Lipid combination

Abstract

The present disclosure discloses lipid combinations. Disclosed are combinations or compositions of mussel lipids and krill oil for use in the treatment of inflammation or pain. A process for preparing krill oil having a phospholipid content of about 50% or greater is also disclosed.

Description

[0001] This application is a divisional application of the application filed on December 21, 2018, with application number 201880014073.2 and invention title "Lipid Combination". field

[0002] This disclosure generally relates to combinations of marine lipids. In particular, this disclosure relates to combinations of lipids obtained from New Zealand green-lipped mussels (Perna canaliculus) and krill, compositions and formulations comprising said combinations, and the use of said combinations and compositions in therapeutic applications. This disclosure also relates to processes for manufacturing krill oil and the use of krill oil in combinations and compositions. background

[0003] References to any prior publications (or information derived therefrom) or to any known substance in this specification are not and should not be construed as an endorsement or acknowledgment or in any way imply that such prior publications (or information derived therefrom) or known substances constitute part of the general knowledge in the field to which this specification is addressed.

[0004] Inflammation is an essential physiological adaptive response to injury and infection; without it, humans and animals cannot survive. Its function is to eliminate the initial cause of injury, remove offending factors, and initiate the repair of tissue structure and function. The early acute phase of inflammation is typically characterized by fever, pain, redness, and swelling. The usual outcome of acute inflammation is the recovery and repair of the injury; however, an uneven acute inflammatory response and a prolonged chronic inflammatory response can be detrimental in conditions such as sepsis.

[0005] Importantly, chronic inflammation is now involved in the pathology of many diseases that affect all tissues and organs, including osteoarthritis, rheumatoid arthritis, cardiovascular disease, cerebrovascular disease, respiratory disease, autoimmune disease, and sarcopenia; in fact, chronic inflammation is involved in the process of aging itself.

[0006] In response to the inflammatory process, a range of drugs have been developed, the most effective being glucocorticoid steroids, which suppress excessive inflammation. However, due to their significant side effects, steroid therapy is limited in its wide clinical use and is generally restricted to short-term use only. A second class of inflammatory drugs, known as nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, has been 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 steroids and can be used for more chronic inflammatory conditions, such as osteoarthritis.

[0007] Table 1: List of commonly used nonsteroidal anti-inflammatory drugs (NSAIDs)

[0008] Prostaglandins play a crucial role in the inflammatory response. A significant increase in the presence of prostaglandins in inflamed tissue leads to pain and fever by raising temperature and dilating blood vessels, causing redness and swelling at the sites where prostaglandins are released. NSAIDs are competitive site inhibitors of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), thereby reducing prostaglandin synthesis. By reducing prostaglandin production, NSAIDs help relieve the discomfort of fever and reduce inflammation and associated pain. NSAIDs are commonly used to treat acute and chronic conditions characterized by pain and inflammation, such as osteoarthritis, rheumatoid arthritis, headaches and migraines, and fever. However, considerable concerns remain regarding the safety of NSAIDs, which can exhibit gastrointestinal, renal, and cardiovascular toxicity. For example, aspirin can cause gastrointestinal bleeding within days of use, and in July 2015, the FDA reiterated previous warnings about cardiac hazards associated with common NSAID painkillers, excluding aspirin. These pain relievers include ibuprofen (Advil, Motrin) and naproxen (Aleve), as well as prescription-only NSAIDs.

[0009] Therefore, there is a need for alternative anti-inflammatory treatments. Overview

[0010] It has now been found that certain combinations of mussel lipids and krill oil can advantageously provide additive or synergistic inhibition of the production or release of one or more pro-inflammatory mediators involved in the inflammatory process. In some embodiments, combinations of the present disclosure can therefore provide novel therapeutic treatments for disorders characterized by inflammation or having inflammatory components. In some embodiments, combinations of the present disclosure can provide novel therapeutic treatments for pain, such as inflammation-related pain.

[0011] Therefore, in one aspect, a combination comprising mussel lipids and krill oil is provided. The combination of mussel lipids and krill oil can be suitable for administration to a subject, either alone or simultaneously. In some embodiments, the combination is a composition comprising mussel lipids and krill oil.

[0012] In another aspect, a combination consisting of mussel lipids and krill oil, or essentially consisting of mussel lipids and krill oil, is provided. This combination of mussel lipids and krill oil can be suitable for administration to a subject, either alone or simultaneously. In some embodiments, the combination is a composition consisting of mussel lipids and krill oil, or essentially consisting of mussel lipids and krill oil.

[0013] In another embodiment, a combination comprising mussel lipids and krill oil is provided for use in treatment. In some embodiments, a combination comprising mussel lipids and krill oil is provided for use in treating inflammation in a subject. A combination comprising mussel lipids and krill oil is also provided for use in treating pain in a subject. The combination of mussel lipids and krill oil may be suitable for administration alone or simultaneously to a subject. In some embodiments, the combination is a composition comprising mussel lipids and krill oil.

[0014] In another aspect, this disclosure provides methods for treating inflammation in a subject in need, including administering to the subject a combination comprising mussel lipids and krill oil. This disclosure also provides methods for treating pain in a subject in need, including administering to the subject a combination comprising mussel lipids and krill oil. The combination of mussel lipids and krill oil may be suitable for administration to the subject alone or simultaneously. In some embodiments, the combination is a composition comprising mussel lipids and krill oil.

[0015] In another aspect, this disclosure provides the use of mussel lipids and krill oil in the manufacture of a combination medicament for treating inflammation. This disclosure also provides the use of mussel lipids and krill oil in the manufacture of a combination medicament for treating pain. The medicament may be suitable for administration alone or in combination. In some embodiments, the combination is a composition comprising mussel lipids and krill oil.

[0016] In another aspect, this disclosure provides a combination for treating inflammation, the combination comprising mussel lipids and krill oil. This disclosure also provides a combination for treating pain, the combination comprising mussel lipids and krill oil. The mussel lipids and krill oil may be suitable for administration alone or simultaneously to a subject. In some embodiments, the combination is a composition comprising mussel lipids and krill oil.

[0017] In some embodiments, mussel lipids are in the form of dried mussel powder. In other embodiments, mussel lipids are in the form of a lipid extract obtained from mussels (“mussel lipid extract”). In still other embodiments, mussel lipids may be in the form of a combination or composition of dried mussel powder and mussel lipid extract.

[0018] In some embodiments, the krill oil has a phospholipid content in the range of about 40-99% w / w, and in other embodiments, it has a phospholipid content in the range of about 50-99% w / w, for example, about 60-80% w / w.

[0019] In some embodiments, combinations of the present disclosure may be useful in treating one or more disorders in a subject, said disorders having an inflammatory component, and whereby inhibition of one or more pro-inflammatory molecules is therapeutically beneficial. In some embodiments, the combinations may be applicable for treating one or more chronic disorders.

[0020] In some implementations, such as for use in the treatment of chronic inflammation, the combination may eliminate, avoid, or otherwise mitigate the extent, severity, or duration of one or more side effects associated with normally available NSAID classes.

[0021] In another aspect, a process is provided for preparing krill oil having a phospholipid content of about 50% or more, for example, about 60% or more, comprising the following steps: (a) Contacting krill biomass feedstock with a mixture of CO2 and ethanol to extract krill oil; and (b) The krill oil is contacted with CO2 to extract at least a certain proportion of nonpolar lipid components, such that the oil has a phospholipid content of at least 50% w / w.

[0022] In some embodiments, the krill oil obtained by this process has a phospholipid content of about 60% w / w or greater, for example, a phospholipid content of at least about 65% w / w, or 70% w / w, or 80% w / w, or 90% w / w.

[0023] This disclosure also relates to a process for enriching the phospholipid content of krill oil.

[0024] Therefore, in another aspect, a process is provided for increasing the phospholipid content of krill oil having a phospholipid content of less than 50% w / w to about 50% w / w or greater, said process comprising the step of contacting the krill oil having a phospholipid content of less than 50% w / w with CO2 to selectively remove nonpolar lipid components.

[0025] In some embodiments, the primary krill oil has a phospholipid content of less than about 50% w / w, for example less than about 40% w / w, less than about 30% w / w, or 20% w / w. In some embodiments, the enriched oil thus 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.

[0026] Another embodiment provides krill oil with a phospholipid content of about 50% w / w or greater (e.g., ≥ 60% w / w) obtained by the process of this disclosure.

[0027] Another embodiment provides krill oil having a phospholipid content of about 50% w / w or 60% w / w or greater for use in the combinations and compositions described herein. Attached Figure

[0028] Figure 1A The diagram illustrates the NO-inhibitory effects of various concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, olive oil, and N-(3-(aminomethyl)benzyl)acetamidine (1400W) in lipopolysaccharide (LPS) and interferon-γ (IFγ) stimulated RAW264.7 cells.

[0029] Figure 1B The diagram illustrates the NO release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, olive oil, and N-(3-(aminomethyl)benzyl)acetamidine (1400W).

[0030] Figure 2A The diagram illustrates the inhibitory effects of various concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, olive oil, and dexamethasone on TNFα inhibition in lipopolysaccharide (LPS) and interferon-γ (IFγ) stimulated RAW264.7 cells.

[0031] Figure 2B The diagram depicts various combinations of concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, and TNFα release (%) of olive oil in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ).

[0032] Figure 3A The diagram illustrates the IL-6 inhibitory effects of various concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, olive oil, and dexamethasone in lipopolysaccharide (LPS) and interferon-γ (IFγ) stimulated RAW264.7 cells.

[0033] Figure 3BThe diagram depicts various combinations of concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, and the IL-6 release (%) of olive oil in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ).

[0034] Figure 4A The diagram illustrates the PGE2 inhibitory effects of various concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, olive oil, and diclofenac in lipopolysaccharide (LPS) and interferon-γ (IFγ) stimulated RAW264.7 cells.

[0035] Figure 4B The diagram depicts various combinations of concentrations of mussel lipid extract and krill oil, each of the mussel lipid extract and krill oil alone, and the PGE2 release (%) of olive oil in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ).

[0036] Figure 5A The diagram illustrates the NO release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) for various concentrations of mussel lipid extract and krill oil (LY90-LY50).

[0037] Figure 5B The diagram illustrates the NO release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) for various concentrations of mussel lipid extract and krill oil (LY50-LY10).

[0038] Figure 6A The diagram illustrates the NO release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) for various concentrations of mussel lipid extract and krill oil (LY75-LY60).

[0039] Figure 6B The diagram illustrates the NO release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) for various concentrations of mussel lipid extract and krill oil (LY60–LY45).

[0040] Figure 7 The diagram depicts the isobologram of synergistic NO inhibition by various combinations of mussel lipid extract and krill oil (LY90-LY10).

[0041] Figure 8AThe diagram illustrates the TNFα release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY90-LY60).

[0042] Figure 8B The diagram illustrates the TNFα release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY60-LY30).

[0043] Figure 8C The diagram illustrates the TNFα release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY40-LY10).

[0044] Figure 9A The diagram illustrates the TNFα release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY70–LY55).

[0045] Figure 9B The diagram illustrates the TNFα release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY50-LY35).

[0046] Figure 10 The diagram illustrates the isotropic effects of synergistic TNFα inhibition by various combinations of mussel lipid extract and krill oil (LY90-LY10).

[0047] Figure 11A The diagram illustrates the IL-6 release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY90-LY50).

[0048] Figure 11B The diagram illustrates the IL-6 release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY50-LY10).

[0049] Figure 12AThe diagram illustrates the IL-6 release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY70-LY50).

[0050] Figure 12B The diagram illustrates the IL-6 release (%) in RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFγ) using various concentrations of mussel lipid extract and krill oil (LY50-LY30).

[0051] Figure 13 The diagram illustrates the isotropic effects of synergistic IL-6 inhibition by various combinations of mussel lipid extract and krill oil (LY90-LY10). describe

[0052] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated integer or step or group of integers, but not to exclude any other integer or step or group of integers or steps.

[0053] Throughout this specification and the appended claims, unless the context otherwise requires, the phrase "consisting essentially of" and variations such as "consistses essentially of" shall be understood to indicate that the recited elements are essential, i.e., necessary, elements of the invention. This wording allows for the presence of other unrecited elements that do not substantially affect the features of the invention, but excludes additional unspecified elements that would affect the fundamental and novel features of the defined invention.

[0054] Unless the context explicitly indicates otherwise, the singular forms “a”, “an”, and “the” include the plural aspect.

[0055] The term "invention" includes all disclosures, aspects, implementations, and embodiments as described herein.

[0056] As used herein, “about” means a quantity, value, or parameter that can vary by up to 10%, 5%, or 2%–1% of the stated quantity, value, or parameter, and includes at least the tolerances accepted in the art. When used with respect to stated integer values, “about” can include a variation of an integer on either side of the stated value; for example, “50%” can include 49% and 51%. When preceding a range of stated values, it is intended to apply to both the upper and lower limits of that range.

[0057] Unless the context otherwise indicates, the features described below can be applied independently to any aspect or implementation.

[0058] As used in this article, "mussel lipids" refers to lipids derived from New Zealand greenlip (NZGL) (or green-shelled) mussels. New Zealand Green Mouth The lipid fraction is extracted or obtained from mussel meat. Mussel lipids may contain one or more of the following: polyunsaturated long-chain fatty acids (PUFAs) such as ALA, ETA, EPA, and DHA; sterols; sterol esters; triglycerides; nonpolar lipid carotenoids; and (NZGL) other components of mussel meat. Mussel lipids may be in the form of dried mussel powder or as a lipid fraction extracted from mussel meat (“mussel lipid extract”). It is also envisioned that “mussel lipids” include a mixture of mussel powder and mussel lipid extract, for example, mussel lipids may be supplemented by adding mussel powder, or vice versa. In some embodiments, mussel lipids are isolated lipid fractions.

[0059] Mussel lipid powder can be prepared from fresh (raw), frozen, or heat-treated NZGL mussel meat by any suitable drying method (e.g., freeze-drying, rapid drying, or vacuum drying) and pulverizing method. In addition to fatty acids (including ALA, ETA, EPA, and DHA), the mussel powder obtained by drying mussel meat will also 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.

[0060] Mussel lipid extracts can be obtained from fresh (raw), frozen, heat-treated, or dried (e.g., freeze-dried, rapid-dried, or vacuum-drum-dried) NZGL mussel meat (e.g., in powder, spray-dried, or pulverized form) by any suitable method, such as solvent extraction (e.g., acetone or ethanol – see, for example, WO2005073354 A1, the contents of which are incorporated herein by reference), enzymatic treatment (see, for example, WO2006128244, the contents of which are incorporated herein by reference), or supercritical fluid extraction. In some embodiments, mussel lipid extracts are advantageously obtained by extraction with supercritical CO2 from dried (e.g., freeze-dried) mussel meat (optionally stabilized to prevent oxidation). Exemplary methods for obtaining mussel lipid extracts are described in WO 97 / 09992 A1, the contents of which are incorporated herein by reference. Other methods will be known in the art.

[0061] In a preferred embodiment, the process is carried out under conditions such as cold processing, in which beneficial components, such as fatty acids, are largely preserved and largely intact.

[0062] An exemplary mussel lipid extract obtained according to the process described in WO 97 / 09992 A1 is also known as PCSO-524. ® (Pharmalink International Limited, Hong Kong). PCSO-524 ® It contains added vitamin E (0.15% w / w, added as an antioxidant preservative) and includes a combination of free fatty acids, triglycerides, sterol esters, nonpolar lipids, and carotenoids (Sinclair, A. J. et al., 2000), and is a source of long-chain ω-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-octadecanoic acid, 5,9,12,16-nonadecanoate, 7,11,14,17-eicosapentaenoic acid, and 5,9,12,15,18-docosapentaenoic acid. PCSO-524 ® (Formulated with olive oil into a packaged oral dosage form) with Lyprinol ® and Omega XL ® (for human consumption) and Antinol ® Sold under the name of (for dogs and cats).

[0063] In some embodiments, the mussel lipid extract used in the combinations described in this disclosure is formulated with vitamin E (e.g., added in amounts 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 lipids are in the form of PCSO-254. ® The form is used, namely, a mussel lipid extract containing 0.15% w / w vitamin E. In some embodiments, the mussel lipids optionally containing vitamin E are further formulated with a carrier oil such as olive oil. Although in some embodiments the mussel lipids are mussel lipid extracts and contain added vitamin E, the addition of vitamin E is optional, and therefore, in some embodiments, the mussel lipid extract is used purely, i.e., without containing any other additional ingredients such as vitamin E.

[0064] Mussel lipids in various forms can also be purchased from commercial suppliers.

[0065] Krill oil can be prepared from any suitable krill species, including Antarctic krill ( Euphausia superba Antarctic krill, Pacific krill Euphausia pacifica (Pacific krill), Northern krill) Maganycitiphanes norvegica (Northern krill) and crystal krill) Euphausia crystallorophias (Ice krill), cold krill) Euphausia frigida ), long-faced krill ( Euphausia longirostris ), three-spined krill ( Euphausia triacantha ) and krill ( Euphausia vallentini In some preferred embodiments, krill oil is obtained from Antarctic krill.

[0066] Marine lipids contain fatty acids, particularly omega-3 fatty acids such as EPA and DHA, in both free and triglyceride forms. Similarly, krill oil is rich in omega-3 fatty acids; however, krill oil contains a significant amount of phospholipids, in which fatty acids are attached to phosphate ester head groups via the glycerol portion. It is this phospholipid-bound form of fatty acids that allows for more efficient uptake into cell membranes and thus greater bioavailability than the triglyceride form. Typical phospholipids found in krill oil can include: phosphatidylcholine, alkyl acyl phosphatidylcholine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine, lysoalkyl acyl phosphatidylcholine, phosphatidylethanolamine, alkyl acyl phosphatidylethanolamine, cardiolipin + N-acyl phosphatidylethanolamine, lysophosphatidylethanolamine, and lysoalkyl acyl phosphatidylethanolamine. Krill oil also contains a considerable amount of astaxanthin, an antioxidant, which is the reason for its red color.

[0067] 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 other 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 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 krill oil has a phospholipid content in the range of about 40-99% w / w. In some other 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 mentioned herein, "enriched" krill oil means krill oil having a phospholipid content of at least about 60% w / w. The phospholipid content can be determined by any suitable means in the art, such as... 31 P NMR analysis.

[0068] Methods 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 KrillBiomass can be extracted using solvents (e.g., alcohols, such as ethanol; ketones, such as acetone; or dimethoxyethane) and / or supercritical fluids (e.g., CO2). Some non-limiting exemplary processes for preparing krill oil are described in U.S. Patent Nos. 9,028,877, 9,375,453, 6,800,299, 8,828,447, 9,150,815, 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 is also available from commercial suppliers.

[0069] 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 other 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. Water and solvents can be removed by any suitable means, such as mild heating for a 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°C, or 50°C, or 40°C, and preferably in such a way that the integrity of the components is substantially not impaired), nitrogen flow, or lyophilization (freeze-drying).

[0070] The combination of these substances, which inhibit the inhibitory activity of one or more inflammatory mediators such as nitric oxide (NO), cytokines such as interleukins (e.g., IL-6), prostaglandins (e.g., PG-E2), and TNFα, can be useful in treating one or more disorders or symptoms in a subject, where inhibition of one or more such molecules is therapeutically beneficial. In particular, the combination of this disclosure can be useful in treating excessive acute or chronic inflammation and / or associated symptoms such as pain, fever, redness, and swelling. In some embodiments, the combination can be useful in treating inflammation in which the pathology includes inflammatory components and / or pain associated with such disorders. Some non-limiting examples of inflammatory disorders include atherosclerosis, allergies, asthma, autoimmune diseases (e.g., celiac disease, psoriasis, rheumatoid arthritis, psoriatic arthritis), fibromyalgia, gout, migraine, osteoarthritis, ulcerative colitis, cancer, impaired cognition including Alzheimer's disease, type 2 diabetes, delayed onset myalgia (DOMS), Crohn's disease, and ankylosing spondylitis. In some embodiments, 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, combinations of the present disclosure may be useful in treating disorders in which inhibition of PGE2 may be beneficial, such as rheumatoid arthritis, migraines, and pain (pain may be nociceptive (somatic or visceral) pain and / or neuropathic pain).

[0071] It should be understood that the combinations described herein can be applied alone or simultaneously. Where suitable for simultaneous application, the combination can be provided and / or applied as an intimate composition or mixture comprising both mussel lipid extract and krill oil, or as a discrete dosage form of each component. Where the mussel lipid extract and krill oil are each provided and / or applied separately, they can be applied simultaneously, sequentially, or at different times.

[0072] In another embodiment, mussel lipids and krill oil may optionally be formulated in combination with one or more pharmaceutically acceptable carriers and / or additives, or formulated alone. Some examples of suitable carriers are edible oils such as olive oil, castor oil, flaxseed oil, grapeseed oil, fish oil (e.g., tuna oil), rapeseed oil, vegetable oils, sunflower oil, chia oil, soybean oil, sesame oil, algae 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, and C), dietary minerals, amino acids, odor and taste masking agents, emulsifiers, pharmaceutically acceptable alcohols (e.g., ethanol, glycerol, propylene glycol, and polyethylene glycol) or other viscosity modifiers, surfactants (e.g., polysorbate), suspending agents, lactose, dextrose, sucrose, mannitol, sorbitol, glucose, lubricants, binders, starch, absorption enhancers, and preservatives, may also be used. Included. The carrier or additive may perform one or more functions. Mussel lipids and / or krill oil may optionally be further supplemented or combined with one or more additionally purified or partially purified components of mussel and krill oil, such as astaxanthin and its esters, fatty acids (e.g., EPA, DHA) in the form of free acids, esters, triglycerides, or phospholipids, sterols, sterol esters, amino acids, peptides and proteins, and glycosaminoglycans (e.g., chondroitin sulfate). Other anti-inflammatory foods such as whole-ground forms or extracts thereof, such as turmeric (curcumin), ginger, garlic, cloves, etc., may also optionally be incorporated.

[0073] The formulated combination can be prepared according to methods known in the art. Such methods include steps that induce a close association between mussel lipid extracts and / or krill oil and a carrier, optionally together with one or more additive components. It will be understood that any carrier or additive will be pharmaceutically acceptable.

[0074] Therefore, in some embodiments, mussel lipids 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, from about 20% w / w to about 80% w / w. In other embodiments, the carrier oil constitutes 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. In some implementations, the weight ratio of the carrier oil to the combined amount of mussel lipids 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, or about 1:1.5, or about 1:2, or about 1:2.5, or about 1:3.

[0075] While any form of administration, such as oral, parenteral, topical, transdermal, or subcutaneous, is contemplated herein, advantageously, in some embodiments, the combinations of this disclosure may be provided and / or administered in oral dosage forms. In some embodiments, the combinations may be present in bulk form, such as 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 a single dose may be measured. Measurement and / or administration may be performed by any means, such as a spoon or ladle, syringe, dropper, or measuring cup. The measured dose may be administered directly to the subject or mixed with, poured onto, or sprinkled on food or beverages.

[0076] 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, sachets, chewable tablets, and capsules (including hard gel capsules and soft gel capsules).

[0077] An example of a suitable unit oral dosage form is a capsule in the form of a hard-shell or soft-shell capsule. The shell may contain one or more of gelatin, pullulan, hydroxypropyl methylcellulose, PVA copolymer, carrageenan, or other sugar components such as starch or cellulose, or mixtures thereof, and may also contain colorants, opacifiers, plasticizers (e.g., sorbitol, xylose, maltitol, and glycerol), etc. Methods for encapsulating marine oils and lipids such as mussel oil and krill oil are known in the art. See, for example, WO2015 / 121378, the contents of which are incorporated herein by reference. In some embodiments, where krill oil is encapsulated separately, the krill oil may be encapsulated in the absence of optional additional agents such as viscosity modifiers; that is, the capsule filler consists essentially of krill oil.

[0078] In some advantageous embodiments, the combination of mussel lipids and krill oil of this disclosure is present in the form of soft gel capsules, such as soft gel capsules containing both mussel lipids and krill oil, or separate soft gel capsules wherein the mussel lipids and krill oil are encapsulated separately, optionally with a suitable carrier and / or additives. Suitable soft gel capsules can be prepared from gelatin (or alternatively, sugar sources such as pullulan and hydroxypropyl methylcellulose), optionally with one or more plasticizers such as sorbitol and glycerol (glycerin), and additives such as colorants and opacifiers. In one example, the soft gel capsule shell may contain gelatin, and one or both of sorbitol and glycerol.

[0079] Microencapsulation is a method in which tiny droplets or particles are surrounded by coating walls or embedded in a matrix to form a powder, wherein the coating or matrix forms a functional barrier that avoids or reduces the tendency for chemical reactions such as oxidation. Furthermore, it can provide potential taste or odor masking. Therefore, in some embodiments, mussel lipids and / or krill oil can be microencapsulated, alone or together, to form a powder. Commonly used microencapsulation methods include emulsification, spray drying, freeze drying, coaxial electrospraying, extrusion, coagulation, supercritical fluid technology, 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, for example, Bakry, A. M. et al.) Comprehensive Reviews in Food Science and Food SafetyReferences to, 15, 143, 2016, and WO2014 / 170464 and WO2014 / 169315, the contents of which are incorporated herein by reference. Mussel lipids and / or krill oil may be microencapsulated with one or more carriers or additives as described above. Microencapsulated mussel lipids and / or krill oil powder may be further encapsulated, for example, in hard-shell capsule unit dosage forms. Powdered microencapsulated lipids or oils may optionally be combined with one or more carriers or additives.

[0080] In some embodiments, the combination of this disclosure can be taken with food or beverage, for example by sprinkling, stirring, mixing, or affixing the combination of mussel lipid extract and krill oil to, or on, food or beverage, or by applying or incorporating the combination of mussel lipid extract and krill oil into, or on, food or beverage. Therefore, the combination can be provided in a form intended for incorporation into, or on, a beverage or food. In some embodiments, the combination can also be formulated in the preparation of food and beverages to provide functional foods.

[0081] In some implementations, mussel lipids and krill oil are formulated alone or together with carrier oils such as olive oil and optionally antioxidants (e.g., vitamin E).

[0082] Subjects to be treated with the combination therapy described in this disclosure include mammalian subjects, such as humans, primates, felines, canines, bovines, equines, porcines, leporines, ovines, and caprines, and include livestock (e.g., cattle, 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 envisioned because they can provide convenient testing systems.

[0083] Any of the dosage forms described above may be used for human or veterinary purposes as appropriate.

[0084] Therapeutic effective amount is intended to include a combination of amounts that, when administered according to the desired dosing regimen, are at least partially effective in achieving the desired therapeutic effect, which includes one or more of the following: reducing, eliminating, or decreasing the duration, severity, and / or frequency of inflammation in the particular disorder or condition being treated, and / or one or more symptoms of inflammation (e.g., heat, pain, swelling, redness); 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) stopping or reversing the occurrence or progression of the particular disorder or condition being treated.

[0085] The appropriate dosage and administration regimen can be determined by the attending physician or veterinarian and may depend on the specific condition / symptom being treated, the severity of the condition, and the subject's general age, health, and weight. Appropriate daily doses of mussel lipids and / or krill oil can be independently determined in the range from about 10 mg to about 10 g, for example, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 g. g, 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, 6.0g, 6.5g, 7.0g, 7.5g, 8.0g, 8.5g, 9.0g or about 9.5g. In some other embodiments, the daily dose of this combination can range from about 20 mg to about 15 g, for example, about 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 g, 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, 6.0g, 6.5g, 7.0g, 7.5g, 8.0g, 8.5g, 9.0g, 9.5 g, 10.0g, 10.5g, 11.0g, 11.5g, 12.0g, 12.5g, 13.0g, 13.5g, 14.0g or 14.5g.

[0086] In some implementations, a single unit dose (e.g., a soft gel capsule) may contain approximately 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 190 mg, or 200 mg. Combinations of 210 mg, 220 mg, 225 mg, 230 mg, 240 mg, 250 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 325 mg, 330 mg, 340 mg, 350 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 425 mg, 430 mg, 440 mg, 450 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 490 mg, or about 500 mg, optionally formulated with a carrier oil (e.g., olive oil). In some other embodiments, the combination also contains vitamin E. In some other embodiments, the composition contains mussel lipids (e.g., as PCSO-524) in an amount ranging from about 10% w / w to about 90% w / w of the total mussel lipids and krill oil, and contains krill oil in an amount ranging from about 90% w / w to about 10% w / w of the total mussel lipids and krill oil, i.e., a mussel lipid to krill oil ratio of about 10:90 to 90:10. The weight ratio of mussel lipids to krill oil is, for example, 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.

[0087] The dosage can be conveniently administered once daily, or the daily dose can be divided and administered multiple times daily (e.g., twice, three, or four times). In some embodiments, the combination of this disclosure can be administered once weekly, twice weekly, three times weekly, or more frequently per week, such as every other day. In some embodiments, treatment can be continuous or long-term, for example, for a period of at least 6-12 months or at least 2-3 years, or continuously.

[0088] The combination of this disclosure, for example at any daily dose, may contain mussel lipids in an amount ranging from about 1% w / w to about 99% w / w of the total 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 mussel lipids and krill oil, i.e., a mussel lipid to krill oil weight ratio from about 1:99 to 99:1. In some embodiments, the combination contains mussel lipids in an amount ranging from about 5% w / w to about 95% w / w of the total mussel lipids and krill oil, and krill oil in an amount ranging from about 95% w / w to about 5% w / w of the total mussel lipids and krill oil, i.e., a mussel lipid to krill oil weight ratio from about 5:95 to 95:5. In some embodiments, the composition contains mussel lipids in an amount ranging from about 10% w / w to about 90% w / w of the total mussel lipids and krill oil, and contains krill oil in an amount ranging from about 90% w / w to about 10% w / w of the total mussel lipids and krill oil, i.e., a weight ratio of mussel lipids to krill oil from about 10:90 to 90:10. In another embodiment, the combination comprises mussel lipids 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.

[0089] While the combinations of the present disclosure can be administered as the sole anti-inflammatory treatment for any one or more disorders, they can also be administered in combination with one or more NSAID regimens, such as celecoxib, diclofenac, diflunisal, etodoxacin, fenprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, telorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxapzin, piroxicam, sulindac, and tometetin. In some embodiments, the combinations of the present disclosure can eliminate or reduce potential side effects associated with NSAIDs, for example, by eliminating or substantially eliminating the need for additional NSAID treatment, or by reducing the dose and / or frequency of administration of the NSAID required to achieve a beneficial therapeutic effect.

[0090] As discussed above, in one or more embodiments, the krill oil used in the combinations disclosed herein can 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 utilize a combination of CO2 and CO2 / ethanol. However, it is generally accepted that the higher the phospholipid content of krill oil, the more viscous it becomes, with krill oil having a content of about 60% or higher 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.

[0091] An exemplary prior art process is described in WO2007123424. This document describes a two-step process whereby the feed material is first extracted with pure CO2 to extract only neutral lipids (i.e., nonpolar triglycerides), leaving a material enriched with phospholipids by removing the nonpolar triglycerides. This phospholipid-enriched material is then extracted with CO2 + ≥10% ethanol co-solvent to extract polar phospholipids and the remaining nonpolar triglycerides together from the krill feed biomass. This method inefficiently utilizes plant capacity on a commercial scale because, during both stages of the process, a large volume of the high-pressure extractor is filled with non-extractable proteins, carbohydrates, and ash components from the feed biomass. This is also fundamentally an inefficient batch operation (compared to a more efficient continuous operation), which can adversely affect process costs. Conversely, enrichment for delivering the final polar phospholipid content to the finished oil can be achieved directly on an industrial-scale, bulk heterogeneous solid feedstock in the first step. Precise final oil enrichment requires accurate prior knowledge of the polar and nonpolar lipid contents of the solids feed, and how well each will be extracted subsequently. In practice, uncertainties at commercial scale can translate into costly over-enrichment, which then necessitates a final blending back to the required specifications. This again negatively impacts the economics of the process.

[0092] US 9,735,453, US 9,078,905, US 9,028,877, US 9,320,765, and US 9,072,752 describe the extraction of neutral lipids (nonpolar triglycerides) from krill using CO2 or CO2 with approximately 5% ethanol, followed by the extraction of krill oil with high amounts of phospholipids, astaxanthin esters, and / or ω-3 fatty acids from the bulk heterogeneous solid material using CO2 / ~20% ethanol. These processes share the disadvantages described above.

[0093] This disclosure now describes a process, in some embodiments, that can reduce, minimize, or eliminate one or more of the disadvantages discussed above when preparing krill oil containing high or enriched phospholipids according to prior art methods, particularly at an industrial or commercial scale (e.g., when producing batches of oil in quantities of at least about 50 kg, 100 kg, 200 kg, 300 kg, or 500 kg and larger). Therefore, this disclosure also provides a process for preparing krill oil having a phospholipid content of at least about 50% w / w or higher, preferably to at least about 60% w / w or higher; and a process for enriching oils with lower phospholipid content (less than about 50% w / w) to at least about 50% w / w or higher, preferably to at least about 60% w / w or higher.

[0094] In some embodiments, this disclosure provides a two-step process for preparing 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 certain proportion of nonpolar lipid components (e.g., triglycerides) from the first krill oil to obtain a second krill oil that is rich in phospholipids (i.e. has a higher phospholipid content) compared to the first krill oil.

[0095] In contrast to the existing technologies discussed above, some implementations of this process begin with the non-selective extraction of oil from krill biomass feedstock. The extraction of both polar lipids (e.g., phospholipids) and non-polar lipids (e.g., triglycerides) from solid feedstock powder can be achieved using a mixture of CO2 and ethanol (e.g., azeotropic ethanol – a water-ethanol mixture containing approximately 95% ethanol).

[0096] In some preferred embodiments, a mass ratio of at least about 15% w / w or about 20% w / w ethanol in CO2 may be used, for example, in the range of about 17-22% w / w. In other embodiments, a mass ratio of at least about 25% w / w ethanol in CO2 may be used, such as at 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 in CO2.

[0097] In some embodiments, including any of the other embodiments discussed in this paragraph, the extraction temperature is at or below about 60°C, such as at or below about 55°C, or at or below about 50°C, or at or below about 45°C, or at or below 40°C, or at or below 35°C, or at or below 30°C, to advantageously reduce or minimize the risk of product degradation. The extraction pressure can be set to ensure supercritical, subcritical, and / or near-critical conditions for the selected temperature and CO2 to ethanol ratio. In some embodiments, the pressure is in the range of about 200-350 bar, for example, about 250-300 bar, although higher pressures, such as 400 bar and above, are technically effective and can also be used. In some embodiments, the pressure value or pressure range produces sufficient solvent density to ensure that the extraction of nonpolar lipids does not become a rate limiter of the process. In some embodiments, the extraction pressure conditions can be adjusted throughout the extraction process to move between supercritical, subcritical, or near-critical conditions. In some embodiments, subcritical and / or near-critical conditions are used, the conditions of which will vary depending on the binary mix ratio of CO2 to ethanol (or the ternary mix ratio in the case of azeotropic ethanol also containing water). The extraction time can be determined by those skilled in the art and can depend particularly on the extraction conditions and desired economic optimization. In some embodiments, the extraction time is typically in the range of about 1-15 hours, for example about 2-10 hours, such as about 2-5 hours, or about 3-6 hours, or about 4-5 hours. This, in turn, can depend particularly on the amount and particle size of the biomass feed material. Larger particles will allow for the use of higher solvent flow rates while still maintaining static biomass and uniform solvent contact. However, larger particles also present an increased diffusion requirement for the solvent to reach the center of the particle, where more solvent is needed. Therefore, in some embodiments, the particle size of the feed material is about 1-5 mm, for example about 2-3 mm.

[0098] In some other implementations, the extraction pressure is about 300 bar and the extraction temperature is about 60°C.

[0099] Oil separation can be carried out at lower temperatures (e.g., about 25-35°C) and pressures (e.g., about 25-60 bar).

[0100] The extracted oil contains both polar lipids (e.g., phospholipids) and nonpolar 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.

[0101] In some advantageous embodiments, the water and ethanol present in the oil can then be removed using any suitable method, such as evaporation under vacuum (optionally with gentle heating, e.g., about 65°C or less), nitrogen flow, or lyophilization. In some embodiments, the oil is optionally subjected to evaporation under vacuum with gentle heating. Further, this is followed by a short residence time (e.g., 1-3 seconds) at a higher temperature (e.g., about 70°C, about 75°C, or about 80°C) to remove the water and ethanol co-solvent from the low-viscosity mixture of polar and nonpolar lipids, the high proportion of nonpolar lipids providing the low viscosity. In some preferred embodiments, the temperature advantageously does not exceed about 60°C throughout the gentle heating under vacuum in order to avoid or minimize degradation of the components of the extracted oil. In other embodiments, the temperature advantageously does not exceed about 55°C, or about 50°C, or about 45°C, or about 40°C, or about 35°C, or about 30°C, or about 25°C throughout the gentle heating under vacuum.

[0102] In some embodiments, the residual volatile content (water and ethanol) after evaporation is about or less than about 3% w / w, as this minimizes the possibility that residual ethanol and water adversely affect lipid separation in subsequent enrichment steps. In other 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.

[0103] At this stage, after evaporation, the oil remains fluid due to the presence of nonpolar lipid components and can be readily analyzed for, for example, phospholipid and / or ω-3 fatty acid content. This is important because precise analysis is required to calculate the desired enrichment and, therefore, the final phospholipid content of the oil achieved in subsequent steps. In particular, if a high final phospholipid content is desired, over-enrichment (i.e., further removal of nonpolar lipids), even slightly, can lead to processing problems attributable to excessive viscosity. The evaporated oil can be thoroughly mixed to ensure homogeneity, optionally after transfer to an intermediate product tank. Optionally, the oil can be gently heated (e.g., at temperatures less than or about 60°C or about 55°C, or about 50°C or about 45°C, or about 40°C, or about 35°C, or about 30°C, or about 25°C) to help maintain a fluid and homogeneous material for analysis. In some embodiments, the lower viscosity of the unenriched oil obtained by the first-step CO2 / EtOH extraction, compared to the first-step bulk solid biomass used in the prior art processes discussed above, can advantageously allow for more accurate compositional analysis because bulk homogeneity can be more easily achieved. In some embodiments, this can advantageously avoid, minimize, or otherwise reduce the over-enrichment of phospholipids in the subsequent selective extraction of nonpolar lipids, which could otherwise result in undesirable viscous or immobile solid products.

[0104] The second step of the process involves the preferred or selective extraction of nonpolar lipids (triglycerides) from the oil obtained in the first step. If the unenriched oil obtained by the first extraction step has been transferred to an intermediate tank, the unenriched oil is returned to the extraction facility. The oil is then subjected to further CO2 extraction, in some preferred embodiments under supercritical conditions, such as at or above about 300 bar and at about 60°C, to selectively extract the nonpolar lipids (triglycerides). In some embodiments, extraction may begin at a lower pressure and then incrementally increased to a desired level (e.g., about 300 bar). Nonpolar lipids (triglycerides) can be extracted stepwise to enrich the remaining liquid until the desired compositional target is achieved, such as a phospholipid content of at least about 50% w / w, or at least about 55% w / w phospholipid, or at least about 60% w / w phospholipid, or at least about 65% w / w phospholipid, or at least about 70% w / w phospholipid, or at least about 75% w / w phospholipid, or at least about 80% w / w phospholipid, or at least about 85% w / w phospholipid, or at least about 90% w / w phospholipid, or at least about 95% 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.

[0105] In some implementations, after the required amount of nonpolar lipids has been extracted to achieve the desired level of phospholipid enrichment, partially depressurizing the extraction vessel allows the residual pressure to facilitate the discharge of the now-enriched (and more viscous) residue as needed. By discharging from the still partially pressurized extractor, the discharge 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.

[0106] In one or more embodiments, the process may allow for semi-continuous processing, where individual extraction containers are replaced during continuous rotation, but one at a time, while other extraction containers continue to operate. In this way, shutdowns for changing multiple batches of extractors can be avoided.

[0107] In other embodiments, the second step described herein can be used to enrich the phospholipid content of any krill oil having a phospholipid content of less than about 50% w / w in order to obtain krill oil having a phospholipid content of at least about 50% w / w.

[0108] In some implementations, 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 implementations, the final enriched oil may have 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, 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.

[0109] 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 0.5 w / w or less. In other 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. In another embodiment, 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 0.5 w / w, or 0.3% w / w, or 0.1% w / w, or less.

[0110] In other embodiments, the final enriched krill oil has a phospholipid content of at least about 60% w / w and a residual solvent content of about 3% w / w or less.

[0111] Other embodiments involving krill oil as described herein may also be applied, as appropriate, to krill oil produced using the processes described in this disclosure.

[0112] 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 above description. Example

[0113] Example 1 Preparation of -62% w / w phospholipid krill oil 1. Extraction of krill oil from krill meal Krill meal was extracted with CO2 / ethanol at 60°C and 300 bar (ethanol to krill meal feed ratio of approximately 3.0-3.5:1 w / w), with the ethanol mass fraction ranging from approximately 17-22% w / w. The duration of the ethanol / CO2 extraction stage was between 10 and 15 hours. The extracted oil / CO2 / EtOH mixture was separated at 45 bar and 25°C.

[0114] Several batches of oil obtained by this method were blended to provide krill oil containing both polar and non-polar components and having a phospholipid content of approximately 42% (see Table 1-1 below).

[0115] Table 1-1: PL in krill oil samples 31P NMR analysis

[0116] 2. Selectively extract triglycerides from the krill oil obtained in step 1.

[0117] 5.9 kg of feed krill oil (with the composition described in Table 1-1 above) was directly loaded into a single 10.7 L extraction vessel (155 mm in diameter). The feed material was extracted with CO2 at a temperature of 60 °C and a pressure of 300 bar.

[0118] The extract, which mainly contains triglycerides, was recovered and was significantly less viscous than the feed krill oil loaded into the extraction vessel.

[0119] After the total mass of extracted material reaches 97% of the theoretical amount of extractable material, the depressurization process of the extraction vessel begins. The facility depressurizes from 300 bar to 100 bar at a constant ramp rate over a 15-minute period, during which CO2 circulation continues, albeit at 50% of the extraction flow rate. During this time, the temperature measured at the extractor outlet decreases from the operating temperature of 60°C to 50°C. The extractor is then further depressurized from 100 bar to 75 bar over an additional 15-minute period without pump operation. Afterward, the contents of the separation vessel are emptied.

[0120] While the extraction container remains at the processing temperature and 75 bar pressure, the enriched krill oil contents are emptied from the bottom of the extractor container, well below the residual liquid surface level, thus avoiding the loss of high-pressure CO2 as the residual oil is discharged. The recovery of the enriched oil takes approximately one hour, during which time the container pressure decreases from 75 bar to 54 bar as the remaining CO2 in the extraction container expands to fill the space previously occupied by the residual oil.

[0121] After the enriched oil has been discharged, it was observed that some enriched krill oil remained in the extraction container on the distributor and bottom surface of the container, which was estimated to be less than 2% of the total mass of the enriched oil. On a commercial scale, any such residual oil is recycled into subsequent batches of krill oil used in the extractor.

[0122] Prior to astaxanthin and phospholipid analysis, the enriched oil was heated in an oven at 55°C for 1 hour. This allowed the sample to be sufficiently fluid for stirring to achieve a homogeneous sample for analysis.

[0123] Table 1-2 summarizes the quality of the feed, extracted oil, and enriched oil, as well as the phospholipid (PL) and astaxanthin (Asta) contents. Very small amounts of phospholipids (< 1 g / 100 g extract) and astaxanthin (< 2 mg / 100 g extract) were co-extracted. Overall, the quality of the extract obtained from the enrichment process was 98% of the theoretical extract required to enrich phospholipids to 62%.

[0124] Table 1-2: Summary of phospholipid (PL) and astaxanthin (Asta) content

[0125] Tables 1-3 and 1-4 summarize the composition and content of the enriched oil.

[0126] Table 1-3: PL in krill oil samples 31 P NMR analysis

[0127] Table 1-4: GC analysis of fatty acids

[0128] Example 2- Mussel lipid extract was prepared according to WO97 / 09992 and with PCSO-524. ® The krill oil is used in the form of (Pharmalink International Limited, Hong Kong). It is prepared by the process of Example 1 and has the compositional content stated in Tables 1-3 and 1-4 above.

[0129] Sample preparation Fresh samples were prepared daily. Samples were mixed by inversion before sampling. Samples were weighed in 1.5 mL centrifuge tubes and prepared as a stock solution at 100 mg / mL with ethanol. The stock solution mixture was prepared by weighing the oil at the correct ratio and then bringing it to the concentration with ethanol. Serial dilutions of the stock solution in ethanol were prepared. The serial dilutions were then diluted in cell culture medium (1 / 100) and then added to cells with the final 1 / 10 dilution (in triplicate). This produced an ethanol concentration of 0.1% for all doses and controls. Krill oil contains approximately 62% w / w phospholipids. Mussel lipid extract was prepared using PCSO-524. ® The form is used.

[0130] The abbreviations used in presenting the results are stated in Table 2-1 below: Table 2-1: Abbreviations

[0131] Measurement: Anti-inflammatory activity was determined in RAW264.7 murine macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFNγ), which were cultured in standard cell culture medium and incubated with LPS and IFNγ in the presence or absence of different test compounds / extracts and positive controls. The production of inflammatory mediators, including NO, PGE2, LTB4, cytokines TNFα, and IL-6, was measured using a commercially available kit and the established method. Each sample was tested at at least three concentrations using relevant internal controls (using three replicate assays, with a maximum concentration of 100 μg / ml) (n=9) (Table 2-2). Cytotoxicity of each sample was also determined by an MTT assay. No cytotoxicity was detected at any of the concentrations tested.

[0132] Table 2-2 summarizes the measurement parameters for each assay. In short, for the anti-inflammatory assay, the cultured RAW264.7 cells were counted and seeded in 96-well plates (0.8 × 10⁻⁶). 5 Cells / well were seeded onto plates and incubated for the specified time. The medium was then aspirated and replaced with fresh medium, followed by the addition of the test compound. The compound was incubated for 1 h before the addition of the stimulant. The plates were then incubated for 4–18 h, and the supernatant was analyzed for the medium of interest. Remaining cell viability was determined by MTT assay.

[0133] Positive controls were selected based on their wide applicability in similar assays, including N-(3-(aminomethyl)benzyl)acetamidine (1400W), a slow, tight-binding inhibitor of inducible nitric oxide synthase (iNOS) (Garvey, EP et al.). J Biol Chem , 1997,21:272(8):4959-63); and dexamethasone, a commonly used cytokine inhibitor. Diclofenac is a common nonsteroidal anti-inflammatory agent and is known as an inhibitor of cyclooxygenase (COX), which produces PGE2.

[0134] Table 2-2: Measurement Parameters

[0135] result 1. Nitric oxide determination NO is a free radical metabolite that has been shown to have numerous physiological functions, acting both as a signaling molecule and as a toxic agent in inflammation (Coleman, 2001). NO is derived from L-arginine via oxidation by three types of nitric oxide synthases (NOS); these three types are the constituents originally described in murine macrophages: neuronal NOS and endothelial NOS, as well as the inducible form iNOS (Nathan & Xie, 1994; Stuehr & Marletta, 1985). The inducible form is sequentially activated upon expression and is therefore regulated at the transcriptional level by NF-κB stimulated by inflammatory molecules such as LPS and IFNγ. NO production via iNOS experiences a lag of several hours before being produced at a sustained level of much higher (nM) NO (Nathan & Xie, 1994). The inducible form of NOS is likely involved in inflammation and is more readily assessed in vitro due to the higher levels of NO produced.

[0136] NO is an unusual signaling molecule. Because there are no specific cell surface receptors for NO, it enters cells indiscriminately, with effects depending on cell type and NO concentration, thus producing a wide range of physiological responses. NO induces increased vascular permeability, vasodilation, and free radical generation, which cause tissue damage and eliminate pathogens (Guzik, Korbut, & Adamek-Guzik, 2003). These physiological changes are associated with inflammation accompanied by increased blood flow, allowing more immune cells to enter affected tissues and destroy pathogens.

[0137] The results of the NO inhibition assay Figure 1A and Figure 1B And as described in Table 2-3.

[0138] Table 2-3: IC50 of NO inhibition 50

[0139] 2. Tumor necrosis factor-α TNFα is a major cell signaling protein (cytokine) involved in the acute inflammatory response. Macrophages are the primary source of TNFα, although it can be released by many other cell types such as CD4+ lymphocytes, natural killer (NK) cells, neutrophils, mast cells, eosinophils, and neurons. TNFα is produced through the activation of MAPK and NF-κB. It increases its own production and the production of other inflammatory cytokines such as interleukin-1β (IL-1β). TNFα induces fever, apoptotic cell death, cachexia, inflammation, and inhibits tumorigenesis and viral replication. Many disease states involve TNFα, 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).

[0140] The results of the TNFα inhibition assay were in Figure 2A and Figure 2B And as described in Table 2-4.

[0141] Table 2-4: IC50 of TNFα inhibition 50

[0142] *No mathematical model fits the dose-response curve 3. Interleukin-6 Similar to TNFα, IL-6 is considered a pro-inflammatory cytokine. IL-6 is secreted by T cells and macrophages that stimulate the immune response. IL-6 causes an increase in the production of neutrophils in the bone marrow. It supports B cell growth and antagonizes the differentiation of T cells into regulatory T cells. It can cross the blood-brain barrier and initiate the synthesis of PGE2 in the hypothalamus, thereby altering the body's temperature set point (Banks, Kastin, & Gutierrez, 1994).

[0143] The results of the IL-6 inhibition assay were in Figure 3A and Figure 3B As depicted in Table 2-5.

[0144] Table 2-5: IC50 of IL-6 inhibition 50

[0145] 4. Prostaglandin E2 Prostaglandin E2 (PGE2) is a lipid mediator produced from arachidonic acid (AA) by the action of the enzyme cyclooxygenase (COX) and is associated with inducing fever, pain sensation, and inflammation. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the biosynthesis of prostaglandins (including PGE2), producing antipyretic, analgesic, and anti-inflammatory effects (Kawahara, K. et al., 2015 and Kawabata A., 2011).

[0146] The result is Figure 4A and Figure 4B And as depicted in Table 2-6.

[0147] Table 2-6: IC50 of PG-E2 inhibition 50

[0148] *No mathematical model fits the dose-response curve Summary of Results In this assay system, mussel lipid extract and krill oil were shown to inhibit NO, TNFα and IL-6 individually at the tested concentrations, but not PGE2.

[0149] In inhibiting NO, TNFα, and IL-6, the combination of mussel lipid extract and krill oil was more effective than either mussel lipid extract or krill oil alone. In PGE2 assays, neither mussel lipid extract nor krill oil alone showed inhibitory activity, but the combination exhibited inhibition.

[0150] Example 3: Synergistic Effect Mussel lipid extract was prepared according to WO97 / 09992 and with PCSO-524. ® The krill oil is used in the form of [missing information]. It is prepared by the process of Example 1 and has the compositional content as stated in Tables 1-3 and 1-4 above.

[0151] Samples, formulations and combinations Before sampling the experimental sample, PCSO-524 ® Stock samples of high-phospholipid krill oil were mixed by inversion. Samples were weighed in 15 mL centrifuge tubes and prepared as a stock solution at 100 mg / mL with ethanol. The mixture was prepared by mixing diluted oil at the correct ratio. Serial dilutions were prepared in ethanol. The serial dilutions were then diluted in cell culture medium (1 / 100) and subsequently added to cells with the final 1 / 10 dilution (in triplicate). This produced a 0.1% ethanol concentration for all doses and controls. These doses were prepared fresh daily. Table 3-1 shows the abbreviations used for each sample.

[0152] Table 3-1: Abbreviations of Sample Names

[0153] Measurement Anti-inflammatory activity was determined in RAW264.7 murine macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFNγ) in standard cell culture medium (DMEM, 5% fetal bovine serum) and incubated with and without different test compounds / extracts and controls. The production of inflammatory mediators, including NO, the cytokine TNFα, and IL-6, was measured using a commercial ELISA kit (suppliers listed in Table 3-2) according to the established method. Each sample was tested at at least six concentrations using relevant internal controls (using three parallel assays, with a maximum concentration of 100 μg / ml) (n=9) (shown in Table 3-2). Cytotoxicity of each tested sample was determined by MTT assay. No cytotoxicity was detected at any of the tested concentrations.

[0154] The measurement parameters used for each assay are summarized in Table 3-2. In short, for NO, TNFα, and IL-6 assays, the cultured RAW264.7 cells were counted and plated in 96-well plates (0.8 × 10⁻⁶). 5 Cells / well were incubated for 48 h. The culture medium was then aspirated and replaced with fresh medium, followed by the addition of the test compound. The compound was incubated for 1 h before the addition of the stimulant. The plate was then incubated for 18 h, and the supernatant was analyzed for the medium of interest. Remaining cell viability was determined by MTT assay.

[0155] Positive controls were selected based on their wide 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.

[0156] Table 3-2: Anti-inflammatory assay and positive control

[0157] Synergistic effect calculation Synergistic effects were expressed as a Combination Index. The Combination Index (CI) and IC50 weights of the synergistic effect plot were calculated using Compsyn software. The dose-response curve generated in Graphpad Prism was transformed into 10 points representing the curve. These 10 points were then input into the Compsyn program, which generated a curve fitting the data points. This method is preferred to closely replicate the complete dose-response curve from the synergistic effect program. The Compsyn fitted curve was then used for synergistic calculations. The pattern of synergistic effects within the tested range can be observed using an equivalence plot, which plots the relative contribution of each component to the activity at IC50. A straight line exists between the two blended drugs (Biavatti, 2009). Values ​​below this line indicate synergistic effects, values ​​above this line are considered additive, and values ​​above this line are antagonistic.

[0158] result 1. Nitric oxide determination Combinations were tested to determine whether they exhibited synergistic inhibition of the inflammatory signaling molecule NO. Combinations were first tested in 10% increments. The most active combination observed was LY60. Further 5% increments around LY60 were then tested.

[0159] The dose-response curves of the tested combination LY90-LY10 were in Figure 5A , Figure 5B , Figure 6A and Figure 6B The results are depicted in Tables 3-3 and 3-4, which present the IC50 values ​​and combination indices for NO inhibition. A combination index less than 1 indicates synergistic effects. The equivalent effect plot for a 10% increment is shown in... Figure 7 It is depicted in the middle.

[0160] Table 3-3: IC50 of NO inhibition by the LY90-LY10 combination 50 value

[0161] Table 3-4: IC50 of NO inhibition for combinations of LY75 to LY45 50 (CI) and portfolio index

[0162] 2. TNFα assay Combinations were tested to determine whether they exhibited synergistic inhibition of the inflammatory cytokine TNFα. Combinations were first tested in 10% increments. The most active combination observed was LY50. Further 5% increments around LY50 were then tested.

[0163] The dose-response curves of the tested combination LY90-LY10 were in Figure 8A , Figure 8B , Figure 8C , Figure 9A and Figure 9B The results are depicted in Tables 3-5 and 3-6, which present the IC50 values ​​and combination indices for TNFα inhibition. A combination index less than 1 indicates synergistic effect. The isodynamic plot for a 10% increment is shown in... Figure 10 It is depicted in the middle.

[0164] Table 3-5: IC50 for TNFα inhibition 50 value

[0165] * - No possible estimate Table 3-6: ICs used for LY70-LY35 50 And CI (n=9)

[0166] 3. IL-6 Measurement Combinations were tested to determine whether they exhibited synergistic inhibition of the inflammatory cytokine IL-6. Combinations were first tested in 10% increments. The most active combination observed was LY60. Further 5% increments around LY60 were then tested.

[0167] The dose-response curves of the tested combination LY90-LY10 were in Figure 11A , Figure 11B , Figure 12A and Figure 12B The results are depicted in Tables 3-7 and 3-8, which present the IC50 values ​​and combination indices for IL-6 inhibition. A combination index less than 1 indicates synergistic effects. The equivalent effect plot for a 10% increment is shown in... Figure 13 It is depicted in the middle.

[0168] Table 3-7: ICs for IL-6 inhibition 50 (n=3)

[0169] Table 3-8: ICs for IL-6 inhibition 50 and combination index

[0170] Summary of Results In terms of inhibiting NO, TNFα and IL-6, the combination of mussel lipid extract and krill oil was demonstrated in this assay system to meet the mathematical criteria for synergistic effects.

[0171] Example 4: Patient Study Patients suffering from various pain / inflammatory conditions were administered a combination of mussel lipid extract (in the form of PCSO-542) and krill oil (61% PL) in capsule form at a ratio of 75:25 (PCSO-542 to krill oil). The composition of the capsules is shown in Table 4-1.

[0172] Table 4-1: Composition of 150mg oil blend capsules

[0173] *Contains 0.15% w / w Vitamin E (Approximately 0.056 mg / capsule) The dosage is typically in the range of 2–8 capsules per day, administered once, twice, or three times. Patients usually already have one or more NSAIDs, including acetaminophen or ibuprofen, to manage pain before starting treatment with this combination. Results are depicted in Table 4-2.

[0174] Table 4-2: Summary of Patient Outcomes

[0175] This disclosure also provides the following: 1) A combination comprising mussel lipids and krill oil, wherein the combination is suitable for application alone or in sequence.

[0176] 2) According to the combination described in 1), the combination is in the form of a composition comprising mussel lipids and krill oil.

[0177] 3) According to the combination described in 1) or 2), wherein the krill oil has a phospholipid content of at least about 40% w / w.

[0178] 4) According to the combination described in 3), wherein the krill oil has a phospholipid content of at least about 60% w / w.

[0179] 5) The combination according to any one of 1)-4), wherein the krill oil has a water content of about 5% w / w or less.

[0180] 6) According to the combination described in 5), wherein the krill oil has a water content of about 3% w / w or less.

[0181] 7) According to the combination described in 5), wherein the krill oil has a water content of about 1% w / w or less.

[0182] 8) The combination according to any one of 1)-7), wherein the krill oil has an extraction solvent content of about 5% w / w or less.

[0183] 9) According to the combination described in 8), wherein the krill oil has an extraction solvent content of about 3% w / w or less.

[0184] 10) According to the combination described in 8), wherein the krill oil has an extraction solvent content of about 1% w / w or less.

[0185] 11) The combination according to any one of 1)-10), wherein the mussel lipid is in the form of mussel powder.

[0186] 12) The combination according to any one of 1)-10), wherein the mussel lipid is in the form of a mussel lipid extract and optionally contains vitamin E.

[0187] 13) The combination according to any one of 1)-12), wherein the weight ratio of mussel lipids to krill oil is in the range of 1:99 to 99:1.

[0188] 14) According to the combination described in 13), wherein the weight ratio of mussel lipids 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.

[0189] 15) The combination according to any one of 1)-14) is in the form of an oral unit dosage form.

[0190] 16) According to the combination described in 15), wherein the oral unit dosage form is a soft gel capsule.

[0191] 17) According to the combination of 15) or 16), wherein the oral unit dosage form comprises mussel lipids from about 10 mg to about 10 g.

[0192] 18) The combination according to 17), wherein the oral unit dosage form comprises about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g, 2.5 g, 2.6 g, 2.7 g, 2.8 g, 2.9 g, 3.0 g, 3.2 g, 3.5 g, 3.7 g, etc. Mussel lipids in the amounts of 4.0 g, 4.5 g, 5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, or approximately 9.5 g.

[0193] 19) The combination according to any one of 15)-18), wherein the oral unit dosage form comprises krill oil from about 10 mg to about 10 g.

[0194] 20) The combination according to 19), wherein the oral unit dosage form comprises about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g, 2.5 g, 2.6 g, 2.7 g, 2.8 g, 2.9 g, 3.0 g, 3.2 g, 3.5 g, 3.7 g, etc. Krill oil in quantities of 4.0 g, 4.5 g, 5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, or approximately 9.5 g.

[0195] 21) The combination according to any one of 15)-20), wherein the oral unit dosage form comprises about 10-500 mg of the combination.

[0196] 22) The combination according to 21), wherein the oral unit dosage form comprises about 50-300 mg of the combination.

[0197] 23) The combination according to any one of 1)-22) further comprises one or more pharmaceutically acceptable carriers and / or additives.

[0198] 24) The combination according to any one of 1)-22), wherein the combination consists of mussel lipids and krill oil or consists essentially of mussel lipids and krill oil.

[0199] 25) A composition comprising mussel lipids and krill oil.

[0200] 26) The composition according to 25) wherein the krill oil has a phospholipid content of at least about 50% w / w.

[0201] 27) The composition according to 25) or 26), wherein the mussel lipids are in the form of mussel lipid extracts and optionally contain vitamin E.

[0202] 28) The composition according to any one of 25)-27), wherein the weight ratio of mussel lipids 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.

[0203] 29) The composition according to any one of 25)-28) further comprises a carrier oil.

[0204] 30) The composition according to 29), wherein the carrier oil constitutes from about 10% w / w to about 90% w / w of the total composition.

[0205] 31) The composition according to 29) wherein the weight ratio of the combined amount of carrier oil to mussel lipids and krill oil is from about 3:1 to about 1:3.

[0206] 32) The composition according to any one of 25)-31) is in unit dosage form.

[0207] 33) The composition according to 32) is encapsulated in a soft gel capsule.

[0208] 34) The composition according to 32) or 33) comprises about 10-500 mg of a combination of mussel lipids and krill oil.

[0209] 35) The composition according to 34) comprises about 50-300 mg of a combination of mussel lipids and krill oil.

[0210] 36) A combination of any one of 1)-24) or a composition of any one of 25)-35) for use in treating inflammation in a subject.

[0211] 37) A method of treating inflammation in a subject in need, the method comprising administering to the subject a combination of any one of 1)-24) or a composition of any one of 25)-35).

[0212] 38) Use of mussel lipids and krill oil in the manufacture of combination drugs for the treatment of inflammation.

[0213] 39) According to the use described in 38), the drug is suitable for administration alone or in combination.

[0214] 40) According to the use described in 39), the drug is in the form of a composition of mussel lipids and krill oil.

[0215] 41) A combination for treating inflammation, the combination comprising mussel lipids and krill oil.

[0216] 42) The agent described in 41) is suitable for use alone or in combination.

[0217] 43) A combination of any one of 1)-24) or a composition of any one of 25)-35) for use in treating pain in a subject.

[0218] 44) A method for treating pain in a subject in need, the method comprising administering to the subject a combination according to any one of 1)-24) or a composition according to any one of 25)-35).

[0219] 45) Use of mussel lipids and krill oil in the manufacture of combination drugs for the treatment of pain.

[0220] 46) According to the use described in 45), the drug is suitable for administration alone or in combination.

[0221] 47) According to the use described in 46), the drug is in the form of a composition of mussel lipids and krill oil.

[0222] 48) A combination for treating inflammation, the combination comprising mussel lipids and krill oil.

[0223] 49) The agent described in 48) is suitable for use alone or in combination.

[0224] 50) A process for preparing krill oil having a phospholipid content of about 50% or more, comprising the following steps: (a) Contacting krill biomass feedstock with a mixture of CO2 and ethanol to extract krill oil; and (b) The krill oil is contacted with CO2 to extract at least a certain proportion of nonpolar lipid components, such that the oil has a phospholipid content of at least 50% w / w.

[0225] 51) According to the process described in 50), the krill biomass feed material is contacted with a mixture of about 15% w / w to about 30% w / w ethanol in CO2.

[0226] 52) The process according to 50) or 51), wherein step (a) is carried out at a temperature of about 60° or less.

[0227] 53) The process according to any one of 50)-52), wherein step (a) is carried out at a pressure of about 300 bar or greater than about 300 bar.

[0228] 54) The process according to any one of 50)-53), wherein step (b) is carried out at a temperature of about 60° or less.

[0229] 55) The process according to any one of 50)-54), wherein step (a) is carried out at a pressure of about 300 bar or greater than about 300 bar.

[0230] 56) The process according to any one of 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.

[0231] 57) The process according to any one of 50)-56), wherein ethanol is removed from the extract oil obtained in step (a).

[0232] 58) According to the process described in 57), the ethanol is removed under vacuum at a temperature of about 60°C or less. References

[0233]

[0234] .

Claims

1. A combination comprising mussel lipids and krill oil, wherein the combination is suitable for application alone or in sequence.

2. The combination according to claim 1, wherein the combination is in the form of a composition comprising mussel lipids 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.

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