wherein step (a) is carried out at a pressure of about 300 bar or greater than about 300 bar.

56. The process of any one of claims 50-55, wherein the oil obtained from step (b) has a phospholipid content in the range of about 60% w/w to about 90% w/w.

57. The process of any one of claims 50-56, wherein ethanol is removed from the extracted oil obtained in step (a).

58. The process of claim 57, wherein the ethanol is removed under vacuum at a temperature of about 60 ℃ or less.