Lipid emulsions with anti-inflammatory action for total parenteral and enteral nutrition

Abstract

The present invention relates to a lipid emulsion for total parenteral and enteral (oral or through gastric or duodenal tube) nutrition. The lipid emulsions have advantageous immunomodulatory, anti-inflammatory and anti-diabetic effects. The lipid emulsion may also comprise a drug, may be used as an antidote, or for reversing negative effects from ischemia-reperfusion injury.

Description

[0001] This application claims the priority of European Patent Application EP22193442.5 filed on September 1, 2022, which is incorporated herein by reference. Technical Field

[0002] The present invention relates to lipid emulsions for total parenteral and enteral (oral or via a gastric or duodenal tube) nutrition and drug administration. The lipid emulsions according to the present invention have advantageous immunomodulatory, anti-inflammatory and antidiabetic effects. The lipid emulsions may contain drugs and may be used as antidotes or for reversing the negative effects caused by ischemia-reperfusion injury. Background Art

[0003] Parenteral lipid emulsions (LEs) are heterogeneous systems consisting of an oil phase uniformly dispersed in an aqueous phase in the presence of an emulsifier. The droplet size (usually between 200 and 350 nm) characterizes these lipid emulsions as suitable for parenteral administration, and they have a physiological pH of about 7, isotonicity and a high ζ potential to prevent instability. Currently commercially available lipid emulsions consist of triglycerides from vegetable oils or fish oils such as soybean oil, olive oil, coconut oil, fish oil, etc. or their blends (Table 1), egg yolk lecithin (emulsifier), glycerol (to provide isotonicity) and water.

[0004] Based on the above prior art, the object of the present invention is to provide an immunomodulatory, anti-inflammatory and antidiabetic lipid emulsion for total parenteral nutrition and enteral nutrition and administration. This object is achieved by the subject matter of the independent claims of this patent specification and by the further advantageous embodiments described in the dependent claims, examples, drawings and general description of this patent specification. Summary of the Invention

[0005] A first aspect of the present invention relates to a lipid emulsion for parenteral administration, wherein the lipid emulsion comprises an oil phase and an aqueous phase, and wherein the oil phase of the lipid emulsion comprises:

[0006] - an ω-3 fatty acid component,

[0007] - an ω-6 fatty acid component,

[0008] - a monounsaturated fatty acid component, and

[0009] - a saturated fatty acid component,

[0010] They are in the ratios and relationships given in the claims, the detailed description and the examples (any % values given throughout the document should be interpreted as mass / mass unless otherwise stated).

[0011] A second aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments, which is used for parenteral or enteral nutrition.

[0012] The third aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments, which is used as an antagonist / detoxifying agent ("lipid pool") for treating poisoning caused by lipophilic drugs.

[0013] The fourth aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments, which is used to protect vital organs from ischemia-reperfusion injury. In certain embodiments, the vital organs are selected from the heart, brain, liver, kidneys, and lungs.

[0014] The fifth aspect of the present invention relates to a lipid emulsion according to the first aspect, which is used for preventing or treating type I and type II diabetes.

[0015] Terms and Definitions

[0016] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any of the definitions set forth below and any document incorporated herein by reference, the defined terms shall prevail.

[0017] As used herein, the terms "comprising", "having", "containing", and "including" and their other similar forms and grammatical equivalents are intended to have equivalent meanings and are open-ended, as one or more items following any of these words are not intended to be an exhaustive listing of such one or more items, nor are they intended to be limited to the listed one or more items. For example, an article "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain not only components A, B, and C, but also one or more other components. Thus, it is intended and understood that the disclosure of "comprising" and its similar forms and their grammatical equivalents includes the disclosure of embodiments "consisting essentially of" or "consisting of".

[0018] Where a range of values is provided, it is understood that, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of that range to one-tenth of the lower limit unit and any other stated value or intermediate value within that stated range are covered by the present disclosure, subject to any specifically excluded limits within that range. Where the range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0019] References herein to "about" a value or parameter include (and describe) variations that are for that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0020] As used herein, including in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and chemical methods.

[0022] In the context of this specification, the term TPN refers to total parenteral nutrition.

[0023] In the context of this specification, the term SDA refers to stearidonic acid.

[0024] In the context of this specification, the term ALA refers to α-linolenic acid.

[0025] The term F3 (see specifically the figures) represents Formulation #3 and is used for the newly generated lipid emulsion having unique physicochemical and biological effects as disclosed herein. In the specification, TPN-F3, F3, VV-TPN, and VV are used synonymously.

[0026] As used herein, the term drug refers to a chemical substance that produces a biological effect when administered to a living organism. A drug is a chemical substance used to treat, cure, prevent, or diagnose a disease or to promote health.

[0027] As used herein, the term toxic compound refers to a chemical substance that can impair the health of a patient or even potentially endanger life.

[0028] As used herein, the term lipophilicity refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and nonpolar solvents.

[0029] As used herein, the term antidote refers to the ability of a chemical substance or mixture to reduce the damage of a drug or compound to a patient's body.

[0030] As used herein, the term ischemic - reperfusion injury refers to tissue damage caused when blood supply returns to tissue after a period of ischemia or lack of oxygen (hypoxia or hypoxemia).

[0031] As used herein, the term pharmaceutical composition refers to the emulsion of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for parenteral or injectable administration.

[0032] As used herein, the term pharmaceutically acceptable carrier includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, absorption delaying agent, salt, preservative, drug, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetening agent, flavoring agent, dye, etc. known to those skilled in the art and combinations thereof (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

[0033] As used herein, the term "treating / treatment" of any disease or disorder (e.g., diabetes) in one embodiment means ameliorating the disease or disorder (e.g., slowing or arresting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" means alleviating or improving at least one physical parameter, including those parameters that may not be discernible by the patient. In yet another embodiment, "treatment" means physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters) or both regulating the disease or disorder. Unless specifically described below, methods for assessing the treatment and / or prevention of diseases are generally known in the art. Detailed Description

[0034] A first aspect of the present invention relates to a lipid emulsion for parenteral administration, wherein the lipid emulsion comprises an oil phase and an aqueous phase, and wherein the oil phase of the lipid emulsion comprises (all % values are mass / mass):

[0035] - an ω - 3 fatty acid component,

[0036] - an ω - 6 fatty acid component,

[0037] - a monounsaturated fatty acid component, and

[0038] - a saturated fatty acid component.

[0039] The mass of the ω-3 fatty acid component accounts for 20% to 50% of the oil phase, the mass of the ω-6 fatty acid component accounts for 3% to 35% of the oil phase, the mass of the monounsaturated fatty acid component accounts for 5% to 40% of the oil phase, and the mass of the saturated fatty acid component accounts for 5% to 45% of the oil phase.

[0040] In certain embodiments, the mass of the ω-3 fatty acid component accounts for 20% to 40% of the oil phase, the mass of the ω-6 fatty acid component accounts for 5% to 25% of the oil phase, the mass of the monounsaturated fatty acid component accounts for 10% to 35% of the oil phase, and the mass of the saturated fatty acid component accounts for 10% to 40% of the oil phase.

[0041] In certain embodiments, the mass of the ω-3 fatty acid component accounts for 25% to 35% of the oil phase, the mass of the ω-6 fatty acid component accounts for 10% to 15% of the oil phase, the mass of the monounsaturated fatty acid component accounts for 18% to 30% of the oil phase, and the mass of the saturated fatty acid component accounts for 20% to 35% of the oil phase.

[0042] In certain embodiments, the mass of the ω-3 fatty acid component accounts for about 32% of the oil phase, the mass of the ω-6 fatty acid component accounts for about 12% of the oil phase, the mass of the monounsaturated fatty acid component accounts for about 27% of the oil phase, and the mass of the saturated fatty acid component accounts for about 29% of the oil phase.

[0043] The ω-3 fatty acid component consists of one or more ω-3 (C 10 -C 24 alkyl-oligo-enoic acid) fatty acids, and the ω-3 fatty acids are characterized by the presence of more than one carbon double bond, where one carbon-carbon cis double bond is three atoms away from the terminal methyl group (exemplary structure of the ω-3 fatty acid α-linolenic acid (C18:3ω-3);

[0044]

[0045] In certain embodiments, the ω-3 fatty acid component consists of one or several members of the group consisting of α-linolenic acid and stearidonic acid.

[0046] The oil phase of the lipid emulsion contains ≥5% stearidonic acid (C18:4ω-3) as part of the ω-3 fatty acid component.

[0047]

[0048] In certain embodiments, the oil phase of the lipid emulsion contains about 10% stearidonic acid as part of the ω-3 fatty acid component.

[0049] The oil phase of the lipid emulsion contains ≥15% α-linolenic acid (ALA) (C18:3ω-3) as part of the ω-3 fatty acid component.

[0050] In certain embodiments, the oil phase of the lipid emulsion comprises about 20% alpha-linolenic acid as part of the omega-3 fatty acid component.

[0051] The omega-6 fatty acid component consists of one or more (C 10 -C 24 -alkyl-oligo-enoic acid) omega-6 fatty acids, which are characterized by the presence of more than one carbon double bond, with one carbon-carbon cis double bond six atoms from the terminal methyl group (exemplary structure of the omega-6 fatty acid linoleic acid (C18:2 omega-6));

[0052]

[0053] In certain embodiments, the omega-6 fatty acid component consists of one or more members of the group consisting of: linoleic acid and gamma-linolenic acid.

[0054] The monounsaturated fatty acid component consists of one or more fatty acids, which are characterized by the presence of one carbon-carbon double bond.

[0055] In certain embodiments, the monounsaturated fatty acid component comprises or consists of oleic acid (CAS number 112-80-1).

[0056] The saturated fatty acid component consists of one or more fatty acids, which are characterized by the absence of carbon-carbon double bonds and only carbon-carbon single bonds.

[0057] In certain embodiments, the saturated fatty acid component comprises or consists of one or more members of the group consisting of: caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, and palmitic acid.

[0058] In certain embodiments, the ratio (m / m) of the omega-6 fatty acid component to the omega-3 fatty acid component is from 1:5 to 2:1. In certain embodiments, the ratio (m / m) of the omega-6 fatty acid component to the omega-3 fatty acid component is from 1:4 to 1:1. In certain embodiments, the ratio (m / m) of the omega-6 fatty acid component to the omega-3 fatty acid component is from 1:3 to 1:2. In certain embodiments, the ratio (m / m) of the omega-6 fatty acid component to the omega-3 fatty acid component is about 1:2.6.

[0059] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0060] - 30% to 60% PUFA (polyunsaturated fatty acids);

[0061] - 5% to 45% MUFA (monounsaturated fatty acids); and

[0062] -5% to 50% SFA (saturated fatty acid).

[0063] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0064] -35% to 55% PUFA;

[0065] -10% to 40% MUFA; and

[0066] -10% to 45% SFA.

[0067] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0068] -40% to 50% PUFA;

[0069] -20% to 30% MUFA; and

[0070] -20% to 35% SFA.

[0071] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0072] -Approximately 44% PUFA;

[0073] -Approximately 27% MUFA; and

[0074] -Approximately 29% SFA.

[0075] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0076] -5% to 35% stearidonic acid (C18:4ω-3);

[0077] -5% to 50% oleic acid (C18:1);

[0078] -2% to 30% linoleic acid (C18:2ω-6);

[0079] -5% to 50% α-linolenic acid (C18:3ω-3); and

[0080] -0.5% to 15% γ-linolenic acid (C18:3ω-6).

[0081] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0082] -5% to 25% stearidonic acid;

[0083] -10% to 40% oleic acid;

[0084] -4% to 20% linoleic acid;

[0085] -10% to 40% alpha-linolenic acid; and

[0086] -1% to 10% gamma-linolenic acid.

[0087] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0088] -5% to 15% stearidonic acid;

[0089] -20% to 30% oleic acid;

[0090] -5% to 15% linoleic acid;

[0091] -20% to 30% alpha-linolenic acid; and

[0092] -2% to 5% gamma-linolenic acid.

[0093] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0094] - Approximately 10% stearidonic acid;

[0095] - Approximately 24% oleic acid;

[0096] - Approximately 9% linoleic acid;

[0097] - Approximately 22% alpha-linolenic acid; and

[0098] - Approximately 3% gamma-linolenic acid.

[0099] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0100] - 0.5% to 15% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid (C6:0, C8:0, C10:0);

[0101] - 3% to 35% lauric acid (C12:0);

[0102] - 1% to 15% myristic acid (C14:0); and

[0103] - 1% to 20% palmitic acid (C16:0).

[0104] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0105] - 1% to 10% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0106] - 5% to 25% lauric acid;

[0107] - 2% to 12% myristic acid; and

[0108] - 3% to 15% palmitic acid.

[0109] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0110] - 2% to 5% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0111] - 10% to 15% lauric acid;

[0112] - 3% to 8% myristic acid; and

[0113] - 5% to 12% palmitic acid.

[0114] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0115] - Approximately 3.5% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0116] - Approximately 12% lauric acid;

[0117] - Approximately 4.5% myristic acid; and

[0118] - Approximately 7.9% palmitic acid.

[0119] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0120] - 8% to 50% olive oil;

[0121] - 8% to 50% coconut oil; and

[0122] - 20% to 90% Buglossoides arvensis oil

[0123]

[0124] In certain embodiments, the oil phase of the lipid emulsion comprises:

[0125] - 12% to 40% olive oil;

[0126] - 12% to 40% coconut oil; and

[0127] - 30% to 70% Buglossoides arvensis oil In certain embodiments, the oil phase of the lipid emulsion comprises:

[0128] - 20% to 30% olive oil;

[0129] - 20% to 30% coconut oil; and

[0130] - 40% to 60% Buglossoides arvensis oil In certain embodiments, the oil phase of the lipid emulsion comprises:

[0131] - Approximately 25% olive oil;

[0132] - Approximately 25% coconut oil; and

[0133] - Approximately 50% Lithospermum arvense seed oil

[0134] In certain embodiments, the lipid emulsion further comprises a stabilizer and / or an antioxidant. In certain embodiments, the lipid emulsion further comprises a stabilizer and / or an antioxidant selected from:

[0135] - EDTA; and / or

[0136] - α-tocopherol.

[0137] In certain embodiments, the lipid emulsion further comprises a stabilizer and / or an antioxidant selected from:

[0138] - Approximately 2.5 μmol / L EDTA; and / or

[0139] - Approximately 200 mg / L α-tocopherol.

[0140] In certain embodiments, the lipid emulsion comprises

[0141] - Egg yolk lecithin;

[0142] - Glycerol; and

[0143] - Water.

[0144] In certain embodiments, the ratio (V / V) between the oil phase and the water phase ranges from 0.1 to 0.9. In certain embodiments, the ratio (V / V) between the oil phase and the water phase ranges from 0.2 to 0.8.

[0145] A second aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments for parenteral nutrition.

[0146] In certain embodiments, the parenteral nutrition is administered to patients in need of short-term or long-term total parenteral nutrition (TPN). In certain embodiments, the parenteral nutrition is administered to TPN patients suffering from metabolic diseases, especially insulin resistance. In certain embodiments, the parenteral nutrition is administered to TPN patients suffering from liver diseases. In certain embodiments, the parenteral nutrition is administered to TPN patients suffering from systemic acute and / or chronic inflammation. In certain embodiments, the parenteral nutrition is administered to TPN patients with impaired immune systems and reduced host defenses. In certain embodiments, the parenteral nutrition is administered to septic patients. In certain embodiments, the parenteral nutrition is administered to TPN patients undergoing chemotherapy.

[0147] An alternative of the second aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments, which is used for enteral nutrition.

[0148] In certain embodiments, enteral nutrition is administered to patients in need of short-term or long-term enteral nutrition. In certain embodiments, enteral nutrition is administered to patients suffering from metabolic diseases, especially insulin resistance. In certain embodiments, enteral nutrition is administered to patients suffering from liver diseases. In certain embodiments, enteral nutrition is administered to patients suffering from systemic acute and / or chronic inflammation. In certain embodiments, enteral nutrition is administered to patients with impaired immune systems and reduced host defenses. In certain embodiments, enteral nutrition is administered to septic patients. In certain embodiments, enteral nutrition is administered to patients receiving chemotherapy.

[0149] In certain embodiments, the lipid emulsion further comprises a drug. In certain embodiments, the drug has a molecular weight of <1000 g / mol, especially <500 g / mol, and complies with the Lipinsky Rules of Five.

[0150] In certain embodiments, the drug is selected from

[0151] - lipophilic drugs, especially where the lipophilic drugs are selected from diazepam, propofol, etomidate, alprostadil, dexamethasone, flurbiprofen, vitamins A, D, E, K, paclitaxel, cyclosporine, clarithromycin, phenobarbital, physostigmine, cinnarizine, chlorambucil, and docetaxel;

[0152] - RNA-based drugs using the lipid emulsion as a vehicle;

[0153] - RNA vaccines or DNA vaccines, and optionally adjuvants.

[0154] The third aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments, which is used as an antagonist / detoxifying agent ("lipid pool") for the treatment of poisoning caused by lipophilic drugs or lipophilic toxic compounds.

[0155] The fourth aspect of the present invention relates to a lipid emulsion according to the first aspect and its embodiments, which is used to protect vital organs from ischemia-reperfusion injury. In certain embodiments, the vital organs are selected from the heart, brain, liver, kidney, and lungs.

[0156] The fifth aspect of the present invention relates to a lipid emulsion according to the first aspect, which is used for the prevention or treatment of type II diabetes.

[0157] In certain embodiments, the lipid emulsion is formulated for parenteral administration.

[0158] In certain embodiments, the lipid emulsion is formulated for enteral or oral administration.

[0159] Medicine, Dosage Forms, and Salts

[0160] Similarly, within the scope of the present invention is a method of treating a patient in need thereof for a condition associated with an inability to take in food, the method comprising administering to the patient a lipid emulsion as described above.

[0161] Similarly, provided is a dosage form for treating a condition associated with an inability to take in food, comprising a non-agonist ligand or an antisense molecule according to any of the above aspects or embodiments of the present invention.

[0162] As used herein, "a condition associated with an inability to take in food" can refer to any condition in which a patient is temporarily or permanently unable to receive nutrients via natural (i.e., ingestion) means. Such conditions include unconsciousness (including coma), inability to swallow (e.g., due to a neurological disorder), having an obstructed esophageal passage (e.g., due to trauma, neoplastic disease, or other conditions in which the esophageal passage is restricted or non-functional).

[0163] Indications for total parenteral nutrition or partial parenteral nutrition cover a wide range of clinical conditions, such as critically ill patients (trauma, surgery, sepsis, shock), patients receiving home parenteral nutrition due to chronic intestinal failure, cachectic cancer patients, patients with inflammatory bowel disease (Crohn's disease, ulcerative colitis), patients with gastrointestinal obstruction, high-output enterocutaneous fistula or short bowel syndrome, (most) elderly patients with acute or chronic debilitating diseases who are unable to meet their nutritional requirements, and patients with intractable nausea and vomiting (hyperemesis gravidarum). In addition, malnutrition (calorie- and / or protein-related) is a common healthcare problem with a high prevalence (20% to 50%) among hospitalized patients, and is clearly associated with higher healthcare costs due to increased complications, longer hospital stays, and higher use of home healthcare services. In critically ill patients, supplemental parenteral nutrition to enteral nutrition aimed at meeting increased caloric requirements under stress is also thought to reduce complication rates and associated healthcare costs.

[0164] Those skilled in the art know that any specifically mentioned pharmaceutical compound herein can exist as a pharmaceutically acceptable salt of the said drug. Pharmaceutically acceptable salts comprise the ionized drug and a counterion of opposite charge. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, benzenesulfonate, tartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, ethoate, fumarate, glucoheptonate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, naphthalenesulfonate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethyl iodide, and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine penicillin, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0165] The dosage form can be used for parenteral administration. Optionally, a pharmaceutically acceptable carrier and / or excipient can be present.

[0166] Pharmaceutical Compositions and Administration

[0167] Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0168] In certain embodiments of the present invention, the compounds of the present invention are generally formulated into pharmaceutical dosage forms to provide an easily controllable drug dosage and to give a refined and easily handled product to the patient.

[0169] The dosage regimen of the compounds of the present invention will vary according to known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health status, medical condition, and weight of the recipient; the nature and degree of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic functions of the patient, and the desired effect. In certain embodiments, the compounds of the present invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses two, three, or four times a day.

[0170] In certain embodiments, for a subject weighing about 50 - 70 kg, the pharmaceutical composition or combination of the present invention may be a unit dose of about 1 - 1000 mg of the active ingredient. The therapeutically effective dose of the compound, pharmaceutical composition, or combination thereof depends on the species, body weight, age, and individual condition of the subject, the disorder or disease being treated, or the severity thereof. A physician, clinician, or veterinarian of ordinary skill can readily determine the effective amount of each active ingredient necessary to prevent, treat, or inhibit the progression of a disorder or disease.

[0171] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization; and / or can contain conventional inert diluents, lubricants, or buffering agents, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents, and buffering agents, etc. They can be produced by standard methods, for example, by conventional mixing, granulation, dissolution, or lyophilization methods. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al., "The Theory and Practice of Industrial Pharmacy", 4th Edition, 2013 (ISBN 8123922892).

[0172] Manufacturing and Treatment Methods According to the Invention

[0173] As a further aspect, the present invention further encompasses the use of the lipid emulsion as identified herein in a method of manufacturing a medicament for treating or preventing a condition associated with the inability to ingest food.

[0174] Similarly, the present invention encompasses a method of treating a patient diagnosed with a disease associated with the inability to ingest food. The method requires administering to the patient an effective amount of the lipid emulsion as identified herein.

[0175] Regardless of whether alternatives for individual separable features (such as, for example, lipid concentration or medical indication) are stated herein as "embodiments", it is to be understood that such alternatives can be freely combined to form discrete embodiments of the present invention disclosed herein. Thus, any alternative embodiment of lipid concentration can be combined with any alternative embodiment of the medical indication mentioned herein.

[0176] The present invention further encompasses the following items.

[0177] Items

[0178] 1. A lipid emulsion for administration to a patient, particularly for parenteral administration, wherein the lipid emulsion comprises an oil phase and an aqueous phase, and wherein the oil phase of the lipid emulsion comprises:

[0179] - an ω-3 fatty acid component,

[0180] o wherein the mass of the ω-3 fatty acid component accounts for 20% to 50% of the oil phase;

[0181] o wherein the ω-3 fatty acid component consists of one or more ω-3 fatty acids, and the ω-3 fatty acids are characterized by the presence of more than one carbon double bond, and one carbon-carbon double bond is three atoms away from the terminal methyl group;

[0182] o and wherein the oil phase of the lipid emulsion contains ≥5% stearidonic acid;

[0183] o and wherein the oil phase of the lipid emulsion contains ≥15% α-linolenic acid (ALA);

[0184] - ω-6 fatty acid component,

[0185] o wherein the mass of the ω-6 fatty acid component accounts for 3% to 35% of the oil phase;

[0186] o wherein the ω-6 fatty acid component consists of one or more ω-6 fatty acids, and the ω-6 fatty acids are characterized by the presence of more than one carbon double bond, and one carbon-carbon double bond is six atoms away from the terminal methyl group;

[0187] - monounsaturated fatty acid component,

[0188] o wherein the mass of the monounsaturated fatty acid component accounts for 5% to 40% of the oil phase;

[0189] o wherein the monounsaturated fatty acid component consists of one or more fatty acids, and the fatty acids are characterized by the presence of one carbon-carbon double bond;

[0190] - saturated fatty acid component,

[0191] o wherein the mass of the saturated fatty acid component accounts for 5% to 45% of the oil phase;

[0192] o wherein the saturated fatty acid component consists of one or more fatty acids, and the fatty acids are characterized by the absence of carbon-carbon double bonds and only carbon-carbon single bonds.

[0193] 2. The lipid emulsion according to item 1, wherein the ω-3 fatty acid component consists of one or several members selected from the group consisting of α-linolenic acid and stearidonic acid.

[0194] 3. The lipid emulsion according to any one of the preceding items, wherein

[0195] - the oil phase of the lipid emulsion contains about 10% stearidonic acid; and

[0196] - The oil phase of the lipid emulsion contains about 20% alpha-linolenic acid (ALA).

[0197] 4. The lipid emulsion according to any one of the preceding items, wherein the ω-6 fatty acid component consists of one or several members of the group consisting of linoleic acid and gamma-linolenic acid.

[0198] 5. The lipid emulsion according to any one of the preceding items, wherein the monounsaturated fatty acid component contains oleic acid or consists of oleic acid.

[0199] 6. The lipid emulsion according to any one of the preceding items, wherein the saturated fatty acid component consists of one or several members of the group consisting of: caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, and palmitic acid or consists of one or several members of the group consisting of the following.

[0200] 7. The lipid emulsion according to any one of the preceding items, wherein

[0201] - The mass of the ω-3 fatty acid component accounts for 20% to 40% of the oil phase;

[0202] - The mass of the ω-6 fatty acid component accounts for 5% to 25% of the oil phase;

[0203] - The mass of the monounsaturated fatty acid component accounts for 10% to 35% of the oil phase;

[0204] - The mass of the saturated fatty acid component accounts for 10% to 40% of the oil phase.

[0205] 8. The lipid emulsion according to any one of the preceding items, wherein

[0206] - The mass of the ω-3 fatty acid component accounts for 25% to 35% of the oil phase;

[0207] - The mass of the ω-6 fatty acid component accounts for 10% to 15% of the oil phase;

[0208] - The mass of the monounsaturated fatty acid component accounts for 18% to 30% of the oil phase;

[0209] - The mass of the saturated fatty acid component accounts for 20% to 35% of the oil phase.

[0210] 9. The lipid emulsion according to any one of the preceding items, wherein

[0211] - The mass of the ω-3 fatty acid component accounts for about 32% of the oil phase;

[0212] - The mass of the ω-6 fatty acid component accounts for about 12% of the oil phase;

[0213] - The mass of the monounsaturated fatty acid component accounts for about 27% of the oil phase;

[0214] - The mass of the saturated fatty acid component accounts for about 29% of the oil phase.

[0215] 10. The lipid emulsion according to any one of the preceding items, wherein the ratio (m / m) of the ω-6 fatty acid component to the ω-3 fatty acid component is from 1:5 to 2:1.

[0216] 11. The lipid emulsion according to any one of the preceding items, wherein the ratio (m / m) of the ω-6 fatty acid component to the ω-3 fatty acid component is from 1:4 to 1:1.

[0217] 12. The lipid emulsion according to any one of the preceding items, wherein the ratio (m / m) of the ω-6 fatty acid component to the ω-3 fatty acid component is from 1:3 to 1:2.

[0218] 13. The lipid emulsion according to any one of the preceding items, wherein the ratio (m / m) of the ω-6 fatty acid component to the ω-3 fatty acid component is about 1:2.6.

[0219] 14. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0220] - 30% to 60% PUFA;

[0221] - 5% to 45% MUFA; and

[0222] - 5% to 50% SFA.

[0223] 15. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0224] - 35% to 55% PUFA;

[0225] - 10% to 40% MUFA; and

[0226] - 10% to 45% SFA.

[0227] 16. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0228] - 40% to 50% PUFA;

[0229] - 20% to 30% MUFA; and

[0230] - 20% to 35% SFA.

[0231] 17. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0232] - Approximately 44% PUFA;

[0233] - Approximately 27% MUFA; and

[0234] - Approximately 29% SFA.

[0235] 18. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0236] - 5% to 35% stearidonic acid;

[0237] - 5% to 50% oleic acid;

[0238] - 2% to 30% linoleic acid;

[0239] - 5% to 50% α-linolenic acid; and

[0240] - 0.5% to 15% γ-linolenic acid.

[0241] 19. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0242] - 5% to 25% stearidonic acid;

[0243] - 10% to 40% oleic acid;

[0244] - 4% to 20% linoleic acid;

[0245] - 10% to 40% α-linolenic acid; and

[0246] - 1% to 10% γ-linolenic acid.

[0247] 20. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0248] - 5% to 15% stearidonic acid;

[0249] - 20% to 30% oleic acid;

[0250] - 5% to 15% linoleic acid;

[0251] - 20% to 30% α-linolenic acid; and

[0252] - 2% to 5% γ-linolenic acid.

[0253] 21. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0254] - Approximately 10% eicosatetraenoic acid;

[0255] - Approximately 24% oleic acid;

[0256] - Approximately 9% linoleic acid;

[0257] - Approximately 22% α-linolenic acid; and

[0258] - Approximately 3% γ-linolenic acid.

[0259] 22. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0260] - 0.5% to 15% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0261] - 3% to 35% lauric acid;

[0262] - 1% to 15% myristic acid; and

[0263] - 1% to 20% palmitic acid.

[0264] 23. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0265] - 1% to 10% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0266] - 5% to 25% lauric acid;

[0267] - 2% to 12% myristic acid; and

[0268] - 3% to 15% palmitic acid.

[0269] 24. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0270] - 2% to 5% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0271] - 10% to 15% lauric acid;

[0272] - 3% to 8% myristic acid; and

[0273] - 5% to 12% palmitic acid.

[0274] 25. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0275] - Approximately 3.5% of a short-chain fatty acid component selected from caproic acid, caprylic acid, and capric acid;

[0276] - Approximately 12% lauric acid;

[0277] - Approximately 4.5% myristic acid; and

[0278] - Approximately 7.9% palmitic acid.

[0279] 26. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0280] - 8% to 50% olive oil;

[0281] - 8% to 50% coconut oil; and

[0282] - 20% to 90% Lithospermum arvense seed oil.

[0283] 27. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0284] - 12% to 40% olive oil;

[0285] - 12% to 40% coconut oil; and

[0286] - 30% to 70% Lithospermum arvense seed oil.

[0287] 28. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0288] - 20% to 30% olive oil;

[0289] - 20% to 30% coconut oil; and

[0290] - 40% to 60% Lithospermum arvense seed oil.

[0291] 29. The lipid emulsion according to any one of the preceding items, wherein the oil phase of the lipid emulsion comprises:

[0292] - Approximately 25% olive oil;

[0293] - Approximately 25% coconut oil; and

[0294] - Approximately 50% Lithospermum arvense seed oil.

[0295] 30. The lipid emulsion according to any one of the preceding items, wherein the lipid emulsion further comprises a stabilizer and / or an antioxidant.

[0296] 31. The lipid emulsion according to any one of the preceding items, wherein the lipid emulsion further comprises a stabilizer and / or an antioxidant selected from:

[0297] - EDTA; and / or

[0298] - α-tocopherol.

[0299] 32. The lipid emulsion according to any one of the preceding items, wherein the lipid emulsion further comprises a stabilizer and / or an antioxidant selected from the following:

[0300] - about 2.5 μmol / L EDTA; and / or

[0301] - about 200 mg / L α-tocopherol.

[0302] 33. The lipid emulsion according to any one of the preceding items, wherein the lipid emulsion comprises

[0303] - egg yolk lecithin;

[0304] - glycerol; and

[0305] - water.

[0306] 34. The lipid emulsion according to any one of the preceding items, wherein the ratio (V / V) between the oil phase and the water phase is in the range of 0.1 to 0.9.

[0307] 35. The lipid emulsion according to any one of the preceding items, wherein the ratio (V / V) between the oil phase and the water phase is in the range of 0.2 to 0.8.

[0308] 36. The lipid emulsion according to any one of the preceding items, which is for parenteral nutrition.

[0309] 37. The lipid emulsion for parenteral nutrition according to item 36, wherein the parenteral nutrition is administered to a patient having one or more of the following indications:

[0310] - patients in need of short-term and long-term total parenteral nutrition (TPN); and / or

[0311] - TPN patients suffering from metabolic diseases, especially insulin resistance; and / or

[0312] - TPN patients suffering from liver diseases; and / or

[0313] - TPN patients suffering from systemic acute and / or chronic inflammation, and / or

[0314] - TPN patients with reduced host defense.

[0315] 38. The lipid emulsion according to any one of the preceding items 1 to 35, which further comprises a drug.

[0316] 39. The lipid emulsion according to item 38, wherein the drug satisfies Lipinski's rule of five and has a molecular weight of < 500 g / mol.

[0317] 40. The lipid emulsion according to item 38 or 39, wherein the drug is selected from

[0318] - lipophilic drugs, especially where the lipophilic drugs are selected from diazepam, propofol, etomidate, alprostadil, dexamethasone, flurbiprofen, vitamins A, D, E, K, paclitaxel, cyclosporine, clarithromycin, phenobarbital, physostigmine, cinnarizine, chlorambucil, and docetaxel;

[0319] - RNA-based drugs using the lipid emulsion as a vehicle;

[0320] - RNA vaccines or DNA vaccines, and optionally adjuvants.

[0321] 41. The lipid emulsion according to any one of the preceding items 1 to 35, which is used as an antagonist / detoxifying agent for treating poisoning caused by lipophilic drugs.

[0322] 42. The lipid emulsion according to any one of the preceding items 1 to 35, which is used to protect vital organs, especially vital organs selected from the heart, brain, liver, kidneys, and lungs, from ischemia-reperfusion injury.

[0323] 43. The lipid emulsion according to any one of the preceding items 1 to 35, which is used to prevent or treat type II diabetes.

[0324] 44. The lipid emulsion according to any one of the preceding items or the lipid emulsion used, wherein the lipid emulsion is formulated for parenteral administration.

[0325] 45. The lipid emulsion according to any one of the preceding items 1 to 35 or 41 to 43 or the lipid emulsion for use, wherein the lipid emulsion is formulated for enteral or oral administration.

[0326] 46. The lipid emulsion according to any one of the preceding items 1 to 35, which is used for enteral nutrition.

[0327] 47. The lipid emulsion for enteral nutrition according to item 46, wherein the enteral nutrition is administered to patients having one or more of the following indications:

[0328] - patients in need of short-term and long-term enteral nutrition; and / or

[0329] - patients suffering from metabolic diseases, especially insulin resistance; and / or

[0330] - patients suffering from liver diseases; and / or

[0331] - patients suffering from systemic acute and / or chronic inflammation, and / or

[0332] - Patients with reduced host defense.

[0333] The present invention is further illustrated by the following examples and drawings, from which further embodiments and advantages can be derived. These examples are intended to illustrate the invention and not to limit its scope. Brief Description of the Drawings

[0334] Figure 1 Shows the fatty acid composition of F3 (a novel lipid emulsion, TPN-F3, F3, VV-TPN, and VV are used synonymously throughout the specification) as determined by gas chromatography.

[0335] Figure 2 Shows lipopolysaccharide-binding protein (LBP, Figure A), the correlation of LBP with the ratio of IL-6 to IL-10 (Figure B), suppressor of cytokine signaling 3 (SOCS3, Figure C), and the correlation of SOCS3 with the ratio of IL-6 to IL-10 (Figure D) in the total liver tissue of mice treated with different types of total parenteral nutrition (TPN) for 7 days. A lower ratio of IL-6 to IL-10 indicates a lower degree of inflammation in the liver tissue. Protein abundance was determined by immunoblotting and normalized relative to vinculin. The Y-axis represents arbitrary relative units. TPN-IL = mice treated with Intralipid-based TPN. TPN-OV = mice treated with Omegaven-based TPN. TPN-F3 = mice treated with F3-based TPN. * Significantly increased compared to TPN-IL, # significantly increased compared to TPN-OV. Box plots show the median and the 25th and 75th percentiles. Bars represent mean ± SD. For TPN-IL, N = 7; for TPN-OV, N = 7; for TPN-F3, N = 6.

[0336] Figure 3 Shows the transcription factors PPARα (Figure A), PPARγ1 (Figure B), and PPARγ2 (Figure C) in the nuclear fraction of the total liver tissue from mice treated with different types of total parenteral nutrition (TPN) for 7 days. Protein abundance was determined by immunoblotting and normalized relative to the nuclear marker TATA-binding protein (TBP). The Y-axis represents arbitrary relative units. TPN-IL = mice treated with Intralipid-based TPN. TPN-OV = mice treated with Omegaven-based TPN. TPN-F3 = mice treated with F3-based TPN. * Significantly different from TPN-IL, # significantly decreased compared to TPN-OV. Bars represent mean ± SD. Box plots show the median plus the 25th and 75th percentiles. For each group, N = 6.

[0337] Figure 4Shows HOMA-IR (Homeostatic Model Assessment of Insulin Resistance, Figure A), blood glucose (Figure B), plasma insulin concentration (Figure C), and liver glycogen levels (Figure D) in mice treated with different types of total parenteral nutrition (TPN) for 7 days. Results were measured by standard methods (see reference #2 for details). Higher HOMA-IR indicates reduced insulin sensitivity. HOMA-IR was calculated from plasma insulin and whole blood glucose using a normalization factor of 14.1, which is the adjustment factor for C57BL / 6J mice. Liver glycogen is a reliable indicator of insulin signaling and insulin sensitivity in the liver: the higher the glycogen level, the more effective the insulin signaling. TPN-IL = mice treated with Intralipid-based TPN. TPN-OV = mice treated with Omegaven-based TPN. TPN-F3 = mice treated with F3-based TPN. # Significantly increased to TPN-OV, * Significantly increased to TPN-IL. Bars represent mean ± SD. Box plots represent median plus 25th and 75th percentiles. For each group, N = 12.

[0338] Figure 5 Shows the abundance of insulin receptor β subunit (IRβ, Figure A), insulin receptor substrate 1 (IRS1, Figure B), and insulin receptor substrate 2 (IRS2, Figure C) in total liver tissue homogenates of mice treated with different types of total parenteral nutrition (TPN) for 7 days. IRβ abundance was measured by enzyme-linked immunosorbent assay. IRS protein abundance was measured by immunoblotting and normalized relative to vinculin. The Y-axis represents arbitrary relative units. TPN-IL = mice treated with Intralipid-based TPN. TPN-OV = mice treated with Omegaven-based TPN. TPN-F3 = mice treated with F3-based TPN. * Significantly increased to TPN-IL, # Significantly increased to TPN-OV. Bars represent mean ± SD. Box plots represent median plus 25th and 75th percentiles. For each group, N = 12.

[0339] Figure 6Shows glycogen synthase (GS, Figure A), phosphorylation of glycogen synthase at serine 641 (Figure B), glucokinase (GCK, Figure C) and its nuclear (inactive) and cytoplasmic (active) fractions (Figure D) in the liver tissue of mice treated with different types of total parenteral nutrition (TPN) for 7 days. Protein abundance was determined by immunoblotting and the nuclear fraction was normalized relative to vinculin or TBP, respectively. Chow = tissue samples from chow-fed mice as control samples. TPN-IL = mice treated with Intralipid-based TPN. TPN-OV = mice treated with Omegaven-based TPN. TPN-F3 = mice treated with F3-based TPN. § Significantly reduced relative to all other groups. * Significantly increased relative to TPN-OV. Box plots represent the median plus the 25th and 75th percentiles. For each group, N = 6.

[0340] Figure 7 Shows the results of the ferrous oxidation-xylenol orange (FOX) assay. The content of primary oxidation products in Intralipid, Omegaven and three different batches of F3 lipid emulsions changes over time.

[0341] Figure 8 Shows the sterol content (Figure A) in Intralipid (IL), Omegaven (OV) and formulation #3 (F3). Phytosterols and phytostanols (phytosterols, Figure C) are plant-derived compounds that are structurally related to cholesterol (Figure B, an animal-derived sterol). The higher level of cholesterol in Intralipid (compared to F3) may be due to the purification process used for egg lecithin in Intralipid (compared to Lipoid 80 used in F3). Stigmasterol (Figure D) is an unsaturated phytosterol commonly present in soybeans. Other phytosterols include campesterol (Figure E) and β-sitosterol (Figure F).

[0342] Figure 9Shows the anti-inflammatory effects of TPN-F3 in key insulin-sensitive tissues. Interleukin-6 (IL6; Figures A, D, G, J), interleukin-10 (IL10; Figures B, E, H, K), and IL6 / IL10 ratio (Figures C, F, I, L) in total tissue homogenates from the liver (Figures A, B, C), skeletal muscle (Figures D, E, F), epididymal white adipose tissue (WAT) (Figures G, H, I), and pancreas (Figures J, K, L) of mice treated with different types of total parenteral nutrition (TPN) for 7 days. Cytokine concentrations were measured by enzyme-linked immunosorbent assay. IV-chow, mice with a chow diet and continuous saline infusion; IL-TPN, mice treated with Intralipid-based TPN; OV-TPN, mice treated with Omegaven-based TPN; VV-TPN, mice treated with TPN-F3-based TPN. #, significantly increased relative to all other groups; @, significantly different from VV-TPN; **, significantly different from IL-TPN; §, significantly decreased relative to IV-chow. Bars represent mean ± SD. N = 6 per group.

[0343] Figure 10 Shows the regulation of glycogenesis in different TPN regimens. Panel A: Glycogen synthase (GS), phosphorylation of GS at serine 641, and representative immunoblots in liver tissues of mice treated with different types of total parenteral nutrition (TPN) for 7 days. Panels B to D: Abundance of glucokinase (GCK) in total tissue lysates (Panel B), cytoplasmic (active) fraction (Panel C), and nuclear (inactive) fraction (Panel D) with representative immunoblots. For total tissue lysates or cytoplasmic fractions, protein expression was normalized to vinculin, or for nuclear fractions, protein expression was normalized to TATA-binding protein (TBP). Chow, chow-fed mice (control samples); IL-TPN, mice treated with Intralipid-based TPN; OV-TPN, mice treated with Omegaven-based TPN; VV-TPN, mice treated with TPN-F3-based TPN. # significantly decreased relative to all other groups; * significantly increased relative to chow; ** significantly increased relative to TPN-OV. Bars represent mean ± SD. N = 6 per group.

[0344] Figure 11Interleukin-10-mediated insulin signaling in TPN based on TPN-F3 is shown. Effects of neutralizing anti-IL10 treatment on IL6, IL10 and their respective ratios (Figure A), hepatic glycogen levels (Figure B), IRS2 protein expression and its tyrosine phosphorylation (Figures C and D), glucokinase (GCK) in total tissue lysates (Figures E and G), and inactive (nuclear) GCK (Figure F) in mice treated with VV-TPN for 7 days. Protein abundance was determined by immunoblotting and normalized to vinculin and TATA-binding protein (TBP) for nuclear fractions, respectively. VV-TPN, mice treated with TPN-F3-based TPN; VV-TPN (IgG), mice treated with VV-based TPN and concomitant isotype control antibody; VV-TPN (anti-IL10), mice treated with VV-based TPN and concomitant neutralizing anti-IL10 treatment. * Significantly different from VV-TPN. # Significantly different from VV-TPN (IgG). Bars represent mean ± SD. For each group, N = 5 - 6. Note: Isotype control antibody IgG has previously been shown to exert some anti-IL10 effects by binding to immune cells via Fc receptors.

[0345] Figure 12 Microbiome analysis of intestinal mucosal samples from the colon of mice treated with IL-TPN, OV-TPN, and VV-TPN for 7 days compared to control mice is shown. Figure A: TPN altered the relative bacterial abundance at the phylum level, significantly expanding Bacteroidetes at the expense of Firmicutes. Figure B: Alpha diversity at the phylum level was significantly affected by TPN containing Intralipid (IL-TPN) and Omegaven (OV-TPN), but significantly less so by TPN-F3 (VV-TPN). Figure C: Verrucomicrobiota (i.e., Akkermansia muciniphila) was significantly increased in mice receiving IL-TPN and OV-TPN compared to mice receiving VV-TPN. chow (C), control mice on chow diet; IV-chow (S), mice on chow diet and continuous saline infusion; IL-TPN (IL), mice treated with Intralipid-based TPN; OV-TPN (OV), mice treated with Omegaven-based TPN; VV-TPN (VV), mice treated with TPN-F3-based TPN. *, significantly different from chow and IV-chow; @, significantly different from VV-TPN, N = 6 per group. Figures were generated using MicrobiomeAnalyst.

[0346] Figure 13 CD4 in the liver tissue of VV-TPN mice is shown+ T cell characteristics. Panel A: Proportion of naive T cells (CD44 低 / CD62L 高 ) and "antigen-experienced" T cells, which are cells that express low levels of CD62L / L-selectin and include effector memory (CD44 高 / CD62L 低 ) T cells. Note that the abundance of central memory (CD44 高 / CD62L 高 ) CD4 + T cells is very low in the liver. Panels B–D: Intracellular staining for cytokine production in activated hepatic CD4 + T cells in response to phorbol myristate acetate (PMA) and ionomycin. Intracellular staining was performed to measure the expression of interferon-γ (IFNγ; Panels B and C) and interleukin-17A (IL17A; Panels D and E) produced by CD4 + T cells. Data are presented as mean fluorescence intensity ratio (MFI ratio; Panels B and D) and percentage of CD4 + T cells that are positively stained (CD4 + T cell %; Panels C and E). Chow, control mice on chow diet; IL-TPN, mice treated with Intralipid-based TPN; VV-TPN, mice treated with TPN-F3-based TPN. Bars represent mean ± SE. Dots represent individual experiments. For each group, N = 3–4.

[0347] Figure 14 Lipid mediators in liver tissue of mice treated with different TPNs are shown. Panel A: Heat map of measured lipid mediators. Columns correspond to study groups, and rows correspond to lipid mediators derived from polyunsaturated fatty acid (PUFA) precursors annotated on the left of the heat map. Data are color-coded according to the z-score representing the relative amount of each lipid mediator. n-3 PUFA precursors are α-linolenic acid (ALA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). n-6 PUFA precursors are dihomo-γ-linolenic acid (DGLA), linoleic acid (LA), and arachidonic acid (AA). The 15-lipoxygenase metabolite of ALA, 9S-hydroxy-9Z,11E,15Z-octadecatrienoic acid (9(S)-HOTrE), is highlighted.

[0348] Figure 15Shows the supplementation of Intralipid-based TPN with 9(S) / 13S-hydroxy-9Z,11E,15Z-octadecatrienoic acid (9(S) / 13(S)-HOTrE) mimicking the phenotype induced by TPN-F3-based TPN. Mice receiving IL-TPN were treated with 9 / 13-hydroxy-octadecatrienoic acid (5 ng / mL) added to the TPN mixture. In total liver tissue homogenates, plasma concentration of interleukin-10 (IL10; Figure A), tissue level of interleukin-6 (IL6; Figure B), tissue level of interleukin-10 (Figure C), IL6 / IL10 ratio (Figure D). Hepatic glycogen content (Figure E), abundance of insulin receptor substrate 2 protein (IRS2; Figure F), abundance of tyrosine-phosphorylated IRS2 (Figure G), and respective ratios (Figure H). IL-TPN, mice treated with Intralipid-based TPN; VV-TPN, mice treated with TPN-F3-based TPN; IL-TPN+HOTrE, mice treated with Intralipid-based TPN supplemented with 9(S) / 13(S)-HOTrE. Bars represent mean ± SD. N = 4 - 6 per group.

[0349] Table 1 shows the composition of the oil phase in commonly used lipid emulsions for total parenteral nutrition (amounts given per 100 mL). The emulsions are 20%, except for Omegaven which is only available as a 10% emulsion. Amounts are given per 100 mL. Intralipid, SMOFlipid, and Omegaven are manufactured by Fresenius Kabi (Bad Homburg, Germany); Lipofundin is manufactured by B. Braun (Melsungen, Germany); ClinOleic is manufactured by Baxter Healthcare Corporation (Deerfield, Illinois, USA). All emulsions use egg yolk lecithin as an emulsifier and glycerol to adjust the osmotic pressure. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.

[0350] Table 2 shows the composition of F3, listing the major fatty acids present in the lipid emulsion

[0351] Examples

[0352] Hereinafter, TPN-F3, F3, VV-TPN, and VV are used synonymously.

[0353] Example 1: Composition of the Oil Phase in a Conventional Lipid Emulsion

[0354] Table 1: Composition of the oil phase in common lipid emulsions for total parenteral nutrition (amounts given per 100 mL)

[0355]

[0356]

[0357] SMOFlipid was created to take into account the recommendations regarding the optimal dietary intake of polyunsaturated fatty acids, whereby the n-6:n-3 ratio should be between 1:1 and 4:1. The oil phase of the newly developed F3 also takes these dietary requirements into account and shows an n-6:n-3 ratio of 1:2.6. However, it consists only of non-GMO (without genetic engineering) vegetable oils, one of which is Ahiflower oil. Ahiflower oil is a rich single plant source of n-3 alpha-linolenic acid (ALA) and stearidonic acid (SDA), as well as n-6 gamma-linolenic acid (GLA) and linoleic acid (LA). Ahiflower oil contains 17% to 20% SDA, the highest level of naturally occurring SDA in any commercially available dietary vegetable oil. Oils with a high SDA content are increasingly considered to be excellent sources of n-3 fatty acids that affect inflammation and metabolism. In addition, the degree of unsaturation of SDA is lower than that of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) present in fish oil, making it more stable when present in lipid emulsions. The oil blend of F3 contains 50% Ahiflower and thus contains approximately 10% SDA and approximately 20% ALA. See details in Table 2 below.

[0358] Table 2 : Composition of F3 listing the major fatty acids present in the lipid emulsion

[0359]

[0360] F3 also contains EDTA (2.5 μM) and tocopherol (160 - 200 mg / L) to stabilize the unsaturated fatty acids and prevent oxidation.

[0361] Example 2: Problems Commonly Associated with TPN

[0362] Infusion of fatty acid-containing lipid emulsions typically causes insulin resistance, promotes lipid accumulation, and enhances tissue inflammation (especially interleukin-6 release). In addition, the use of lipid emulsions in TPN is associated with impaired immune responses and a higher incidence of infections. For a detailed review, see Lucchinetti et al., "Novel Strategies to Prevent Total Parenteral Nutrition-Induced Gut and Liver Inflammation, and Adverse Metabolic Outcomes," Mol Nutr Food Res. 2021;65(5):e1901270. doi:10.1002 / mnfr.201901270. PMID:32359213. F3 is a newly developed lipid emulsion that has unique beneficial effects on inflammation, insulin signaling, and immune responses and has reduced side effects, especially when used in the context of TPN.

[0363] Example 3: TPN-F3 Elicits a Potent Anti-Inflammatory Effect in Key Metabolic Insulin-Sensitive Tissues during TPN Compared to a Standard Lipid Emulsion Figure 9

[0364] After 7 days of TPN, body weights in the TPN group were similar and consistent with previous reports using the same TPN mouse model. Compared with IL-TPN and OV-TPN, VV-TPN increased the plasma concentration of IL10. VV-TPN further decreased the concentration of the pro-inflammatory cytokine IL6 in liver tissue and, concomitantly, increased the production of the anti-inflammatory cytokine IL10, significantly reducing the IL6 / IL10 ratio ( Figure 9 ). The IL6 / IL10 ratio was higher in the liver tissue of IL-TPN and OV-TPN mice. A similar pattern of the IL6 / IL10 ratio was observed in skeletal muscle, epididymal white adipose (eWAT), and pancreatic tissues ( Figure 2 ). Compared with IL-TPN, VV-TPN also decreased TNFα in liver tissue. VV-TPN, but not IL-TPN or OV-TPN, increased the production of lipopolysaccharide-binding protein (LBP) in the liver, a protein that detoxifies endotoxin (LPS) via high-density lipoprotein (HDL) and chylomicrons. Higher LBP levels are associated with lower inflammation, as demonstrated by the lower IL6 / IL10 ratio, which implies that LBP clears endotoxin released from the leaky gut, resulting in reduced production of IL6 in the liver ( Figure 2A and 2B). Similarly, suppressor of cytokine signaling 3 (SOCS3), a protein that inhibits IL6 signaling, was increased in the livers of VV-TPN mice and was negatively correlated with the IL6 / IL10 ratio ( Figure 3 C and 2D). Similar to OV-TPN, VV-TPN elevated nuclear PPARα, a transcription factor known to increase hepatic fatty acid oxidation and reduce fat-induced liver inflammation, while no differences in nuclear PPARγ1 were observed among the different TPN groups ( Figure 3 A and 3B). Only VV-TPN decreased nuclear PPARγ2, a transcription factor known to promote lipid accumulation and pro-inflammatory cytokine production in the liver and cause steatohepatitis ( Example 4: F3 Promotes Insulin Signaling, Particularly in the Liver, and Further Improves Systemic Glucose Tolerance during TPN C). Compared with IL-TPN, VV-TPN and OV-TPN also reduced the LPS content in epididymal white adipose tissue (eWAT), but only VV-TPN significantly reduced the protein expression of pro-inflammatory NF k B, consistent with its reduced IL6 / IL10 ratio. Finally, VV-TPN also increased anti-inflammatory IL4 in liver and pancreatic tissues. In summary, compared with IL-TPN and OV-TPN, VV-TPN exerted the strongest anti-inflammatory effect in multiple key metabolic insulin-sensitive tissues.

[0365] Figure 4 Figure 4

[0366] TPN impairs insulin signaling, leading to systemic insulin resistance. Compared with TPN-IL, TPN-F3 and TPN-OV improved the systemic insulin response, as evaluated by HOMA-IR (homeostatic model assessment of insulin resistance) ( Figure 5 A to 4C). However, only TPN-F3 preserved insulin signaling in the liver, as demonstrated by the normalized level of glycogen, a reliable indicator of hepatic insulin sensitivity ( Figure 5 D). Compared with TPN-IL, TPN-F3 and TPN-OV increased the abundance of insulin receptors in liver tissue ( Figure 10 A), but only TPN-F3 increased the abundance of insulin receptor substrate 2 (IRS2), while IRS1 remained unchanged ( Figure 10 B and 5C and Table 4). TPN-IL significantly reduced the abundance of glycogen synthase in liver tissue, while TPN-F3 and TPN-OV did not ( Figure 10 A). No differences in the phosphorylation status of hepatic glycogen synthase at Ser461, the phosphorylation site that causes its inhibition, were noted among the three TPNs ( Figure 10 A). However, compared with TPN-OV, TPN-F3 showed a higher abundance of glucokinase ( Figure 11B), which is the rate-limiting step in glycogen formation in the liver, and a higher proportion of active cytosolic glucokinase in TPN-F3 ( Figure 11 C), which explains the higher glycogen storage in the liver tissues of mice treated with F3. Collectively, these findings highlight the enhanced insulin sensitivity of TPN-F3 in the liver compared to TPN-IL or TPN-OV. Administration of a neutralizing IL-10 antibody to TPN-F3 increased the IL-6 / IL-10 ratio to the value observed in TPN-IL ( Figure 11 A), and decreased hepatic glycogen storage due to an increased nuclear fraction of glucokinase ( Example 5: F3, but Not IL or OV, Promotes a Potent Host Defense against Invasive Bacteria Such as Akkermansia muciniphila during TPN F), which is consistent with impaired insulin signaling. Overall, these findings highlight the crucial role of elevated IL-10 production in liver tissues for maintaining insulin signaling in mice treated with TPN-F3 and further mechanistically link the metabolic benefits of TPN-F3 to its anti-inflammatory cytokine profile. Figure 13 B), which is consistent with impaired insulin signaling. Overall, these findings highlight the crucial role of elevated IL-10 production in liver tissues for maintaining insulin signaling in mice treated with TPN-F3 and further mechanistically link the metabolic benefits of TPN-F3 to its anti-inflammatory cytokine profile.

[0367] Table 4: Metabolic data

[0368]

[0369]

[0370] https: / / www.microbiomeanalyst.ca /

[0371] TPN causes depletion of immune cells and a dysfunctional cytokine response. However, in contrast to TPN-IL, F3-TPN induces an "activated" phenotype of resident liver macrophages (Kupffer cells) with M1-like polarization and increases the percentage of CD4+ T cells producing INFγ and IL17 ( Figure 12 ), which is consistent with the previously reported higher immune responses of insulin-sensitive macrophages and CD4+ T cells compared to insulin-resistant macrophages and CD4+ T cells. Consistent with this observation, the hepatic tissue concentration of INFγ (a key cytokine for effective host defense) was also elevated in mice treated with TPN-F3 compared to those treated with TPN-IL or TPN-OV.

[0372] A detailed gut microbiome analysis based on 16S RNA sequencing was performed. For this purpose, mucosal samples were collected from the colon at the end of the 7-day experimental period. DNA was extracted according to the protocol of the ZymoBIOMICS DNA Miniprep kit (D4300, ZymoResearch Corp; Irvine, CA, USA). Amplification of the V3-V4 hypervariable region was performed with the standard primers 341F and 805R. Libraries were prepared at the Fasteris facility (Genesupport / Fasteris SA, Plan-les-Ouates, Switzerland) using the Metafast protocol. 16S rRNA gene sequences were clustered into operational taxonomic units (OTUs) and mapped to the SILVA database. All data analyses were performed using the web-based tool MicrobiomeAnalyst.ca( Figure 12 Figure 13 ). The following results were obtained. As shown previously, TPN decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes( Figure 13 ). This analysis further revealed significant changes in the Verrucomicrobiota. This phylum encompasses and is defined by Akkermansia, with the mucin-degrading bacterium Akkermansia muciniphila being the only and most prominent member. Akkermansia muciniphila is a symbiotic bacterium that becomes pathogenic during gut dysbiosis and IL-10 deficiency. Importantly, in contrast to mice treated with TPN-IL or TPN-OV, it was inhibited from invading the intestinal wall in mice treated with TPN-F3, providing evidence for stronger host defense when treated with TPN-F3( Figure 13 ).

[0373] To better understand the effect of TPN containing different lipid emulsions on host defense, the abundance and phenotype of immune cells in tissues were determined. IL-TPN was chosen for direct comparison with VV-TPN because its immunometabolic phenotype is significantly different from that of OV-TPN, which exhibits an intermediate phenotype. TPN generally causes a decrease in leukocytes (CD45+) in various organs including the intestine, which typically undergoes significant atrophy. Analysis of the immune cell profiles of the spleen, mesenteric lymph nodes, and liver showed a defect in TNFα response in PMA / ionomycin-stimulated CD4+ T cells (except for VV-TPN in the liver) compared to mice fed a diet. However, in contrast to IL-TPN, VV-TPN increased antigen experience, namely primed effector and effector memory CD4+ T cells (CD44 低 / CD62L 低 , CD44 高 / CD62L 低 )( Example 6: TPN-F3 Elicits a Distinct Profile of Lipid Mediators in Liver Tissue, and 9-Hydroxy-Octadecatrienoic Acid (a Lipid Mediator of 18-Carbon ALA) Contributes to the TPN-F3-Induced Immune-Metabolic Phenotype A) and IFNγ-( (18-Carbon ALA Lipid Mediator) B and 13C) and IL17-producing CD4+ T cells( Figure 14The percentages of activated B cells (CD80+ / CD86+), and along with increased percentages of M1-like resident (CD11b 低 / CD11c+) and non-resident (CD11b 高 / CD11c+) macrophages in the liver, while decreasing M2-like macrophages (CD11b 低 / CD206+, CD11b 高 / CD206+). Activation of B cells was associated with higher levels of IgG against endotoxin (LPS) in VV-TPN compared to OV-TPN and IL-TPN. Based on cell findings, the hepatic tissue concentration of IFNγ (a key cytokine for effective host defense) was higher in mice treated with VV-TPN compared to IL-TPN and OV-TPN. Although we could not reliably measure intracellular IL10 expression in immune cells by flow cytometry, probably due to low intracellular expression, both T cells and macrophages collected seven days after VV-TPN showed expression of IL10 as measured by immunoblotting. Expression of IL6, IFNγ, and TNFα in macrophages, as measured by flow cytometry, was similar in chow-fed, IL-TPN, and VV-TPN mice.

[0374] Figure 15 Figure 15

[0375] Oxylipins are oxidative metabolites of fatty acids produced in the liver, including eicosanoids (prostaglandins and leukotrienes) and specialized pro-resolving mediators, which are known as potent regulators of metabolism and immune responses. To test whether providing higher amounts of 18-carbon n-3 fatty acids (i.e., ALA and SDA released during VV-TPN) causes a unique distribution of oxylipins in the liver, lipid mediators were determined using a targeted lipidomics approach (UHPLC-MS / MS). While OV-TPN increased many oxylipins derived from long-chain n-3 fatty acids (i.e., EPA and DHA), which have known (predominantly) anti-inflammatory effects, VV-TPN did not, and showed no increase in many common pro-inflammatory oxylipins derived from arachidonic acid, as observed in IL-TPN. 9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOTrE), a monohydroxy polyunsaturated fatty acid produced from ALA by 12 / 15-lipoxygenase, was increased only in VV-TPN mice ( Example 7: Additional Benefits of F3 Compared to Commercially Available Lipid Emulsions)。To test whether hydroxy-octadecatrienoic acid contributes to the immunometabolic phenotype induced by VV-TPN, 9 / 13-HOTrE was added to IL-TPN. Providing these lipid mediators increased the plasma and hepatic tissue concentrations of IL10 after TPN, resulting in a similar low IL6 / IL10 ratio as observed in the hepatic tissue of VV-TPN mice ( Figure 7 D). The decreased IL6 / IL10 ratio was further accompanied by increased IRS2 protein expression and its tyrosine phosphorylation in the liver and higher glycogen content ( Figure 8 F). When added to IL-TPN, the supplementation of HOTrE also increased the IFNγ concentration in the hepatic tissue, while the TNFα concentration remained elevated as in IL-TPN. These observations provide evidence that HOTrE does contribute to the VV-TPN-induced immunometabolic phenotype.

[0376] Example 8: Clinical Application Areas of the Novel Lipid Emulsion F3 with Beneficial Anti-Inflammatory, Hepatoprotective, Anti-Diabetic, and Host Defense-Enhancing Effects

[0377] Sustainability

[0378] F3 does not contain fish oil, which is a concern due to overfishing of the oceans. Additionally, there are no toxins (such as radioactive substances in the 2011 Fukushima nuclear disaster) that accumulate in the food chain.

[0379] Antioxidation / Degradation

[0380] F3 has fatty acids with only 4 double bonds, as opposed to the 5 or 6 double bonds present in fish oil, and is thus more resistant to oxidation / degradation (extended shelf life). Notably, F3 shows 2.5 times lower primary oxidation products than Omegaven over an extended period (see Example 9: Other Application Areas of the Novel Lipid Emulsion F3 ). The secondary oxidation products (TBARS) at 300 days after production are 18 μmol(kg oil) in F3 -1 , higher than Intralipid (4 μmol(kg oil) -1 ), but significantly lower than OV (25 μmol(kg oil) -1 ).

[0381] Reduced total sterol load

[0382] F3 has the lowest amount of total sterols, including phytosterols plus cholesterol, as measured by mass spectrometry (see Example 10: Drugs Administered in a Lipid Emulsion Used as a Vehicle , 4 independent measurements of sterols in Intralipid, Omegaven, and F3). Notably, F3 also contains less stigmasterol (a phytosterol associated with liver inflammation) than Intralipid.

[0383] Example 11: RNA Therapy Using a Lipid Emulsion as a Vehicle Example 12: Vaccine Therapy Using a Lipid Emulsion as a Vehicle and Adjuvant

[0384] Patients in need of short - term and long - term total parenteral nutrition (TPN)

[0385] TPN is a life - saving nutritional therapy in situations where enteral feeding is contraindicated or insufficient. TPN is provided to millions of patients who cannot orally ingest or digest and absorb the daily essential amounts of nutrients (partial or TPN). Although parenteral nutrition is transient in many cases, lasting from days to weeks, thousands of patients in the United States alone require home - based long - term (> 3 months) parenteral nutrition each year. Additionally, in the United States, 500,000 infants (including premature and low - birth - weight infants) rely on TPN. The indications for total or partial parenteral nutrition cover a wide range of clinical conditions, such as critically ill patients (trauma, surgery, sepsis, shock), patients receiving home parenteral nutrition due to chronic intestinal failure, cachectic cancer patients, patients with inflammatory bowel disease (Crohn's disease, ulcerative colitis), patients with gastrointestinal obstruction, high - output enterocutaneous fistulas or short bowel syndrome, (most) elderly patients with acute or chronic debilitating diseases who cannot meet their nutritional needs, and patients with intractable nausea and vomiting (hyperemesis gravidarum). Furthermore, malnutrition (calorie - and / or protein - related) is a common healthcare problem with a high prevalence (20% to 50%) among inpatients.

[0386] Patients in need of short - term and long - term enteral nutrition

[0387] Enteral and parenteral formulations are designed for subjects / patients who cannot meet their nutritional needs through the consumption of ordinary food due to their specific conditions. The details of the patient's medical condition inform the route of application of nutritional support. Parenteral lipid emulsions can also be administered via the enteral route (as part of a complete enteral formula) because the requirements for this route of administration are less stringent in terms of sterility, osmotic pressure, and pH. The use of lipid emulsions in enteral applications has significant advantages in minimizing the adverse reactions of parenteral applications (such as liver disease, metabolic disruption, immunosuppression, and intestinal atrophy, see the detailed review: Lucchinetti et al., Molecular Nutrition & Food Research, March 2021; 65(5):e1901270). Since the enteral administration of lipid emulsions results in the complete absorption, uptake, and distribution of lipid emulsions and their compounds throughout the body, the same biological effects as those observed with parenteral administration are expected, particularly with regard to the inflammatory, metabolic, and immune systems.

[0388] Patients with metabolic diseases, especially insulin resistance

[0389] The most prevalent metabolic disease in the world is glucose intolerance (also known as prediabetes), with an estimated prevalence of 25% to 30% in Western populations. Patients with type I and type II diabetes (up to 10%). All metabolic conditions that concern insulin resistance.

[0390] Patients with reduced host defense

[0391] Many patients suffer from a dysregulation of their immune system due to metabolic disorders, autoimmune disorders, or infections.

[0392] Patients with liver diseases

[0393] In Western societies, the prevalence of NASH (non-alcoholic steatohepatitis and cirrhosis) is between 30% and 50%. Other liver diseases that would benefit from the new lipid emulsion are Alagille syndrome, alcohol- and drug-related liver diseases, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cholestasis, Crigler-Najjar syndrome, galactosemia, Gilbert syndrome, hemochromatosis, hepatic encephalopathy, hepatitis A, hepatitis B, hepatitis C, hepatorenal syndrome, intrahepatic cholestasis of pregnancy, lysosomal acid lipase deficiency, liver cysts, liver cancer, neonatal jaundice, primary biliary cholangitis, primary sclerosing cholangitis, progressive familial intrahepatic cholestasis, Reye syndrome, type I glycogen storage disease, Wilson's disease, etc.

[0394] Patients with systemic acute and / or chronic inflammation

[0395] This includes conditions with infectious diseases, and also includes conditions with sterile inflammation, such as rheumatoid arthritis, autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, psoriasis, ankylosing spondylitis, etc.

[0396] Example 13: Other Applications of Lipid Emulsions as "Rescue Therapy" in Medicine

[0397] Lipid emulsions can be used as drug delivery systems (vehicles) for the parenteral (injectable) or enteral administration (lipophilic) of drugs, including biologics, and more recently for nucleic acid-based therapies (i.e., using nucleic acids and related compounds to alter gene expression for therapeutic purposes) and for vaccines. Other applications of lipid emulsions in medicine are the treatment of (lipophilic) drug overdose / intoxication (the emulsion serves as an antidote) and the prevention of ischemia-reperfusion injury (protection of vital organs).

[0398] Discussion

[0399] The application of lipid emulsions in parenteral or enteral drug delivery has the following distinct advantages: 1) alleviating pain, irritation, and thrombophlebitis, 2) reducing toxicity, 3) improving stability and solubility due to reduced degradation, and 4) primarily targeting drug delivery to the liver. Examples of currently marketed drugs formulated with injectable lipid emulsions are diazepam, propofol, etomidate, alprostadil, dexamethasone, flurbiprofen, vitamins A, D, E, K, paclitaxel, and cyclosporine. Many other drugs would be more stable in lipid emulsions, but specific formulations have not been created for the healthcare market to date. These include clarithromycin, phenobarbital, physostigmine, cinnarizine, chlorambucil, docetaxel, etc.

[0400] In principle, all highly lipophilic drugs can be administered intravenously using lipid emulsions as a safe vehicle.

[0401] Materials and Methods

[0402] This is an emerging field where lipid carrier systems are used as vehicles in gene therapy. RNA-lipid delivery systems have been and are being used in clinical trials. For example, siRNA-EphA2-DOPC targeting EPHA2 is used in advanced cancer (NCT 01591356), ALN-VSP02 targeting KSP and VEGF is used in solid cancer therapy (NCT 00882180), and TKM-ApoB targeting ApoB is used in the treatment of hypercholesterolemia (NCT 00927459).

[0403] https: / / www.microbiomeanalyst.ca /

[0404] Liposomes are ideal carriers in combination vaccines targeting several antigens and enhance antibody induction and cell-mediated immunity. For example, in the production of a pentavalent combined vaccine against hepatitis A and B, diphtheria, tetanus, and influenza A / B, it has good immunogenicity and excellent tolerance.

[0405] ​

[0406] Lipid emulsions can act as a "lipid sink" in patients poisoned by lipophilic drugs such as local anesthetics, β-blockers, tranquilizers, calcium blockers, etc. Additionally, they can be used to protect vital organs such as the heart, brain, liver, kidneys, and lungs from ischemia-reperfusion injury.

[0407] ​

[0408] The development of a novel lipid emulsion for TPN use was driven by the unsatisfactory clinical outcomes (such as hepatotoxicity, diabetes-like metabolic conditions, and infection risks associated with immunosuppression) of patients reliant on life-saving TPN that uses currently available lipid emulsions. The lipid emulsion newly designed and engineered by the present inventors contains large amounts of two shorter-chain 18-carbon n-3 fatty acids, namely alpha-linolenic acid (ALA) and stearidonic acid (SDA), and is optimized for the recommended 1:2.5 n-6 / n-3 ratio. It is more resistant to oxidation and hydrolysis than 20 / 22-carbon n-3 fatty acid-based lipid emulsions, facilitating a longer shelf life, and also contains lower amounts of potentially toxic phytosterols such as stigmasterol than other vegetable oil-based lipid emulsions. The emulsion uses only vegetable oils instead of algae, krill, or fish oil and is thus referred to herein as TPN-F3(VV), making it more sustainable in terms of overfishing the oceans and having a lower risk of exposure to bioaccumulating marine toxins such as dioxins, mercury, and radionuclides. A detailed comparison by the present inventors of two commonly used lipid emulsions, namely soybean oil-based Intralipid and fish oil-based Omegaven, revealed that TPN-F3 has a unique combination of anti-inflammatory, insulin-sensitizing, and immune-enhancing properties that are unmatched by currently available lipid emulsions. Specifically, we demonstrated that VV-TPN, which releases 18-carbon n-3 fatty acids, mediates its beneficial effects by enhancing IL10-dependent insulin signaling and by enhancing immunity.

[0409] During TPN, the immune system is continuously assaulted by large amounts of bacterial toxins, namely endotoxin (LPS), which leak from the gut into the portal venous system. Although the total bacterial load in the gut is reduced due to the lack of oral nutrients, Gram-negative and invasive bacteria dominate the gut microbiome during TPN. At the same time, the immune system is affected by a catabolic state driven by insulin resistance. A recent study in mice showed that insulin resistance plays a key role in the development of immune cell dysfunction because insulin and T cell receptor signaling converge on the same downstream kinase Akt. Using activated CD4+ T cells lacking the insulin receptor, reduced proliferation and cytokine production, namely IFNγ, and impaired differentiation of Th1 and Th17 T cells have been shown in mice. Intact insulin signaling appears to contribute to the metabolism of T cells, thus enabling a more effective immune response against pathogens. Another study in rats showed insufficient IL10 production in CD4+ T cells in the absence of the insulin receptor. Insulin resistance also promotes the activation of impaired "lazy" M2-like macrophages. Thus, it is conceivable that VV-TPN improves insulin signaling and thus enhances immune cells, such as by increased primed effector CD4+ T cells (CD44 低 / CD62L 低) as evidenced by the number of

[0410] Compared with IL-TPN, we did not observe an increase in the production of IL6, IFNγ, and TNFα in hepatic macrophages isolated from VV-TPN mice. Thus, in contrast to VV-TPN mice, the increase in IL6 and TNFα expression in the liver tissue of IL-TPN mice may be due to activated hepatocytes. Further, macrophages, which are crucial for both innate non-specific host defense and adaptive specific immune responses, are activated by IFNγ released from T cells. IFNγ reprograms the metabolism of macrophages to maintain their viability and pro-inflammatory activity, including ROS production, by switching energy metabolism from OXPHOS to glycolysis, while IL10 reverses the energy metabolism back to OXPHOS and inhibits the activation of the NLRP3 inflammasome. This specific cytokine microenvironment may indeed be the basis for the unique anti-inflammatory but immune-enhancing phenotype that we observed in VV-TPN mice. In a clinical study of cancer patients, parenteral nutrition supplemented with lipid emulsions based on olive oil (n-9 fatty acids) and fish oil (long-chain n-3 fatty acids) was compared. The functions of the innate and adaptive immune systems were higher in patients treated with olive oil, suggesting that olive oil has neurogenic effects on inflammation and immune stimulation. Patients treated with fish oil showed signs of immunosuppression, manifested as a decrease in the number of PMA-stimulated CD4+ T cells producing IFNγ, while the number of regulatory T cells was relatively higher. Interestingly, in the studies of the present inventors themselves, the production of anti-endotoxin (LPS) IgG1 was higher in mice treated with VV-TPN, which is consistent with helper T cell-mediated B cell activation. These observations are consistent with previous nutritional studies using vegetable oils rich in 18-carbon ALA and SDA. Patel et al. compared the offspring of pregnant Sprague-Dawley rats fed a maternal diet rich in SDA with the offspring of pregnant rats fed a control diet. The SDA-rich diet led to higher B cell function, as measured by IgG1 production, a higher number of activated helper T cells, and spleen cells stimulated with endotoxin showed decreased IL6 and TNFα but increased IL10 production. Finally, in a nutritional study conducted in human volunteers, daily oral intake of 10 mL of Borago officinalis seed oil rich in ALA and SDA for 4 weeks increased the production of IL10 in endotoxin-stimulated whole blood.

[0411] Hepatic toxicity is a major problem with TPN, and cases of severe steatohepatitis and portal fibrosis have also been reported in patients on fish oil-based TPN. Although we observed the priming of immune cells in the liver tissue, the actual number of leukocytes in the liver tissue was reduced during TPN compared to chow-fed mice. In fact, compared with standard lipid emulsions, VV-TPN showed many liver-protective features, namely the combination of a decrease in pro-inflammatory IL6 and an increase in anti-inflammatory IL10. IL10 is a protective factor against high-fat diet-induced insulin resistance in the liver. Mice fed a high-fat diet and treated with neutralizing anti-IL10 antibodies showed increased expression of pro-inflammatory cytokines, mitochondrial-dependent apoptotic signaling, and disruption of insulin signaling, with downregulation of IRS2 and reduced glycogen content in the liver. LBP is a protein that specifically detoxifies endotoxin produced by hepatocytes and was upregulated only in VV-TPN. Similarly, SOCS3, a potent inhibitor of IL6 signaling, was upregulated only in VV-TPN. The increased abundance of LBP and SOCS3 was negatively correlated with the IL6 / IL10 ratio, indicating a contribution to the mechanism of reducing liver inflammation. Additionally, VV-TPN upregulated anti-inflammatory PPARα in the liver tissue similarly to OV-TPN, but only downregulated pro-inflammatory and adipogenic PPARγ2. Finally, VV-TPN increased the concentration of the anti-inflammatory helper T cell cytokine IL4 in the liver tissue and especially in the pancreatic tissue, and IL4 is known to be beneficial in affecting β-cell function as well as lipid and glucose metabolism.

[0412] The immune-metabolic interactions during TPN are ultimately the result of specific fatty acid species released from the administered lipid emulsion. While most TPN formulations have shown a reduction in the number of total T cells in patients, TPN that predominantly releases long-chain n-6 fatty acids further impairs T cell function. The provision of specific fatty acids alters the composition of the phospholipid bilayer of the cell membrane in T cells, a process termed "lipid remodeling", thereby altering the function of key membrane-associated receptor proteins including the T cell receptor. In the studies of the present inventors, the provision of shorter-chain 18-carbon n-3 fatty acids primed subsets of CD4+ effector T cells expressing IFNγ and IL17, B cells, and macrophages. Detection of IL10 expression in these primed immune cells suggested that T cells, macrophages, and possibly other cell types such as hepatocytes were the likely sources of elevated plasma and tissue IL10 concentrations. In fact, the occurrence of anti-IL10 effects in the presence of an isotype IgG control antibody, presumably by binding to Fc-receptors, clearly indicated that immune cells were the major source of elevated IL10. Different lipid emulsions used for TPN also induced different profiles of bioactive lipid mediators. Lipid mediators, also known as oxylipins, are the major sources of fatty acid-induced biological actions in many tissues and cells, including T cells and macrophages. In the studies of the present inventors, the heatmap of lipid mediators measured in liver tissue did indeed show significant differences between the three TPNs studied. Liver tissue from VV-TPN mice lacked typical n-6 arachidonic acid-derived pro-inflammatory mediators but also lacked many long-chain n-3-derived anti-inflammatory mediators, except for a few EPA-derived mediators. Apparently, VV-TPN induced its own characteristic profile of oxylipins, which could not be simply attributed to the bioconversion of shorter-chain n-3 fatty acids (i.e., ALA and SDA) to long-chain n-3 fatty acids (EPA, DHA), as we did not observe an increase in the formation of DHA-derived lipid mediators. In fact, similar observations have been reported in ALA-treated macrophages, where the 18-carbon lipid mediator, i.e., 9 / 13-HOTrE rather than EPA- or DHA-derived lipid mediators, was associated with reduced IL6 production in M1-polarized macrophages and increased IL10 production and enhanced phagocytosis in M2-polarized macrophages. Importantly, in the TPN mouse model of the present inventors, supplementation of IL-TPN with 9 / 13-HOTrE mimicked many of the features of the immunometabolic phenotype induced by VV-TPN. It has previously been shown that 9 / 13-HOTrE increases IL10 in murine peritoneal macrophages, inhibits the NLRP3 inflammasome, and increases survival in murine endotoxin and cecal ligation sepsis models. Recent studies have also proposed the possibility that activated lymphocytes and possibly other immune cells increase the synthesis of their own lipid mediators, which can directly stimulate their activation and proliferation in an autocrine manner.

[0413] Although we reported the beneficial biological effects of a novel TPN based on short-chain n-3 fatty acids, additional studies are needed to provide more mechanistic insights into 18-carbon n-3 fatty acid-derived lipid mediators and their specific immunometabolic roles in TPN. In addition, the translatability of the observed beneficial immunometabolic effects from mouse models to patients must be demonstrated.

[0414] In summary, the findings of the present inventors suggest that a novel lipid emulsion based on 18-carbon n-3 fatty acids has significant anti-inflammatory, antidiabetic, and immunopotentiating properties and acts as "immunonutrition" during TPN. This unique feature is unparalleled by currently available lipid emulsions and may be particularly beneficial for susceptible patients at risk of infection, septic patients with "immunoparalysis", and cancer patients.

[0415] ​

[0416] This study complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and conformed to the ARRIVE guidelines (https: / / www.nc3rs.org.uk / arrive-guidelines). The experimental protocol (AUP000002007) used in this study was approved by the Animal Policy and Welfare Committee of the University of Alberta.

[0417] Preparation of the novel lipid emulsion TPN-F3 (F3 or VV)

[0418] Wet 1.2% lecithin (LIPOID E80, egg-derived phospholipids containing 80% phosphatidylcholine, suitable for parenteral application, Lipoid GmbH, Ludwigshafen, Germany) in purified water (<20% final volume; NANOpure Diamond Barnstead, Thermo Scientific, Waltham, MA, USA, or Endotoxin-free Ultra Pure Water, EMD Millipore Corp., Billerica, MA, USA) in a water bath at 45 °C for 2 h. Use a Polytron high-shear mixer (Polytron PT6000 drive unit, PT-DA 3012 / 2TS dispersing aggregate, Kinematica AG, Malters, Switzerland) to promote dispersion at 20,000 rpm for 60 s. Add 2.5 μM final concentration of EDTA (EDTA disodium salt dihydrate, Carl Roth GmbH+Co.KG, Karlsruhe, Germany, ≥99%) and 2.2% glycerol (Acros Organics, NJ, USA, 99+%) for isotonicity to the lecithin / water phase. Add 20% of the pre-mixed lipid phase, which consists of 50% Ahiflower oil (NaturesCrops International, Kensington, Prince Edward Island, Canada), 25% olive oil (LIPOID refined olive oil, European Pharmacopoeia, Lipoid AG, Steinhausen, Switzerland), and 25% coconut oil (Bioriginal, Saskatoon, Saskatchewan, Canada) (Table 4) to the lecithin / water phase. Supplement with 0.016% α-tocopherol (Sigma-Aldrich, St. Louis, MO, USA, type V, approximately 1000 IU / g), and add purified water to reach the final volume. Subsequently, homogenize the coarse emulsion using a PL300 or HL60 high-pressure homogenizer (Dyhydromatics, Maynard, MA, USA) equipped with a 75.1T reaction chamber and a 200.2L backpressure module (only for PL300) to obtain a preferred droplet size between 260 - 300 nm (six cycles at a pressure of 18 kpsi (PL300) to 22 kpsi (HL60)). Adjust the pH to >8.5 with 1M NaOH until the ζ-potential >|30| mV. Fill aliquots into 50 mL glass bottles (Muller+Krempel AG, Bulach, Switzerland), load the headspace with inert argon gas (PanGas AG, Dargmersellen, Switzerland, argon 5.0), and crimp the vials. Sterilize the emulsion at 121 °C and 2 bar for 15 min (Systec DE-23, Systec GmbH, Linden, Germany).Confirmation of the absence of microbial growth (membrane filtration method according to Ph Eur 2.6.1) and endotoxins (< 0.1 IU / ml, gel-clot LAL gel-clot assay according to Ph Eur 2.6.14) (Bioexam AG, Lucerne, Switzerland). The droplet size of the sterilized emulsion was further tested by dynamic light scattering (Malvern Zetasizer 3000HS A, Malvern Instruments, Malvern, UK), and the primary (mFOX assay) and secondary (TBARS assay) oxidation products, as well as unesterified fatty acids, were quantified by fluorescence detection after labeling with a fluorophore and separation by high-pressure liquid chromatography (FL-RP-HPLCREF). Cholesterol and phytosterols were determined using UPLC-MRM / MS, and the composition of each lipid emulsion was finally verified by gas chromatography (Agilent 6890 GC system). The absence of in vitro toxicity to T cells was tested before each in vivo application.

[0419] Metabolic data of mice receiving 7-day total parenteral nutrition (IL-TPN, OV-TPN, VV-TPN) compared to conventionally fed mice receiving (IV-chow) or not receiving saline infusion (chow).

[0420] Data are presented as mean (SD) or median (25th, 75th percentile). N = 6.

[0421] GLP-1, glucagon-like peptide-1; HOMA-IR, homeostasis model assessment of insulin resistance; IRβ, insulin receptor, β subunit; IRS2, insulin receptor substrate 2; pY-IRS2, pan-tyrosine phosphorylated IRS2; OD, optical density; chow, group-housed C57BL / 6J mice without instrumentation in static cages; IV-chow, fed mice receiving heparinized saline solution; IL-TPN, Intralipid-based total parenteral nutrition; OV-TPN, Omegaven-based total parenteral nutrition; VV-TPN, TPN-F3-based total parenteral nutrition.

[0422] Analysis of variance (ANOVA) was performed only on the lipid infusion groups, and differences were considered significant (bold) if the overall P < 0.05. Multiple comparison procedures were then performed appropriately. *, significantly decreased compared to OV-TPN and VV-TPN; #, significantly increased compared to IL-TPN and OV-TPN; +, significantly increased compared to OV-TPN; §, significantly increased compared to IL-TPN.

[0423] TPN mouse model, treatment groups, formulations and administration of TPN

[0424] The TPN mouse model used in this study has been previously described in detail (Lou et al. Molecular Nutrition & Food Research 2021; 65: e2000412). Briefly, male C57BL / 6 mice (22–25 g) were equipped with a tunneled jugular vein catheter (JVC) and a magnetic vascular access button. A magnetic tether (VABM1T / 25; Instech Laboratories Inc., Plymouth Meeting, PA, USA) was attached to a swivel to ensure freedom of movement, and TPN was administered using a programmable infusion pump (SAI Infusion Technologies, Lake Villa, IL, USA). Mice were housed in individual open conventional shoebox cages and maintained under controlled lighting conditions (12-hour light / dark cycle) at a constant temperature of 21 °C and 60% relative humidity, with free access to autoclaved water and a conventional chow diet (5L0D PicoLab Laboratory Rodent Diet; Canadian Lab Diets, Inc., Leduc County, Alberta, Canada). Mice were acclimated for 4 days prior to the experiment and then randomly assigned to receive TPN containing Intralipid (IL-TPN), TPN containing Omegaven (OV-TPN), or TPN containing TPN-F3 (VV-TPN). Intralipid and Omegaven were purchased from Fresenius Kabi (AG, Klins, Switzerland). On day 5, the infusion was started. Mice assigned to TPN received a continuous infusion of the TPN solution, starting at 0.25 mL hr -1 −1 (6 mL per day on day 1 of the infusion), followed by an increase to a maximum of 0.32 mL hr -1 −1 (7.7 mL per day on days 4 to 7 of the infusion). Mice receiving 0.9% saline and heparin (10 U mL -1 ) at 6 mL per day and with free access to water and chow were used as controls (IV-chow). Age-matched instrument-free C57BL / 6 mice were housed in conventional cages for 7 days (3 mice per cage, with free access to water and chow) and used as additional chow-fed controls (chow). Some mice receiving VV-TPN were treated with 100 mg / 24 h of anti-mouse interleukin-10 (IL10) antibody (BioXCell #BE0049) added to the TPN mixture or its IgG1 isotype control antibody (BioXCell #BE0290). Some mice receiving IL-TPN were treated with 9 / 13-hydroxy-octadecatrienoic acid (5 ng / mL) added to the TPN mixture. Nutritional requirements for protein were provided as an amino acid solution (4 kcal g -1 −1). Carbohydrates were provided as glucose (3.4 kcal g -1) is provided, and lipids are provided as lipid emulsions (10 kcal / g). -1 ) Mice receiving TPN received isocaloric (150 kcal / 100 mL), isonitrogenous TPN solutions containing Intralipid, Omegaven, or TPN-F3. 8 TPN provided 13% of total calories from amino acids, 71% from glucose, and 16% from lipids, with a non-protein energy to nitrogen ratio of 170. Calculated values were to meet the nutrient and energy requirements of mice weighing 24 g. For details of TPN composition and dosage, see Table 3. On day 8, tail blood glucose concentration was measured using a OneTouch VerioIQ (LifeScan Canada Ltd., Burnaby, British Columbia, Canada). Mice were disconnected from the infusion line, weighed, anesthetized with isoflurane, and euthanized by cervical dislocation prior to blood and tissue collection. Blood samples were obtained by cardiac puncture and processed immediately, while tissues were immediately frozen in liquid nitrogen and stored at -80 °C until analysis or further processing for immune cell isolation.

[0425] Table 3: Composition of total parenteral nutrition (TPN) formulations for in vivo mouse experiments

[0426]

[0427] The final formulations also contained sodium phosphate (13.4 mmol / L), electrolytes, and heparin (100 U / 10 mL). All TPN formulations provided similar energy (150 kcal / 100 mL), 13% from amino acids, 16% from lipids, and 71% from carbohydrates (glucose).

[0428] IL-TPN, Intralipid-based total parenteral nutrition; OV-TPN, Omegaven-based total parenteral nutrition; VV-TPN, TPN-F3-based total parenteral nutrition.

[0429] Hormone measurements, glycogen content, insulin receptor, and phosphorylated (pY) insulin receptor substrate 2 (IRS2) and phosphoenolpyruvate carboxykinase (PEPCK) activities

[0430] Plasma insulin, glucagon, and glucagon-like peptide 1 (GLP-1) were measured from heparinized or EDTA-treated blood collected by cardiac puncture using the following ELISA kits: Mercordia #10-1247-01 (insulin), Mercordia #10-1281-01 (glucagon), Crystal Chem #81508 (GLP-1). Liver glycogen content was measured from tissue powder using the Sigma glycogen assay kit #MAK016. Total liver insulin receptor protein β subunit was determined using the insulin receptor ELISA kit #KHR9111 (Thermo Fisher Scientific). Tyrosine phosphorylation of IRS2 and total IRS2 was measured in liver lysates using an in-house ELISA. PEPCK activity of liver tissue was performed using the PEPCK activity kit (Abcam #ab239714) according to the manufacturer's recommendations.

[0431] Cytokine profile

[0432] Interferon-γ (IFNγ) #DY485, interleukin-6 (IL6) #DY406, IL10 #DY417, tumor necrosis factor-α (TNFα) #DY410), and interleukin-4 (IL4) #DY405 were measured from tissue powder using the R&D DuoSet ELISA kits according to the manufacturer's instructions. 10 mg of powder was rinsed twice in ice-cold PBS to remove residual blood and then homogenized in 100 mL of ice-cold lysis buffer #6 (R&D Systems) using a Qiagen TissueLyser II (Qiagen) device. The homogenate was placed on ice for 15 minutes and then centrifuged at 2,000 g for 5 minutes. The supernatant was collected and aliquoted for DC protein assay (Bio-Rad Laboratories) and DuoSet ELISA assay (R&D Systems), and then stored at -80 °C. All cytokine measurements were normalized to the sample protein concentration. Sample dilutions were performed if necessary to maintain a final urea concentration of 1 M in all samples before adding to the plate. Plasma IL10 was measured using the R&D Quanbtikine ELISA kit #M1000B.

[0433] Immunoblotting

[0434] Total tissue homogenates and nuclear / cytoplasmic fractions were prepared as previously reported (Lou et al., Molecular Nutrition & Food Research 2021; 65: e2000412). Protein concentrations were determined by Bradford or DC protein assay (Bio-Rad Laboratories). Equal protein loads were separated by SDS-PAGE and transferred to nitrocellulose membranes for probing with antibodies of interest. Immunoreactivity was visualized using ECL reagent (PerkinElmer) and quantified by ImageJ software. All immunoreactivities were normalized relative to total liver lysates against vinculin or actin, and for nuclear fractions, relative to TATA-binding protein (TBP). Glucokinase was detected by immunoblotting in both cytoplasmic and nuclear fractions, and for each band, the intensity of the band was normalized relative to vinculin, actin, or TBP, respectively.

[0435] Immune cell isolation

[0436] Immune cells from the liver, spleen, mesenteric lymph, large intestine, and small intestine were isolated as previously described (Tsai et al., Cell metabolism 2018; 28: 922 - 34e4). EasySep TM Mouse T Cell Isolation Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada) was used for negative selection of T cells by removing non-T cells with biotinylated antibodies against non-T cells and streptavidin-coated magnetic particles. EasySep TM Mouse F4 / 80 Positive Selection Kit (STEMCELL Technologies) was used to isolate macrophages.

[0437] Flow cytometry analysis

[0438] Cells obtained from each tissue (1 - 2 x 10 6 / sample) were stained with fluorophore-conjugated antibodies against innate immune cells and lymphocytes, run on an LSR Fortessa-SORP flow cytometer, and analyzed using FlowJo V10 software (BD, Ashland, Oregon, USA).

[0439] Lipopolysaccharide (LPS) measurement and plasma anti-LPS IgG antibody concentration

[0440] PyroGene TMRecombinant Factor C Endotoxin Detection Fluorescent Kit (Lonza #50-658U), with a minimum detection limit of 0.005 endotoxin (LPS) units (EU) / mL, was used to measure the endotoxin levels in (epididymal) white adipose tissue. A commercially available ELISA kit (Chondrex #6106) was used to measure the concentration of murine plasma anti-LPS IgG antibodies against Escherichia coli O111:B4 lipopolysaccharide.

[0441] Microbiome analysis using 16S rRNA sequencing

[0442] Mucosal samples were collected from the colon wall at the end of the 7-day experimental period. DNA was extracted according to the protocol of the ZymoBIOMICS DNAMiniprep kit (D4300, Zymo Research Corp; Irvine, CA, USA). Amplification of the V3-V4 hypervariable region was performed using the standard primers 341F (CCTACGGGNGGCWGCAG, SEQ ID NO:1) and 805R (GACTACHVGGGTATCTAATCC, SEQ ID NO:2). Libraries were prepared at the Fasteris facility (Genesupport / Fasteris SA, Plan-les-Ouates, Switzerland) using the Metafast protocol. 16S rRNA gene sequences were clustered into operational taxonomic units (OTUs) and mapped to the SILVA database. All data analysis was performed using the web-based tool MicrobiomeAnalyst.ca( ​ , Lucchinetti et al. ClinNutrition ESPEN 2022).

[0443] Quantitative analysis of lipid mediators using UHPLC-MS / MS

[0444] Lipid mediators were quantified using internal standards, calibrators, and quality controls as previously described in detail (Hartling et al. Clinical chemistry and laboratory medicine 2021; 59:1811-23). Lipid mediators were extracted from liver tissue powder using methanol and solid-phase extraction. Samples were evaporated under nitrogen and reconstituted for UHPLC-MS / MS injection.

[0445] Statistics

[0446] Data were summarized as mean (SD) or median (25th, 75th percentile), depending on the underlying data distribution (normal vs skewed) of the specified number of independent observations (N). Comparisons were focused on the TPN groups because the study design aimed to directly compare each TPN group. Depending on the underlying data distribution, significance of differences between groups was determined by ANOVA, followed by post hoc analysis with the Tukey method or by non-parametric methods (Kruskal-Wallis test, followed by post hoc analysis with the Dunn test). For lipid mediator statistics, all concentrations were log-transformed and converted to z-scores for principal component analysis (PCA). Samples with concentrations below the limit of detection were set to 1 / 2 the limit of detection if the corresponding lipid mediator was detected in other samples of the same matrix. Depending on the data distribution, unpaired t-tests or Mann-Whitney U tests were used to compare two groups if deemed necessary. Differences were considered statistically significant if the overall p < 0.05 (two-sided). SigmaPlot (version 14.0; Systat Software Inc, San Jose, California) was used for analysis.

Claims

1. A lipid emulsion for administration to a patient, wherein the lipid emulsion comprises an oil phase and an aqueous phase, and wherein the oil phase of the lipid emulsion comprises: - An ω-3 fatty acid component, o wherein the mass of the ω-3 fatty acid component accounts for 25% to 35% of the oil phase; o wherein the ω-3 fatty acid component consists of one or more ω-3 fatty acids, and the ω-3 fatty acids are characterized by the presence of more than one carbon-carbon double bond, and one carbon-carbon double bond is three atoms away from the terminal methyl group; o and wherein the oil phase of the lipid emulsion comprises ≥5% stearidonic acid; o and wherein the oil phase of the lipid emulsion comprises ≥15% α-linolenic acid (ALA); - An ω-6 fatty acid component, o wherein the mass of the ω-6 fatty acid component accounts for 10% to 15% of the oil phase; o wherein the ω-6 fatty acid component consists of one or more ω-6 fatty acids, and the ω-6 fatty acids are characterized by the presence of more than one carbon-carbon double bond, and one carbon-carbon double bond is six atoms away from the terminal methyl group; - A monounsaturated fatty acid component, o wherein the mass of the monounsaturated fatty acid component accounts for 18% to 30% of the oil phase; o wherein the monounsaturated fatty acid component consists of one or more fatty acids, and the fatty acids are characterized by the presence of one carbon-carbon double bond; - A saturated fatty acid component, o wherein the mass of the saturated fatty acid component accounts for 20% to 35% of the oil phase; o wherein the saturated fatty acid component consists of one or more fatty acids, and the fatty acids are characterized by the absence of carbon-carbon double bonds and only carbon-carbon single bonds.

2. The lipid emulsion according to claim 1, wherein the ratio (m / m) of the ω-6 fatty acid component to the ω-3 fatty acid component ranges from 1:3 to 1:

2.

3. The lipid emulsion according to any one of the preceding claims, wherein the oil phase of the lipid emulsion comprises: - 40% to 50% PUFA (polyunsaturated fatty acid); - 20% to 30% MUFA (monounsaturated fatty acid); and - 20% to 35% SFA (saturated fatty acid).

4. The lipid emulsion according to any one of the preceding claims, wherein the oil phase of the lipid emulsion comprises: - 5% to 15% stearidonic acid; - 20% to 30% oleic acid; - 5% to 15% linoleic acid; - 20% to 30% α-linolenic acid; and - 2% to 5% gamma-linolenic acid.

5. The lipid emulsion according to any one of the preceding claims, wherein the oil phase of the lipid emulsion comprises: - 2% to 5% of a short-chain fatty acid component selected from caproic acid, caprylic acid and capric acid; - 10% to 15% lauric acid; - 3% to 8% myristic acid; and - 5% to 12% palmitic acid.

6. The lipid emulsion according to any one of the preceding claims, wherein the oil phase of the lipid emulsion comprises: - 20% to 30% olive oil; - 20% to 30% coconut oil; and - 40% to 60% Lithospermum arvense seed oil.

7. The lipid emulsion according to any one of the preceding claims, wherein the lipid emulsion further comprises a stabilizer and / or antioxidant selected from the following: - EDTA; and / or - α - tocopherol.

8. The lipid emulsion according to any one of claims 1 to 7 preceding, which further comprises a drug.

9. The lipid emulsion according to any one of claims 1 to 7 preceding, which is used as an antagonist / detoxifying agent for the treatment of poisoning caused by lipophilic drugs or lipophilic toxic compounds.

10. The lipid emulsion according to any one of claims 1 to 8 preceding, which is used to protect vital organs from ischemia - reperfusion injury.

11. The lipid emulsion according to any one of claims 1 to 8 preceding, which is used for the prevention or treatment of type I or type II diabetes.

12. The lipid emulsion according to any one of claims 1 to 8 preceding, which is used for parenteral nutrition, wherein the parenteral nutrition is administered to a patient applicable to one or more of the following indications: - Patients in need of short - term and long - term total parenteral nutrition (TPN); and / or - TPN patients with metabolic diseases; and / or - TPN patients with insulin resistance; and / or - TPN patients with liver diseases; and / or - TPN patients with systemic acute and / or chronic inflammation, and / or - TPN patients with reduced host defense, and / or - Septic patients, and / or - TPN patients receiving chemotherapy.

13. The lipid emulsion according to any one of claims 1 to 8 preceding, which is used for enteral nutrition, wherein the enteral nutrition is administered to a patient applicable to one or more of the following indications: - Patients in need of short - term or long - term enteral nutrition; and / or - Patients with metabolic diseases; and / or - Patients with insulin resistance; and / or - Patients with liver diseases; and / or - Patients with systemic acute and / or chronic inflammation, and / or - Patients with reduced host defense, and / or - Septic patients, and / or - Patients receiving chemotherapy.

14. The lipid emulsion according to any one of claims 1 to 8 preceding, or the lipid emulsion used according to claims 9 to 12, wherein the lipid emulsion is formulated for parenteral administration.

15. The lipid emulsion according to any one of claims 1 to 9 preceding, or the lipid emulsion for use according to claims 9 to 11 or 13, wherein the lipid emulsion is formulated for enteral or oral administration.

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