RU2783166C2 - Methods and compositions related to emulsions containing fish oil and/or omega-3 fatty acids - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to emulsion compositions containing fish oil and medium chain triglycerides (hereinafter – MCT), and/or omega-3 fatty acids and MCT. The emulsion composition contains: fish oil and MCT in a ratio between, but not including, from 30:70 to about 70:30; or omega-3 fatty acids and MCT in a ratio from about 30:70 to about 70:30; wherein the total triglyceride + diglyceride content of fish oil and/or omega-3 fatty acids contains no more than 10% of diglyceride. A method for reduction in the risk of development or treatment of inflammatory reactions includes administration of the emulsion composition to a subject who needs it. A method for reduction in the risk of development or treatment of a liver disease includes administration of the emulsion composition to a subject who needs it. A method for reduction in the risk of development or treatment of inflammatory reactions and a liver disease includes administration of the emulsion composition to a subject who needs it. The use of the emulsion composition for provision of parenteral nutrition to a subject who needs it is also described.

EFFECT: invention allows for obtaining a stable emulsion, which even further reduces the risk of development of inflammatory reactions and/or liver diseases, can improve treatment of a number of patients, for whom normal nutrition is not recommended or is not sufficient.

89 cl, 18 dwg, 4 tbl

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) under U.S. Provisional Application No. 62/543,437, filed August 10, 2017, the contents of which are hereby incorporated by reference in its entirety.

GOVERNMENTAL SUPPORT

This invention was made with government support under Grants No. 1F32DK104525-01; 5T32HL007734-22; and T35HL110843 awarded by the National Institutes of Health. The government has certain rights to the invention.

FIELD OF TECHNOLOGY

The technology described herein relates to lipid emulsions for administration to patients, eg, orally or parenterally.

BACKGROUND OF THE INVENTION

Long-term and short-term PN dependent patients may be vulnerable to inflammatory strokes. In long-term PN-dependent patients, chronic disease states and a long-term implanted central venous catheter to deliver PN can induce a pro-inflammatory state. Short-term PN-dependent patients, such as trauma patients, postoperative patients, and patients with acute disease requiring intensive care, are also prone to pro-inflammatory problems.

In addition to using the PN itself, a particular lipid emulsion administered through the PN can modulate the inflammatory response and influence the inflammatory status of PN-dependent patients. Soy oil-based lipid emulsions are rich in pro-inflammatory omega-6 fatty acids, while fish oil-based lipid emulsions contain an abundance of more anti-inflammatory omega-3 fatty acids. Fish oil lipid emulsions have been used to treat parenteral nutrition-associated liver disease (PNALD) in patients whose condition is characterized by liver inflammation and cholestasis.

SUMMARY OF THE INVENTION

The development of lipid emulsions that further reduce the risk of inflammatory reactions and/or liver disease may improve the management of a number of patients for whom a normal diet is not recommended or insufficient. As described herein, the inventors have found that mixed emulsions of a) MCTs and b) fish oils and/or omega-3 fatty acids (e.g., omega-3-rich oils) provide improved anti-inflammatory properties compared to fish alone. fat or MCT. Further, this document describes the unexpected effectiveness obtained with certain ratios of a) MCT and b) fish oil and/or omega-3 fatty acids (eg, oils with a predominance of omega-3 fatty acids).

In particular, it is demonstrated herein that mixed FO and MCT emulsions provide improved anti-inflammatory effects in response to an inflammatory stimulus compared to FO alone. In particular, mixtures in ratios from 30:70 to 70:30 provided an unexpectedly better balance of liver protection and blunting the inflammatory response compared to less balanced mixtures. Surprisingly, a ratio of 50:50 was optimal for this effect. Used in this document, the ratio of MCT to fish oil or omega-3 fatty acids relative to each other are given in units of wt./wt. The amount of MCT, fish oil and/or omega-3 fatty acids in wt./about. in the present compositions is described elsewhere in this document.

In addition, many prior art formulations explicitly require vegetable oils, as their developers believe that the absence of vegetable oils will lead to an essential fatty acid deficiency (EFAD) (see, for example, US Patent Publication 2010/0233280). In contrast, embodiments of the formulations and methods described herein do not contain vegetable oil, and are not predominantly omega-6 fatty acids, and still do not induce EFAD despite expert claims that such a result should have been would be observed.

In one aspect of any of the embodiments described herein, the invention is an emulsion composition comprising: a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 30:70 to about 70:30; or a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 30:70 to about 70:30. In one aspect of any of the embodiments of the invention described herein is an emulsion composition containing: a combination of fish oil and medium chain triglycerides (MCTs) or a combination of omega-3 fatty acids and MCTs in a ratio between 30:70 and 70:30, but not including them. In some embodiments of any of the aspects, the composition comprises a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40; or a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40. In some embodiments of any of the aspects, the composition comprises a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 50:50; or a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 50:50. In some embodiments of any of the aspects, the composition contains a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of 50:50; or a 50:50 combination of omega-3 fatty acids and medium chain triglycerides (MCTs). In some embodiments of any of the aspects, the omega-3 fatty acid composition is provided as an omega-3 fatty acid predominant oil. In some embodiments of any of the aspects, the omega-3 fatty acid composition is provided as an omega-3 fatty acid-dominant oil that has not been reesterified.

As used herein, "omega-3-rich oil" refers to a fatty acid oil that contains a higher proportion of omega-3 fatty acids than omega-6 fatty acids. In some embodiments of any of the aspects, the fatty acids in the omega-3 fatty acid-dominated oil are greater than 50%, e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90 %, greater than 95% or greater than 98% omega-3 fatty acids (the above percentages are % fatty acids in fatty acid oil by weight).

In some embodiments of any of the aspects, the composition further comprises alpha-tocopherol. In some embodiments of any of the aspects, alpha-tocopherol is present in an amount of at least 100 mg/l of the total composition. In some embodiments of any of the aspects, alpha-tocopherol is present in an amount of at least 120 mg/l. In some embodiments of any of the aspects, the emulsion composition contains alpha-tocopherol and other forms of vitamin E (e.g., beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and/or delta- tocotrienol) in a ratio of at least 2:1 by weight of the entire composition. In some embodiments of any of the aspects, the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 10:1. In some embodiments of any of the aspects, the emulsion composition does not contain other forms of vitamin E other than alpha-tocopherol.

In some embodiments of any of the aspects, phytosterols are not present in the composition. In some embodiments of any of the aspects, phytosterols are present in the composition. In some embodiments of any of the aspects, the phytosterols are present in the composition at a concentration of less than 50 mg/L. Phytosterols may include plant sterols and plant stanols, eg beta-sitosterol, campesterol, stigmasterol, ergosterol, sitostanol, campestanol, avenasterol and/or stigmastanol.

In some embodiments of any of the aspects, arachidonic acid is present in the composition at a concentration of at least 900 mg/L. In some embodiments of any of the aspects, docosahexaenoic acid is present in the composition at a concentration of at least 13.4 g/L. In some embodiments of any of the aspects, eicosapentaenoic acid is present in the composition at a concentration of at least 11.6 g/L.

In some embodiments of any of the aspects, the composition is formulated for oral administration. In some embodiments of any of the aspects, the composition is formulated for parenteral or intravenous administration. In some embodiments of any of the aspects, the composition further comprises the addition of one or more additional fatty acids, or mixtures thereof. In some embodiments of any of the aspects, the supplement contains one or more fatty acids that are therapeutic for a disease.

In one aspect of any of the embodiments described herein, the method comprises administering an emulsion composition as described herein to a subject in need thereof. In some embodiments of any of the aspects, the administration is parenteral administration. In some embodiments of any of the aspects, the administration is total parenteral administration. In some embodiments of any of the aspects, the administration is oral administration. In some embodiments of any of the aspects, the subject is a subject in need of treatment or prevention of a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; atresia of the biliary tract; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemia-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; anginal condition; asthmatic condition; epileptic state; lacunar state; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal failure associated liver disease (IFALD), parenteral nutrition-associated cholestasis (PNAC), essential fatty acid deficiency (EFAD), parenteral nutritional dependence complicated by soy allergy or allergy to lipid emulsions containing ingredients other than MCTs and fish oil and/or omega-3 fatty acids. In some embodiments of any of the aspects, the subject is a subject in need of treatment or prevention of parenteral nutrition-associated liver disease (PNALD).

In some embodiments of any of the aspects, the subject is in need of parenteral nutrition. In some embodiments of any of the aspects, the subject is in need of total parenteral nutrition. In some embodiments of any of the aspects, the patient is not receiving oral nutrition. In some embodiments of any of the aspects, the patient is not receiving other parenteral formulations. In some embodiments of any of the aspects, the patient does not receive sufficient oral nutrition to maintain nutritional balance. In some embodiments of any of the aspects, the patient is not receiving other parenteral formulations sufficient to maintain nutritional balance. In some embodiments of any of the aspects, the patient is not receiving other topical formulations sufficient to maintain nutritional balance. In some embodiments of any of the aspects, the patient is not receiving other nutritional sources of fatty acids. In some embodiments of any of the aspects, the patient is not receiving other parenteral nutritional sources of fatty acids. In some embodiments of any of the aspects, the patient is not receiving other nutritional sources of essential fatty acids. In some embodiments of any of the aspects, the patient is not receiving other parenteral nutritional sources of essential fatty acids.

In some embodiments of any of the aspects, the emulsion formulation is administered as monotherapy. In some embodiments of any of the aspects, the emulsion formulation is administered as a monotherapy for therapeutic benefit. In some embodiments of any of the aspects, the emulsion composition is administered as monotherapy for nutritional needs (meeting nutritional needs).

In some embodiments of any of the aspects, the patient is a patient in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; atresia of the biliary tract; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemia-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; anginal condition; asthmatic condition; epileptic state; lacunar state; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis; psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal failure associated liver disease (IFALD), parenteral nutrition-associated cholestasis (PNAC), essential fatty acid deficiency (EFAD), parenteral nutritional dependence complicated by soy allergy or allergy to lipid emulsions containing ingredients other than MCTs and fish oil and/or omega-3 fatty acids.

In some embodiments of any of the aspects, the dose administered is from about 0.5 g total fatty acids in emulsion/kg/day to about 5 g total fatty acids in emulsion/kg/day. In some embodiments of any of the aspects, the dose administered is from about 1 g total fatty acids in emulsion/kg/day to about 3 g total fatty acids in emulsion/kg/day. In some embodiments of any of the aspects, the dose administered is about 2 g total fatty acids in emulsion/kg/day. In the above doses, "kg" refers to the weight of the composition administered to the subject. In some embodiments of any of the aspects, the method further comprises administering an additive of one or more additional fatty acids, or a mixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

On FIG. 1A-1B demonstrate that there are no differences in height (FIG. 1A) or organ weights (FIG. 1B) among treatment groups. N=9-10 mice per group, statistical analysis one-way ANOVA.

On FIG. 2A-2C depict serum levels of essential fatty acids. Fig. 2A: Biochemical EFAD did not occur in any of the fat-sourced groups. Only in the PN-fed saline group did EFAD occur. *=p<0.05 compared to food. Fig. 2B: SO and SO+AT contain more omega-6 fatty acids than the FO-treated groups. *=p<0.05 compared to FO. Fig. 2C: FO and FO+P contain more omega-3 fatty acids compared to SO-treated groups. 8=p<0.05 compared to FO. All statistical analysis was performed using one-way ANOVA.

On FIG. 3A-3B demonstrate that alpha-tocopherol confers hepatoprotective properties for SO. SO+AT resulted in normalization of liver architecture (Fig. 3A, bottom row, middle bar) and reduced liver fat accumulation (Fig. 3B, bottom row, middle bar) compared to SO (Figures 3A and 3B, top row, middle column). Phytosterols (FO+P) do not impair the hepatoprotective properties of FO (FIGS. 3A and 3B, right column, both rows).

On FIG. 4 shows the gene expression profiles for PPARg and ACC2. Fat-free PN mice and PN-fed mice injected with SO had increased expression of PPARg and ACC2. The FO-treated groups had normal expression of these genes, as did the SO+AT group. *=p<0.05 versus food by one-way ANOVA. The results are shown as a fold difference compared to the food with the control diet.

On FIG. 5A-5B depict PPARg and ACC protein expression. Fig. 5A: Western blot images for ACC, PPARg and beta actin housekeeping gene. Fig. 5B: Quantification of protein expression for each group, normalized to beta-actin and compared to food.

On FIG. 6A shows liver staining with Hemotoxylin and Eosin, and FIG. 6B shows Oil Red O staining. FO, 70:30 and 50:50 show normal liver architecture similar to food. 30:70 has improved liver architecture compared to the saline and SO groups, with moderate steatosis (Fig. 6A). FO, 70:30, 50:50 and 30:70 have no detectable liver fat accumulation (FIG. 6B).

On FIG. 7 demonstrates that FO, SO and all mixtures of FO and MCT effectively prevent biochemical EFAD. *=p<0.0001 compared to food according to one-way ANOVA.

On FIG. 8A TNFa levels and in FIG. 8B depicts IL-6 levels after saline or LPS tests. 3A: *=p<0.05 compared to food, and #=p<0.05 compared to FO according to one-way ANOVA 3B: *=p<0.002 compared to food and #=p<0.05 compared with FO according to one-way ANOVA.

On FIG. 9A shows hematoxylin and eosin staining, and FIG. 9B depicts Oil Red O liver analysis from each treatment group. Mice treated with saline and SO developed hepatic steatosis (4A) and accumulated liver fat (Fig. 9B). FO, FOE, FO/MCT, and FOE/MCT retained normal liver architecture (FIG. 9A), and did not accumulate detectable liver fat (FIG. 9B).

On FIG. 10A depicts serum TNFa and FIG. 10B depicts serum IL-6 in mice treated with LPS. Both markers decreased compared to FO in the FOE, FO/MCT and FOE/MCT treated groups. There was no significant difference between the FOE, FO/MCT and FOE/MCT groups. *=p<0.003 compared to food, and #=p<0.006 compared to FO according to one-way ANOVA.

On FIG. 11A-11D demonstrate that there is no difference in growth parameters between the PN diet and laboratory formulated fat emulsion groups. Fig. 11A: Body weight was observed every other day during the course of PN without any statistical difference in height between groups. Masses of the liver (Fig. 11B), spleen (Fig. 11C) and right kidney (Fig. 11D) at euthanasia after 19 days of the PN diet. There were no differences in organ mass between groups. N=10 mice per treatment group. Statistical analysis was performed using one-way ANOVA.

On FIG. 12A-12C depict the serum fatty acid profiles after 19 days of the PN diet fed with laboratory-formulated fat emulsions. On FIG. 12A: Triene to tetraene ratios. Only mice without fat source (PN + saline) met the biochemical criteria for EFAD. The distribution of major omega-6 fatty acids (FIG. 12B) and omega-3 fatty acids (FIG. 12C) reflects the source of fat administered to each group. N=3 samples per treatment group, randomly selected. Statistical analysis was performed using one-way ANOVA.

On FIG. 13A-13B depict normalization of liver architecture and liver fat accumulation when α-tocopherol is added to SO as an intravenous fat source with the PN diet. Images of Representative Hematoxylin and Eosin (H&E, Fig. 13A) and Oil Red O (Fig. 13B) demonstrate liver architecture and liver fat accumulation, respectively. N=10 samples per treatment group for H&E (FIG. 13A), and n=3 representative samples for Oil Red O (FIG. 13B). Images are 100X magnification (10X objective, 10X eyepiece).

On FIG. 14A-14B depict the expression of ACC (FIG. 14A) and PPARγ (FIG. 14B), which is a deregulated fat-free PN diet and a PN diet with SO as a fat source, and normalized for FO and for the addition of α-tocopherol to FO as fat sources. . Gene expression is measured as a fold difference compared to the fed group. N=5 samples per processing group, each made in a technical duplicate. Statistical analysis was performed using one-way ANOVA. The results are shown as a fold difference compared to the food with the control diet.

On FIG. 15A-15C depict ACC protein levels (FIG. 15A) and PPARγ (FIG. 15B) activated by a low fat PN diet and a PN diet with SO as a fat source and normalized for FO and for the addition of α-tocopherol to SO as fat source. The protein level was quantified by normalizing each group for the corresponding beta-actin level and compared with the food group. Western blots were performed in biological duplicates for each group. Statistical analysis was performed using one-way ANOVA. Fig. 15C: Western blot image.

On FIG. 16 demonstrates that laboratory-formulated fat emulsions replicate the effects of similar commercially available intravenous fat emulsions in a mouse model of PN-induced liver injury. Fat-free PN leads to the development of steatosis within 19 days (lower left panel). Formulated with FO in the laboratory (PN+FO) and commercially available FO emulsion (OM) retain normal liver architecture with a PN diet, while formulated with SO in the laboratory (PN+SO) and commercially available SO (IL) do not .

On FIG. 17 and FIG. 18 shows tables of content and particle size of the fat emulsions used in Example 3.

DETAILED DESCRIPTION

As described herein, the inventors have found that specific emulsions of a) fish oil or omega-3 fatty acids and b) medium chain triglycerides (MCTs) provide unexpected efficacy as a nutritional source and work better, for example, in preventing and reducing inflammatory responses. than any of the components taken separately. In addition, the emulsions described herein have improved stability. Finally, in contrast to earlier work in this field, the emulsions described herein do not cause deficiency of essential fatty acids and can be used as a monotherapy for the nutritional sources of fat for the patient. In one aspect of any of the embodiments of the invention described herein is an emulsion composition containing: a) a combination of fish oil and medium chain triglycerides (MCT) in a ratio of from about 70:30 to about 30:70; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 70:30 to about 30:70.

As used herein, the term "emulsion" refers to a heterogeneous system containing at least two or more substantially immiscible liquids, one liquid being dispersed in the other liquid as droplets. By way of example only, emulsions can be two-phase systems containing two immiscible liquid phases thoroughly mixed and dispersed with each other. Examples of an emulsion include, but are not limited to, water-in-oil emulsions, oil-in-water emulsions, water-in-water emulsions, water-in-oil-in-water emulsions, and oil-in-water-in-oil emulsions. In some embodiments of any of the aspects, the continuous phase of the emulsion is water.

In some embodiments of any of the aspects, the emulsion contains water. In some embodiments, the emulsion has about 20% total fat in water by weight/volume. In some embodiments, the emulsion has from about 10% total fat in water by weight/volume to about 50% total fat in water by weight/volume. In some embodiments, the emulsion has about 20% total fat in water by weight/volume. In some embodiments, the emulsion has from 10% total fat in water by weight/volume to 50% total fat in water by weight/volume.

Emulsions are generally unstable mixtures and do not form spontaneously, so energy is required to mix the continuous and dispersed phases and form an emulsion. This energy can be applied, for example, by shaking, mixing, homogenizing, spraying, high pressure pumping and ultrasonic emulsification. The emulsion composition described herein can be obtained by mixing the fat components listed herein with any proteins, carbohydrates and/or other additional additives and homogenizing the mixture until a stable emulsion is obtained. In some embodiments of any of the aspects, both the MCT and the fish oil and/or omega-3 fatty acids are emulsified, eg, rather than simply blended or blended into an emulsion of the other. However, over time, the resulting emulsion may tend to return to a stable state of separate oil and water layers. Accordingly, in some embodiments, the emulsion composition described herein may further comprise any natural or synthetic emulsifier known in the art. The addition of an emulsifier can increase the kinetic stability of emulsions so that, once formed, the emulsion does not change significantly during long-term storage.

The emulsion described herein can be prepared using a number of conventional techniques known to those skilled in the art. For example, the core lipid(s) is first mixed with one or more emulsifiers and an antioxidant, if used. An emulsion is then prepared by slowly adding this oil phase to water with constant stirring. If an osmolality modifier is used, it is added to the water before mixing with the oil phase. At this stage, if necessary, you can adjust the pH, and if required, the final volume with water.

In some embodiments of any of the aspects, the particle size of the oil globules in the emulsion, eg, when formulated for parenteral administration, is within or below the size range of a naturally occurring chylomicron, which is 0.4-1.0 microns. If the particle size is larger than this, lipid particles can be deposited in the liver, spleen, and lungs, resulting in a significant fat load after infusion (Rahui C.M., et I al., Am. Hosp. Pharm. 1992, 49:2749-2755 ). Lipids with small particle sizes are better dispersed in emulsions and tend to form safer and more stable emulsions. The choice of suitable conditions for the preparation of emulsions according to the present invention is within the normal skill of a worker in the art.

In some embodiments, the emulsion is a stable emulsion. As used herein, the term "stable emulsion" refers to an emulsion in which the droplets remain substantially evenly distributed throughout the continuous phase (or carrier liquid) for an extended period of time (e.g., at least about 1 month or longer), including reasonable periods of storage and use. For example, droplets do not aggregate or settle after a long period of time (eg, at least about 1 month or longer).

As used herein, the term "substantially immiscible" refers to two or more liquids that do not form a homogeneous mixture when they are in contact with each other. In some embodiments, when two or more substantially immiscible liquids are in contact with each other, one of the liquids may have a partial solubility (eg, no more than 10% or less) in the other essentially immiscible liquid. The term "homogeneous mixture" as used herein means that all of the components and/or liquids in the mixture are wholly present in one phase. For example, one or more components and/or liquids do not separate into distinct phases even when the mixture is left stationary for an extended period of time (e.g., at least about 6 hours or longer, including, e.g., at least about 12 hours, at least about 18 hours, at least about 24 hours, or longer). When referring to the miscibility of droplets and carrier liquid, the term "substantially immiscible" refers to a liquid (e.g., a thin liquid layer) forming at least the outer surface of the droplets and a carrier liquid that do not form a homogeneous mixture when they are in contact with each other.

The term "drop", as used here and throughout the description, refers to the final volume of a substance containing at least one liquid or at least one liquid phase, including, for example, at least two or more liquids or liquid phases. The droplets may be of any size, configuration and/or shape. In some embodiments of the various aspects described herein, the drops may have a droplet size that is smaller (eg, at least 50% smaller) than the inside diameter of the needle that is used to administer the emulsion containing the drops. As will be appreciated by one of skill in the art, droplets typically show a distribution of droplet sizes around a specified "size". Unless otherwise indicated, the term "droplet size" or "size" as used herein refers to the type of droplet size distribution, i.e., the value that occurs most frequently in the size distribution. Methods for measuring droplet size are known to the person skilled in the art, e.g. by dynamic light scattering (such as photocorrelation spectroscopy, laser diffraction, small angle laser light scattering (LALLS) and medium angle laser light scattering (MALLS)), light shading methods (such as the analysis method according to Coulter), or by other techniques (such as rheology and light or electron microscopy).

In some embodiments of any of the aspects, the emulsion formulation is about 10-50% of the emulsion, which means that the MCTs and fish oils and/or omega-3 fatty acids described herein are present in an amount of 10 to 50 g per 100 ml composition. In some embodiments of any of the aspects, the composition of the emulsion is about 20-40% of the emulsion.

In some embodiments of any of the aspects, the composition of the emulsion is about 10-50% of the emulsion. In some embodiments of any of the aspects, MCTs and the fish oil and/or omega-3 fatty acids described herein provide 1.1 kcal/mL to 5 kcal/mL of the formulation.

While the compositions for total enteral or parenteral nutrition described herein are intended to be administered in the form of emulsions, other formulation forms are also within the scope of the invention. For example, the compositions described herein as emulsions can also be made into powder form by increasing the percentage of total solids in the formula using procedures well known to those skilled in the art. The concentrate or powder may be reconstituted for administration by adding water (tap or deionized sterilized water) to form an emulsion.

As used herein, the term "fish oil" refers to an oil derived from fish or fish tissue. Fish oil is available commercially, for example, 10% (w/w) fish oil triglycerides are available from Nisshin Flour Milling Co. located in Nisshin City, Japan. OMEGAVEN (Fresnius Kabi) is suitable for use in the methods and compositions described herein. In some embodiments of any of the aspects, the fish oil may contain omega-3 fatty acids, DHA and/or EPA. In some embodiments of any of the aspects, the fish oil may be a fish oil derived from one or more cold water fish known to be high in omega-3 fatty acids. Non-limiting examples of cold water fish can be deep sea fish, shark, salmon, cod, salmon, bonito, mackerel, Atlantic mackerel, haddock, herring, mahi-mahi, menhaden, mackerel, caplin, tilapia, Pacific saury, krill, anchovies, pollock, trout , whitefish, tuna, smelt, shad and sardines. In some embodiments of any of the aspects, the fish oil may be a fish oil derived from one or more cold water salt water fish. In some embodiments of any of the aspects, the fish oil may be a fish oil derived from shark, salmon, cod, salmon, bonito, mackerel, Atlantic mackerel, haddock, herring, mahi-mahi, menhaden, mackerel, caplin, tilapia, Pacific saury, krill, anchovy, pollock, trout, whitefish, tuna, smelt, shad, sardine, or any combination thereof.

As used herein, the term "fatty acid" includes fatty acids such as unsaturated (e.g., monounsaturated, polyunsaturated) or saturated fatty acids, as well as pharmaceutically acceptable esters, free acids, mono-, di- and triglycerides, derivatives, conjugates , precursors, salts and mixtures thereof.

As used herein, the term "omega-3 fatty acids" includes natural and synthetic omega-3 fatty acids, as well as pharmaceutically acceptable esters, free acids, triglycerides, derivatives, conjugates, precursors, salts, and mixtures thereof. Omega-3 fatty acids may include, but are not limited to, hexadecatrienoic acid (HTA); α-Linolenic acid (ALA); Stearidonic acid (SDA); eicosatrienoic acid (ETA); eicosatetraenoic acid (ETA); eicosapentaenoic acid (EPA); Geneicosapentaenoic acid (HPA); docosapentaenoic acid (DPA); clupanodonic acid; docosahexaenoic acid (DHA); Tetracosapentaenoic acid and Tetracosahexaenoic acid (Nizinic acid). Omega-3 fatty acids for use in the emulsions described herein may have a high content of eicosapentaenoic acid (EPA) as well as docosahexaenoic acid (DHA). Omega-3 fatty acids can be marine or synthetic. For example, a suitable source of omega-3 fatty acids is fish oil or seal oil. Suitable fish oil sources include deep sea fish, shark, salmon, cod, salmon, bonito, mackerel, Atlantic mackerel, haddock, herring, mahi-mahi, menhaden, mackerel, caplin, tilapia, Pacific saury, krill, anchovies, pollock, trout, whitefish, tuna, smelt, shad and sardine, cold water fish as described elsewhere herein, and the like.

Fatty acids in accordance with the present description can be obtained from animal oils and/or non-animal oils. In some embodiments of the present disclosure, the fatty acids are derived from at least one oil selected from marine oil, algae oil, vegetable oil, and microbiological oil. Marine animal oils include, for example, fish oils such as tuna fish oil, krill oil, and a lipid composition derived from fish. Vegetable oils include, for example, linseed oil, rapeseed oil, mustard seed oil and soybean oil. Microbiological oils include, for example, Martek products. In at least one embodiment of the present disclosure, the fatty acids are obtained from a marine animal oil, such as fish oil. In at least one embodiment, the marine oil is a purified fish oil.

In some embodiments of any of the aspects, the omega-3 fatty acids in the emulsion described herein may contain EPA, DHA, or a combination thereof. In some embodiments of any of the aspects, the omega-3 fatty acids in the emulsion described herein may consist essentially of EPA, DHA, or a combination thereof. In some embodiments of any of the aspects of the omega-3 fatty acids in the emulsion described herein, may consist of EPA, DHA, or combinations thereof.

Examples of additional omega-3 fatty acids (e.g., omega-3-rich oils) and mixtures thereof covered by this disclosure include omega-3 fatty acids as defined in European Pharmacopoeia Omega-3 Triglycerides or the monograph Fish oil rich in omega-3 acids; which are incorporated herein by reference in their entirety. Examples of additional omega-3 fatty acids (e.g. omega-3 predominant oils) and mixtures thereof covered by this specification include omega-3 fatty acids as defined in European Pharmacopoeia Omega-3 Triglycerides, European Pharmacopoeia Omega-3 acid Ethyl Esters 60 or monograph Fish oil rich in omega-3; which are incorporated herein by reference in their entirety.

Commercial examples of omega-3 fatty acids suitable for the present invention include various mixtures of fatty acids (e.g., which can be in the form of triglycerides (TG), ethyl esters (EE), free-form fatty acids (FA) and/or in as phospholipids), including but not limited to: Incromega™ marine animal omega-3 oil concentrates such as Incromega™ El070, Incromega™ TG7010 SR, Incromega™ E7010 SR, Incromega™ TG6015, Incromega™ EPA500TG SR, Incromega™ E400200 SR, Incromega™ E4010, Incromega™ DHA700TG SR, Incromega™ DHA700E SR, Incromega™ DHA500TG SR, Incromega™ TG3322 SR, Incromega™ E3322 SR, Incromega™ TG3322, Incromega™ E3322, Incromega™ Trio TG/EE (Croda International PLC, Yorkshire, England); EPAX6000FA, EPAX5000TG, EPAX4510TG, EPAX2050TG, EPAX7010EE, EPAX5500EE, EPAX5500TG, EPAX5000EE, EPAX5000TG, EPAX6000EE, EPAX6000TG, EPAX6000FA, EPAX6500EE, EPAX6500TG, EPAX4510TG, EPAX1050TG, EPAX2050TG, EPAX 7010TG, EPAX7010EE, EPAX6015 TG/EE, EPAX4020TG, и EPAX4020EE (ЕРАХ is a wholly owned subsidiary of the Norwegian company AUSTEVOLL Seafood ASA); Meg-3® EPA/DHA fish oil concentrates (Ocean Nutrition Canada); DHA FNO "Functional Nutritional Oil" and DHA CL "Clear Liquid" (Lonza); Superba™ Krill Oil (Aker); omega-3 products containing DHA, manufactured by Martek; krill oil Neptune (Neptune); fish oil products and fish oil anti-reflux concentrate (TG) manufactured by Mnllers; Lysi Omega-3 Fish oil; Seven Seas Triomega® Cod Liver Oil Blend (Seven Seas); and Fri Flyt Omega-3 (Vesteralens).

The fish oil and/or omega-3 fatty acids used herein may be purified, for example, to meet quality standards for parenteral administration. In some embodiments of any of the aspects, fish oil and/or omega-3 fatty acids may be fortified with other or additional omega-3 fatty acid triglycerides, e.g., to increase omega-3 fatty acid triglycerides to fish oil and/or omega-3 fatty acids. 3 fatty acids can be supplemented with purified or synthesized omega-3 triglycerides from another source. Methods for extracting and purifying oils are well known in the art. There is no need to re-esterify oils in order to refine, extract or refine them.

In some embodiments of any of the aspects, the fish oil and/or omega-3 fatty acids described herein may contain from 0-100 percent DHA and 0-100 EPA.

In some embodiments of any of the aspects described herein, the omega-3 fatty acids and/or fish oil may be highly refined, e.g., highly enriched beyond the original content of omega-3 fatty acids and their triglyceride compound as part of this specific procedure. . In some embodiments of any of the aspects, these compositions may contain at least 95% by weight, eg, 96% by weight, 97% by weight, 98% by weight or more monomeric triglycerides. In some embodiments of any of the aspects, these compositions may contain less than 1% by weight of oxidized triglycerides, less than 0.2% by weight of trimeric and oligomeric triglycerides, and less than 0.8% by weight of dimeric polyglycerides, and less than 1.5% by weight of non-emulsifiable individual carbohydrates and sterane. In some embodiments of any of the aspects, the total content of eicosapentaenoic acid and docosahexanoic acid in fish oil and/or omega-3 fatty acids is about 25%-50% by weight. In some embodiments of any of the aspects, the total content of eicosapentaenoic acid and docosahexanoic acid in fish oil and/or omega-3 fatty acids is about 35%-50% by weight. While fish oils can typically have a cholesterol content of 4,000 to 12,000 ppm, the cholesterol content of fish oils and/or omega-3 fatty acids used here (e.g., after purification or fortification) is less than 2,500 ppm, eg less than 1500 ppm.

In some embodiments of any of the aspects, the fish oils and/or omega-3 fatty acids in the compositions described herein are natural triglycerides, eg, they have not been distilled or interesterified. Distillation and/or transesterification of triglycerides is used in some compositions known in the art (e.g., to remove myristic or palmitoleic acids), but this document specifically provides that such procedures alter triglycerides, causing toxicity in them and increasing the presence of diglycerides, monoglycerides and free fatty acids. In some embodiments of any of the aspects of the omega-3 fatty acids of the compositions described herein are triglycerides, eg they are not diglycerides as described elsewhere. For example, the omega-3 fatty acids of the compositions described herein may contain no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, not more than 4%, not more than 3%, not more than 2%, not more than 1% or less, or not contain diglycerides, eg as a proportion of total diglycerides + triglycerides.

As used herein, the term "medium chain triglycerides" or "MCTs" refers to triglycerides having fatty acids with 6-12 carbon atoms in the chain. MCTs have saturated fatty acids. Triglyceride ("TG") (also known as triacylglycerol or triacylglyceride) is a glyceride in which glycerol is esterified with three fatty acids. MCTs may contain fatty acids selected from, for example, caprylic acid, capric acid, lauric acid and caproic acid. MCTs can be obtained from a plant, such as a fruit or vegetable, for example, from a variety of plants. The description of MCTs for use in this invention may, for example, comply with the requirements of the EP 0868 monograph entitled "Triglycerides, Medium Chain" (Triglycerida saturate media) (EP 0868, 2008); which is included in this document in its entirety.

In some embodiments of any of the aspects, the MCT may contain 90% or more C8 and/or C10 fatty acids. In some embodiments of any of the aspects, the MCT may contain 95% or more C8 and/or C10 fatty acids. In some embodiments of any of the aspects, the MCT may contain 30%-50% C10 fatty acids and 45%-65% C8 fatty acids. In some embodiments of any of the aspects, the MCT may contain about 41% C10 fatty acids and about 54% C8 fatty acids.

In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCT) in a ratio of from 70:30 to 30:70; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of 70:30 to 30:70. In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of from about 60:40 to about 30:70; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 60:40 to about 30:70. In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCT) in a ratio of from 60:40 to 30:70; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of 60:40 to 30:70.

In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of from about 50:50 to about 30:70; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 50:50 to about 30:70. In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCT) in a ratio of from 50:50 to 30:70; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of 50:50 to 30:70.

In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of from about 40:60 to about 60:40; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40. In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCT) in a ratio of from 40:60 to 60:40; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of 40:60 to 60:40.

In some embodiments of any of the aspects, the emulsion may contain a) a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 50:50; or b) a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 50:50. In some embodiments of any of the aspects, the emulsion may comprise a) a 50:50 combination of fish oil and medium chain triglycerides (MCTs); or b) a 50:50 combination of omega-3 fatty acids and medium chain triglycerides (MCTs).

In some embodiments of any of the aspects, the emulsion compositions described herein contain from about 5% wt./about. up to about 50% wt./about. from the total amount of fat in the total volume of the composition. Total fat may refer to MCTs and fish oil/omega-3 fatty acids combined. In some embodiments of any of the aspects, the emulsion compositions described herein contain from about 15% wt./about. up to about 25% wt./about. from the total amount of fat in the total volume of the composition. In some embodiments of any of the aspects, the emulsion compositions described herein contain from about 5% wt./about. up to about 50% wt./about. from the total amount of fat in the total volume of the composition. In some embodiments of any of the aspects, the emulsion compositions described herein contain from about 20% wt./about. the total amount of fat in the total volume of the composition. In some embodiments of any of the aspects, the emulsion compositions described herein contain from 5% wt./about. up to 50% w/v from the total amount of fat in the total volume of the composition. In some embodiments of any of the aspects, the emulsion compositions described herein contain from 15% wt./about. up to 25% w/v from the total amount of fat in the total volume of the composition. In some embodiments of any of the aspects, the emulsion compositions described herein contain from 5% wt./about. up to 50% w/v from the total amount of fat in the total volume of the composition. In some embodiments of any of the aspects, the emulsion compositions described herein contain 20% wt./about. the total amount of fat in the total volume of the composition.

In some embodiments of any of the aspects, the emulsions described herein may additionally contain alpha-tocopherol. As used herein, the term "alpha-tocopherol" refers to the form of vitamin E having the structure of Formula I, in any of its stereoisomers.

In some embodiments of any of the aspects, alpha-tocopherol is present in the emulsion in an amount of at least 50 mg/L. In some embodiments of any of the aspects, alpha-tocopherol is present in the emulsion in an amount of at least 75 mg/L. In some embodiments of any of the aspects, alpha-tocopherol is present in the emulsion in an amount of at least 100 mg/L. In some embodiments of any of the aspects, alpha-tocopherol is present in the emulsion in an amount of at least 120 mg/L. In some embodiments of any of the aspects, alpha-tocopherol is present in the emulsion in an amount of at least 150 mg/L. In some embodiments of any of the aspects, alpha-tocopherol is present in the emulsion in an amount of at least 200 mg/L.

In some embodiments of any of the aspects, the emulsion composition contains alpha-tocopherol and other forms of vitamin E (e.g., beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and/or delta- tocotrienol) in a ratio of at least 2:1 by weight. In some embodiments of any of the aspects, the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 3:1 by weight. In some embodiments of any of the aspects, the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 5:1 by weight. In some embodiments of any of the aspects, the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 10:1 by weight. In some embodiments of any of the aspects, the emulsion composition does not contain other forms of vitamin E other than alpha-tocopherol.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs and b) fish oils and/or omega-3 fatty acids. In some embodiments of any of the aspects, the emulsion as described herein may consist essentially of a) MCTs and b) fish oils and/or omega-3 fatty acids. In some embodiments of any of the aspects, the emulsion described herein may be composed of a) MCTs and b) fish oils and/or omega-3 fatty acids. In some embodiments of any of the aspects, the omega-3 fatty acid is provided as an omega-3 fatty acid predominant oil.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oil and/or omega-3 fatty acids, and c) one or more emulsifiers (e.g. egg phospholipid), phospholipids, glycerol and sodium oleate. In some embodiments of any of the aspects, the emulsion as described herein may consist essentially of a) MCTs and b) fish oils and/or omega-3 fatty acids. In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oils and/or omega-3 fatty acids, and c) one or more emulsifiers (e.g., phospholipids and/or egg phospholides) , glycerin and sodium oleate. In some embodiments of any of the aspects, the emulsion described herein may be composed of a) MCTs and b) fish oils and/or omega-3 fatty acids.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oil and/or omega-3 fatty acids, and c) one or more emulsifiers, phospholipids, egg phospholipids, glycerol, and sodium oleate.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oils and/or omega-3 fatty acids, and c) alpha-tocopherol. In some embodiments of any of the aspects, the emulsion described herein may consist essentially of a) MCTs, b) fish oil and/or omega-3 fatty acids, and c) alpha-tocopherol. In some embodiments of any of the aspects, the emulsion as described herein may be composed of a) MCTs, b) fish oil and/or omega-3 fatty acids, and c) alpha-tocopherol.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oil and/or omega-3 fatty acids, c) alpha-tocopherol, and d) one or more emulsifiers, phospholipids, egg phospholipids , glycerin and sodium oleate. In some embodiments of any of the aspects, the emulsion described herein may consist essentially of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) alpha-tocopherol, and d) one or more emulsifiers (e.g. ., phospholipids or egg phospholipids), glycerol and sodium oleate. In some embodiments of any of the aspects, the emulsion described herein may consist of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) alpha-tocopherol, and d) one or more emulsifiers (e.g., phospholipids and/or egg phospholide), glycerol and sodium oleate.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) one or more of egg phospholipid, glycerol, sodium oleate and sodium hydroxide, and optionally e) alpha-tocopherol and/or phytosterols. In some embodiments of any of the aspects, the emulsion described herein may consist essentially of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) one or more of egg phospholipid, glycerin , sodium oleate and sodium hydroxide, and optionally e) alpha-tocopherol and/or phytosterols. In some embodiments of any of the aspects, the emulsion described herein may consist of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) one or more of egg phospholipid, glycerol, oleate sodium and sodium hydroxide, and optionally e) alpha-tocopherol and/or phytosterols.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) egg phospholipid, glycerin, sodium oleate and sodium hydroxide, and optionally, e) alpha-tocopherol and/or phytosterols. In some embodiments of any of the aspects, the emulsion described herein may consist essentially of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) egg phospholipid, glycerol, sodium oleate, and sodium hydroxide, and optionally e) alpha-tocopherol and/or phytosterols. In some embodiments of any of the aspects, the emulsion described herein may be composed of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) egg phospholipid, glycerol, sodium oleate, and sodium hydroxide and optionally e) alpha-tocopherol and/or phytosterols.

In some embodiments of any of the aspects, the emulsion described herein may contain a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) egg phospholipid, glycerin, sodium oleate and sodium hydroxide, and e) alpha-tocopherol. In some embodiments of any of the aspects, the emulsion described herein may consist essentially of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) egg phospholipid, glycerol, sodium oleate, and sodium hydroxide; and e) alpha-tocopherol. In some embodiments of any of the aspects, the emulsion described herein may be composed of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) water, d) egg phospholipid, glycerol, sodium oleate and sodium hydroxide and e) alpha-tocopherol.

Under certain conditions, eg during TPN, phytosterols (eg β-sitosterol, campesterol, stigmasterol, sitostanol and campestanol) can induce inflammatory conditions and PNALD. In some embodiments of any of the aspects, the emulsion does not contain phytosterols. In some embodiments of any of the aspects, the emulsion does not contain omega-6 fatty acids. In some embodiments of any of the aspects, the emulsion does not contain fats and/or fatty acids derived from a vegetable source. In some embodiments of any of the aspects, the emulsion does not contain long chain fatty acids derived from a vegetable source.

As described herein, the compositions of the present invention can counteract the negative side effects of phytosterols (eg, β-sitosterol, campesterol, stigmasterol, sitostanol and campestanol omega). Accordingly, in some embodiments of any of the aspects, phytosterols and/or omega-6 fatty acids are present in the emulsions described herein. In some embodiments of any of the aspects, the phytosterols are present in the emulsion at a concentration of less than 50 mg/L. In some embodiments of any of the aspects, the omega-6 fatty acids are present in the emulsion at a concentration of less than 50 mg/L.

In some embodiments of any of the aspects, the phytosterols are present in the emulsion at a concentration of less than 100 mg/l. In some embodiments of any of the aspects, the omega-6 fatty acids are present in the emulsion at a concentration of less than 100 mg/l.

In some embodiments of any of the aspects, the phytosterols are present in the emulsion at a concentration of less than 25 mg/L. In some embodiments of any of the aspects, the omega-6 fatty acids are present in the emulsion at a concentration of less than 25 mg/L.

In some embodiments of any of the aspects, fats and/or fatty acids derived from a vegetable source are present in the emulsion at a concentration of less than 100 mg/l, e.g., less than 100 mg/l, less than 50 mg/l, or less than 25 mg/l.

As used herein, the term "phytosterol" refers to sterols and stanols of plant origin, eg, phytosteroids. Non-limiting examples of phytosterols may include P-sitosterol, campesterol, stigmasterol, sitostanol and campestanol.

As used herein, "omega-6 fatty acid" includes natural and synthetic omega-6 fatty acids, as well as pharmaceutically acceptable esters, free acids, triglycerides, derivatives, conjugates, precursors, salts, and mixtures thereof. Omega-6 fatty acids may include, but are not limited to, Linoleic acid (LA); Gamma-linolenic acid (GLA); calendic acid; Eicosadienoic acid, Dihomo-gamma-linolenic acid (DGLA); Arachidonic acid (AA, ARA); Docosadienoic acid; Adrenic acid; osbondic acid; tetracosatetraenoic acid; and tetracosapentaenoic acid. Omega-6 fatty acids can be of vegetable or synthetic origin.

In some embodiments of any of the aspects, arachidonic acid is present in the composition at a concentration of at least 900 mg/l. In some embodiments of any of the aspects, docosahexaenoic acid is present in the composition at a concentration of at least 13.4 grams/L. In some embodiments of any of the aspects, eicosapentaenoic acid is present in the composition at a concentration of at least 11.6 grams/L.

In some embodiments of any of the aspects described herein, the emulsions may additionally contain one or more emulsifiers (eg, phospholipids and/or egg phospholides), glycerol, and sodium oleate. In some embodiments of any of the aspects described herein, the emulsion may contain 10-30% MCT and fish oil and/or omega-3 fatty acids, 0.5-2.5% egg phospholipid, 0.5-5.0% glycerin and 0.005-0.1% sodium oleate. In some embodiments of any of the aspects described herein, the emulsion may contain about 20% MCT and fish oil and/or omega-3 fatty acids, about 1.2% egg phospholipid, about 2.5% glycerol, and about 0.03% sodium oleate. In some embodiments of any of the aspects described herein, the emulsion may contain 20% MCT and fish oil and/or omega-3 fatty acids, 1.2% egg phospholipid, 2.5% glycerol, and 0.03% sodium oleate.

The emulsion described herein can be administered, for example, orally, parenterally or intravenously. In some embodiments of any of the aspects described herein, the emulsion is formulated for oral administration. In some embodiments of any of the aspects described herein, the emulsion is formulated for parenteral and/or intravenous administration.

In some embodiments of any of the aspects of the compositions described herein, can be obtained by first emulsifying each component as a separate emulsion, and then mixing or combining these emulsions together, e.g., emulsifying an oily source of omega-3 fatty acids (e.g., omega-3-dominant oils) and/or fish oils, emulsifying MCTs, and then mixing or combining the two emulsions together to form the final combined emulsion of MCTs and fish oils (or omega-3 fatty acids) in the above combinations.

In some embodiments of any of the aspects, each emulsion can be individually formulated as follows by high pressure homogenization: First, a lipid dispersion is created using an egg phospholipid emulsifier that is added to heated (75-90°C) sterile USP-grade water for injection ( SWFI) under high shear mixing conditions. Allow the temperature to drop to 40-45°C. Sodium oleate is then added and stirring is continued at 3900-4000 rpm for 40 minutes. The heated SWFI is then added sequentially to maintain the temperature at 40-45°C. Glycerin is added under continuous shear mixing. The result is a dispersion consisting of 12% egg phospholipid, 25% glycerol and 0.3% sodium oleate. The crude dispersion is then transferred to a homogenizer and homogenized at 9000 psi and 40-45°C for 20 cycles, filtered through a 0.45 µm membrane and the pH adjusted to 10.4 with 0.5N HCl. sodium hydroxide (NaOH). All steps are performed under a nitrogen atmosphere. For compounding emulsions, an oil (eg, fish oil or MCT oil) is added to the dispersing agent in a thin stream under continuous shear mixing at 3500-4500 rpm for 40-45 minutes while maintaining the temperature at 40-45°C. The resulting crude emulsions are transferred to a homogenizer and homogenized at 5000 psi and 40-45° C. for at least 9 emulsion cycles. The pH of the emulsions is buffered to >8.8 using 0.1 N sodium hydroxide. NaOH. All steps of the compounding process are performed under a nitrogen atmosphere. The finished emulsions are aliquoted into glass serum vials and filled with nitrogen gas prior to sealing. All vials were subjected to heat sterilization. This allows each type of oil to be optimally homogenized to improve compliance with United States Pharmacopeia (USP) Chapter <729>, which states that parenteral fat emulsions have an average fat globule size <500nm and a fat globule percentage >5µm (PFAT5) ≤0.05% .

In some embodiments of any of the aspects described herein, the lipid emulsions may be a mixture of two or more emulsions. This approach may have the advantage that additional components can be easily added to the mixture after the initial lipid emulsification. As a non-limiting example, a 50:50 blend of 20% fish oil emulsion in combination with 20% MCT oil emulsion may also be supplemented with, e.g., additional fish oil emulsion, triglycerides, DHA, linoleic acid, or other lipids as needed for a particular patient condition. at the time of insertion, not at the time of manufacture. This allows the clinician more flexibility in titrating the oil blend for specific patient conditions and reduces the need for practitioners to maintain multiple combinations of pre-mixed MCTs and fish oils (or omega-3 fatty acids).

In some embodiments of any of the aspects, the compositions described herein can be prepared by first mixing the components together and then preparing the mixture to emulsify, e.g., by mixing a source of omega-3 fatty acids and/or fish oil with MCTs, and then emulsifying the mixture. In some embodiments of any of the aspects described herein, the lipid emulsions may be an emulsification of a mixture of lipid preparations.

For the preparation of lipid emulsions in accordance with the present invention, one or more emulsifying agents may be mixed with, for example, a source of omega-3 fatty acids or MCTs. Emulsifiers for this purpose are usually phospholipids of natural, synthetic or semi-synthetic origin. Various suitable emulsifiers are known in the art. Examples of suitable emulsifying agents include, but are not limited to, egg phosphatidylcholine, egg lecithin, L-α-dipalmitoylphosphatidylcholine (DPPC), DL-α-dipalmitoylphosphatidylethanolamine (DPPE), and dioleoylphosphatidylcholine (DOPC). In accordance with the present invention, the total concentration of diglycerides and monoglycerides, as well as free fatty acids in the emulsifier should be low in order to minimize the contribution to the total oil concentration of the emulsion. In one embodiment of the present invention, the total concentration of triglycerides as well as free fatty acids in the emulsifier is less than about 3.5%. In some embodiments of any of the aspects, lecithin is used as an emulsifier in lipid emulsions. In addition, egg lecithin can be used as an emulsifier. Egg lecithin, containing 80-85% phosphatidylcholine and less than 3.5% fat, can also be used as an emulsifier. One skilled in the art will appreciate that other components may be present in the egg lecithin without adversely affecting the emulsifying properties. For example, egg lecithin may contain one or more of phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomeilin, and other natural ingredients.

In some embodiments of any of the aspects, the emulsion described herein contains from about 0.5% to about 5% (w/v) of an emulsifying agent. In some embodiments of any of the aspects, the emulsion described herein contains from about 0.6% to about 2% (w/v) of an emulsifying agent. In some embodiments of any of the aspects, the emulsion described herein contains from about 0.8% to about 1.8% (w/v) of an emulsifying agent. In some embodiments of any of the aspects, the emulsion described herein contains from about 1.0% to about 1.5% (w/v) of an emulsifying agent. In some embodiments of any of the aspects, the emulsion described herein contains about 1.2% (w/v) of an emulsifying agent.

The ratio of lecithin to oil (eg MCT, fish oil and/or omega-3 fatty acids) in the emulsion is important in determining the size of the oil globules formed in the emulsion. In some embodiments of any of the aspects, the ratio of lecithin to oil is from about 1:4 to about 1:20. In some embodiments of any of the aspects, the ratio is from about 1:4 to about 1:18. In some embodiments of any of the aspects, the ratio is from about 1:4 to about 1:15. In some embodiments of any of the aspects, the ratio is from about 1:4 to about 1:10.

The lipid emulsion of the present invention may further contain additional components such as antioxidants, chelating agents, osmolality modifiers, buffers, neutralizing agents, and the like, which improve the stability, uniformity and/or other properties of the emulsion. Suitable antioxidants that can be added to lipid emulsions include, but are not limited to, alpha-tocopherol (vitamin E) and tocotrienols. As known in the art, tocotrienols are a natural mixture of tocotrienols and vitamin E extract concentrated from rice bran oil distillate. Tocotrienols have a similar structure to vitamin E and contain three double bonds in the carbon side chain of the molecule. In some embodiments of any of the aspects, the concentration of the antioxidant added to the emulsion is typically from about 0.002 to about 1.0% (w/v). In some embodiments of any of the aspects, the concentration of the antioxidant used in the emulsion is from about 0.02% to about 0.5% (w/v). In some embodiments of any of the aspects, tocotrienols are added to the emulsion as an antioxidant. In some embodiments of any of the aspects, about 0.5% (w/v) tocotrienols are added to the emulsion. In some embodiments of any of the aspects, vitamin E is added to the emulsion as an antioxidant. In some embodiments of any of the aspects, about 0.02% (w/v) vitamin E is added to the emulsion.

The emulsion may further contain a chelating agent to improve emulsion stability and reduce the formation of oxidized fatty acids. Suitable chelating agents are known in the art and are those generally recognized as safe (GRAS) compounds. Examples include, but are not limited to, EDTA. In some embodiments of any of the aspects, the emulsion contains EDTA. In some embodiments of any of the aspects, the emulsion contains concentrations of EDTA from about 1 x 10 ^-6 M to about 5 x 10 ^-5 M.

An osmolality modifier may also be included in the emulsion to adjust the osmolality of the emulsion to a value suitable for parenteral administration. The amounts and types of osmolality modifiers for use in parenteral emulsions are well known in the art. An example of a suitable osmolality modifier is glycerol. The concentration of the osmolality modifier is typically from about 2% to about 5% (w/v). In some embodiments of any of the aspects, the amount of osmolality modifier added to the emulsion is from about 2% to about 4%. In some embodiments of any of the aspects, the amount of osmolality modifier added to the emulsion is from about 2% to about 3%. In some embodiments of any of the aspects, about 2.25% (w/v) glycerol is added to the emulsion as an osmolality modifier. In some embodiments of any of the aspects, the final product is isotonic to allow infusion of the emulsion through a central or peripheral venous catheter.

The pH of the emulsion can be adjusted with buffers or neutralizing agents. It has been shown that emulsions with pH values close to or above physiological pH are less susceptible to fatty acid peroxidation. One skilled in the art will appreciate that the pH of the emulsions can be adjusted by using an appropriate base that neutralizes the negative charge of the fatty acids, by using an appropriate buffer, or a combination thereof. Various bases and buffers are suitable for use with the emulsions of the present invention. One skilled in the art will appreciate that adding a buffer to an emulsion will affect not only the final pH but also the ionic strength of the emulsion. High ionic strengths can adversely affect the zeta potential of the emulsion (ie, the surface charge of the oil globules) and are therefore undesirable. It is believed that the selection of a suitable buffer strength to provide a suitable pH is within the normal skill of a person skilled in the art. In some embodiments of any of the aspects, the pH of the emulsion is adjusted with sodium hydroxide. In some embodiments of any of the aspects, the pH is adjusted with a buffer. In some embodiments of any of the aspects, the buffer is a phosphate buffer. In some embodiments of any of the aspects, both sodium hydroxide and a phosphate buffer are added to the emulsion. In some embodiments of any of the aspects, the final pH of the emulsion is from about 6.0 to about 9.0. In some embodiments of any of the aspects, the pH of the emulsion is from about 7.0 to about 8.5. In some embodiments of any of the aspects, the pH of the emulsion is from about 7.0 to about 8.0.

In some embodiments of any of the aspects, the lipid emulsion may further comprise components to regulate the stability of the emulsion, eg amino acids or carbohydrates such as fructose or glucose. The lipid emulsion may also be formulated to include nutrients such as glucose, amino acids, vitamins, or other parenteral nutritional supplements. The composition of the lipid emulsion to include a therapeutic agent is also considered to be within the scope of the present invention. As used herein, the term “therapeutic agent” refers to a physiologically or pharmacologically active substance that produces a localized or systemic effect or effects in animals and refers generally to drugs, nutritional supplements, vitamins, minerals, enzymes, hormones, proteins, polypeptides, antigens and other therapeutically or diagnostically useful compounds.

In some embodiments of any of the aspects, the emulsion composition may further comprise the addition of one or more additional fatty acids, or mixtures thereof. In some embodiments of any of the aspects, the methods described herein may further include adding an additive of one or more additional fatty acids, or mixtures thereof. The supplement may contain another fatty acid that is therapeutic for a disease, eg a disease for which the subject is in need of treatment. For example, DHA may be therapeutic for patients with cystic fibrosis, and a supplement for a subject with cystic fibrosis may contain DHA, for example, in excess of the amount needed for nutritional balance. In some embodiments of any of the aspects, the supplement contains fatty acids in a ratio or mixture that is therapeutic. In some embodiments of any of the aspects, the supplement is provided at a dosage of nanograms to grams/kg/day. In some embodiments of any of the aspects, the supplement is provided at a dose of 1 nanogram to 10 grams/kg/day. In some embodiments of any of the aspects, the supplement is provided at a dose of 1 nanogram to 100 grams/kg/day. In some embodiments of any of the aspects, the supplement is provided at a dose of 1 nanogram to 1000 grams/kg/day.

In some embodiments of any of the aspects, the composition comprises a mixture of a) an emulsion of an additive (e.g., additional fatty acid(s)) and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs. This approach, i.e. mixtures of emulsions, allows the mixture to be prepared at the time of or just before administration (e.g. at the bedside or in the pharmacy on site) and therefore to adapt the concentration and/or identity of the additive to be adapted to the needs of the patient according to clinical indications, eg, based on their symptoms, pathology and/or age. In some embodiments of any of the aspects, the supplement may be, for example, an additional fish oil emulsion, trigylcerides, DHA, linoleic acid, or other lipids, as desired.

In one aspect, this document describes a kit containing a composition as described herein, eg, an emulsion, as described herein. A kit is any article (eg, package or container) containing at least one reagent, eg, an emulsion, that is promoted, distributed, or sold as a unit to perform the methods described herein. The kits described herein may optionally contain additional components suitable for performing the methods described herein, eg, needles, tubing, etc., suitable for administration by the desired route. By way of example, the kit may contain fluids (eg, buffers) suitable for use with the emulsions described herein, educational material that describes the operation of the method as described herein, and the like. The kit may further comprise devices and/or reagents for delivering the composition as described herein. In addition, the kit may contain an instruction sheet and/or may contain information about dosage, frequency of administration, etc.

The kits of the invention comprise one or more packages or containers containing an emulsion in combination with a set of instructions, usually written instructions, regarding the use and dosage of the emulsion. The kits may further comprise additional containers containing one or more nutrients, or therapeutic or diagnostic compounds, which may be added to the emulsion prior to administration. Packages containing the emulsion may be in the form of unit doses or pharmacy packs. Doses can be packaged in such a format that each dose is associated with, for example, a day of the week. The kit may also be associated with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of biological products, which notice reflects the approval of the manufacturing, use, or sale agency for administration to a human or animal.

The design of the container is also an important factor in the production of fat emulsions. For example, if the emulsion is packaged in glass, the container may be filled with nitrogen before the actual emulsion is added. After adding the emulsion, the glass container can be refilled with nitrogen to remove dead space when the lid is closed. This filling with nitrogen prevents the formation of peroxide. If the product is packaged in plastic, a DEHP-free container that is gas-tight can be used. The container may also be suitably coated to minimize the formation of peroxide in lipids as well as leaching of plasticizer from the container into the product itself. Also, if plastic is used, a desiccant may be included in the bag, as well as an indicator that shows if there is an air leak in the outer wrap. In some embodiments of any of the aspects, the container may be latex-free.

In one aspect of any of the embodiments, a method is described herein comprising administering an emulsion composition as described herein to a subject in need thereof. A subject in need of an emulsion composition as described herein may be a subject in need of supplemental nutrition (oral or parenteral), a subject in need of parenteral nutrition, total parenteral nutrition, a subject in need of treatment for an inflammatory condition, and/or a subject in need of treatment or reduction of inflammation. One of skill in the art can easily identify subjects who are in need of an emulsion composition as described herein, e.g., subjects receiving parenteral nutrition who have obvious symptoms of inflammation and/or symptoms that are taken as indicators that traditional PN or TPN must be reduced or stopped due to the risk of side effects (including inflammation and/or liver disease). Such subjects and their symptoms are detailed, for example, in Guidelines for the Provision and Assessment of Nutrition Support Therapy in the Adult Critically 111 Patient: Society of Critical Care Medicine (SCCM) and American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.), Taylor et al., which is incorporated herein by reference in its entirety (available on the World Wide Web at journals.lww.com/ccmjournal/Fulltext/2016/02000/Guidelines_for_the_Provision_and_Assessment_of.20.aspx).

In some embodiments of any of the aspects, the patient to whom the emulsion composition described herein is administered may be a subject in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; atresia of the biliary tract; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemic-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; status anginosus; status asthmaticus; status epilepticus; status lacunaris; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal insufficiency associated with liver disease (IFALD), cholestasis associated with parenteral nutrition (PNAC), deficiency of essential fatty acids (EFAD), dependence on parenteral nutrition complicated by allergy to soy or allergies to lipid emulsions containing ingredients other than MCTs and fish oil and/or omega-3 fatty acids. In some embodiments of any of the aspects, the patient to whom the emulsion composition described herein is administered may be a subject who has or has been diagnosed with a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; atresia of the biliary tract; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemic-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; status anginosus; status asthmaticus; status epilepticus; status lacunaris; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal insufficiency associated with liver disease (IFALD), cholestasis associated with parenteral nutrition (PNAC), deficiency of essential fatty acids (EFAD), dependence on parenteral nutrition complicated by allergy to soy or allergies to lipid emulsions containing ingredients other than MCTs and fish oil and/or omega-3 fatty acids.

In some embodiments of any of the aspects, the subject to whom the emulsion is administered as described herein has or is in need of treatment for a liver disease, eg, fatty liver disease. As used herein, the term "fatty liver disease" refers to a disease in which fat (hepatocytes) accumulate excessively in the liver and can cause severe diseases such as chronic hepatitis and cirrhosis of the liver. In patients with fatty liver disease, lipids, especially neutral fat, accumulate in hepatocytes to the extent that their amount exceeds the physiologically acceptable range. From a biochemical point of view, the standard for assessing fatty liver is that the mass of neutral fat is about 10% (100 mg/g wt) or more of the wet weight of liver tissue. Fatty liver disease is usually detected by observation of elevated serum levels of liver-specific enzymes such as ALT and AST transaminases, which are indicators of hepatocyte damage, and by symptoms that include fatigue and pain in the liver region, although a definitive diagnosis is often a biopsy is required.

In some embodiments of any of the aspects, the subject to whom the emulsion is administered as described herein has or is in need of treatment for PN-associated or PN-induced liver disease. This disease includes both biochemical changes, ie. elevated serum aminotransferase, bilirubin, and alkaline phosphatase; and histological changes such as steatosis, steatohepatitis, lipidosis, cholestasis, fibrosis, and cirrhosis. The disease may progress and worsen over the course of PN administration and appears to be more common in the pediatric population. Additional risk factors for this condition include prematurity, low birth weight, long-term use, lack of concomitant oral intake, sepsis, and multiple operative procedures. In general, the severity of PN-induced liver pathology is believed to be inversely proportional to the age of the patient.

In some embodiments of any of the aspects, the emulsion described herein is administered by parenteral administration (PN). In some embodiments of any of the aspects described herein, the emulsion is administered by total parenteral administration (TPN). In some embodiments of any of the aspects, the subject to whom the emulsion described herein is administered requires parenteral administration (PN). In some embodiments of any of the aspects, the subject who is administered the emulsion described herein requires total parenteral administration (TPN). Methods for administering lipid emulsions to patients for PN application or therapeutic effect are known in the art. Typically, the emulsions are administered by infusion over a suitable period of time. Appropriate dosages and administration; regimens can be easily determined by one of ordinary skill in the medical field.

In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any oral nutrition. In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any oral nutrition, including fats and/or fatty acids.

In some embodiments of any of the aspects, the subject is administered the emulsion described herein, during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter any other parenteral drugs. In some embodiments of any of the aspects, the subject is administered the emulsion described herein, during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter any other parenteral drugs, containing fats and/or fatty acids.

In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other nutritional sources of fats and/or fatty acids.

In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other nutritional sources of essential fatty acids. In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other oral nutritional sources of essential fatty acids. In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other parenteral nutritional sources of essential fatty acids.

In some embodiments of any of the aspects, the emulsion described herein is administered orally.

In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other food sources that would be sufficient to maintain nutritional balance. In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other oral/enteral food sources that would be sufficient to maintain nutritional balance. In some embodiments of any of the aspects, the subject is administered the emulsion described herein during the period of treatment in which he is administered the emulsion (e.g., the period in days or weeks in which he is administered the emulsion), do not enter and / or allow any other sources of parenteral nutrition, which will be sufficient to maintain nutritional balance. As used herein, the term nutritional balance refers to maintaining growth, development and the absence of nutritional deficiencies by providing adequate nutrition. The nutritional balance meets the needs of each person without over-providing any particular nutrient that can lead to adverse effects.

As described elsewhere herein, the emulsions described herein are demonstrated to be suitable for monotherapy, eg they do not cause essential fatty acid deficiency, inflammation and/or other nutritional deficiencies when administered as monotherapy. This property is not characteristic of all compositions of fat/fatty acids, which may otherwise be suitable for administration (eg, parenteral administration). Accordingly, in some embodiments of any of the aspects, the emulsion described herein is administered as monotherapy for a condition in which a subject is in need of treatment. In some embodiments of any of the aspects, the emulsion described herein is administered as monotherapy for nutritional requirements, e.g., an anti-inflammatory agent without significant nutritional value may be administered simultaneously, but the emulsion will still be monotherapy for nutritional requirements. In some embodiments of any of the aspects, the emulsion described herein is administered as monotherapy for fatty acids, eg, no other source of fatty acids is administered to or consumed by the subject.

In some embodiments of any of the aspects, the emulsion described herein may be administered at a dose of about 0.5 g fatty acids/kg/day to about 5 g fatty acids/kg/day. In some embodiments of any of the aspects, the emulsion described herein may be administered at a dose of 0.5 g fatty acids/kg/day to 5 g fatty acids/kg/day. In some embodiments of any of the aspects described herein, the emulsion can be administered at a dose of from about 1 g fatty acids/kg/day to about 3 g fatty acids/kg/day. In some embodiments of any of the aspects, the emulsion described herein can be administered at a dose of 1 g fatty acids/kg/day to 3 g fatty acids/kg/day. In some embodiments of any of the aspects, the emulsion described herein may be administered at a dose of about 2 g fatty acids/kg/day.

In some embodiments of any of the aspects, the administration of the emulsion as described herein is continued for at least 3 days, e.g., 3 or more days, 4 or more days, 5 or more days, 7 or more days, 2 or more than weeks, 3 or more weeks, 4 or more weeks, 6 or more weeks, 2 months or more, or 3 months or more. In some embodiments of any of the aspects, the administration of the emulsion as described herein is continued for at least 3 weeks. In some embodiments of any of the aspects, the administration of the emulsion as described herein is continued for at least 6 weeks. In some embodiments of any of the aspects, the administration of the emulsion as described herein continues for at least 3 months.

The compositions and methods described herein may be administered to a subject having or diagnosed as having the condition described herein. In some embodiments, the methods described herein include administering an effective amount of the compositions described herein, eg, an emulsion, to a subject to alleviate a symptom of the condition described herein. As used herein, the term "symptom relief" means improvement in any condition or symptom associated with a condition. Compared to an equivalent untreated control, this reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard methodology. Those skilled in the art are aware of various methods of administering to subjects the compositions described herein. Such methods may include, but are not limited to, oral, parenteral, or intravenous administration.

As used herein, the term "effective amount" refers to the amount of the emulsion described herein necessary to relieve at least one or more symptoms of the disease or disorder, and refers to the amount of the pharmacological composition sufficient to provide the desired effect. The term "therapeutically effective amount", therefore, refers to the amount of the emulsion described herein, which is sufficient to provide a certain effect (eg, nutritional or anti-inflammatory effect) when administered to a typical subject. As used herein, an effective amount in various contexts may also include an amount sufficient to delay the development of a symptom of a disease, reverse the course of a symptom of a disease (for example, but without limitation, slow the progression of a symptom of a disease), or reverse a symptom of a disease. Thus, it is generally not possible in practice to determine an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. to determine LD50 (dose lethal in 50% of the population) and ED50 (dose therapeutically effective in 50% of the population). The dosage may vary depending on the dosage form used and the route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50/ED50 ratio. Compositions and methods that exhibit superior therapeutic performance are preferred. A therapeutically effective dose can be initially estimated from cell culture assays. In addition, the dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the emulsion or its components at which half-maximal symptom inhibition is achieved) as determined in cell cultures, or in an appropriate animal. models. Plasma levels can be measured, for example, using high performance liquid chromatography. The effects of any particular dosage may be monitored by appropriate bioassays, eg inflammation or liver function assays, among others. The dosage may be determined by the physician and, if necessary, adjusted according to the observed effects of the treatment.

In some embodiments, the technology described herein refers to a pharmaceutical composition containing the emulsion described herein as described herein, and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition contain the emulsion described herein as described herein. In some embodiments of the invention, the active ingredients of the pharmaceutical composition consist essentially of the emulsion described herein, as described herein. In some embodiments of the invention, the active ingredients of the pharmaceutical composition consist of the emulsion described herein, as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository waxes; (9) glycols such as propylene glycol; (10) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline solution; (17) Ringer's solution; (18) ethyl alcohol; (19) pH buffer solutions; (20) polyesters, polycarbonates and/or polyanhydrides; (21) excipients such as polypeptides and amino acids (22) serum component such as serum albumin, HDL and LDL; (23) C ₂ -C ₁₂ alcohols such as ethanol; and (24) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavors, flavors, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments of the invention, the carrier inhibits the degradation of active agents, as described herein. It is noted that the emulsion itself may contain water. The emulsion can be administered with other components used in parenteral nutrition solutions (eg dextrose, crystalline amino acids, trace elements, multivitamins, electrolytes and minerals).

In some embodiments of the invention, the pharmaceutical composition containing the emulsion described herein, as described herein, may be a parenteral dosage form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contamination, parenteral dosage forms are preferably sterile or may be sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, ready-to-dissolve or suspendable in a pharmaceutically acceptable injectable vehicle, dry products, injectable suspensions, and emulsions. In addition, controlled release parenteral dosage forms may be formulated for administration to a patient, including, but not limited to, ^DUROS® type dosage forms and dose dumping.

Pharmaceutical compositions containing the emulsion described herein may also be formulated to be suitable for oral administration, e.g., in discrete dosage forms such as, but not limited to, tablets (including, but not limited to, scored tablets or tablets with coated), pills, caplets, capsules, chewable tablets, powder sachets, sachets, troches, cachets or liquids such as, but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, non-aqueous liquid, oil-in-oil emulsion water or water-in-oil emulsion. Such compositions contain a predetermined amount of a pharmaceutically acceptable salt of the disclosed compounds and can be prepared by pharmaceutical methods well known to those skilled in the art. In general, see Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

The methods described herein may further include administering a second agent and/or treatment to the subject, eg, as part of a combination therapy. As a non-limiting example, if the subject is to be treated for pain or inflammation in accordance with the methods described herein, the subject may also be administered a second agent and/or treatment known to be beneficial to subjects suffering from pain or inflammation. . Examples of such agents and/or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (eg, cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclomethasone); methotrexate; sulfasalazine; leflunomide; drugs against TNF; cyclophosphamide; pro-permissive drugs; mycophenolate; or opiates (eg, endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

In certain embodiments of the invention, an effective dose of the composition containing the emulsion described herein, as described herein, can be administered to the patient once. In certain embodiments of the invention, an effective dose of a composition containing an emulsion described herein may be administered to a patient multiple times, e.g., daily or several times a day over a period of time of at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, or at least 3 months .

The dosage of the composition, as described herein, may be determined by the physician and, if necessary, adjusted based on the observed effects of treatment. With regard to duration and frequency of treatment, it is common practice for qualified clinicians to monitor subjects to determine when treatment is providing therapeutic benefit and to determine whether to increase or decrease dosage, increase or decrease frequency of administration, discontinue treatment, reinitiate treatment, or make other changes to the regimen. treatment. The dosing schedule may vary from weekly to daily, depending on a number of clinical factors, such as the sensitivity of the subject to the components of the emulsion. The desired dose or amount of activation may be administered at one time or divided into sub-doses, eg 2-4 sub-doses, and administered over a period of time, eg at appropriate intervals throughout the day or other suitable schedule. In some embodiments of the invention, the administration may be chronic, eg, one or more doses and/or treatments daily for several weeks or months. Examples of dosing and/or treatment regimens are administration daily, twice a day, three times a day, or four or more times a day over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months , 4 months, 5 months or 6 months or more. The composition containing the emulsion described herein can be administered over a period of time, such as a period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

Dose ranges for administering the emulsion described herein according to the methods described herein depend on, for example, the form of the emulsion, its potency, and the degree to which symptoms, markers, or indicators of the condition described herein are desired. decrease. The dosage should not be so high as to cause side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and may be determined by one of skill in the art. The dosage may also be adjusted by the individual physician in case of any complications.

The effectiveness of the emulsion described herein, eg, in treating the condition described herein or inducing a response as described herein, can be determined by a qualified physician. However, a treatment is considered an "effective treatment" as the term is used herein if one or more of the signs or symptoms of the condition described herein are changed in a favorable manner, other clinically acceptable symptoms are improved or even resolved, or the desired response is induced, eg, at least 10% after treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring the marker, indicator, symptom, and/or incidence of the disease being treated according to the methods described herein, or any other suitable measurable parameter, such as, for example, nutritional balance. , inflammation and/or liver function. Efficacy can also be measured by the individual's absence of deterioration, which is assessed by hospitalization or by the need for medical interventions (ie, the progression of the disease stops). Methods for measuring these indicators are known to those skilled in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include a human or animal) and includes: (1) inhibition of the disease, eg, preventing worsening of symptoms (eg, pain or inflammation); or (2) alleviating the severity of the disease, eg, inducing regression of symptoms. An effective amount to treat a disease means that amount which, when administered to a subject in need thereof, is sufficient to provide effective treatment, as the term is defined herein, for that disease. The effectiveness of an agent can be determined by evaluating physical indicators of the condition or desired response. One of ordinary skill in the art may well monitor the effectiveness of administration and/or treatment by measuring any of these parameters or any combination of parameters. Efficacy can be evaluated in animal models of the condition described herein. When using an experimental animal model, the effectiveness of treatment is confirmed by a statistically significant change in the marker.

Provided herein are assays in in vitro models and in animals that allow evaluation of a given emulsion dose as described herein. As a non-limiting example, the effects of an emulsion dose can be assessed by administering the emulsion orally or parenterally and then assessing serum fatty acid levels, markers of inflammation (eg, circulating TNF-alpha and/or IL-6), and histology of the liver, spleen, and/or or kidneys.

For convenience, the meanings of certain terms and phrases used in the description, examples and appended claims are given below. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings given below. The definitions are provided to assist in describing specific embodiments of the invention, and are not intended to limit the claimed invention, as the scope of the invention is only limited by the claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. If there is an apparent inconsistency between the use of a term in the art and its definition given in this document, the definition given in the description takes precedence.

For convenience, certain terms used herein, in the description, examples and appended claims, are collected herein.

The terms "decrease", "reduced", "decrease" or "inhibit" are all used herein to mean a decrease by a statistically significant value. In some embodiments, "reduce", "reduce", or "reduce" or "inhibit" generally means a reduction of at least 10% from a control level (e.g. no given treatment or agent) and may include, for example , a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. "Decrease" or "inhibition" as used herein does not encompass complete inhibition or reduction from a control level. "Total inhibition" is 100% inhibition compared to the control level. The reduction may be preferable to a level within the normal range for an individual without the disorder.

The terms "increased", "increase", "enhance" or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments of the invention, the terms "increased", "increase", "enhance" or "activate" may mean an increase of at least 10% compared to the control level, for example, an increase of at least about 20%, or at least at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80% , or at least about 90% or up to and including an increase of 100% or any increase of 10-100% over the control level, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase of 2 to 10 times or more over the control level. In the context of a marker or symptom, "increase" is a statistically significant increase in that level.

As used herein, the term "subject" means a human or animal. Typically, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, Javanese macaques, spider monkeys and macaques, for example, Rhesus. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, felines, eg domestic cat, canines, eg dogs, foxes, wolves, birds, eg chickens, emu, ostrich, and fish, eg. , trout, catfish and salmon. In some embodiments, the subject is a mammal, eg, a primate, eg, a human. The terms "individual", "patient" and "subject" are used interchangeably herein.

Preferably, the subject is a mammal. The mammal may be a human, non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects for animal models of the diseases and conditions described herein. The subject may be a male or a female.

The subject may be one who has previously been diagnosed with or identified as suffering from or having a condition in need of treatment, or one or more complications associated with such a condition, and optionally already treated for that condition, or one or more complications associated with state. Alternatively, the subject may also be one who has not previously been diagnosed with the condition or one or more complications associated with the condition. For example, a subject may be one who exhibits one or more risk factors for a condition or one or more complications associated with a condition, or a subject who does not exhibit risk factors.

A "subject in need" of treatment for a particular condition may be a subject having the condition, diagnosed as having the condition, or at risk of developing the condition.

As used herein, the terms "treat", "treatment", "cure" or "improvement" refer to therapeutic methods of treatment in which the goal is to reverse, alleviate, ameliorate, inhibit, slow or halt the progression or severity of a condition, associated with a disease or disorder, eg, the condition, disease or disorder described herein. The term "treatment" includes the reduction or alleviation of at least one adverse effect or symptom of a condition, disease or disorder. Treatment is usually "effective" if one or more symptoms or clinical markers improve. Alternatively, the treatment is "effective" if the progression of the disease is reduced or stopped. That is, "treatment" includes not only improvement in symptoms or markers, but also stopping or at least slowing the progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of one or more symptoms, improvement in disease severity, stabilization (i.e., no worsening) of the disease state, delay or retardation of disease progression, improvement or mitigation of the disease state, remission (partial or total) and/or reduction in mortality, detectable or not. The term "treating" a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care).

As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, eg a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms which, under sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier may be an emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier may be an artificial or engineered carrier, eg, a carrier in which the active ingredient does not occur naturally.

As used herein, the term "administering" refers to placing a compound disclosed herein in a subject in a manner or route that results in at least partial delivery of the agent to the desired site. Pharmaceutical compositions containing the compounds disclosed herein may be administered by any suitable route that results in effective treatment of the subject.

The term "statistically significant" or "significant" refers to statistical significance and usually means two standard deviations (2SD) or a large difference.

Except in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about". The term "about" when used with percentages can mean ±1%.

As used herein, the term "comprising" means that other elements may also be present in addition to the specific elements presented. The use of the word "comprising" indicates an inclusion, not a limitation.

The term "consisting of" refers to compositions, methods and their respective components, as described herein, which exclude any element not specified in this description of the embodiment of the invention.

Used in this document, the term "consisting essentially of" refers to those elements that are necessary for this embodiment of the invention. This term allows for the presence of additional elements that do not significantly affect the main and new or functional characteristic(s) of this embodiment of the invention.

The terms "a", "an" and "the" in the singular include plural references unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. While methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. Abbreviation "ex." comes from the Latin example gratia and is used in this document to refer to a non-limiting example. Thus, the abbreviation "ex." is synonymous with "for example".

The groups of alternative elements or embodiments of the invention disclosed herein should not be construed as limitations. Each group member may be listed and claimed individually or in any combination with other group members or other elements provided herein. One or more members of the group may be included or removed from the group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the description herein is deemed to contain the modified group, thus fulfilling the written description of all Markush groups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure pertains. It should be understood that this invention is not limited to the particular methodology, protocols, reagents, etc. described herein, and as such may vary. The terminology used herein is only intended to describe specific embodiments and is not intended to limit the scope of the present invention, which is solely defined by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter ef al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN ^1936113414 ); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), content which is incorporated into the present description by reference in its entirety.

Other terms are defined herein in the description of various aspects of the invention.

All patents and other publications; including literary references, issued patents, published patent applications and concurrent patent applications; those cited herein are specifically incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this respect shall be construed as an admission that the inventors are not entitled to precede such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the content of these documents are based on information available to the applicants and do not constitute any admission as to the correctness of the dates or content of these documents.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. While specific embodiments and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be appreciated by those skilled in the art. For example, although the steps or functions of the method are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The disclosure guidelines provided herein may be applied to other procedures or methods as appropriate. The various embodiments described herein may be combined to provide additional embodiments. Aspects of the disclosure may be modified, if necessary, to use the compositions, functions, and concepts of the aforementioned references and uses to provide still further embodiments of the disclosure. These and other changes may be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the above embodiments of the invention may be combined or replaced by elements in other embodiments of the invention. Furthermore, while the benefits associated with some embodiments of the disclosure have been described in the context of those embodiments, other embodiments may also exhibit such benefits, and not all embodiments need to exhibit such benefits to fall within the scope of the disclosure.

The technology described herein is further illustrated by the following examples, which should in no way be construed as further limitations.

Some embodiments of the technology described herein may be defined in accordance with any of the following numbered paragraphs:

1. An emulsion composition containing:

a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 30:70 to about 30:70; or

a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 30:70 to about 30:70.

2. Emulsion composition according to paragraph 1, containing:

a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40; or

a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40.

3. Emulsion composition according to paragraph 1, containing

a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of about 50:50; or

a combination of omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 50:50.

4. Emulsion composition according to paragraph 1, containing

a combination of fish oil and medium chain triglycerides (MCTs) in a ratio of 50:50; or

a 50:50 combination of omega-3 fatty acids and medium chain triglycerides (MCTs).

5. An emulsion composition according to any one of paragraphs 1-4, wherein the composition further comprises alpha-tocopherol.

6. An emulsion composition according to any one of paragraphs 1-5, wherein alpha-tocopherol is present in an amount of at least 100 mg/L.

7. An emulsion composition according to any one of paragraphs 1-6 wherein alpha-tocopherol is present in an amount of at least 120 mg/l.

8. An emulsion composition according to any one of paragraphs 1-7, wherein the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 2:1.

9. An emulsion composition according to any one of paragraphs 1-8, wherein the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 10:1.

10. An emulsion composition according to any one of paragraphs 1-9, wherein the emulsion composition does not contain other forms of vitamin E other than alpha-tocopherol.

11. An emulsion composition according to any one of paragraphs 1-10, wherein phytosterols are present in the composition.

12. An emulsion composition according to any one of paragraphs 1-11, wherein the phytosterols are present in the composition at a concentration of less than 50 mg/L.

13. An emulsion composition according to any one of paragraphs 1-12 formulated for oral administration.

14. An emulsion composition according to any one of paragraphs 1-12 formulated for parenteral or intravenous administration.

15. An emulsion composition according to any one of paragraphs 1-14, further comprising the addition of one or more additional fatty acids, or mixtures thereof.

16. The emulsion composition of paragraph 15, wherein the supplement contains one or more fatty acids which are therapeutic for the disease.

17. A method comprising administering the emulsion composition of any of paragraphs 1-16 to a subject in need thereof.

18. The method of paragraph 17, wherein the administration is parenteral administration.

19. The method of paragraph 17, wherein the administration is total parenteral administration.

20. The method of paragraph 17, wherein the administration is oral administration.

21. The method of any of paragraphs 17-20 wherein the subject is in need of parenteral nutrition.

22. The method of any of paragraphs 17-21 wherein the subject is in need of total parenteral nutrition.

23. The method of any of paragraphs 17-22, wherein the patient is not receiving oral nutrition.

24. The method of any of paragraphs 17-23, wherein the patient is not receiving other parenteral formulations.

25. The method of any one of paragraphs 17-24, wherein the patient is not receiving sufficient oral nutrition to maintain nutritional balance.

26. The method of any of paragraphs 17-25, wherein the patient is not receiving other parenteral formulations that are sufficient to maintain nutritional balance.

27. The method of any of paragraphs 17-26, wherein the patient is not receiving other nutritional sources of fatty acids.

28. The method of any one of paragraphs 17-27, wherein the patient is not receiving other sources of fatty acid parenteral nutrition.

29. The method of any of paragraphs 17-28, wherein the patient is not receiving other nutritional sources of essential fatty acids.

30. The method of any one of paragraphs 17-29, wherein the patient is not receiving other sources of parenteral nutrition of essential fatty acids.

31. The method of any of paragraphs 17-30, wherein the emulsion composition of any of paragraphs 1-14 is administered as monotherapy.

32. The method of any of paragraphs 17-31, wherein the emulsion composition of any of paragraphs 1-14 is administered as monotherapy for nutritional needs.

33. The method according to any one of paragraphs 17-32, in which the patient is a patient in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver diseases associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); intestinal failure associated liver disease (IFALD), parenteral nutrition-associated cholestasis (PNAC), essential fatty acid deficiency (EFAD), parenteral nutritional dependence complicated by soy allergy or allergy to lipid emulsions containing ingredients other than MCTs and fish oil and/or omega-3 fatty acids.

34. The method of any one of paragraphs 17-33, wherein the dose administered is from about 0.5 g fatty acids/kg/day to about 5 g fatty acids/kg/day.

35. The method of any one of paragraphs 17-34, wherein the dose administered is from about 1 g fatty acids/kg/day to about 3 g fatty acids/kg/day.

36. The method of any one of paragraphs 17-35, wherein the dose administered is about 2 g fatty acids/kg/day.

37. The method of any of paragraphs 17-36, further comprising adding an additive of one or more additional fatty acids, or a mixture thereof.

38. The method of paragraph 37, wherein the supplement contains one or more fatty acids that are therapeutic for the disease.

Some embodiments of the technology described herein may be defined in accordance with any of the following numbered paragraphs:

1. An emulsion composition containing:

fish oil and medium chain triglycerides (MCTs) in a ratio between, but not including, from 30:70 to about 70:30; or

omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 30:70 to about 70:30.

2. Emulsion composition according to paragraph 1, containing:

fish oil and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40; or

omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40.

3. Emulsion composition according to paragraph 1, containing

fish oil and medium chain triglycerides (MCTs) in a ratio of about 50:50; or

omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 50:50.

4. Emulsion composition according to paragraph 1, containing

fish oil and medium chain triglycerides (MCTs) in a ratio of 50:50; or omega-3 fatty acids and medium chain triglycerides (MCTs) in a 50:50 ratio.

5. An emulsion according to any one of paragraphs 1-4, wherein the emulsion is an oil-in-water emulsion.

6. An emulsion composition according to any one of paragraphs 1-5, wherein the composition further comprises alpha-tocopherol.

7. An emulsion composition according to any one of paragraphs 1-6 wherein alpha-tocopherol is present in an amount of at least 100 mg per liter of emulsion composition.

8. An emulsion composition according to any one of paragraphs 1-7, wherein alpha-tocopherol is present in an amount of at least 120 mg per liter of emulsion composition.

9. An emulsion composition according to any one of paragraphs 1-8, wherein the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 2:1.

10. The emulsion composition of any one of paragraphs 1-9, wherein the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 10:1.

11. The emulsion composition of any one of paragraphs 1-10, wherein the emulsion composition does not contain other forms of vitamin E other than alpha-tocopherol.

12. An emulsion composition according to any one of paragraphs 1-11, wherein phytosterols are present in the composition.

13. The emulsion composition of any one of paragraphs 1-12, wherein the phytosterols are present in the composition at a concentration of less than 50 mg per liter of the emulsion composition.

14. An emulsion composition according to any one of paragraphs 1-13, wherein arachidonic acid is present in the composition at a concentration of at least 900 mg/l.

15. An emulsion composition according to any one of paragraphs 1-14, wherein docosahexaenoic acid is present in the composition at a concentration of at least 13.4 g/l.

16. An emulsion composition according to any one of paragraphs 1-15, wherein eicosapentaenoic acid is present in the composition at least 11.6 g/l.

17. An emulsion composition according to any one of paragraphs 1-16, wherein the composition comprises a mixture of a fish oil and/or omega-3 fatty acid emulsion and an MCT emulsion.

18. The emulsion composition of any one of paragraphs 1-17, wherein the composition comprises an emulsion of a mixture of fish oil and/or omega-3 fatty acids and MCTs.

19. An emulsion composition according to any one of paragraphs 1-18, wherein the fish oil and/or omega-3 fatty acid oil has not been distilled or transesterified.

20. An emulsion composition according to any one of paragraphs 1-19, wherein the total content of triglycerides + diglycerides in fish oil and/or omega-3 fatty acid oil is not more than 10% diglyceride.

21. An emulsion composition according to any one of paragraphs 1-20 formulated for parenteral or intravenous administration.

22. An emulsion composition according to any one of paragraphs 1-21, further comprising the addition of one or more additional fatty acids, or mixtures thereof.

23. The emulsion composition of paragraph 22, wherein the supplement contains one or more fatty acids which are therapeutic for the disease.

24. An emulsion composition according to any one of paragraphs 1-23, wherein the composition comprises a mixture of a) an additive emulsion and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs.

25. An emulsion composition according to any one of paragraphs 1-24, further comprising one or more of egg phospholipid, glycerin, sodium oleate, and sodium hydroxide.

26. An emulsion composition according to any one of paragraphs 1-25, additionally containing egg phospholipid, glycerin, sodium oleate and sodium hydroxide.

27. A method comprising administering the emulsion composition of any of paragraphs 1-26 to a subject in need thereof.

28. The method of paragraph 27, wherein the administration is parenteral administration.

29. The method of paragraph 28, wherein the administration is total parenteral administration.

30. The method of any of paragraphs 27-29 wherein the subject is in need of parenteral nutrition.

31. The method of any of paragraphs 27-30 wherein the subject is in need of total parenteral nutrition.

32. The method of any of paragraphs 27-31, wherein the patient is not receiving oral nutrition.

33. The method of any of paragraphs 27-32, wherein the patient is not receiving other parenteral preparations.

34. The method of any of paragraphs 27-33, wherein the patient is not receiving sufficient oral nutrition to maintain nutritional balance.

35. The method of any of paragraphs 27-34, wherein the patient is not receiving other parenteral preparations that are sufficient to maintain nutritional balance.

36. The method of any of paragraphs 27-35, wherein the patient is not receiving other nutritional sources of fatty acids.

37. The method of any of paragraphs 27-36, wherein the patient is not receiving other sources of fatty acid parenteral nutrition.

38. The method of any of paragraphs 27-37, wherein the patient is not receiving other nutritional sources of essential fatty acids.

39. The method of any one of paragraphs 27-38, wherein the patient is not receiving other sources of parenteral nutrition of essential fatty acids.

40. The method of any of paragraphs 27-39, wherein the emulsion composition of any of paragraphs 1-26 is administered as monotherapy.

41. The method of any of paragraphs 27-40, wherein the emulsion composition of any of paragraphs 1-26 is administered as monotherapy for nutritional needs.

42. The method according to any one of paragraphs 27-41, in which the patient is a patient in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; biliary atresia; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemic-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; status anginosus; status asthmaticus; status epilepticus; status lacunaris; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal insufficiency associated with liver disease (IFALD), cholestasis associated with parenteral nutrition (PNAC), deficiency of essential fatty acids (EFAD), dependence on parenteral nutrition complicated by allergy to soy or allergies to lipid emulsions containing ingredients other than MCTs and fish oil and/or oil, which is dominated by omega-3 fatty acids and/or omega-3 fatty acids.

43. The method of any one of paragraphs 27-42, wherein the dose administered is from about 0.5 g fatty acids/kg/day to about 5 g fatty acids/kg/day.

44. The method of any one of paragraphs 27-43, wherein the dose administered is from about 1 g fatty acids/kg/day to about 3 g fatty acids/kg/day.

45. The method of any one of paragraphs 27-44, wherein the dose administered is about 2 g fatty acids/kg/day.

46. The method of any one of paragraphs 27-45, wherein the dose administered is from about 0.5 g fish oil/kg/day to about 5 g fish oil/kg/day.

47. The method of any one of paragraphs 27-46, wherein the dose administered is from about 1 g fish oil/kg/day to about 3 g fish oil/kg/day.

48. The method of any one of paragraphs 27-47, wherein the dose administered is about 2 g fish oil/kg/day.

49. The method of any one of paragraphs 27-48, further comprising adding an additive of one or more additional fatty acids, or a mixture thereof.

50. The method of paragraph 49, wherein the supplement contains one or more fatty acids that are therapeutic for the disease.

51. The method of any one of paragraphs 27-50, wherein the emulsion composition comprises a mixture of a) an additive emulsion and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs.

52. The method according to paragraph 51, in which the mixture is prepared at the time or at the site of administration.

53. The method according to paragraph 52, further comprising preparing the mixture in accordance with clinical indications.

54. An emulsion composition according to any one of paragraphs 1-26, intended for parenteral nutrition to a subject in need thereof.

55. The emulsion composition of paragraph 54, wherein the emulsion composition is administered by parenteral administration.

56. The emulsion composition of any one of paragraphs 54-55, wherein the emulsion composition is administered by total parenteral administration.

57. The emulsion composition of any of paragraphs 54-56 wherein the subject is in need of total parenteral nutrition.

58. The emulsion composition of any of paragraphs 54-57, wherein the patient is not receiving oral nutrition.

59. The emulsion composition of any of paragraphs 54-58, wherein the patient is not receiving other parenteral preparations.

60. The emulsion composition of any of paragraphs 54-59 wherein the patient is not receiving sufficient oral nutrition to maintain nutritional balance.

61. The emulsion composition of any of paragraphs 54-60, wherein the patient is not receiving other parenteral formulations that are sufficient to maintain nutritional balance.

62. The emulsion composition of any of paragraphs 54-61, wherein the patient is not receiving other nutritional sources of fatty acids.

63. The emulsion composition of any of paragraphs 54-62, wherein the patient is not receiving other parenteral nutritional sources of fatty acids.

64. The emulsion composition of any of paragraphs 54-63, wherein the patient is not receiving other nutritional sources of essential fatty acids.

65. The emulsion composition of any of paragraphs 54-64, wherein the patient is not receiving other parenteral nutritional sources of essential fatty acids.

66. The emulsion composition of any of paragraphs 54-65, wherein the emulsion composition of any of paragraphs 1-26 is administered as monotherapy.

67. An emulsion composition according to any one of paragraphs 54-66, wherein the emulsion composition according to any one of paragraphs 1-26 is administered as monotherapy for nutritional needs.

68. The emulsion composition according to any one of paragraphs 54-67, where the patient is a patient in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; biliary atresia; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemic-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; status anginosus; status asthmaticus; status epilepticus; status lacunaris; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal insufficiency associated with liver disease (IFALD), cholestasis associated with parenteral nutrition (PNAC), deficiency of essential fatty acids (EFAD), dependence on parenteral nutrition complicated by allergy to soy or allergies to lipid emulsions containing ingredients other than MCTs and fish oil, oil, which is dominated by omega-3 fatty acids, and/or omega-3 fatty acids.

69. The emulsion composition of any one of paragraphs 54-68, wherein the dosage administered is from about 0.5 g fatty acids/kg/day to about 5 g fatty acids/kg/day.

70. The emulsion composition of any one of paragraphs 54-69, wherein the dosage administered is from about 1 g fatty acids/kg/day to about 3 g fatty acids/kg/day.

71. An emulsion composition according to any one of paragraphs 54-70, wherein the dose administered is about 2 g fatty acids/kg/day.

72. The emulsion composition of any one of paragraphs 54-71, wherein the dosage administered is from about 0.5 g fish oil/kg/day to about 5 g fish oil/kg/day.

73. The emulsion composition of any one of paragraphs 54-72, wherein the dosage administered is from about 1 g fish oil/kg/day to about 3 g fish oil/kg/day.

74. The emulsion composition of any one of paragraphs 54-73, wherein the dose administered is about 2 g fish oil/kg/day.

75. The emulsion composition of any one of paragraphs 54-74, wherein the patient is additionally administered an additive of one or more additional fatty acids, or mixtures thereof.

76. The emulsion composition of any one of paragraphs 54-74, wherein the emulsion composition further comprises the addition of one or more additional fatty acids, or mixtures thereof.

77. The emulsion composition of any of paragraphs 54-76, wherein the supplement contains one or more fatty acids that are therapeutic for the disease.

78. The emulsion composition of any one of paragraphs 54-77, wherein the emulsion composition comprises a mixture of a) an additive emulsion and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs.

79. The emulsion composition of paragraph 78, wherein the mixture is prepared at the time or site of administration.

80. The emulsion composition of paragraph 79, further comprising preparing a mixture as indicated clinically.

EXAMPLES

EXAMPLE 1 Role of alpha-tocopherol and phytosterols in fish oil-mediated protection against parenteral nutrition-associated liver damage.

Parenteral nutrition-associated liver disease (PNALD) is a risk of long-term dependence on parenteral nutrition (PN) characterized by the development of liver inflammation and cholestasis. The use of soybean oil-based intravenous lipid emulsions as sources of parenteral fat may exacerbate the risk of developing PNALD, but intravenous fat cannot be eliminated in PN-dependent patients due to the risk of essential fatty acid deficiency. Intravenous fish oil lipid emulsions can effectively treat PNALD while providing adequate amounts of essential fatty acids. However, the mechanisms by which fish oil protects the liver are not fully understood. Two important differences between fish oil and soy oil are the phytosterol and alpha-tocopherol content. As described herein, intravenous lipid emulsions were formulated in the laboratory to investigate the role of phytosterols and alpha-tocopherol in modulating liver protection in a mouse model of parenteral nutrition-associated liver injury. Using lipid emulsions developed in the laboratory, soybean oil (SO) emulsion was unable to protect against PN-induced hepatosteosis in mice, while soybean oil emulsion supplemented with α-tocopherol (SO+AT) maintained normal liver structure. Fish oil emulsion (FO) and fish oil emulsion to which phytosterols were added (FO+P) were both able to protect against PN-induced steatosis. Expression of key genes for liver adipose tissue metabolism, acetyl-CoA carboxylase (ACC) and peroxisome profilator-activated receptor gamma (PPARγ), was increased in SO treated animals, while ACC and PPARγ levels were comparable to food-fed controls. treated with SO+AT, FO and FO+P. This study demonstrates the hepatoprotective role of α-tocopherol in PN-induced liver injury and that phytosterols do not appear to impair the hepatoprotective effects of fish oil.

Parenteral nutrition (PN) is the intravenous administration of macronutrients and micronutrients, including carbohydrates, protein in the form of amino acids, lipids, vitamins, and trace elements. PN is a critical component of therapy for patients with intestinal insufficiency (IF) who are unable to absorb sufficient amounts of oral nutrients due to insufficient bowel length or bowel dysfunction. Although PN is life-sustaining for patients with IF, there are complications associated with intravenous feeding. One such complication is the development of parenteral nutrition-associated liver disease (PNALD), which is characterized by cholestatic liver disease that can progress to cirrhosis and end-stage liver disease requiring liver transplantation. Traditionally, the progression of PNALD could only be stopped if patients could wean from PN and achieve enteral autonomy. More recently, it has been demonstrated that the use of fish oil as a source of parenteral oil can prevent PN-induced liver injury in animal models and reverse cholestasis and halt or slow the progression of liver disease in patients with PNALD.

Fat is an important component of PN. The fat in PN is an energy-dense source of calories as well as a source of long-chain polyunsaturated essential fatty acids (EFAs), which include the omega-3 and omega-6 fatty acid families. The introduction of low fat PN requires excess carbohydrate calories to meet the calorie requirement. Providing PN without fat also leads to the development of essential fatty acid deficiency (EFAD), which can be characterized by dermatitis, hair loss, developmental delay and growth failure. PN dependent patients can be biochemically monitored for EFAD by serum fatty acid profiling and measuring the ratio of the non-essential omega-9 fatty acid, which is triene, to the essential omega-6 fatty acid arachidonic acid, which is tetraene. The biochemical definition of EFAD is the ratio of triene to tetraene greater than 0.2.

The fat in PN is administered as an oil-in-water emulsion in which the oil is dispersed as globules surrounded by a phospholipid monolayer in an aqueous medium. The globules must be small enough to move through the bloodstream without causing embolic events. In the United States, the United States Pharmacopeia (USP) sets standards that intravenous fat emulsions must have an average globule size of less than 500 nm in diameter and a percentage of fat globules greater than 5 µm in diameter (PFAT5) of no more than 0.05 %. The types and proportions of fatty acids introduced are determined by the composition of the oils used to prepare the emulsion. The oils may also contain naturally occurring non-triglyceride components or additives that are included in emulsions formulated with such oils.

Soybean oil based fat emulsions are the most commonly used sources of parenteral fat. In the United States, the only sources of parenteral fat approved by the Food and Drug Administration (FDA) contain soybean oil. Exposure to intravenous soybean oil (SO) emulsions may exacerbate the risk of developing PNALD. Intravenous fish oil (FO) emulsions have been shown to prevent PN-induced liver damage in animal models. When administered as the sole source of parenteral fat to patients who develop PNALD, FO can reverse cholestasis and halt the progression of liver disease. Although the mechanisms of the hepatoprotective properties of FO and the hepatotoxic properties of SO are not fully understood, there are several differences between fish oil and soybean oil that may make an important contribution to the differential effects of SO and FO on the liver.

The purpose of this study is to test the hypothesis that α-tocopherol provides hepatoprotective properties, and phytosterols - hepatotoxic properties of intravenous fat emulsions. To test this hypothesis, it is not possible to use commercially available intravenous fat emulsions, as SO and FO with different levels of phytosterols and α-tocopherol do not exist. Therefore, emulsions were prepared in the laboratory, allowing to control both the amount of α-tocopherol and phytosterols in each emulsion, and the uniformity of all components of the emulsion, varying only by the type of oil. Laboratory-formulated SO and FO are safe and well tolerated by mice. Laboratory-derived FO and SO have the same effects on the liver as their commercial counterparts in a mouse model of PN-induced liver injury. In this paper, it was tested whether the addition of phytosterols to fish oil confers FO hepatotoxicity and whether the addition of α-tocopherol to soybean oil confers SO hepatoprotective properties.

Ways:

Fish oil may protect against parenteral nutrition-induced hepatosteatosis in a mouse model. The approach was used to test whether the addition of phytosterols to fish oil impairs the hepatoprotective properties of intravenous fish oil emulsions and/or whether alpha-tocopherol in soybean oil makes soybean oil emulsions more hepatoprotective.

In the laboratory, 20% oil-in-water emulsions containing soybean oil (SO), fish oil (FO), soybean oil supplemented with 200 mg/l alpha-tocopherol (SO+AT), or fish oil, to which was added 450 mg/l phytosterols (85% beta-sitosterol, 15% stigmasterol) (FO+P). Emulsions were prepared using high pressure homogenization. All final emulsions contained 20% oil, 1.2% egg phospholipid, 2.5% glycerol and 0.03% sodium oleate. Table 2 shows the levels of phytosterol and α-tocopherol in emulsions prepared with these oils, as well as in commercially available FO (OM) and SO (IL). Phytosterol levels in emulsions formulated with SO, SO+AT and FO+P were comparable. Alpha-tocopherol levels in emulsions prepared with FO, FO+P and SO+AT were comparable. The average globule size and PFAT5 analysis for all emulsions were in accordance with USP standards (Table 1).

In the examples provided herein, Crystalpure 28/12 TG™ fish oil (Product No. 30572344; BASF Pharma, Florham Park NJ) was used, which contains a mixture of approximately 38% EPA + DHA, where 11.4% is DHA and 26.9 % - EPA (by area). Unless otherwise indicated, each component of the emulsions used in each of the Examples described herein was of pharmaceutical, food grade, and/or industrial grade.

The emulsions were used as sources of fat in a mouse model of PN-induced liver injury. Mice were placed on a standard food diet or an oral liquid diet consisting of low fat PN, which is equivalent to what patients receive. Mice treated with PN were injected with saline (no source of fat) or one of the prepared emulsions (2.4 g/kg/day intravenously by tail vein injection). After 19 days on their respective diets, the mice were sacrificed. Livers were harvested for histology (hematoxylin and eosin and oil red O), gene expression analysis by RT-PCR, and protein expression analysis by Western blot. The right kidneys and spleens were also removed for histology.

Composition of Lipid Emulsion

Emulsion Materials: Sterile Water for Injection (SWFI, Hospira, Lake Forest, IL), Egg Phospholipid (Lipoid LLC, Newark, NJ), Sodium Oleate (Lipoid LLC, Newark NJ), and Glycerin (Sigma-Aldrich, St. Louis, MO) were used to compile the variance. The oils used were USP grade soybean oil (Spectrum Chemicals, New Brunswick, NJ) and CrystalPure EPA 28/12 TG fish oil (BASF Ludwigshafen, Germany). Additives used were α-tocopherol (Sigma-Aldrich, St. Louis, MO), beta-sitosterol (Sigma-Aldrich, St. Louis, MO) and stigmasterol (Sigma-Aldrich, St. Louis, MO). Commercial emulsions used for the assays included Omegaven (Fresenius Kabi, Bad Homburg, Germany) and Intralipid (Fresenius Kabi, Uppsala, Sweden).

FO+P oil preparation: CrystalPure EPA 28/12 TG fish oil was heated to maintain the temperature between 50-60° C. under constant stirring conditions. Phytosterols (85% beta-sitosterol, 15% stigmasterol) were added to a final concentration of 2.25 mg phytosterols per gram of oil and mixed until dissolved. When used to prepare a 20% emulsion, the calculated concentration of phytosterol is 450 mg of phytosterols per liter of emulsion.

Preparation of oil SO+AT: Soybean oil was heated to maintain the temperature between 50-60°C under constant stirring conditions. Alpha-tocopherol was added to a final concentration of 1 mg α-tocopherol per gram of oil and mixed for 10-15 minutes. When used to prepare a 20% emulsion, the calculated α-tocopherol content is 200 mg α-tocopherol per liter of emulsion.

Composition of the Emulsion: The emulsion was prepared by high pressure homogenization as described previously (Fell et al. JPEN 2017 41:181-187). All steps were performed at 40-45°C unless otherwise noted. All steps were performed under a nitrogen atmosphere.

First, a dispersion was prepared by adding frozen egg phospholipid to SWFI heated to 75-90°C under high shear mixing conditions and allowing the mixture to equilibrate at 40-45°C. Sodium oleate was added and shear mixing was continued (4000-4100 rpm) for 40 minutes after which glycerol was added. The crude dispersion was homogenized (Panda Plus™ Homogenizer, GEA Niro Saovi, Columbia, MD) at 9000 psi for 20 cycles. The dispersion was filtered through a 0.45 µm membrane and the pH was adjusted to 10.4 with 0.5N HCl. sodium hydroxide. The final dispersion consisted of 12% egg phospholipid, 25% glycerol and 0.3% sodium oleate. One batch of dispersion was enough to prepare five 1-liter emulsions.

Emulsions were prepared by adding oil to an appropriate volume of dispersion under high shear mixing conditions (3800-4200 rpm, adjusted to avoid foaming), while stirring was continued for 40-45 minutes and slowly adjusted to a final volume of 500 ml using SWFI keeping the temperature at 40-45°C. The crude emulsion was homogenized at 5000 psi for at least 9 cycles. The pH of the final emulsion was adjusted to 9-9.5 using 0.1N. sodium hydroxide, packaged in 20 ml serum vials with headspace filled with nitrogen gas, and the packaged emulsions were autoclaved. The final emulsion composition consisted of 20% oil, 1.2% egg phospholipid, 2.5% glycerol and 0.03% sodium oleate.

All emulsions were tested for mean globule size and PFAT5 (Micro Measurements, Deerfield, IL) in accordance with USP <401> standards.

Determination of Phytosterol and Alpha-Tocopherol Levels in Emulsions: To determine phytosterol levels, samples were saponified with 2 mol/l ethanolic KOH and sterols were extracted with n-heptane. The extracts were evaporated and separated on a column for capillary gas chromatography. Detection was carried out using a flame ionization detector. Quantitative determination was carried out using epicoprostanol as an internal control. Alpha-tocopherol levels were determined as described (Xu et al. Eur. J. Lipid Sci Technol. 2015 117:15-22), however, external calibration was used instead of internal calibration.

Mouse Model of PN-Induced Liver Injury. All animal experiments were approved by the Boston Children's Hospital Institutional Animal Care and Use Committee. Six week old C57BL/6 mice (Jackson Labs, Bar Harbor, ME) were fed either a standard chow diet or a liquid diet consisting of PN administered to patients at Boston Children's Hospital (20% Dextrose, 2% amino acid, 30 mEq/L sodium , 20 mEq/L potassium, 15 mEq/L calcium, 10 mEq/L Magnesium, 10 mol/L phosphate, 36.67 mEq/L chloride, 19.4 mEq/L acetate, Pediatric multivitamins, Pediatric trace elements). Mice injected with PN were given intravenous (IV) saline, IV FO, IV FO+P, IV SO, or IV SO+AT (2.4 g/kg/day via tail vein injection). After 19 days, the animals were sacrificed by asphyxiation with carbon dioxide. Blood was collected for serum collection. The liver, spleen and right kidney were removed for further analysis. This experiment was performed twice with different batches of emulsions for each experiment. The first experiment used 5 mice per treatment group and the second experiment used 10 mice per treatment group.

Organ Processing and Histology. Spleens, kidneys and one portion of each liver were placed in 10% formalin and stored at 4°C for 24 hours, then transferred to 70% ethanol. Samples were embedded in paraffin and sectioned for Hematoxylin and Eosin (H&E) staining to assess liver structure. The second portion of each liver was placed in Optimum Cutting Temperature (OCT) medium (Fisher Scientific, Pittsburgh, RA) and frozen in liquid nitrogen. Samples were frozen cut and stained with oil red O to assess liver fat accumulation. Visualization was performed using a Zeiss Axiophot™ microscope (Oberkochen, Germany). The sections were analyzed by a certified pathologist who was unaware of the treatment groups. A third of each liver was flash frozen in liquid nitrogen and stored at -80°C for analysis of gene and protein expression.

Serum Fatty Acid Profiling: Extraction of fatty acids from serum was performed as previously described (Meisel et al. J Pediatr Surg 2011 46:666-673). Briefly, serum samples (30 μl per sample) with tricosanoic acid added as an internal standard were subjected to extraction with chloroform and methanol in a ratio of 2:1 to isolate the lipid fraction. The samples were saponified with 0.5 N. methanolic sodium hydroxide. Samples were incubated in 14% BF3/methanol for 30 minutes at 100°C. The steps were performed under nitrogen gas to minimize oxidation. Analysis was performed by gas liquid chromatography (Hewlett Packard 6890™) and detection with a flame ionization detector. An external fatty acid methyl ester standard (NuCheck Prep™, Elysian, MN) was used to identify the fatty acid peaks in the sample.

Gene Expression Analysis. Livers were cut to 25 mg per sample and RNA extracted using the Qiagen AUPrep™ DNA/RNA/Protein kit (Gaithersburg, MD) according to the manufacturer's instructions. Taqman primers (Invitrogen, Carlsbad, CA) and reagents (Agilent Technologies, Santa Clara, CA) were used for each reaction according to manufacturer's instructions with 200 ng of RNA. The ABI One Step Plus™ cycler used a two-step RT-PCR protocol. An initial reverse transcription step of 30 minutes at 50°C and 10 minutes at 95°C was followed by an amplification step of 15 seconds at 95°C and 1 minute at 60°C, cycled 40 times. The expression of the target gene was normalized to the GAPDH gene and compared to a food-fed control group using the 2-ΔΔCt method.

Protein analysis. The liver was cut to 25 mg per sample and homogenized in radioimmunoprecipitation assay buffer (RIPA) with protease inhibitor and phosphatase inhibitor using stainless steel beads in a Bullet Blender™. Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA). Ten milligrams of protein per sample was separated using a 4-12% Bis Tris polyacrylamide gel (Invitrogen, Carlsbad, CA) before being transferred to a nitrocellulose membrane. The membranes were blocked in 5% skim milk for 1 hour. The membranes were incubated in the primary antibody overnight and in the secondary antibody for 1 hour. ACC and PPARγ antibodies were obtained from Cell Signaling technologies (Danvers, MA). Anti-beta actin antibody was obtained from Santa Cruz Biotechnologies (Paso Robles, CA).

Results:

All emulsions met USP criteria for mean globule size and PF AT5 (Table 1). FO, FO and SO+AT had similar levels of alpha-tocopherol, while SO contained little alpha-tocopherol, similar to the commercial SO (IL) emulsion (Table 2). In the analysis of phytosterol levels, SO, SO+AT and FO+P contained similar amounts of phytosterols, while FO and commercial emulsion of OM with fish oil contained very little phytosterols (Table 2).

There were no adverse clinical effects with any of the emulsions used and the animals tolerated all emulsions well. There were no differences in growth parameters between treatment groups and there were no differences in organ weight scores between groups (FIGS. 1A-1B).

Unstable intravenous emulsions can lead to organomegaly, especially enlargement of the spleen and liver due to the deposition of fat globules. There were no signs of organomegaly in any of the treatment groups. Liver (Fig. 1B), spleen (Fig. 1C) and kidney (Fig. 1D) masses were similar in all treatment groups.

Serum fatty acid profiles were performed to confirm that each emulsion was capable of preventing EFAD and delivering the expected EFA complement for the oil used in the emulsion. SO contains omega-6 fatty acids and is low in omega-3 fatty acids, while FO is high in omega-3 fatty acids and low in omega-6 fatty acids. These EFA balances should be reflected in the sera of the animals in each respective treatment group. All emulsions prevented biochemical deficiency of essential fatty acids (FIG. 2A). FO and FO+P emulsions resulted in lower serum levels of the omega-6 fatty acid arachidonic acid and higher serum levels of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compared to SO and SO+ emulsions. AT (Fig. 2C).

Serum fatty acid analysis indicates that only mice fed PN without a source of fat developed an essential fatty acid deficiency, defined as a triene to tetraene ratio of greater than 0.2 (FIG. 2A). The balance of essential fatty acids reflects the source of fat received by each group. Mice treated with SO and SO+AT had higher serum levels of omega-6 fatty acids than the FO and FO+P groups (Figure 2B), and mice treated with FO and FO+P had higher levels of omega-6 fatty acids. 3 fatty acids than mice treated with SO and SO+AT.

To evaluate the effect of each emulsion on the development of PN-induced steatosis, a histological analysis of the liver was performed. SO did not prevent PN-induced steatosis (Fig. 3A). However, the addition of α-tocopherol to SO(SO+AT) resulted in the maintenance of normal liver architecture in PN-fed animals (FIG. 3A). FO and FO+P also retained normal liver architecture (FIG. 3A), indicating that the addition of phytosterols to FO does not interfere with FO's ability to protect the liver from PN-induced steatosis. In the oil red O liver fat accumulation assay, SO+AT resulted in a reduction in liver fat accumulation compared to SO (FIG. 3B), indicating that α-tocopherol confers the hepatoprotective properties of SO. Both FO and FO+P had minimal liver fat accumulation, again indicating that the addition of phytosterols to FO did not impair the hepatoprotective properties of FO (FIG. 3B). Histologically, the PN and saline treated group developed hepatosteatosis as previously shown. The SO emulsion is unable to protect the liver from PN-induced steatosis. FO, FO+P and SO+AT were able to prevent PN-induced hepatosteatosis (FIGS. 3A-3B).

Since the PN diet leads to hepatic steatosis and liver fat accumulation, it has been hypothesized that these histological changes may be due to altered expression of liver oil genes and that fish oil may retain the normal expression of such genes. When analyzing gene expression, Peroxisome Proliferator Activated Receptor Gamma (PPARg), which is a transcriptional regulator of fat processing in the liver; and Acetyl CoA Carboxylase 2 (ACC), which catalyzes the rate-limiting step of de novo lipogenesis, were increased by diet-free PN and SO-fed PN mice, but were normalized to similar levels of chow-fed controls in mice treated with FO, FO+P and SO+AT (Fig. 4). This pattern was also observed at the level of protein expression (FIGS. 5A-5B). This indicates that the addition of α-tocopherol may confer the hepatoprotective properties of SO. Both FO and FO+P resulted in normalized gene (FIG. 4) and protein (FIG. 5) expression of ACC2 and PPARγ, indicating that, at the molecular level, the addition of phytosterols does not impair the hepatoprotective properties of FO.

This mouse model histologically demonstrates steatosis in response to a PN+SO diet. However, the clinical correlate, PN-associated liver disease, is characterized by cholestasis. Thus, it is hypothesized that phytosterol-containing intravenous fat may cause disturbances in the expression of genes that regulate bile synthesis and transport. This paper demonstrates that:

a. Addition of phytosterols does not impair the hepatoprotective properties of fish oil in this mouse model of PN-induced liver injury

b. The addition of alpha-tocopherol confers hepatoprotective properties to soybean oil both histologically and at the molecular level, suggesting that alpha-tocopherol contributes to the hepatoprotective properties of fish oil.

With. Molecular markers (PPARg and ACC) have been identified that characterize a more hepatoprotective lipid emulsion and distinguish such emulsions from more hepatotoxic lipid emulsions.

SHP (Small Heterodimeric Partner) acts with FXR to regulate bile acid transport and its expression is upregulated in the presence of phytosterols, while the presence of alpha-tocopherol drives it towards normalization. Interestingly, FXR itself is downregulated in expression in the presence of phytosterols. MRP3 (also MRP2 and BSEP) is a target for FXR-mediated gene expression. MRP3 is downregulated by SO, AT cannot normalize MRP3 levels, but phytosterols do not appear to modulate this downregulation since FO+P administration does not decrease MRP3 expression. Cyp7a1 is usually reduced in conditions of cholestasis. The treatment regimens described herein are reduced in all mice receiving PN, regardless of the source of fat. Those receiving PN+SO or PN+FOP have a greater reduction in expression than the other groups, and alpha-tocopherol appears to be able to partially normalize expression in SO+AT-treated animals. But it appears that the addition of phytosterols to FO (FO+P versus FO) negatively affects Cyp7a1 expression. (Some data not shown).

In this study, an intravenous lipid emulsion prepared in the laboratory using soybean oil supplemented with alpha-tocopherol was able to maintain normal liver structure and normal expression of 2 important genes involved in liver fat in a mouse model of PN-induced liver injury. An intravenous lipid emulsion prepared in a laboratory using soybean oil containing no added α-tocopherol is unable to protect against PN-induced hepatosteatosis and dysregulation of liver fat management. In this document, α-tocopherol was added to soybean oil prior to the preparation of emulsions.

This study also identified PPARγ and ACC2 as genes that are not regulated by the PN diet, normalized by FO and α-tocopherol-containing SO, but not by SO alone. PPARγ is a transcriptional regulator of systemic and hepatic metabolism, as well as inflammation. Interestingly, this study found no hepatotoxic effect from the addition of phytosterols to fish oil in PN-induced liver injury. One possible conclusion is that the phytosterols in soybean oil are not responsible for soybean oil-associated hepatotoxic effects in a mouse model of PN-induced liver injury. An alternative explanation is that phytosterols have hepatotoxic properties but are unable to overcome the hepatoprotective properties of fish oil. The omega-3 fatty acids present in fish oils are precursors to anti-inflammatory lipid mediators, and α-tocopherol is also abundant in fish oils. These properties may provide hepatoprotection that cannot be overcome by the presence of phytosterols. A third possible explanation is that specific phytosterols at specific concentrations or a specific balance of phytosterols are required for phytosterol-related hepatotoxic properties to occur. In this study, the composition of phytosterols added to fish oil approximated the types and amounts of phytosterols found in soybean oil.

All emulsions formulated in this study are protected from the development of EFAD. Traditionally, linoleic (LA) and alpha-linolenic (ALA) acids, the parent omega-6 and omega-3 fatty acids respectively, are considered EFAs. Interestingly, this study showed that serum ARA, EPA and DHA reflect the balance of EFA provided by the injected emulsion rather than serum LA and ALA levels.

The ability of α-tocopherol to make SO less hepatotoxic in a PN-induced liver injury model indicates that α-tocopherol may be useful in the clinical management of PNALD and other similar liver pathologies.

EXAMPLE 2 - Formulation of Lipid Emulsions with Anti-Inflammatory Action for PN-Dependent Patients.

Long-term and short-term PN dependent patients may be vulnerable to inflammatory processes. For long-term PN-dependent patients, chronic disease and long-term indwelling central venous catheter delivery of PN may precipitate a pro-inflammatory state. Short-term PN-dependent patients are also prone to pro-inflammatory issues and include trauma patients, postoperative patients, and acutely ill patients who require an intensive care unit.

Parenteral lipid emulsions can modulate the inflammatory response and influence the inflammatory status of PN-dependent patients. Soy oil based lipid emulsions are rich in pro-inflammatory omega-6 fatty acids, while fish oil lipid emulsions contain more anti-inflammatory omega-3 fatty acids. Fish oil lipid emulsions have been used to treat parenteral nutrition-associated liver disease (PNALD) in patients with liver inflammation and cholestasis. However, studies on the effect of oral fat sources on the inflammatory response have shown that fish oil, as the sole source of fat, does not provide a greater anti-inflammatory effect than soybean oil. Rather, mixtures of fish oil and medium chain triglycerides (MCTs) had an increased anti-inflammatory effect, as the ratio of fish oil to MCT was reduced.

Although alpha-tocopherol is an important component of fish oil to prevent oxidative degradation of fatty acids, it remains unknown whether alpha-tocopherol may play a role in attenuating the inflammatory response in PN-dependent patients. Because there are many PN-dependent patient populations that benefit from blunting the inflammatory response, the aim of this study was to develop intravenous lipid emulsions that provide anti-inflammatory effects for the PN-dependent population while still providing adequate amounts of essential fatty acids.

It has been verified that this is the case:

a. Mixed fish oil and MCT emulsion formulations provide more anti-inflammatory effect than fish oil alone in response to lipopolysaccharide loading in a mouse model b. Emulsion formulations containing fish oil with additional alpha-tocopherol provide more anti-inflammatory effects than fish oil alone

Ways.

Compositions of 20% oil-in-water emulsion of soybean oil (SO), fish oil (FO) or MCT. SO, FO, and various FO:MCT ratios were tested as fat sources in a mouse model of PN-induced liver injury and their effect on inflammatory markers, Tumor Necrosis Factor alpha (TNFa) and Interleukin-6 (IL-6), after exercise. lipopolysaccharide (LPS). LPS loading was performed as described in Ling PR, Malkan A, Le HD, Puder M, Bistrian BR. Arachidonic acid and docosahexaenoic acid supplemented to an essential fatty acid-deficient diet alters the response to endotoxin in rats. Metabolism. March 2012; 61(3):395-406.

The MCTs used were MCT OIL™ obtained from Nestle Health Science (Bridgewater, NJ; HCPCS Code B4155), which is an oil emulsion containing medium chain triglycerides derived from coconut and/or palm kernel oil. Fatty acids are: <1% shorter than C8, 54% C8 (octanoic), 41% C1O (decanoic), and less than 5% longer than C1O.

Emulsion compositions containing either 20% SO, 20% FO or 20% MCT were prepared using high pressure homogenization. All emulsions contained 20% oil, 1.2% egg phospholipid, 2.5% glycerol and 0.03% sodium oleate.

Mice were administered either a standard chow or an oral liquid diet consisting of fat-free PN patients. PN-fed mice were administered saline (no fat) or one of the following fat emulsion formulations (2.4 g/kg/day, intravenously via tail vein injection: SO, FO, 70% FO: 30% MCT (70 :30) 50% FO: 50% MCT (50:50), 30% FO: 70% MCT (70:30) After 19 days of treatment, animals were administered either saline or LPS (150 µg/kg ip). Animals were sacrificed four hours after ip injection.Sera were collected for fatty acid analysis and measurement of TNFa and IL-6 by ELISA.Livers, spleens and right kidneys were removed for histological analysis.

The 20% oil-in-water emulsion formulations were formulated from 100% pure oil formulations SO, FO, FO to which 500 mg/l alpha-tocopherol (FOE) or MCT was added. Mixtures of the above in various ratios were then prepared using a volume of eg 100 ml of a 50:50 MCT:FO mixture as described herein, prepared by combining 50 ml of 20% MCT emulsion and 50 ml of 20% FO emulsion. Thus, the emulsion compositions used in the examples given herein are always a composition with 20% fat, measured in wt./about.

SO, FO, FOE and mixtures of 50%:50% FO:MCT and FOE:MCT were tested as fat sources in a mouse model of PN-induced liver injury and their effect on inflammatory markers TNFa and IL-6 after LPS loading testing.

Emulsions containing 20% SO, 20% FO, 20% FOE or 20% MCT were prepared using high pressure homogenization. All emulsions contained 20% oil, 1.2% egg phospholipid, 2.5% glycerol and 0.03% sodium oleate. The rest of the emulsion compositions contained water.

Mice were administered either standard chow or an oral liquid diet consisting of fat-free PN patients. PN-fed mice were administered saline (no fat) or one of the following fat emulsion formulations (2.4 g/kg/day, intravenously via tail vein injection: SO, FO, FOE, 50% FO: 50% MCT (FO/MCT) or 50% FOE: 50% MCT (FOE/MCT) After 19 days of treatment, animals were injected with LPS (15 μg/kg ip) Animals were sacrificed four hours after ip injection Serum was collected for analysis of fatty acids and measurements of TNFa and IL-6 by ELISA Livers, spleens and right kidneys were removed for histological analysis.

Results:

All emulsions met USP criteria for mean globule size and percentage of particles greater than 0.5 µm in diameter (PFAT5) (Table 3).

Histologically, PN without a fat source and mice treated with PN with SO as a fat source developed hepatic steatosis (Fig. 6A) and accumulation of fat in the liver on oily red O (Fig. 6B), mice treated with PN, treated with FO or any FO:MCT ratio, improved hepatic architecture (FIG. 6A) and minimal liver fat accumulation (FIG. 6B). While FO, 70:30 and 50:50 showed perfectly normal liver architecture, 30:70 showed trace amounts of steatosis (FIG. 6B).

Since MCT does not contain essential fatty acids, it was checked whether the FO:MCT mixtures contained sufficient concentrations of essential fatty acids to prevent essential fatty acid deficiency (EFAD). SO, FO and all FO:MCT mixtures were able to prevent the development of biochemical EFAD (FIG. 7).

When assessing serum TNFa and IL-6 in mice injected with saline before euthanasia, there was no significant increase in either of these markers (Fig. 8A and 8B, left graphs). In LPS-injected mice, IL-6 and TNFa levels decreased after LPS injection with a decrease in the FO:MCT ratio (FIGS. 8A and 8B, right graphs). Both 50:50 and 30:70 showed significantly lower levels of IL-6 than FO, and 30:70 also had significantly lower TNFa compared to FO.

All emulsions met USP criteria for mean globule size and PFAT5 (Table 4). Based on the above results, it was decided to use a ratio of 50% FO: 50% MCT in this experiment for mixed emulsion compositions of FO or FOE and MCT. The 50:50 group provided the most favorable balance of normal liver structure and anti-inflammatory effect in the above results.

Based on the above results, additional FO:MCT ratios were tested, including 40:60 and 60:40. Above 30% fish oil, liver architecture improved and further improvements were found as the percentage of fish oil was increased up to 50%. The inclusion of 30% or more MCTs provided an improvement in the anti-inflammatory profile measured as described above. Accordingly, it is contemplated herein that FO and MCT formulations with greater than 30% and less than 70% fish oil (and less than 70% and greater than 30% MCT, respectively) provide an unexpectedly beneficial combination of liver architecture and anti-inflammatory activity. .

On histological analysis, animals fed PN without fat as well as PN supplemented with SO developed hepatosteatosis (FIG. 9A) and fat accumulation in the liver when stained with oil red O (FIG. 9B). The FO, FOE, FO/MCT and FOE/MCT groups retained normal liver architecture similar to the fed control group (FIG. 9A) and did not have significant liver fat accumulation (FIG. 9B).

When assessing serum TNFa and IL-6 after LPS loading, FOE, FO/MCT and FOE/MCT had significantly lower levels of both markers compared to FO. There was no significant difference in the levels of any of these markers between the FOE, FO/MCT and FOE/MCT groups (FIGS. 10A-10B).

This paper demonstrates that:

a. Mixed emulsion formulations of FO and MCT provide an anti-inflammatory effect in response to an inflammatory stimulus compared to FO alone. A 50:50 mixture of FO and MCT resulted in the best balance between preserved liver protection and blunting the inflammatory response.

b. Alpha-tocopherol added to FO results in an improved anti-inflammatory effect in response to an inflammatory stimulus compared to FO alone

EXAMPLE 3

Parenteral nutrition-associated liver disease (PNALD) is a risk factor for dependence on parenteral nutrition (PN). Intravenous soybean oil as a source of parenteral fat may exacerbate the risk of developing PNALD, while intravenous fish oil may halt the progression of liver disease. However, the mechanisms by which soybean oil harms the liver and fish oil protects the liver are unclear. Two properties that distinguish soybean oil and fish oil are α-tocopherol and phytosterol content. Soybean oil is rich in phytosterols and low in α-tocopherol, while fish oil is high in α-tocopherol and contains only minor amounts of phytosterols. This study aimed to test whether α-tocopherol confers hepatoprotective properties and phytosterols confers hepatotoxic properties to intravenous fat emulsions. Using lipid emulsions developed in the laboratory, the soybean oil (SO) emulsion failed to protect against PN-induced hepatosteatosis in mice, while the soybean oil emulsion to which α-tocopherol (SO+AT) was added preserved normal liver structure. Fish oil emulsion (FO) and fish oil emulsion to which phytosterols have been added (FO+P) are both able to protect against PN-induced steatosis. Expression of genes and proteins of key genes that control liver adipose tissue, acetyl-CoA carboxylase (ACC) and peroxisome proliferator-activated receptor gamma (PPARγ), was increased in SO-treated animals, while ACC and PPARγ levels were comparable to control animals fed SO+AT, FO and FO+P. This study demonstrates the hepatoprotective role of α-tocopherol in PN-induced liver injury and that phytosterols do not appear to impair the hepatoprotective effects of fish oil.

Parenteral nutrition (PN) is the intravenous administration of macronutrients and micronutrients, including carbohydrates, protein in the form of amino acids, lipids, vitamins, and trace elements. PN is a critical component of therapy for patients with intestinal insufficiency (IF) who are unable to absorb sufficient amounts of oral nutrients due to insufficient length of the intestine or its violation. Although PN is life-sustaining for patients with IF, there are complications associated with the administration of intravenous nutrients. One such complication is the development of parenteral nutrition-associated liver disease (PNALD), which is characterized by cholestatic liver disease that can progress to cirrhosis and end-stage liver disease requiring liver transplantation. Traditionally, the progression of PNALD could only be stopped if the patient could be weaned from PN and achieve enteral autonomy. More recently, it has been demonstrated that the use of fish oil as a source of parenteral oil can prevent PN-induced liver injury in animal models (1, 2) and reverse cholestasis and halt the progression of liver disease in patients with PNALD (3-9).

Fat is an important component of PN. The fat in PN is an energy-dense source of calories as well as a source of long-chain polyunsaturated essential fatty acids (EFAs), which include the omega-3 and omega-6 fatty acid families. Fat-free PN administration requires excess carbohydrate calories to meet caloric requirements. Providing fat-free PN also leads to the development of essential fatty acid deficiency (EFAD), which can be characterized by dermatitis, hair loss, developmental delay, and growth retardation (10, 11). PN dependent patients can be biochemically monitored for EFAD by serum fatty acid profiling and measuring the ratio of non-essential omega-9 fatty acid, which is triene, to essential arachidonic omega-6 fatty acid, which is tetraene. The biochemical definition of EFAD is the ratio of triene to tetraene greater than 0.2 (12).

The fat in PN is administered as an oil-in-water emulsion in which the oil is dispersed as globules surrounded by a phospholipid monolayer in an aqueous medium. The globules must be small enough to move through the bloodstream without causing embolic events. In the United States, the United States Pharmacopeia (USP) has standards that require intravenous fat emulsions to have an average globule size of less than 500 nm in diameter and a percentage of fat globules greater than 5 µm in diameter (PFAT5) of no more than 0.05% (13, fourteen). The types and proportions of fatty acids introduced are determined by the composition of the oils used to prepare the emulsion. The oils may also contain natural non-triglyceride components or additives that are included in emulsions formulated with such oils.

Soybean oil based fat emulsions are the most commonly used sources of parenteral fat. In the United States, the only sources of parenteral fat approved by the Food and Drug Administration (FDA) contain soybean oil. Exposure to intravenous soybean oil (SO) emulsions may exacerbate the risk of developing PNALD (15). Intravenous fish oil (FO) emulsions have been shown to prevent (1, 2) PN-induced liver injury in animal models. When administered as the sole parenteral source of fat to patients who develop PNALD, FO can reverse cholestasis and halt the progression of liver disease (3-9). Although the mechanisms of the hepatoprotective properties of FO and the hepatotoxic properties of SO are not fully understood, there are several differences between fish oil and soybean oil that may make an important contribution to the differential effects of SO and FO on the liver.

Soybean oil is high in phytosterols, which are plant sterol compounds. Commercially available SO contains approximately 450 mg/L phytosterols (16). The predominant phytosterol in commercial SO is beta-sitosterol, accounting for ~70% of total phytosterols (16). Stigmasterol and campesterol are present in smaller but significant amounts, ~15% and ~13%, respectively (16). In vitro studies have shown that stigmasterol can inhibit the expression of the farsenoid X receptor (FXR) of the bile acid transporter, as well as genes modulated by FXR (17). In a mouse model of PNALD, stigmasterol can exacerbate liver injury, suppress the activation of bile acid transporters, and induce the activation of hepatic macrophages (18). Unlike SO, FO contains only trace amounts of phytosterols.

Alpha tocopherol is an antioxidant that is an important fish oil supplement to prevent oxidation of long chain polyunsaturated omega-3 fatty acids. Soybean oil contains less omega-3 fatty acids than fish oil and does not require the addition of α-tocopherol to maintain stability.

What is described herein is a test of the hypothesis that α-tocopherol enhances the hepatoprotective properties and phytosterols enhance the hepatotoxic properties of intravenous fat emulsions. To test this hypothesis, it is not possible to use commercially available intravenous fat emulsions, since SO and FO with different levels of phytosterols and α-tocopherol do not exist. Thus, emulsion compositions were formulated in the laboratory to ensure both the control of the amount of α-tocopherol and phytosterols in each emulsion composition, and the uniformity of all emulsion components with a change only in the type of oil. Laboratory-derived SO and FO are safe and well tolerated in mice (22). Emulsions of FO and SO made in the laboratory have the same effect on the liver as their commercial counterparts in a mouse model of PN-induced liver injury (Fig. 16). In this paper, it was tested whether FO makes hepatotoxic the addition of phytosterols to fish oil and whether SO makes hepatoprotective the addition of α-tocopherol to soybean oil.

Results.

Emulsion Analysis. The following oils have been used to prepare 20% oil-in-water emulsions: fish oil, fish oil to which phytosterols have been added (FO+P), soybean oil and soybean oil to which α-tocopherol has been added (SO+AT) . On FIG. 17 shows the levels of phytosterol and α-tocopherol in emulsions made with these oils as well as commercially available FO(OM) and SO(IL). Phytosterol levels in emulsions prepared with SO, SO+AT and FO+P were comparable. Alpha-tocopherol levels in emulsions prepared with FO, FO+P and SO+AT were comparable. The mean globule size and PFAT5 analysis for all emulsions met USP standards (FIG. 18).

Growth Parameters and Fatty Acid Profiles. There were no adverse clinical effects with any of the emulsions used and the animals tolerated all emulsions well. There were no differences in height between treatment groups (FIG. 11A).

Unstable intravenous emulsions can lead to organomegaly, especially enlargement of the spleen and liver due to the deposition of fat globules (23). There were no signs of organomegaly in any of the treatment groups. Liver (Fig. 11B), spleen (Fig. 11C), and kidney (Fig. 11D) masses were similar in all treatment groups.

Serum fatty acid profiles were performed to confirm that each emulsion was capable of preventing EFAD and delivering the expected EFA complement for the oil used in the emulsion. SO is rich in omega-6 fatty acids and low in omega-3 fatty acids, while FO is rich in omega-3 fatty acids and low in omega-6 fatty acids. These EFA balances should be reflected in the sera of the animals in each respective treatment group. All emulsions prevented biochemical deficiency of essential fatty acids (FIG. 12A). FO and FO+P emulsions resulted in lower serum levels of arachidonic omega-6 fatty acid and higher serum levels of omega-3 fatty acids, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), compared to SO and SO emulsions. +AT (FIGS. 12B and 12C).

Histological Analysis. Histological analysis of the liver was performed to evaluate the effect of each emulsion on the development of PN-induced steatosis. SO failed to prevent PN-induced steatosis (FIG. 13A top row, middle panel). However, the addition of α-tocopherol to SO(SO+AT) resulted in the maintenance of normal liver architecture in PN-fed animals (FIG. 13A, bottom row, middle panel). FO and FO+P also retained normal liver architecture (FIG. 13A, right panels), suggesting that the addition of phytosterols to FO does not compromise the ability of FO to protect the liver from PN-induced steatosis. When analyzed with oil red O to assess liver fat accumulation, SO+AT resulted in a reduction in liver fat accumulation compared to SO (FIG. 13B, middle panels), indicating that α-tocopherol confers SO hepatoprotective properties. Both FO and FO+P had minimal liver fat accumulation, again indicating that the addition of phytosterols to FO did not impair the hepatoprotective properties of FO (Fig. 13B, right panels).

Molecular assessment

Given that mice fed a PN diet with SO as a fat source develop steatosis while mice fed a PN diet with FO as a fat source do not, it was hypothesized that SO and FO might affect the processing of fat in the liver in different ways. To test the effect of each emulsion on liver fat processing, gene expression analysis of key liver fat processing genes was performed. Acetyl CoA Carboxylase 2 (ACC2), which catalyzes the rate-limiting step in de novo lipogenesis, and Peroxisome Proliferator Activated Receptor Gamma (PPARγ), which is a transcriptional regulator when working with liver fat, have been identified as upregulated genes. on a fat-free PN diet, is normalized by providing FO but not by SO (FIGS. 14A and 14B). SO+AT showed normalization of ACC2 and PPARγ levels in gene expression (FIGS. 14A and 14B) and protein expression (FIGS. 15A-15C), indicating that addition of α-tocopherol may confer SO hepatoprotective properties. Both FO and FO+P resulted in normalized gene (FIGS. 14A and 14B) and protein (FIGS. 15A-15C) expression of ACC2 and PPARγ, indicating that, at the molecular level, phytosterol supplementation does not impair the hepatoprotective properties of FO.

Discussion

Serum and liver phytosterol levels have been demonstrated to be higher in PN-dependent patients receiving intravenous lipid emulsions containing soybean oil than in patients weaned from PN (24). In addition, among PN-dependent patients, serum and liver phytosterol levels are positively correlated with laboratory tests of liver function, as well as with the degree of portal inflammation and liver fibrosis on histological analysis (24). In neonatal PN-dependent patients, serum phytosterol levels are higher in patients who meet the biochemical criteria for PNALD than in patients without PNALD (25). In vitro studies have shown that stigmasterol, one of the major phytosterols in SO (16), inhibits the expression of target genes for the bile acid-responsive nuclear receptor FXR (17). In contrast, Ng et al found no adverse effect on bile acid clearance when beta-sitosterol and stigmasterol were added to commercial FO in the PNALD model for preterm piglets (21). Studies by Muto et al in the PNALD neonatal piglet model found no improvement in bile flow, serum bile acid concentrations, or direct serum bilirubin levels when α-tocopherol was added to a commercially available SO emulsion (26).

In this study, an intravenous lipid emulsion formulated in the laboratory using soybean oil to which α-tocopherol was added was able to maintain normal liver structure and normal expression of 2 important genes involved in the liver in a mouse model of PN-induced liver injury. An intravenous lipid emulsion made in a laboratory using soybean oil containing no added α-tocopherol is unable to protect against PN-induced hepatosteatosis and liver fat dysregulation. These results are consistent with the findings of Ng et al.; albeit in a different model of PN-induced liver injury using laboratory-made intravenous lipid emulsions. The formulation of intravenous lipid emulsions in the laboratory allowed for control over the formulation protocol and the use of the same tools and ingredients to ensure that all emulsions used in the study were made in exactly the same way. Here, α-tocopherol was added to soybean oil prior to formulation of the emulsions, which mimics the preparation of commercial FO emulsions and is the way α-tocopherol is integrated into commercially available emulsions.

This study also identified PPARγ and ACC2 as genes that are not regulated by the PN diet, normalized by FO and α-tocopherol-containing SO, but not by SO alone. PPARγ is a transcriptional regulator of systemic and hepatic fat, as well as inflammation. ACC codes for an enzyme that catalyzes the rate-limiting step of de novo lipogenesis. It has been demonstrated that the PPARγ agonist rosiglitazone can reduce liver inflammation and associated biomarkers in a methionine- and choline-deficient dietary mouse model of non-alcoholic steatohepatitis (27). In mice lacking the LDL receptor, rosiglitazone has been shown to improve hepatosteatosis induced by a high-fat diet (28). Other studies have reported a positive correlation between increased PPARγ expression and the development of hepatosteatosis and accumulation of hepatic triglycerides in a mouse model of non-alcoholic fatty liver disease (29-31). Liver triglyceride-accumulating STAT5 knockout mice also show reduced liver fat levels with PPARγ antagonism (32). In vitro studies also indicate an adipogenic effect associated with increased PPARγ expression (33). Interestingly, beta-sitosterol, one of the major phytosterols in SO, has also been shown to enhance PPARγ expression in a rat model of radiation-induced oxidative stress (34). ACC2 expression has been shown to be upregulated in response to a high fructose state and normalized by treatment with docosahexaenoic (DHA) omega-3 fatty acid in primary murine hepatocytes (35).

Interestingly, this study found no hepatotoxic effect from the addition of phytosterols to fish oil in PN-induced liver injury. One possible conclusion is that the phytosterols in soybean oil are not responsible for soybean oil-associated hepatotoxic effects in a mouse model of PN-induced liver injury. An alternative explanation is that phytosterols have hepatotoxic properties but are unable to overcome the hepatoprotective properties of fish oil. The omega-3 fatty acids rich in fish oil are precursors to anti-inflammatory lipid mediators (36, 37), and fish oil is also rich in α-tocopherol. These properties may provide hepatoprotection that cannot be overcome by the presence of phytosterols. While results consistent with those of this study were observed in the PNALD model in preterm piglets (21), other in vivo and in vitro studies suggest that phytosterols do indeed have hepatotoxic properties (17, 18). A third possible explanation is that specific phytosterols at specific concentrations or a specific balance of phytosterols are required for phytosterol-associated hepatotoxic properties to occur. In this study, the composition of phytosterols added to fish oil approximated the types and amounts of phytosterols found in soybean oil.

All emulsions formulated in this study are protected from the development of EFAD. Traditionally, linoleic (LA) and alpha-linolenic (ALA) acids, the parent omega-6 and omega-3 fatty acids respectively, are considered EFAs. More recent evidence suggests that providing LA and ALA metabolites such as arachidonic acid (ARA), eicosapentaenoic acid (EPA) and DHA is sufficient to prevent the development of EFAD (38). Interestingly, this study showed that serum ARA, EPA and DHA reflect the balance of EFA provided by the injected emulsion rather than serum LA and ALA levels.

The ability of α-tocopherol to make SO less hepatotoxic in a PN-induced liver injury model implies that α-tocopherol is useful in the clinical management of PNALD and other similar liver pathologies. Currently, FO can be used to treat PNALD, however, FO is not available for all patients. The benefits of α-tocopherol in the treatment of PNALD have yet to be clinically tested, and it is likely that FO has additional properties such as excess omega-3 fatty acids that make FO more beneficial than α-tocopherol alone. However, the results of this study indicate that α-tocopherol is an option in the prevention or treatment of PNALD in patients for whom FO is not available.

Experimental Procedures

Formulation of Lipid Emulsion

Emulsion Materials: Sterile Water for Injection (SWFI, Hospira, Lake Forest, IL), Egg Phospholipid (Lipoid LLC, Newark, NJ), Sodium Oleate (Lipoid LLC, Newark NJ), and Glycerin (Sigma-Aldrich, St. Louis, MO) were used to compile the variance. The oils used were USP grade soybean oil (Spectrum Chemicals, New Brunswick, NJ) and CrystalPure EPA 28/12 TG fish oil (BASF). Additives used were α-tocopherol (Sigma-Aldrich, St. Louis, MO), beta-sitosterol (Sigma-Aldrich, St. Louis, MO), and stigmasterol (Sigma-Aldrich, St. Louis, MO). Commercial emulsions used for the assays included Omegaven (Fresenius Kabi, Bad Homburg, Germany) and Intralipid (Fresenius Kabi, Uppsala, Sweden).

Preparation of FO+P oil: CrystalPure EPA 28/12 TG fish oil was heated to maintain the temperature between 50-60°C under constant stirring conditions. Phytosterols (85% beta-sitosterol, 15% stigmasterol) were added to a final concentration of 2.25 mg phytosterols per gram of oil and mixed until dissolved. When used to prepare a 20% emulsion, the calculated concentration of phytosterol is 450 mg of phytosterols per liter of emulsion.

Preparation of SO+AT oil. Soybean oil was heated to maintain the temperature between 50 and 60°C under constant stirring conditions. Alpha-tocopherol was added to a final concentration of 1 mg α-tocopherol per gram of oil and mixed for 10-15 minutes. When used to prepare a 20% emulsion, the calculated α-tocopherol content is 200 mg α-tocopherol per liter of emulsion.

Making an emulsion. The emulsion was prepared by high pressure homogenization as described previously (22). All steps were performed at 40-45°C unless otherwise noted. All steps were performed under a nitrogen atmosphere.

First, a dispersion was prepared by adding frozen egg phospholipid to SWFI heated to 75-90°C under high shear mixing conditions and allowing the mixture to equilibrate at 40-45°C. Sodium oleate was added and shear mixing was continued (4000-4100 rpm) for 40 minutes after which glycerol was added. The crude dispersion was homogenized (Panda Plus homogenizer, GEA Niro Saovi, Columbia, MD) at 9000 psi for 20 cycles. The dispersion was filtered through a 0.45 μm membrane and the pH was adjusted to 10.4 with 0.5N. sodium hydroxide. The final dispersion consisted of 12% egg phospholipid, 25% glycerol and 0.3% sodium oleate. One batch of dispersion was enough to prepare 5 1 liter emulsions.

Emulsions were prepared by adding oil to an appropriate volume of dispersion under high shear mixing conditions (3800-4200 rpm, adjusted to avoid foaming), while stirring was continued for 40-45 minutes and slowly adjusted to a final volume of 500 ml using SWFI keeping the temperature at 40-45°C. The crude emulsion was homogenized at 5000 psi for at least 9 cycles. The pH of the final emulsion was adjusted to 9-9.5 using 0.1N. sodium hydroxide, packaged in 20 ml serum vials with headspace filled with nitrogen gas, and the packaged emulsions were autoclaved. The final emulsion composition consisted of 20% oil, 1.2% egg phospholipid, 2.5% glycerol and 0.03% sodium oleate.

All emulsions were tested for mean globule size and PFAT5 (Micro Measurements, Deerfield, IL) in accordance with USP <401> standards.

Determination of Phytosterol and Alpha Tocopherol Levels in Emulsions. To determine the levels of phytosterol, the samples were saponified with 2 mol/l ethanolic KOH and the sterols were extracted with n-heptane. The extracts were evaporated and separated on a column for capillary gas chromatography. The detection was with a flame ionization detector. Quantitative determination was carried out using epicoprostanol as an internal control.

Alpha-tocopherol levels were determined as described (39), however an external calibration was used instead of an internal calibration.

Mouse Model of PN-Induced Liver Injury.

All animal experiments have been approved by the Animal Care and Use Committee of Boston Children's Hospital. Six week old C57BL/6 mice (Jackson Labs, Bar Harbor, ME) were fed either a standard chow diet or a liquid diet consisting of PN administered to patients at Boston Children's Hospital (20% Dextrose, 2% amino acid, 30 mEq/L sodium, 20 meq/l potassium, 15 meq/l calcium, 10 meq/l magnesium, 10 mmol/l phosphate, 36.67 meq/l chloride, 19.4 meq/l acetate, Pediatric multivitamins, Pediatric trace elements). Mice injected with PN were given intravenous (IV) saline, IV FO, IV FO+P, IV SO, or IV SO+AT (2.4 g/kg/day via tail vein injection). After 19 days, the animals were sacrificed by asphyxiation with carbon dioxide. Blood was taken for serum collection. The liver, spleen and right kidney were removed for further analysis. This experiment was performed twice with different batches of emulsions for each experiment. The first experiment used 5 mice per treatment group and the second experiment used 10 mice per treatment group.

Organ Processing and Histology. Spleens, kidneys and one portion of each liver were placed in 10% formalin and stored at 4°C for 24 hours, then transferred to 70% ethanol. Samples were embedded in paraffin and sectioned for Hematoxylin and Eosin (H&E) staining to assess liver structure. The second portion of each liver was placed in Optimum Cutting Temperature (OCT) medium (Fisher Scientific, Pittsburgh, RA) and frozen in liquid nitrogen. Samples were frozen cut and stained with oil red O to assess liver fat accumulation. Visualization was performed using a Zeiss Axiophot™ microscope (Oberkochen, Germany). The sections were analyzed by a certified pathologist who was blind to the treatment groups. One third of each liver was flash frozen in liquid nitrogen and stored at -80°C for analysis of gene and protein expression.

Serum Fatty Acid Profiling. Serum fatty acid extraction was performed as previously described (2). Briefly, serum samples (30 μl per sample) with tricosanoic acid added as an internal standard were subjected to extraction with chloroform and methanol in a ratio of 2:1 to isolate the lipid fraction. The samples were saponified with 0.5N. methanolic sodium hydroxide. Samples were incubated in 14% BF ₃ /methanol for 30 minutes at 100°C. The steps were performed under nitrogen gas to minimize oxidation. Analysis was performed by gas liquid chromatography (Hewlett Packard 6890) and detection by a flame ionization detector. An external standard, fatty acid methyl ester (NuCheck™ Prep, Elysian, MN), was used to identify patterns of fatty acid peaks.

Gene Expression Analysis. Livers were cut to 25 mg per sample and RNA was extracted using the Qiagen AllPrep™ DNA/RNA/Protein Kit (Gaithersburg, MD) according to the manufacturer's instructions. Taqman™ primers (Invitrogen, Carlsbad, CA) and reagents (Agilent Technologies, Santa Clara, CA) were used for each reaction according to the manufacturer's instructions with 200 ng of RNA. The ABI One Step Plus cycler used a two-step cyclic RT-PCR protocol. An initial reverse transcription step of 30 minutes at 50°C and 10 minutes at 95°C was followed by an amplification step of 15 seconds at 95°C and 1 minute at 60°C, cycled 40 times. Expression of the target gene was normalized to the GAPDH gene and compared to a fed control group using the 2 ^-ΔΔCt method (40).

Protein analysis. The livers were cut to 25 mg per sample and homogenized in radioimmunoprecipitation assay buffer (RIPA) with protease inhibitor and phosphatase inhibitor using stainless steel balls in a Bullet Blender. Protein concentrations were determined using the Bradford Assay (Bio-Rad, Hercules, CA). Ten milligrams of protein per sample was separated using a 4-12% Bis Tris polyacrylamide gel (Invitrogen, Carlsbad, CA) before being transferred to a nitrocellulose membrane. The membranes were blocked in 5% skim milk for 1 hour. The membranes were incubated in primary antibodies overnight and in secondary antibodies for 1 hour. ACC and PPARγ antibodies were obtained from Cell Signaling technologies (Danvers, MA). Anti-beta actin antibody was obtained from Santa Cruz Biotechnologies (Paso Robles, CA).

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EXAMPLE 4

In Examples 1-3, oil emulsion composition blends were prepared as follows: Oil-in-water emulsions of the desired concentration were developed using Panda Plus GEA Niro Soavi™. Using a Silverson high shear mixer, the phospholipid was dispersed, while still frozen, in a volume of water for injection preheated to 100 degrees Celsius, approximately 3 times the weight of the oil. Sodium Oleate was then added and dissolved with stirring until it finely separated and formed a viscous liquid. Glycerin was then added to the dispersion and mixed using a Silverson high shear mixer until smooth at 3000 rpm for 10 minutes. This solution was then passed through a Panda Plus GEA Niro Soavi™ homogenizer set at 500 psi (2nd stage) and 4500 psi (1st stage) at a total pressure of 5000 psi (+ or - 400 psi) based on recirculation over time. , equivalent to 10 full passes of this volume of dispersion through the homogenizer. During this process, the temperature of the dispersion was maintained at about 40 degrees Celsius. The pH was then recorded and adjusted to 10.2-10.8 at 40° C. (range 38-42° C.) with 0.5 normal sodium hydroxide solution. The oil was heated to approximately 40 degrees Celsius under a nitrogen atmosphere and added as a thin stream to a pH adjusted dispersion of approximately 200 ml water for injection (heated to 100 degrees Celsius) using a Silverson high shear mixer and mixed at 3000 rpm for for a total of 10 minutes, then at 4000 rpm for a total of 5 minutes. Water for injection (heated to 100 degrees Celsius) was then added to the crude emulsion concentrate to 100% by volume of the final emulsion. The diluted emulsion was then added to a Panda Plus GEA Niro Soavi™ hopper and passed through a homogenizer at 900 psi (2nd stage) and 8100 psi (1st stage) at a total pressure of 9000 psi (pressure ratio 1:10, 2nd stage to the 1st stage) for a time not less than required for 9 consecutive full passes. The emulsion in the hopper was continuously circulated with a Heidolph RZR 2020™ mixer set at power 1 and speed 8. The temperature of the emulsion was kept constant at about 40 degrees with a Thermo Cube™ water cooler set at 38 degrees Celsius. After the 9th pass, the emulsion was transferred to a clean glass container. The pH was recorded at 40 degrees Celsius (range 38-40 degrees Celsius) and adjusted to 9 (range 8.8-9.2) with 0.1 normal sodium hydroxide solution. All components of this emulsion were maintained under nitrogen gas in the headspace above all vessels during manufacture. The vials were made from 316 stainless steel containers. After adjusting the pH, the emulsion was transferred to sterile washed storage vials, autoclaved for 15 minutes, and stored in a refrigerator kept at a temperature not exceeding 30 degrees Celsius.

In embodiments where combinations of MCT, FO and/or SO were used, emulsions of said oil were prepared as in the previous paragraph at a concentration of 20% w/v. in water. These emulsions were then mixed together to give various ratios. For example, 100 ml of a 50:50 MCT:FO emulsion composition was prepared by mixing 50 ml of 20% MCT emulsion and 50 ml of 20% FO emulsion. Thus, the final emulsion compositions were 20% total fat/volume.

Claims (96)

1. An emulsion composition containing: fish oil and medium chain triglycerides (MCTs) in a ratio between, but not including, from 30:70 to about 70:30; or omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 30:70 to about 70:30; moreover, the total triglyceride + diglyceride content of fish oil and/or omega-3 fatty acids contains no more than 10% diglyceride. 2. The emulsion composition according to claim 1, containing: fish oil and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40; or omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 40:60 to about 60:40. 3. The emulsion composition according to claim 1, containing fish oil and medium chain triglycerides (MCTs) in a ratio of about 50:50; or omega-3 fatty acids and medium chain triglycerides (MCTs) in a ratio of about 50:50. 4. Emulsion composition according to any one of paragraphs. 1-3, where the emulsion is an oil-in-water emulsion. 5. Emulsion composition according to any one of paragraphs. 1-4, where the composition additionally contains alpha-tocopherol. 6. An emulsion composition according to claim 5, wherein alpha-tocopherol is present in an amount of at least 100 mg per liter of emulsion composition. 7. Emulsion composition according to any one of paragraphs. 5, 6, where alpha-tocopherol is present in an amount of at least 120 mg per liter of emulsion composition. 8. Emulsion composition according to any one of paragraphs. 5-7, wherein the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 2:1. 9. Emulsion composition according to any one of paragraphs. 5-8, wherein the emulsion composition contains alpha-tocopherol and other forms of vitamin E in a ratio of at least 10:1. 10. Emulsion composition according to any one of paragraphs. 5-9, wherein the emulsion composition does not contain forms of vitamin E other than alpha-tocopherol. 11. Emulsion composition according to any one of paragraphs. 1-10, and the composition contains phytosterols. 12. The emulsion composition of claim 11 wherein the phytosterols are present in the composition at a concentration of less than 100 mg per liter of the emulsion composition. 13. Emulsion composition according to any one of paragraphs. 11, 12, in which phytosterols are present in the composition at a concentration of less than 50 mg per liter of emulsion composition. 14. Emulsion composition according to any one of paragraphs. 1-13, in which arachidonic acid is present in the composition at a concentration of at least 900 mg/l. 15. Emulsion composition according to any one of paragraphs. 1-14, in which docosahexaenoic acid is present in the composition at a concentration of at least 13.4 g/l. 16. Emulsion composition according to any one of paragraphs. 1-15, where eicosapentaenoic acid is present in the composition at a concentration of at least 11.6 g/l. 17. Emulsion composition according to any one of paragraphs. 1-16, wherein the composition comprises a mixture of a fish oil and/or omega-3 fatty acid oil emulsion and an MCT emulsion. 18. Emulsion composition according to any one of paragraphs. 1-17, wherein the composition comprises an emulsion of a mixture of fish oil and/or omega-3 fatty acid oil and MCT. 19. Emulsion composition according to any one of paragraphs. 1-18 in which the fish oil and/or omega-3 fatty acid oil has not been distilled or interesterified. 20. Emulsion composition according to any one of paragraphs. 1-19, in which the omega-3 fatty acids are presented as an omega-3 fatty acid-dominated oil that has not been reesterified. 21. Emulsion composition according to any one of paragraphs. 1-20, in which fish oil and/or omega-3 fatty acids do not contain diglycerides. 22. Emulsion composition according to any one of paragraphs. 1-21, wherein the composition contains a) fish oil and MCT at a concentration of 10 g to 50 g per 100 ml, b) or omega-3 fatty acids and MCT at a concentration of 10 g to 50 g per 100 ml. 23. Emulsion composition according to any one of paragraphs. 1-22 formulated for parenteral or intravenous administration. 24. Emulsion composition according to any one of paragraphs. 1-23, additionally containing the addition of one or more additional fatty acids or mixtures thereof. 25. The emulsion composition of claim 24, wherein the supplement contains one or more fatty acids that are therapeutic for the disease. 26. Emulsion composition according to any one of paragraphs. 24, 25, wherein the composition comprises a mixture of a) an additive emulsion and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs. 27. Emulsion composition according to any one of paragraphs. 1-26, additionally containing one or more of egg phospholipid, glycerin, sodium oleate and sodium hydroxide. 28. Emulsion composition according to any one of paragraphs. 1-27, additionally containing egg phospholipid, glycerin, sodium oleate and sodium hydroxide. 29. Emulsion composition according to any one of paragraphs. 1-28, wherein the composition consists essentially of or consists of a) MCTs, b) fish oils and/or omega-3 fatty acids, and c) one or more emulsifiers. 30. Emulsion composition according to any one of paragraphs. 1-29, wherein the composition consists essentially of or consists of a) MCTs, b) fish oil and/or omega-3 fatty acids, c) alpha-tocopherol, and d) one or more emulsifiers. 31. Emulsion composition according to any one of paragraphs. 1-30, in which omega-6 fatty acids are present in the emulsion at a concentration of less than 50 mg/ml. 32. A method for reducing the risk of developing or treating inflammatory reactions, wherein the method includes administering an emulsion composition according to any one of paragraphs. 1-31 to the subject in need of it. 33. A method for reducing the risk of developing or treating a liver disease, the method comprising administering an emulsion composition according to any one of paragraphs. 1-31 to the subject in need of it. 34. A method for reducing the risk of developing or treating inflammatory reactions and liver disease, wherein the method includes administering an emulsion composition according to any one of paragraphs. 1-31 to the subject in need of it. 35. The method according to any one of paragraphs. 32-34, where the administration is parenteral administration. 36. The method of claim 35, wherein the administration is total parenteral administration. 37. The method according to any one of paragraphs. 32-36 where the subject is in need of parenteral nutrition. 38. The method according to any one of paragraphs. 32-37 where the subject is in need of total parenteral nutrition. 39. The method according to any one of paragraphs. 32-38 where the patient is not receiving oral nutrition. 40. The method according to any one of paragraphs. 32-39, where the patient is receiving no other parenteral formulations. 41. The method according to any one of paragraphs. 32-40, where the patient does not receive oral nutrition that is sufficient to maintain nutritional balance. 42. The method according to any one of paragraphs. 32-41, where the patient is not receiving other parenteral formulations that are sufficient to maintain nutritional balance. 43. The method according to any one of paragraphs. 32-42, where the patient does not receive other dietary sources of fatty acids. 44. The method according to any one of paragraphs. 32-43, where the patient is not receiving other parenteral dietary sources of fatty acids. 45. The method according to any one of paragraphs. 32-44 where the patient is not receiving other dietary sources of essential fatty acids. 46. The method according to any one of paragraphs. 32-45 where the patient is not receiving other parenteral nutritional sources of essential fatty acids. 47. The method according to any one of paragraphs. 32-46, where the emulsion composition is administered as monotherapy. 48. The method according to any one of paragraphs. 32-47, where the emulsion composition is administered as monotherapy for nutritional needs. 49. The method according to any one of paragraphs. 32-48, where the patient is a patient in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; atresia of the biliary tract; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemia-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; anginal condition; asthmatic condition; epileptic state; lacunar state; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal failure associated liver disease (IFALD), parenteral nutrition-associated cholestasis (PNAC), essential fatty acid deficiency (EFAD), parenteral nutritional dependence complicated by soy allergy or allergy to lipid emulsions containing ingredients other than MCTs and fish oil, oil, which is dominated by omega-3 fatty acids, and/or omega-3 fatty acids. 50. The method according to any one of paragraphs. 32-49 wherein the dosage administered is from about 0.5 g fatty acids/kg/day to about 5 g fatty acids/kg/day. 51. The method according to any one of paragraphs. 32-50, wherein the dose administered is from about 1 g fatty acids/kg/day to about 3 g fatty acids/kg/day. 52. The method according to any one of paragraphs. 32-51, where the dose administered is about 2 g fatty acids/kg/day. 53. The method according to any one of paragraphs. 32-52 wherein the dosage administered is from about 0.5 g fish oil/kg/day to about 5 g fish oil/kg/day. 54. The method according to any one of paragraphs. 32-53 wherein the dosage administered is from about 1 g fish oil/kg/day to about 3 g fish oil/kg/day. 55. The method according to any one of paragraphs. 32-54, where the dose administered is about 2 g fish oil/kg/day. 56. The method according to any one of paragraphs. 32-42 or 47-55, further comprising adding an additive of one or more fatty acids or mixtures thereof. 57. The method of claim 56, wherein the supplement contains one or more fatty acids that are therapeutic for the disease. 58. The method according to any one of paragraphs. 32-57, wherein the emulsion composition comprises a mixture of a) an additive emulsion and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs. 59. The method according to p. 58, where the mixture is prepared at the time or at the site of administration. 60. The method of claim 59, further comprising preparing the mixture as indicated clinically. 61. The method according to any one of paragraphs. 32-60, where the administration is chronic. 62. The use of the emulsion composition according to any one of paragraphs. 1-31 to provide parenteral nutrition to a subject in need thereof. 63. Use according to claim 62, wherein the emulsion composition is administered by parenteral administration. 64. Application according to any one of paragraphs. 62, 63, where the emulsion composition is administered by total parenteral administration. 65. Application according to any one of paragraphs. 62-64, where the subject is in need of total parenteral nutrition. 66. Application according to any one of paragraphs. 62-65, where the patient does not receive oral nutrition. 67. Application according to any one of paragraphs. 62-66, where the patient does not receive other parenteral formulations. 68. Application according to any one of paragraphs. 62-67, where the patient does not receive oral nutrition that is sufficient to maintain nutritional balance. 69. Application according to any one of paragraphs. 62-68, where the patient is not receiving other parenteral formulations that are sufficient to maintain nutritional balance. 70. Application according to any one of paragraphs. 62-69, where the patient does not receive other dietary sources of fatty acids. 71. Application according to any one of paragraphs. 62-70, where the patient is not receiving other parenteral dietary sources of fatty acids. 72. Application according to any one of paragraphs. 62-71, where the patient does not receive other dietary sources of essential fatty acids. 73. Application according to any one of paragraphs. 62-72, where the patient is not receiving other parenteral dietary sources of essential fatty acids. 74. Application according to any one of paragraphs. 62-73, where the emulsion composition is administered as monotherapy. 75. Application according to any one of paragraphs. 62-74, where the emulsion composition is administered as monotherapy for nutritional purposes. 76. Application according to any one of paragraphs. 62-75, where the patient is a patient in need of treatment for a condition selected from the group consisting of: hepatic steatosis; intestinal failure; liver disease associated with parenteral nutrition (PNALD); sepsis; cystic fibrosis; sickle cell anemia; pancreatitis; inflammatory bowel disease; Crohn's disease; atresia of the biliary tract; primary sclerosing cholangitis; inflammatory infection; inflammatory condition; systemic inflammatory response syndrome (SIRS); hypertriglyceridemia; severe hypertriglyceridemia; severe hepatic steatosis; retinopathy of prematurity; acute tubular necrosis; IgA nephropathy; ischemia-reperfusion injury; traumatic brain injury; multisystem organ failure; respiratory distress syndrome; acute myocardial infarction; myocardial infarction; anginal condition; asthmatic condition; epileptic state; lacunar state; inflammatory bowel disease; regional enteritis; ulcerative colitis; severe or debilitating arthritis; arthritis; psoriasis; severe psoriasis; burns; burns of the third degree; pancreatitis; acute pancreatitis; intestinal failure associated liver disease (IFALD), parenteral nutrition-associated cholestasis (PNAC), essential fatty acid deficiency (EFAD), parenteral nutritional dependence complicated by soy allergy or allergy to lipid emulsions containing ingredients other than MCTs and fish oil, oil, which is dominated by omega-3 fatty acids, and/or omega-3 fatty acids. 77. Application according to any one of paragraphs. 62-76 wherein the dosage administered is from about 0.5 g fatty acids/kg/day to about 5 g fatty acids/kg/day. 78. Application according to any one of paragraphs. 62-77, wherein the dose administered is from about 1 g fatty acids/kg/day to about 3 g fatty acids/kg/day. 79. Application according to any one of paragraphs. 62-78 where the dose administered is about 2 g fatty acids/kg/day. 80. Application according to any one of paragraphs. 62-79 wherein the dosage administered is from about 0.5 g fish oil/kg/day to about 5 g fish oil/kg/day. 81. Application according to any one of paragraphs. 62-80 wherein the dosage administered is from about 1 g fish oil/kg/day to about 3 g fish oil/kg/day. 82. Application according to any one of paragraphs. 62-81, where the dose administered is about 2 g fish oil/kg/day. 83. Application according to any one of paragraphs. 62-69 or 74-82, where the patient is additionally administered an additive of one or more fatty acids or mixtures thereof. 84. Application according to any one of paragraphs. 62-83, where the emulsion composition additionally contains the addition of one or more fatty acids or mixtures thereof. 85. Application according to any one of paragraphs. 62-84, where the supplement contains one or more fatty acids that are therapeutic for the disease. 86. Application according to any one of paragraphs. 62-85, wherein the emulsion composition comprises a mixture of a) an additive emulsion and b) one or more emulsions of i) fish oil and/or omega-3 fatty acids and ii) MCTs. 87. Use according to claim 86, where the mixture is prepared at the time or at the site of administration. 88. Use according to claim 87, additionally containing the preparation of the mixture in accordance with clinical indications. 89. Application according to any one of paragraphs. 62-88 where administration is chronic.

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