Stable diglyceride emulsions and methods for treating organ damage - Patents.com

Abstract

The present invention provides compositions and methods comprising a stable omega-3 diglyceride oil-in-water emulsion for rapid treatment and / or prevention of tissue or organ damage. The compositions provide protection against cell death and are used in patients requiring neuroprotection. For example, the compositions are used to treat ischemic reperfusion injury, such as ischemic stroke. The compositions are also particularly useful in treating trauma, such as traumatic brain injury or spinal cord injury. The compositions have a wide time window, making them effective after the onset of traumatic or ischemic injury (e.g., after stroke), and may be administered in combination with other treatments.

Description

[Background technology]

[0001] Stroke is the leading cause of long-term disability and the fifth leading cause of death in the United States. Currently, tissue plasminogen activator (t-PA) is the only FDA-approved agent for the treatment of acute ischemic stroke. However, its use is limited to a narrow time window of 3 to 4.5 hours.

[0002] Omega-3 (n-3) fatty acids (FAs) are candidates for rapid neuroprotection after stroke. Evidence suggests that n-3 FAs act as bioactive non-saturated lipids with pleiotropic effects and exhibit neuroprotective properties in animal models of stroke. Numerous biological mechanisms may be affected by n-3 FAs, including: (i) reduced mitochondrial reactive oxygen species (ROS) production; (ii) Mitochondrial Ca 2+ uptake and preservation of homeostasis; (iii) modulation of receptor-mediated signaling and inhibition of apoptotic pathways; (iv) an increase in potent n-3 FA-derived resolvins and protectins; and (v) Reduced inflammatory response. Acting individually or cooperatively, these mechanisms may contribute to the neuroprotection of n-3 FAs in ischemic injury, reducing cell death while promoting repair processes.

[0003] However, to be effective in neuroprotection, appropriate levels of n-3 FA must be rapidly delivered to cells at risk of cell death or cell damage.In certain aspects, the present disclosure provides compositions and methods for the rapid delivery of n-3 FA for the treatment of organ damage, including for neuroprotection.The present invention is particularly used as an emergency drug for the treatment of stroke, myocardial infarction, traumatic brain injury, and ischemic organ damage. Summary of the Invention

[0004] The present invention provides compositions and methods comprising stable n-3 diglyceride (DG) oil-in-water emulsions for rapid treatment of and / or prevent organ damage. The compositions provide protection against cell death, particularly in patients in need of neuroprotection or organ protection, including neuroprotection or organ protection against ischemic stroke, myocardial infarction, and traumatic brain injury. The compositions have a wide time window during which administration is effective after the onset of injury (e.g., after the onset of stroke). The compositions may be administered in combination with other treatments, and the compositions may be administered during the recovery phase to further improve outcomes.

[0005] In various aspects and embodiments, the composition is an emulsion that can be stored in a stable form for emergency use.For example, in various embodiments, the composition is delivered by emergency medical professionals at the scene.The emulsion described herein is substantially stable for at least 6 months, or at least 1 year, or at least 24 months.The composition is suitable for parenteral delivery, such as intravenous (iv) delivery or intraarterial delivery, and delivery by gastric tube or duodenal tube, and the physical properties of the emulsion facilitate the rapid delivery of n-3 FA for uptake by injured tissue, including brain tissue.

[0006] According to the present invention, the esterified FA of DG may be mainly n-3 FA. For example, in some embodiments, DG contains at least about 50% n-3 FA, or at least about 75% n-3 FA, or at least about 90% n-3 FA, or about 100% n-3 FA. In some embodiments, the n-3 FA is a long-chain n-3 FA, including one or more of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA). In some embodiments, the n-3 fatty acid is DHA and EPA.

[0007] In various embodiments, the stable emulsion has an average particle size of 200 nm or less and a zeta potential (ZP) of about -30 mV or more negative than -30 mV. In some embodiments, the average particle size of the emulsion is about 190 nm or less, or about 180 nm or less, or about 160 nm or less, or about 140 nm or less. In various embodiments, the polydispersity index (PDI) is 0.3 or less. In various embodiments, the zeta potential of the emulsion is at least about -45 mV or more negative, or at least about -50 mV or more negative, or at least about -55 mV or more negative, or at least about -60 mV or more negative.

[0008] The stable emulsions are suitable for parenteral or gastrointestinal administration, for example, for rapid delivery of n-3 FAs to damaged cells and tissues, including the brain in some embodiments. The lipid component of the emulsions generally comprises about 10% to about 50% by weight of the composition. In some embodiments, at least about 20% by weight of the composition is DG oil, and in some embodiments, about 23% to about 30% by weight of the composition is DG oil. The compositions also comprise an emulsifier, optionally including a co-emulsifier, as described herein.

[0009] The composition contains one or more emulsifiers to achieve desired physical properties. In various embodiments, the emulsifiers can include one or more of phospholipid emulsifiers, phosphoglyceride emulsifiers, and medium-chain and / or long-chain fatty acid emulsifiers. In various embodiments, the composition contains less than about 1% by weight of emulsifiers.

[0010] One example of a composition of the present disclosure is a composition suitable for intravenous or intraarterial injection, comprising a stable diglyceride (DG) oil-in-water emulsion, the emulsion comprising at least 20% by weight of DG oil, the esterified fatty acids of which are at least about 90% n-3 fatty acids, including DHA and EPA, the emulsion having an average particle size of 200 nm or less, a polydispersity index of 0.3 or less, and a zeta potential of about -40 mV or more negative than -40 mV.

[0011] In various embodiments, the composition is approximately isotonic with human blood and optionally includes one or more polyols, such as glycerol, sorbitol, xylitol, and / or glucose. In some embodiments, the composition includes one or more antioxidants, such as one or more of α-tocopherol, β-tocopherol, γ-tocopherol, and ascorbyl esters. In exemplary embodiments, the antioxidant includes α-tocopherol and / or ascorbyl esters, which are optionally ascorbyl palmitate. In some embodiments, the composition includes a metal chelator, which is optionally ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0012] The emulsion may also be co-formulated with other lipophilic active chemicals to facilitate the delivery of these otherwise difficult-to-deliver therapeutic agents, which may result in synergistic effects with other mechanisms of action.For example, in some embodiments, the emulsion is co-formulated with glibenclamide or statins.The DG composition may be administered with one or more additional neuroprotective agents.In yet other embodiments, these additional agents are administered separately from the emulsion as a combined therapy.

[0013] In another aspect, the present invention provides methods for treating patients in need of cell protection from cell death, including acute and chronic injury to various organs or tissues, such as the brain, spinal cord, and newly transplanted organs, among others. In some embodiments, the patient is in need of treatment for ischemic organ injury or in need of protection against damage from ischemic organ injury.

[0014] Thus, in some embodiments, the patient suffers from stroke.The compositions described herein can be administered after stroke onset to provide neuroprotection, i.e., inhibit the cellular process that leads to cell death.The physical and chemical properties of emulsions make them effective even if they are delivered later than desired after stroke onset.For example, in various embodiments, the patient is administered the composition within about 12 hours from stroke onset.

[0015] The composition is suitable for treating both ischemic and hemorrhagic strokes, and therefore may be administered by emergency personnel prior to brain imaging to detect or visualize clots or potential bleeding. If there is no bleeding, the patient may receive thrombolytic therapy (e.g., t-PA) to dissolve the clot. While t-PA is traditionally administered to stroke victims within approximately the first 4.5 hours after the stroke occurs, in accordance with the present disclosure, patients receive such thrombolytic therapy approximately 4.5 hours after the onset of the stroke. By administering the emulsion composition as quickly as possible during an emergency, more time is available to determine whether thrombolytic therapy is appropriate. In yet another embodiment, thrombectomy is performed. The compositions described herein can widen the therapeutic window in which thrombectomy is successful.

[0016] In some embodiments, in connection with ischemic stroke, a subject may receive a dose of DG emulsion as soon as possible after the subject's stroke occurs, and generally within about 24 hours, or about 12 hours, or about 10 hours, or about 8 hours, or about 6 hours. The patient may receive subsequent oral supplementation of DG emulsion and / or n-3 DG and / or n-3 triglycerides (TG) over the next few days, weeks, or months to aid in recovery.

[0017] In other embodiments, the patient has suffered from a traumatic brain injury (TBI) or is at risk of TBI. For example, the patient may be administered the composition within 1 to 24 hours after brain injury to reduce long-term tissue damage from TBI. In some embodiments, after the initial administration, the patient is administered the composition at a frequency of about once every 4 hours to about once per week to aid in recovery. The patient may optionally receive oral supplementation of n-3 DG and / or n-3 TG over the next few days, weeks, or months to aid in recovery.

[0018] In yet another embodiment, the patient suffers from post-traumatic stress disorder (PTSD).For example, the patient can be administered the composition at least about once a week for a period of time to promote recovery.The patient can optionally receive oral supplementation of n-3 DG and / or n-3 TG to help recovery.

[0019] The present invention provides protection for other organs or tissues, including ischemic tissue injury and traumatic tissue injury (including spinal cord injury (SCI)). In such embodiments, the patient may be administered the composition immediately after the emergency injury. In some embodiments, the patient is administered the composition at least once per day to once per week after the initial administration. The patient may optionally receive oral supplementation with n-3 DG and / or n-3 TG over the next few weeks or months to aid in recovery. For example, the patient may receive oral supplementation at least once per day.

[0020] Furthermore, in some embodiments, the patient is a recipient of an organ transplant, such as a liver, kidney, heart, intestinal, or lung transplant. In some embodiments, the patient is administered the composition at least once during the perioperative period. After the perioperative period, the patient may be administered the composition at a frequency ranging from about once every four hours to once a week to aid in recovery. In yet other embodiments, the patient is being treated for acute organ failure, including acute renal failure, liver failure, or heart failure. The patient may optionally receive oral supplementation of n-3 DG and / or n-3 TG for a period of at least one week to at least one month after transplantation to aid in recovery.

[0021] In some embodiments, the patient is suffering from a neurodegenerative disease. For example, a patient with a neurodegenerative disease is administered the composition at least once a week to slow the progression of the disease and / or after a disease recurrence.

[0022] Other aspects and embodiments of the present invention will be apparent from the examples that follow. [Brief explanation of the drawings]

[0023] [Figure 1] The physical properties of a DG emulsion prepared according to the present disclosure, which contains both DHA and EPA and a triglyceride (TG) emulsion, are shown. The left side of Figure 1 shows the average particle size (diameter). The right side of Figure 1 shows the zeta potential (ZP). [Figure 2A-B] Figure 2A shows the total free fatty acids (FFA) released upon hydrolysis of 200 μg of DG or TG emulsions, measured colorimetrically and expressed in nanomoles. Figure 2A: Tri-DHA 10% PL 1.2% vs. DG 10% PL 1.2%. Figure 2B: Tri-DHA 20% PL 1.2% vs. DG 20% PL 1.2%. [Figure 3] 1 shows TLC analysis of n-3 DG oil to test purity and integrity and to identify 1,2-1,3DG molecular species. [Figure 4A-B]Infarct volumes in neonatal mice (10 days old) subjected to HI injury and treated with saline (black bars) as vehicle, or emulsions (n-3 TG (gray bars), n-3 DG (dark gray bars), or n-6 DG (light gray bars)) are shown (Figure 4A). N = 15–17. Values ​​are means ± standard deviations; (Figure 4B) TTC-stained brain sections. *p < 0.05 compared to saline; ** < 0.01 (ANOVA). Dose = 0.375 mg / g body weight. [Figure 5A-B] Figure 5A shows infarct volumes in adult mice subjected to middle cerebral artery occlusion (MCAo) and treated with saline or n-3 DG emulsion. Values ​​are means ± standard error. N = 5 (Figure 5B) TTC-stained brain sections. *p < 0.05 compared to the saline group (Student's t-test). Dose per mouse = 100 mg. [Figure 6A-B] The therapeutic time window (Figure 6A) is shown in rats subjected to MCAo and treated with saline or n-3 TG emulsion 6 or 8 hours after ischemia. Values ​​are mean ± standard error. N = 6; (Figure 6B) TTC-stained brain sections. *p < 0.05 compared with the saline group (ANOVA). Dose per rat = 150 mg. [Figure 7] Shown are the mean infarct volumes in mice treated immediately after ischemic injury with either saline, DHA, EPA, DHA-EPA, or ARA (all DG emulsion doses 0.375 g DG / Kg). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides compositions and methods comprising a stable n-3 DG oil-in-water emulsion for rapid treatment to treat and / or prevent tissue or organ damage. The compositions provide protection from cell death and are used in patients in need of neuroprotection or organ protection. For example, the compositions are used to treat ischemia-reperfusion injury, such as ischemic stroke and myocardial infarction. The compositions are also particularly useful in treating trauma, such as traumatic brain injury or spinal cord injury. The compositions have a wide time window during which they are effective after the onset of traumatic or ischemic injury (e.g., after the onset of stroke) and may be administered in combination with other therapies.

[0025] In various aspects and embodiments, the composition is a stable emulsion that can be stored in a stable form for emergency use.For example, in various embodiments, the composition is delivered on-site by emergency medical professionals.In various embodiments, the emulsion described herein is substantially stable for at least 6 months, or at least 1 year, or at least 18 months, or at least 2 years.The composition is suitable for parenteral delivery, such as intravenous or intra-arterial delivery.In addition, in some embodiments, the physical properties of the emulsion, such as an average particle diameter of 200 nm or less, facilitate the delivery of n-3 fatty acids to and / or the uptake by brain tissue.Without being bound by theory, it is believed that this small particle size will improve the rapid delivery to the brain, which is important for neuroprotection in pathologies such as stroke.

[0026] Emulsions are inherently unstable and therefore do not form spontaneously. Energy input, for example by shaking, stirring, or homogenizing, is required to form an emulsion. Over time, emulsions tend to revert to the stable state of the emulsion-containing phase. However, nanoemulsions can be kinetically stable.

[0027] An emulsion is said to be stable if the size and distribution of the emulsion droplets do not change substantially or significantly over a desired time frame (e.g., at least about six months). That is, emulsion stability refers to the emulsion's ability to resist changes in its properties over time. Emulsion instability can be observed, for example, as aggregation, creaming / sedimentation, and coalescence. When attractive forces exist between droplets, aggregation occurs, forming flocs. Coalescence occurs when droplets merge to form larger droplets, increasing the average droplet size over time. Emulsions can also cream, for example, when droplets rise to the top of the emulsion under the influence of buoyancy. Sedimentation, the opposite phenomenon of creaming, is commonly observed in water-in-oil emulsions. Sedimentation occurs when the dispersed phase is denser than the continuous phase, and gravity pulls the denser globules to the bottom of the emulsion. Like creaming, sedimentation follows Stokes' law.

[0028] Emulsifiers are substances that stabilize emulsions by increasing their kinetic stability. Emulsifiers include surface-active agents or surfactants. Surfactants can increase the kinetic stability of emulsions so that the droplet size does not change significantly over time. Emulsion stability can be assessed by zeta potential, which indicates the repulsion between droplets or particles. Emulsifiers are typically compounds that have a polar or hydrophilic (i.e., water-soluble) portion and a non-polar (i.e., hydrophobic or lipophilic) portion. Surfactants are a type of emulsifier that physically interact with both oil and water, thus stabilizing the interface between oil and water droplets in suspension.

[0029] The present invention delivers n-3 FAs to cells as stable DG emulsions. The term "n-3 FA" refers to polyunsaturated FAs in which one of the carbon-carbon double bonds is between the third and fourth carbon atoms from the distal end of the hydrocarbon chain. Examples of n-3 FAs include α-linolenic acid (18:3n-3; α-ALA; Δ 3,6,9 ), eicosapentaenoic acid (20:5n-3; EPA; Δ 5,8,11,14,17 ), docosahexaenoic acid (22:6n-3; DHA; Δ 4、7,10,13,16,19) and docosapentaenoic acid (22:5n-3; DPA; Δ 7,10,13,16,19 n-3 FAs having at least 20 carbon atoms are referred to as "long-chain n-3 FAs." The source of n-3 FAs may be from any suitable source, such as fish oil, algae oils, and other oils and fats, or may be synthetic.

[0030] A number of biological mechanisms are affected by n-3 FAs that may be beneficial in acute injuries, including: (i) reduced mitochondrial ROS generation; (ii) increased mitochondrial Ca 2+ (iii) modulation of receptor-mediated signaling and inhibition of apoptotic pathways; (iv) increase in potent n-3 FA-derived resolvins and protectins; and (v) reduction of inflammatory responses. Acting individually or cooperatively, these mechanisms may contribute to n-3 FA neuroprotection in ischemic injury, attenuating cell death while promoting repair processes.

[0031] DG consists of two FAs esterified to the trihydric alcohol glycerol. An example of a method for synthesizing DG molecules is by lipase-catalyzed glycerolysis (i.e., transesterification) of n-3 long-chain FAs. In various embodiments, the compositions described herein are substantially DG, i.e., such compositions do not contain significant amounts of triglycerides. In some embodiments, the emulsion composition is at least about 75%, or at least about 85%, or at least about 90%, or at least about 95% DG emulsion, based on the total amount of DG and TG present in the composition.

[0032] According to the present invention, the FA of DG can be mainly n-3 FA. In various embodiments, DG contains at least about 50% n-3 FA, or at least about 75% n-3 FA, or at least about 90% n-3 FA, or at least 95% n-3 FA, or about 100% n-3 FA. In some embodiments, the n-3 FA is a long-chain n-3 FA, including one or more of DHA, EPA, and DPA.

[0033] In various embodiments, n-3 FA comprises DHA, EPA, and / or DPA. For example, in some embodiments, n-3 FA comprises DHA. In some embodiments, n-3 FA is at least about 50% DHA, or at least about 60% DHA, or at least about 75% DHA, or at least about 90% DHA. In some embodiments, n-3 FA comprises EPA. For example, n-3 FA can be at least about 50% EPA, or at least about 60% EPA, or at least about 75% EPA, or at least about 90% EPA. In some embodiments, n-3 FA comprises DPA. For example, n-3 FA can be at least about 50% DPA, or at least about 60% DPA, or at least about 75% DPA, or at least about 90% DPA. In some embodiments, the n-3 FA comprises DHA and EPA, which are optionally present in a ratio of about 2:1 to about 1:2 (e.g., about 1:1). As shown herein, a DG emulsion having a small particle size and containing DHA+EPA exhibits significantly enhanced neuroprotective properties. See Figure 7.

[0034] In various embodiments, the DG molecule comprises 1,3-DG and 1,2-DG. In some embodiments, the DG is predominantly 1,3-DG.

[0035] In various embodiments, the emulsion has an average particle size of 200 nm or less and a zeta potential of about -30 mV or more negative than about -30 mV. In some embodiments, the average particle size of the emulsion is about 190 nm or less, or about 180 nm or less, or about 170 nm or less, or about 160 nm or less, or about 150 nm or less, or about 140 nm or less, or about 120 nm or less, or about 100 nm or less, or about 90 nm or less, or about 80 nm or less. In some embodiments, the average particle size is about 140 nm, about 120 nm, about 110 nm, or about 100 nm, and the polydispersity index is less than about 0.3, or less than about 0.25, or less than about 0.2. In some embodiments, the average particle size is about 110 nm to about 180 nm, or about 120 nm to about 180 nm, and the polydispersity index is less than about 0.3. In various embodiments, the zeta potential of the emulsion is at least as negative as about -35 mV, or at least as negative as about -40 mV, or at least as negative as about -50 mV, or at least as negative as about -55 mV. Emulsions according to these embodiments are stable, meaning that these parameters are maintained for at least 6 months, or in some embodiments, for at least 1 year, at least 18 months, or at least 2 years. According to the present disclosure, stability is determined by storage at 4°C.

[0036] The stable emulsion is suitable for intravenous administration, for example, to rapidly deliver n-3 FAs to damaged tissue, including the brain in some embodiments. That is, in such embodiments, the composition is an injectable composition. The lipid component generally comprises about 10% to about 50% by weight of the composition. In some embodiments, the lipid component of the composition comprises about 10% to about 30%, or about 15% to about 25%. In some embodiments, the lipid component comprises 20% to about 40%, or about 20% to about 30% by weight of the composition. For example, the lipid component may comprise at least about 10% by weight of the composition, or at least about 15%, or at least about 20% by weight of the composition, or at least about 25% by weight of the composition, or at least about 30% by weight of the composition. In such embodiments, the composition comprises at least about 10% by weight of DG fat, or at least about 15% by weight of DG fat, or at least about 20% by weight of DG fat, or at least about 23% by weight of DG fat, or at least about 25% by weight of DG fat, or at least about 27% by weight of DG fat, or at least about 30% by weight of DG fat. In some embodiments, the composition comprises about 10% by weight of DG fat. In some embodiments, the composition comprises 22-27% by weight of DG fat.

[0037] Polydispersity index (PDI) is a measure of particle size distribution within a given sample. PDI values ​​range from 0.0 (for a sample with a completely uniform particle size distribution) to 1.0 (for a highly polydisperse sample with a population of particle sizes). For lipid-based carriers, such as emulsions, a PDI of 0.3 is desirable, indicating a sufficiently uniform particle size distribution. In some embodiments, the PDI of the emulsion is less than about 0.30, such as about 0.25 or less, 0.20 or less, or about 0.15 or less.

[0038] The composition includes one or more emulsifiers to achieve desired physical properties. In various embodiments, the emulsifier may include one or more of a phospholipid emulsifier, a phosphoglyceride emulsifier, and a medium-chain and / or long-chain fatty acid emulsifier. In various embodiments, the composition includes about 0.5% to about 2.4% by weight (e.g., about 0.5% to about 2%) of an emulsifier (e.g., a phospholipid emulsifier), and optionally less than about 1.0% by weight of an emulsifier, and optionally 0.5% to 0.8% by weight of an emulsifier (e.g., a phospholipid emulsifier).

[0039] In some embodiments, the emulsion comprises one or more phospholipid emulsifiers and / or one or more phosphoglyceride emulsifiers. The phosphoglyceride emulsifier may be selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid. In some embodiments, the composition comprises a phosphatidylcholine emulsifier. In various embodiments, the ratio (by weight) of phospholipid and / or phosphoglyceride emulsifier to DG is 1:8 or less, or 1:10 or less, or 1:12 or less, or 1:15 or less. In some embodiments, the emulsifier comprises at least about 70% or at least about 80% phosphatidylcholine. For example, the emulsifier (including any co-emulsifier) ​​may comprise from about 60% to about 80% phosphatidylcholine.

[0040] The composition may further comprise one or more medium- or long-chain FAs as a co-emulsifier. For example, the composition may optionally comprise a long-chain FA selected from C16 to C24 FAs, which is optionally a C18 FA. In some embodiments, the co-emulsifier comprises a saturated FA, optionally selected from lauric acid, myristic acid, palmitic acid, and stearic acid. In some embodiments, the co-emulsifier comprises an unsaturated FA, optionally selected from oleic acid or linoleic acid. The co-emulsifier may be added as an alkali metal salt, which optionally comprises sodium oleate. In exemplary embodiments, the co-emulsifier is present in an amount of about 0.01% to 5% of the total weight of the composition. For example, the co-emulsifier may be present in an amount of about 0.01% to 2% of the total weight of the composition, or about 0.01% to about 1% of the total weight of the composition, or about 0.01% to about 0.05% of the weight of the composition.

[0041] In various embodiments, the composition is approximately isotonic with human blood and optionally contains one or more polyols, such as glycerol, sorbitol, xylitol, and / or glucose. For example, the composition may contain from about 2% to about 10% glycerol by weight of the composition, or from about 2% to about 7% glycerol by weight of the composition.

[0042] In some embodiments, the composition comprises one or more antioxidants, such as one or more of α-tocopherol, β-tocopherol, γ-tocopherol, and ascorbyl esters. In exemplary embodiments, the antioxidant comprises α-tocopherol and / or an ascorbyl ester, which optionally is ascorbyl palmitate.

[0043] In some embodiments, the composition includes a metal chelator, which optionally can be EDTA or EGTA. For example, the emulsion can include about 5 mM to about 15 mM EDTA or EGTA. For example, in some embodiments, the emulsion includes about 10 mM EDTA.

[0044] In various embodiments, a stable emulsion can be prepared according to a process that includes: (1) preparing a mixture of water, glycerol, and EDTA having a temperature of about 50°C to about 80°C (e.g., about 60°C); (2) adding a phosphatidylcholine emulsifier (e.g., at least about 75% PC; which may be derived from egg yolk lecithin), a co-emulsifier (e.g., sodium oleate), and DG oil; (3) homogenizing at a temperature of about 50°C to about 80°C (e.g., about 60°C); (4) Processing through a microfluidizer or, for larger volumes, through a high-pressure homogenizer (i.e., high-shear liquid processor). The pressure applied during this process ranges from 300 to 2000 bar, and in some embodiments, from about 500 to about 1000 bar, such as from about 600 to about 1000 bar. For example, the mixture may be processed using a microfluidizer at about 60°C and a pressure of about 950 bar. The emulsion may be processed for a time and under conditions necessary to achieve the target particle size. This process may involve co-formulation with other lipid-soluble substances, as described below.

[0045] The emulsions may be co-formulated with other lipid-soluble active chemicals to enhance their delivery and provide synergistic effects with other mechanisms of action. For example, in some embodiments, the emulsions are co-formulated with glibenclamide (Simard et al., "Glibenclamide in cerebral ischemia and stroke," Neurocrit Care, 2014, 20(2):319-333). However, glibenclamide is inefficiently delivered to the brain and is generally difficult to deliver given its lipid-soluble nature. Therefore, the present disclosure provides DG emulsions to improve the delivery of glibenclamide for neuroprotection. Because glibenclamide is a lipid-soluble active substance, it can be easily incorporated into emulsions and delivered in smaller amounts than without emulsions, thereby ensuring that the activity is delivered within its therapeutic window.

[0046] The emulsions may also be co-formulated with other lipid-soluble agents, such as statins, thereby extending the benefits of these emulsions in aiding recovery and preventing and / or treating chronic conditions, including those in which patients are at risk for ischemic injury, such as atherosclerosis, and at risk for myocardial infarction. Examples of lipid-soluble statins include atorvastatin, fluvastatin, lovastatin, simvastatin, and cerivastatin.

[0047] In some embodiments, the emulsion further comprises one or more neuroprotective agents. In some embodiments, one or more neuroprotective agents are administered separately as a combination therapy. Examples of neuroprotective agents include glutamate antagonists. Examples of neuroprotective agents include 17β-estradiol, ginsenosides, progesterone, simvastatin, and memantine. Lipid-soluble neuroprotective agents (e.g., 17β-estradiol, simvastatin, or progesterone) may be incorporated into the emulsion.

[0048] In some embodiments, the emulsion further comprises one or more metabolites of EPA, DHA, and / or DPA, such as one or more resolvins or protectins.Resolvins are polyunsaturated fatty acid (PUFA) metabolites derived from omega-3 fatty acids, including EPA, DHA, and DPA.Resolvins (such as RvD and / or RvE) can promote the recovery of normal cell function after tissue inflammation.Proteins, such as neuroprotectin D1 (NPD1), are also PUFA metabolites with strong anti-inflammatory, anti-apoptotic, and neuroprotective activities.In some embodiments, the emulsion comprises DHA and EPA (as described) together with NPD1.

[0049] In various embodiments, the pH of the composition is from about 6 to about 10, and optionally from about 6.5 to about 10, and optionally from about 9 to about 10 (eg, 9.5).

[0050] In various embodiments, the composition has a volume of about 500 mL or less, or about 300 mL or less, or about 100 mL or less, or about 50 mL or less, or about 25 mL or less. In various embodiments, the composition is contained in a pre-filled syringe, optionally having an injection volume of about 1 mL to about 50 mL. In some embodiments, the composition is filled into a vial in a volume of about 25 mL to about 100 mL.

[0051] In another aspect, the present invention provides methods for treating patients in need of protection from cell death, including acute and chronic injury to various organs or tissues, such as the brain, spinal cord, and kidneys, in particular. In some embodiments, the patient is in need of treatment for ischemic or traumatic organ injury. The methods generally involve administering to a patient in need thereof an effective amount of a composition described herein.

[0052] In various embodiments, the patient is in need of neuroprotection. In some embodiments, the patient is at risk of ischemia-reperfusion injury. Ischemia-reperfusion injury is tissue damage that occurs when blood supply returns to tissue after a period of ischemia or oxygen deprivation. The absence of oxygen and nutrients from blood during ischemia creates a pathological condition in which the restoration of circulation leads to inflammation and oxidative damage through the induction of oxidative stress.

[0053] For example, cerebral hypoxia-ischemia (or "stroke") of sufficient duration to deplete high energy reserves in neurons initiates a cascade of events over hours to days of reperfusion that leads to widespread death, both necrotic and apoptotic. These events include ROS generation and cellular oxidative damage, release of inflammatory mediators, and initiation of a sustained inflammatory response, as well as progressive apoptosis that can continue for weeks to months.

[0054] That is, in some embodiments, the patient has experienced a stroke. Stroke is a major cause of morbidity and mortality throughout all stages of life, including infants born prematurely, children in intensive care units, and elderly people with cerebrovascular disease. In some embodiments, the stroke is an ischemic stroke. However, the present invention can also be used to treat hemorrhagic stroke and neonatal stroke. In some embodiments, the subject suffers from or is at risk of hypoxic-ischemic encephalopathy (HIE); hypoxic-ischemic encephalopathy (HIE) is a form of neonatal brain damage caused by oxygen deprivation and reduced blood flow. Infants and children who survive HIE exhibit lifelong neurological handicaps, including cerebral palsy, mental retardation, epilepsy, and learning disabilities (Vannucci, RC (2000), "Hypoxic-ischemic encephalopathy," American Journal of Perinatology 17(3):113-120). Cerebral hypoxia-ischemia commonly occurs in critically ill children, particularly in association with cardiopulmonary arrest.

[0055] The compositions described herein can be administered after a stroke to provide neuroprotection, i.e., inhibit cellular processes leading to cell death. The physical and chemical properties of the emulsions allow them to be effective even when delivered later than desired after the onset of a stroke. For example, in various embodiments, the patient is administered the composition within about 1 to about 24 hours of the onset of a stroke. For example, in some embodiments, the composition is administered about 6 hours after the onset of a stroke, or about 8 hours after the onset of a stroke, or about 10 hours after the onset of a stroke, or about 12 hours after the onset of a stroke, or about 15 hours after the onset of a stroke. In some embodiments, the composition is administered about 10 hours after the onset of a stroke but within 24 hours of the onset of a stroke. The composition can prevent substantial cell death despite delayed emergency treatment. In some embodiments, the patient is administered the composition within about 2 hours after the onset of a stroke or within about 4 hours after the onset of a stroke, which provides substantial protection from cell damage and / or cell death.

[0056] The composition is suitable for the treatment of both ischemic stroke and hemorrhagic stroke, and can therefore be administered by emergency personnel before brain imaging to detect or visualize thrombus or potential bleeding. In the absence of bleeding, patients may receive thrombolytic therapy (e.g., t-PA) to dissolve clots. t-PA is the main enzyme involved in the destruction of clots, catalyzing the conversion of plasminogen to plasmin. t-PA is traditionally administered to stroke patients approximately within the first 4.5 hours after the onset of stroke. In some embodiments, patients receive such thrombolytic therapy approximately 4.5 hours after the onset of stroke, approximately 6 hours after the onset of stroke, or approximately 8 hours after the onset of stroke, and delivery of t-PA together with the DG emulsion extends the therapeutic window of thrombolytic therapy. By administering the emulsion composition as quickly as possible during an emergency, more time is available to determine whether thrombolytic therapy is appropriate. Thrombolytic therapy cannot be applied to patients experiencing hemorrhagic stroke because this therapy worsens the bleeding.

[0057] In yet another embodiment, thrombectomy is performed. Thrombectomy is an interventional procedure that removes blood clots (thrombi) from blood vessels. It is usually performed in cerebral arteries (interventional neuroradiology). The compositions described herein can expand the time window in which thrombectomy can be performed successfully. For example, thrombectomy is performed about 10 hours after the onset of stroke, or about 12 hours after the onset of stroke. In some embodiments, thrombectomy is performed about 18 hours after the onset of stroke, or about 24 hours after the onset of stroke.

[0058] In some embodiments, the patient may receive 1 to 5 doses of the composition within the first 24 hours, with at least one dose prior to thrombolytic therapy or thrombectomy and at least one dose after thrombolytic therapy or thrombectomy is administered.

[0059] The composition is generally delivered parenterally, such as intravenously or intraarterially.In some embodiments, the composition is delivered by intrathecal delivery.In some embodiments, the composition is administered intranasally, which allows rapid delivery to the brain.In some embodiments, the composition is administered by intra-arterial delivery, selectively to the brain that has previously suffered from low circulation.

[0060] In some embodiments, for ischemic stroke, the subject receives a dose of DG emulsion as soon as possible after the onset of the stroke, generally within about 24 hours, about 15 hours, about 12 hours, about 10 hours, about 8 hours, or about 6 hours after the onset of the stroke. The patient may receive subsequent doses over the following days to weeks to aid in recovery. For example, the patient may receive at least four doses of the stable DG emulsion, or at least eight doses of the stable DG emulsion. In some embodiments, the patient receives 1 to 10 or 1 to 4 doses within a period of 1 week to 1 month after the stroke to aid in recovery.

[0061] In other embodiments, the patient has suffered from or is at risk of traumatic brain injury (TBI). Traumatic brain injury typically results from a forceful blow or impact to the head or body. Objects penetrating brain tissue, such as bullets or skull fragments, can also cause traumatic brain injury. Mild traumatic brain injury may temporarily affect brain cells. More severe traumatic brain injury may result in bruising, tissue lacerations, bleeding, and other physical damage to the brain. These injuries may lead to long-term complications or death. In some embodiments, the patient is administered the composition within 1-5 hours or 1-2 hours of brain injury to reduce long-term tissue damage from TBI. In some embodiments, the patient is administered the composition within about 12 hours or about 24 hours of brain injury. In some embodiments, the patient may receive at least four doses of the stable DG emulsion, or at least eight doses of the stable DG emulsion. In some embodiments, after the initial administration, the patient is administered the composition at least four times or at least ten times at a frequency ranging from about once every four hours to once per week to aid in recovery.

[0062] In yet another embodiment, the patient suffers from post-traumatic stress disorder (PTSD). PTSD is a serious condition that a person develops after experiencing or facing a traumatic or frightening event in which serious physical harm has occurred or there is a threat of harm. PTSD is the persistent result of a traumatic or frightening experience that causes intense fear, helplessness, or terror, such as sexual or physical assault, the unexpected death of a loved one, accident, war, or natural disaster. In various embodiments, the compositions described herein provide therapeutic value for PTSD. In some embodiments, the patient is administered the composition at least once a week during the period that promotes recovery.

[0063] The present invention provides uses for protecting other organs or tissues, including spinal cord injury (SCI). In such embodiments, the patient may be administered the composition within about 24 hours of injury, or within about 15 hours of injury, or within about 12 hours of injury, or within about 6 hours of injury, or within about 2 hours of injury, or within about 1 hour of injury. In some embodiments, the patient is administered the composition at least once per day or at least once per week after the initial administration. In some embodiments, the patient may receive at least four doses of the stable DG emulsion, or at least eight doses of the stable DG emulsion. In some embodiments, after the initial administration, the patient is administered the composition at a frequency ranging from once every four hours to once per week (e.g., for at least four weeks) to aid in recovery.

[0064] Furthermore, in some embodiments, the patient is a recipient of an organ transplant, such as a liver, kidney, heart, or lung. In some embodiments, the patient is administered the composition during the perioperative period (e.g., within about 24 hours before and / or within about 24 hours after transplant surgery). In some embodiments, the patient receives at least four doses of the stable DG emulsion, or may receive at least eight doses of the stable DG emulsion. In some embodiments, after the initial dose, the patient is administered the composition at a frequency ranging from once every four hours to once per week to aid in recovery.

[0065] In some embodiments, the patient has acute organ failure, such as acute kidney failure, liver failure, or heart failure. In some embodiments, the patient is administered the composition 1 to 10 times or 1 to 4 times at a frequency ranging from about once every 4 hours to once per week to reduce organ damage and / or organ function decline.

[0066] In some embodiments, the patient suffers from neurodegenerative diseases such as ALS, multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.For example, the patient is administered the composition at least once a week to slow down disease progression, and / or (for example, in the case of MS) ​​during disease relapse, the composition is administered to reduce the severity and duration of relapse and / or slow down disease progression.

[0067] In these and other embodiments, patients in need of neuroprotection may also, or in some embodiments, receive oral supplementation of n-3 fatty acids, which may optionally be in the form of DG or n-3 TG. Oral supplementation may be administered at least once a day and up to three times a day. Oral supplementation may be provided for a period of one week or more or one month or more as needed to aid recovery from acute events, or may be administered indefinitely to aid recovery and prevent recurrence or relapse of the condition. In some embodiments, oral supplementation is provided by n-3 DG oil, which may be administered in the form of capsules. In some embodiments, oral supplementation is provided by diet, for example, by providing n-3 DG oil as a food ingredient. In some embodiments, oral supplementation is provided by n-3 DG emulsion.

[0068] Patients who need neuroprotection may further receive one or more neuroprotective agents, for example, as a combination therapy.Examples of neuroprotective agents include glutamate antagonists.Examples of neuroprotective agents include 17β-estradiol, ginsenosides, progesterone, simvastatin, and memantine.These therapeutic agents, together with n-3 DG emulsion therapy and / or n-3 DG oral supplementation as described herein, provide synergistic protection from brain injury.

[0069] As used herein, the term "about" means ±10% of the associated numerical value.

[0070] Other aspects and embodiments of the present invention will be apparent from the examples described below.

[0071] Example Omega-3 (n-3) fatty acids (FAs) are candidates for rapid neuroprotection after stroke. Multiple biological mechanisms may be affected by n-3 FAs, including: (i) reduced mitochondrial reactive oxygen species (ROS) production; (ii) Mitochondrial Ca 2+ uptake and preservation of homeostasis; (iii) modulation of receptor-mediated signaling and inhibition of apoptotic pathways; (iv) an increase in potent n-3 FA-derived resolvins and protectins; and (v) Reduced inflammatory response. Acting individually or cooperatively, these mechanisms may contribute to n-3 FA neuroprotection in ischemic injury, reducing cell death while promoting repair processes.

[0072] However, to be effective in neuroprotection, adequate levels of n-3 FAs must be rapidly delivered to cells at risk of cell death or injury and therefore must be delivered in a manner that effectively crosses the blood-brain barrier (BBB). Nanoparticle uptake through the BBB can occur through two major endocytic mechanisms: clathrin-mediated endocytosis and caveolin-mediated endocytosis. Emulsion nanoparticles with diameters of 200 nm or less should more efficiently cross the BBB via these endocytic processes. Furthermore, increasing the level of n-3 FAs in emulsions with small particle sizes (e.g., in conjunction with PC emulsions) may further facilitate the direct delivery of n-3 FAs to the brain and other tissues.

[0073] The present disclosure provides shelf-stable compositions and methods for the rapid delivery of n-3 fatty acids for the treatment of ischemic stroke, traumatic brain injury, and other acute organ injuries as described in detail elsewhere herein. Specifically, the following experiments provide compositions and methods for achieving stable omega-3 diglyceride (DG) oil-in-water emulsions for their use as rapid therapeutics to treat and / or prevent organ injury.

[0074] Preparation and characterization of n-3 diglyceride emulsions A DG emulsion formulation was developed to prepare stable emulsions with small particle sizes and increased n-3 FA payloads. As detailed in Table 1, stable DG emulsions were prepared by mixing solubilized egg yolk phosphatidylcholine (PC) with DG oils. DG oils contain at least 90% FA as DHA and / or EPA. Oils with different n-3 FA compositions, i.e., pure DG-DHA, pure DG-EPA, or a mixture of DHA and EPA, were prepared. DG emulsions containing n-6 AA were also prepared. Each oil was analyzed by thin-layer chromatography (TLC) to determine the purity and integrity of the samples. TG emulsions were also prepared using the same process with emulsions containing soybean oil or fish oil triglycerides containing only DHA (Tri-DHA) as their fatty acid.

[0075] [Table 1]

[0076] The emulsion preparation protocol involves mixing HO (containing 0.25 mM EDTA) and glycerin at 60°C. Then, PC (Lipoid E80) and sodium oleate are mixed with moderate stirring for 2 minutes using a very gentle vortex. DG oil is added to the aqueous phase at 60°C. This pre-emulsion is then mixed with a homogenizer at 60°C for 3 minutes. The final step involves processing the pre-emulsion in a high-shear liquid processor (Microfluidizer, Model LV1) at 60°C and 965 bar pressure (equivalent to 14,000 psi) for 3-5 times. Following this method, we obtained a maximum volume of 8 ml per treatment. For larger volumes, higher pressures may be used. The emulsion appeared as a white / milky liquid. This emulsion was stored under argon at 4°C for up to 14 months after preparation. Using PC emulsifier, this process was successfully utilized to prepare stable 10% emulsions (10 g DG / 100 ml) and stable 20% and 24% emulsions (20 g DG / 100 ml and 24 g DG / 100 ml). As described in detail below, stable emulsions with small particle sizes were also obtained using 0.6–0.8 wt% PC emulsifier and 10% and 20% DG oil. Emulsions with DG oil levels higher than 20% in particular have the potential to significantly improve n-3 FA payload.

[0077] Particle size and polydispersity index (PDI) were assessed by dynamic laser scattering (DLS). Data were analyzed for composition, mean particle distribution, and uniformity. A representative DG emulsion is shown in Figure 1 (left), where n-3 DG has a particle size substantially less than 200 nm (approximately 110 nm), while the TG emulsion is larger, approximately 240 nm in this example. We also analyzed the zeta potential; it showed a strong repulsion force for the DG emulsion (which was substantially higher than that of TG), resulting in a more stable system (Figure 1, right). The DG oil used contained approximately equal amounts of 1,2 and 1,3 DG (based on fatty acid position), as shown by TLC (Figure 3).

[0078] After 14 months the same samples were analyzed and the PDI and mean size values ​​remained unchanged, indicating a high degree of stability of the DG emulsion over this period.

[0079] We hypothesized that DG contributes to its own emulsification and that, due to its higher hydrophilicity, DG will penetrate biological membranes more than TG. To test this hypothesis, we compared the effects of fatty acids delivered by either DG or TG as carriers in a model of biological membrane interaction. We evaluated the solubility of DG and TG in a phosphatidylcholine (PC) membrane system and used NMR analysis to investigate how DG or TG can incorporate into lipid mixtures and change the properties of PC membranes. We found that omega-3 DG incorporated into the model membranes to a much greater extent than omega-3 TG, which likely contributes to its multiple and superior biological effects compared to TG. Importantly, these results suggest that n-3 DG, significantly different from TG, partially promotes its own emulsification with PC.

[0080] Based on these results, we next decided to reduce the amount of PL emulsifier from 1.2% (as in our initial preparation) to 0.8–0.6% by weight of emulsifier (i.e., by weight of the composition before adding DG oil). This was to demonstrate whether DG was stable at low amounts of PL. Visual inspection revealed that the DG emulsions showed no oil droplets on the surface, whereas the TG emulsions did. These findings are consistent with the hypothesis that DG itself acts as an emulsifier, and therefore stable emulsions containing a high percentage of DG oil appear to be appropriate. We suggest that DG emulsions of at least 25 wt% are appropriate. Furthermore, the same small particle size and PDI of 0.220 or less are observed when the PL is reduced. Importantly, reducing the amount of PL in the emulsion allows for an increased n-3 FA payload per dose, which would result in significant improvements in neuroprotection.

[0081] To establish that rapid hydrolysis facilitates the clearance of DG emulsions, in vitro lipolysis studies were performed. FA release was assessed by lipoprotein lipase (LpL)-mediated hydrolysis of n-3 DG versus n-3 TG. The activity of purified LpL was 300–400 U / mg protein. LpL was diluted 1:20 in 0.9% NaCl (pH 8.6) immediately prior to incubation with the emulsions. Experiments were performed with increasing amounts of LpL (0–20 μL of the 1:20 dilution) over a fixed time period (30 min). We observed that n-3 DG emulsions (both 10% and 20% emulsions) had more efficient hydrolysis compared to n-3 TG (see representative experiments, Figure 2A,B). These results emphasize that the DG structure facilitates the conversion of emulsions into remnant-like particles in vivo, contributing to faster release and uptake of n-3 FAs. A higher percentage of n-3 FAs in the emulsions can deliver higher levels of these chemicals to cells and tissues (compare FFAs released in Figure 2A with Figure 2B).

[0082] Cells internalize significant amounts of n-3 TG via an absorptive endocytosis pathway that does not involve canonical cellular receptors [31, 35, 36]; on the other hand, the n-6 TG uptake mechanism involves both apoE-dependent and LDL receptor (LDLr)-dependent pathways [31, 37-39]. Differences in TG composition, particle size, and hydrophilicity may, in part, explain the different uptake processes [34, 39, 40]. The overall ability of DG particles to cross the blood-brain barrier (BBB) ​​may depend on their physical and chemical properties and specific transporters. This disclosure predicts that the increased chaotic dynamics at the PL surface of DG emulsions and their small particle size may, in part, facilitate more rapid and greater in vitro uptake of n-3 FAs via a "non-canonical" pathway.

[0083] Animal models of neuroprotection Initial exploratory data showed that patients treated with n-3 DG emulsion (containing over 90% total FA as EPA and DHA, approximately 10 wt% DG oil) We demonstrated that neonatal mice treated with n-3 DG emulsion showed a significant reduction in cerebral infarct volume 24 hours after ischemic injury, and that n-3 DG emulsion was significantly more effective than n-3 TG emulsion (Figures 4A and 4B). n-6 DG treatment did not exert neuroprotective effects after ischemic injury. Next, we investigated whether n-3 DG emulsion protects the brain from ischemia in an adult mouse model of stroke (C57Bl / 6 strain, 10-14 weeks old) using 60-minute transient right middle cerebral artery occlusion (MCAo). Adult mice treated with n-3 DG emulsion (approximately 10 wt% DG oil) immediately after MCAo and during the early reperfusion period had significantly smaller infarcts than control mice (Figure 5A and B). Mice treated with a rapid bolus injection of DG emulsion showed no adverse effects and did not report "signaling shock" due to excessive activation of DG downstream pathways.

[0084] In mice, n-3 TG emulsion has been reported to have a therapeutic time window of 2 hours after stroke.

[23] However, we now find that in an adult rat model of stroke (transient right middle cerebral artery occlusion (MCAo)), intravenous injection of TG emulsion (10 wt% TG oil) up to 6 hours after ischemia significantly reduced infarct volume, suggesting a longer time window for these chemicals in larger mammalian species. (Figure 6(A, B))

[0085] Taken together, our exploratory data highlight the potential of n-3 FA delivered in DG emulsions to be potent neuroprotective agents, providing increased efficacy over n-3 TG in our rodent stroke model and offering a long therapeutic window following acute injury.

[0086] We also investigated the neuroprotective effects provided by n-3 DG emulsions (DG-DHA, DG-EPA, and DG-EPA+DHA) compared with n-6 DG (AA). The emulsions contained approximately 10 wt% DG oils and fats, with over 90% of the fatty acids being n-3 FAs. The data show that neonatal mice treated with DG emulsions formulated with individual fatty acids (DHA or EPA) or DG-DHA+EPA showed a significant reduction in cerebral infarct volume when administered immediately after ischemic injury. In stark contrast, n-6 DG (AA) treatment did not exert a neuroprotective effect after ischemic injury (Figure 7). The infarct volume % was further reduced with the DHA+EPA DG emulsion compared with DG emulsions containing DHA alone and DG emulsions containing EPA alone. This improvement, demonstrated for DG emulsions containing both DHA and EPA (>90% fatty acids, 10 wt% DG oil), and with small particle size and highly negative zeta potential as shown herein, may provide substantial therapeutic advantages over even our earlier DG preparations. Increasing the levels of DG oils in these emulsions will likely provide further therapeutic improvements by increasing the amount of n-3 FAs rapidly delivered.

[0087] Consideration Stroke is a leading cause of long-term disability and the fifth leading cause of death in the United States. Currently, t-PA is the only drug approved by the FDA for the treatment of acute ischemic stroke, but its use is limited to a narrow time window of 3 to 4.5 hours [2-4]. The studies presented herein suggest that n-3 FAs act as bioactive unsaturated lipids with pleiotropic effects and exhibit neuroprotective properties in animal models of stroke. N-3 FAs administered rapidly as TG emulsions can provide neuroprotection after ischemic brain injury. N-3 TG emulsions administered immediately after ischemic injury can result in long-term neurological and tissue morphological recovery in the brain. However, a much more stable neuroprotective effect is achieved when n-3 FAs are delivered as n-3 DGs and immediately infused as DG lipid emulsions after ischemic brain injury. By optimizing the composition of these DG emulsions and their physical properties as described herein, n-3 DG emulsions have the potential to provide a highly effective and room-temperature-stable therapeutic agent for acute organ injury, including but not limited to stroke.

[0088] Adequate levels of n-3 FAs make neural membranes more fluid and promote the active interaction of receptors, ion channels, and protein complexes [10, 11]. This study evaluates the enhanced neuroprotection of n-3 DG containing both EPA and DHA administered as an intravenous lipid emulsion with a small particle size and high negative zeta potential to further enhance the brain delivery of n-3 FAs and their efficacy in ischemic stroke. DG, composed of a glycerol backbone and two fatty acyl groups, possesses a small, electrically neutral polar head group, which confers a pronounced cone-shaped morphology and rapid transbilayer movement. The biophysical properties and physiological effects of DG are regulated by the composition of their fatty acyl groups. As an integral component of cell membranes and lipid droplets, DG may play an important role as a second messenger in cell signaling [12-16]. Phospholipids are known to incorporate a small amount of long-chain fatty acid triglyceride (LCT) emulsions (2.6 mol%) with a favorable orientation of the carbonyl groups located at the water / phospholipid interface [17, 18]. Few studies have highlighted the properties of DG in bilayer organization and mobility [19-22]. The experiments described herein demonstrate that n-3 DG emulsions have more efficient hydrolysis compared to n-3 TG (Figure 2A, B). These results emphasize that the DG structure promotes the conversion of emulsions into remnant-like particles and free fatty acids in vivo, contributing to faster uptake of n-3 FAs and facilitating the onset of cytoprotective effects.

[0089] Rapid treatment with DHA administered as a TG emulsion immediately after ischemic injury significantly reduces brain damage [5, 23]. Data indicate that n-3 DG emulsion exhibits more stable neuroprotective effects than n-3 TG emulsion (suggesting a different metabolism of DG versus TG particles), and that n-3 DG has distinct biological properties and specifically triggers and accelerates important neuroprotective pathways in the early phase of stroke. This should also contribute to the extended therapeutic window of n-3 DG emulsion. Due to the potential of DG to affect the structural and mobility dynamics in phospholipid bilayers, DG emulsion is potentially an "improved" carrier of n-3 FAs to enhance their bioavailability to the brain and accelerate their molecular action in modulating neuroprotective pathways.

[0090] Despite the considerable potential pathways by which n-3 FAs may reduce morbidity and mortality from cardiovascular disease, the results of clinical trials using long-term n-3 FA supplementation have been conflicting and controversial [24-27]. This disclosure challenges existing clinical paradigms by rapidly providing n-3 FAs after injury (e.g., ischemic brain and cardiac injury). The rate of tissue enrichment of n-3 FAs (e.g., EPA and DHA) after oral supplementation is slow, particularly in the brain. Furthermore, free FAs should not be administered directly via parenteral routes because they act as surfactants and have toxic side effects, including encephalopathy

[29] . According to this disclosure, n-3 FAs (ideally including DHA and EPA) are incorporated into n-3 FA DGs as stable intravenous lipid emulsions with small particle size and high negative zeta potential.

[0091] The average particle size influences the stability and in vivo fate of emulsions. Zeta potential, the potential charge difference between mobile particles and the surrounding dispersant layer, is used as an indicator of emulsion stability. According to the present disclosure, reduced average particle size and zeta potential are believed to enhance the stability of DG versus TG emulsions by reducing separation and aggregation phenomena. n-3 DG oil (DHA / EPA, approximately 1.3 / 1, w / w) was incorporated into the emulsion. As shown in Figure 1, a smaller average particle size (approximately 110 nm) of n-3 DG was observed compared to the TG emulsion (approximately 240 nm), which may provide a larger surface-to-core ratio of DG, enabling faster and more extensive uptake and delivery of these n-3 DG emulsions to the brain via endocytosis, as well as more rapid hydrolysis. See Figure 2 (A, B). The zeta potential of TG was -35 mV, while the zeta potential of DG was -51 mV (Figure 1). The net negative charge at the interface in both emulsions is sufficient to prevent flocculation and aggregation through strong electrostatic repulsion, but the more electronegative nature of DG leads to a higher stability of the DG emulsion.

[0092] The optimization of DG emulsion formulation was screened by average particle size, PDI as a homogeneity index, zeta potential, and electron microscopy. The advantages of the oil / water interface may favorably affect the interaction of n-3 DG emulsion demonstrated herein with cellular endocytosis and catabolic pathways. In fact, our study shows that DG has a significantly faster lipolysis rate than TG. See Figure 2 (A, B).

[0093] Following injection, n-3 FAs are more efficiently taken up into the brain than shorter-chain FAs

[10] . However, there is no information regarding the brain delivery of DHA or EPA injected as n-3 DG emulsions. Previous data have shown a significant increase in plasma TG levels within 30 minutes after injection of radiolabeled n-3 TG emulsions

[23] , with less than 0.5% of particles entering the brain. A significant increase in mitochondrial n-3 FA levels was observed, but not in the whole brain [5]. The liver was the organ with the highest n-3 TG particle uptake (>50%)

[31] . This suggests that the neuroprotection observed with n-3 TG emulsions may depend on their delivery, "repackaging," and metabolism in other organs prior to their direct effect in the brain. The DG emulsions disclosed herein, which contain more than 90% FAs as DHA and EPA and have particle sizes less than 200 nm, are likely to allow for more rapid catabolic uptake and have additional uptake pathways, including direct uptake into the brain. After rapid administration, n-3 DG is expected to be partially taken up by the liver, then repackaged into either free FAs or liver-produced lipoproteins and transported directly to the brain. However, uptake of intact emulsion particles into the brain may also contribute to DG emulsion clearance in vivo. The DG emulsion described herein is expected to be a more efficient carrier for delivering n-3 FAs to the brain, providing an alternative and / or additional therapeutic approach for stroke.

[0094] According to the present disclosure, the DG emulsions described herein are predicted to increase n-3 FA clearance and brain uptake compared to TG emulsions or DG emulsions with larger particle sizes (e.g., greater than 200 nm) or containing only DHA or EPA. The inventors predict that after ischemia, whole, intact DG particles will also cross the BBB and promote n-3 FA uptake into the brain. The inventors predict stronger neuroprotective potential from the n-3 DG emulsions described herein compared to n-3 TG emulsions and compared to DG emulsions with larger particle sizes or containing only DHA or EPA. The inventors therefore predict that the n-3 DG emulsions described herein will exhibit an extended neuroprotective time window (e.g., greater than 6 hours in rats) after ischemic injury, demonstrating the superiority of these n-3 DG emulsions. References 1. Benjamin EJ, et al. Heart Disease and Stroke Statistics-2018 Update: A Report from the American Heart Association. 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Claims

1. 1. A composition comprising a stable diglyceride (DG) oil-in-water emulsion, the emulsion having an average particle size of 110-180 nm and a zeta potential (ZP) of about -30 mV or more negative than -40 mV; the emulsion comprises about 10% by weight of DG oil, the esterified fatty acids of which are at least 90% n-3 fatty acids and contain docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); A composition wherein the emulsion further comprises a phosphatidylcholine emulsifier in the range of about 0.5 to about 1.0 wt % and a medium or long chain fatty acid co-emulsifier.

2. 2. The composition of claim 1, wherein the n-3 FA comprises docosahexaenoic acid (DHA) and / or eicosapentaenoic acid (EPA) and / or docosapentaenoic acid (DPA).

3. 3. The composition of claim 2, wherein the n-3 FA comprises DHA and EPA.

4. 4. The composition of claim 3, wherein the n-3 FA is at least about 50% DHA, or at least about 60% DHA, or at least about 75% DHA.

5. The composition of any one of claims 1 to 4, wherein the average particle size of the emulsion is about 180 nm or less, or about 150 nm or less, or about 120 nm or less.

6. The composition according to any one of claims 1 to 5, wherein the zeta potential of the emulsion is at least about -45 mV negative, or at least about -50 mV negative, or at least about -55 mV negative.

7. A composition according to any one of claims 1 to 6, wherein the co-emulsifier is oleic acid, which is added as sodium oleate.

8. The composition of any one of claims 1 to 7, wherein the emulsion is stable for at least about 6 months, or at least about 1 year, or at least about 18 months, or at least about 2 years.

9. The composition of any one of claims 1 to 8, which is suitable for intravenous or intra-arterial delivery.

10. 10. The composition of any one of claims 1 to 9 for use in treating a patient in need of protection from cell death, said use comprising administering to said patient in need an effective amount of said composition.

11. The composition of claim 10, wherein the patient is in need of neuroprotection.

12. 12. The composition of claim 10 or 11, wherein the patient is at risk for ischemia-reperfusion injury.

13. The composition of claim 12, wherein the patient has experienced a stroke.

14. 14. The composition of claim 13, wherein the patient has experienced a pediatric stroke.

15. 15. The composition of claim 14, wherein the subject is suffering from or at risk for hypoxic-ischemic encephalopathy (HIE).

16. 11. The composition of claim 10, wherein the patient has suffered from or is at risk for traumatic brain injury.

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