Methods for treating hepatic steatosis with a bioavailable formulation of oleoylethanolamide - Patent Application 20070233334

Abstract

Compositions and methods for delivering oleoylethanolamide (OEA) in a bioavailable formulation that reduces lipid accumulation in the liver of a subject. Described herein is a novel OEA formulation that allows the transport of the compound to the liver parenchyma, where this natural lipid amide exerts an anti-steatotic effect. This formulation has significantly higher oral bioavailability than either unformulated OEA or a different formulation of OEA (Levagen®-OEA). Furthermore, oral administration of the novel OEA formulation attenuates hepatic steatosis occurring in a mouse model of human metabolic syndrome. The composition is useful for treating human non-alcoholic hepatic steatosis.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 263,314, filed December 13, 2021, the entire contents of which are incorporated by reference into this application. [Background technology]

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in Western countries. It is predicted to replace hepatitis C as the leading indication for liver transplantation. NAFLD is associated with obesity and may progress to nonalcoholic steatohepatitis (NASH) and end-stage liver disease. There are no approved pharmacological treatments for NAFLD or NASH. Animal and human studies suggest that oleoylethanolamide (OEA) may find application in the treatment of NAFLD, but the clinical use of this naturally occurring molecule is largely limited by poor bioavailability due to unfavorable physicochemical properties combined with rapid enzyme-mediated degradation.

[0003] After a meal, the absorptive epithelium of the upper small intestine diverts a portion of the oleic acid obtained from hydrolysis of dietary lipids to generate OEA (Schwartz et al., 2008, DiPatrizio and Piomelli, 2015). OEA acts as a local messenger in the gut to reduce food intake through mechanisms involving the recruitment of peripheral sensory afferents (Rodriguez de Fonseca et al., 2001) and the activation of central pathways that utilize oxytocin and histamine as neurotransmitters (Gaetani et al., 2010, Provensi et al., 2014). Several lines of evidence suggest that OEA initiates this response by associating with the ligand-gated transcription factor peroxisome proliferator-activated receptor-α (PPAR-α). First, OEA is one of the most potent naturally occurring PPAR-α agonists identified to date (affinity constant, KD, ≈40 nM; median effective concentration, EC50, ≈120 nM) (Fu et al., 2003, Astarita et al., 2006a). Second, the satiety-inducing effect of OEA is abolished by PPAR-α deletion and mimicked by synthetic PPAR-α agonists and is associated with increased PPAR-α-regulated transcription in the intestinal mucosa (Fu et al., 2003). Third, virus-mediated enhancement of OEA production in the rat small intestine reduces food intake and concomitantly increases the local expression of PPAR-α target genes (Fu et al., 2008). Finally, OEA levels in the small intestine of various vertebrate species - including fish, snakes, and rodents - increase from ≦50 nM in the fasted state to ≧250 nM after feeding (Astarita et al., 2006b, Fu et al., 2007, Tinoco et al., 2014, Igarashi et al., 2017), a concentration range compatible with the activation of PPAR-α (Fu et al., 2003, Astarita et al., 2006a). In summary, available data indicate that OEA is a physiologically relevant endogenous agonist of intestinal PPAR-α involved in the control of satiety (DiPatrizio and Piomelli, 2015).

[0004] Feeding also regulates OEA production in the liver, but in the opposite direction to that seen in the intestine: hepatic OEA levels increase in the fasting state and decrease after feeding (Fu et al., 2007, Izzo et al., 2010). The molecular basis and physiological significance of these changes have been investigated in detail. Studies have shown that fasting stimulates extrahepatic mast cells to secrete histamine, which enters the liver via the portal circulation and activates G protein-coupled H1 receptors to induce local OEA biosynthesis (Misto et al., 2018). Genetic or pharmacological manipulations that disrupt this process do not affect lipolysis but significantly attenuate fasting-induced ketogenesis, thereby revealing a previously unsuspected regulatory role of mast cell-derived histamine and hepatic OEA production in systemic lipid homeostasis (Misto et al., 2018). Consistent with such a role, subchronic intraperitoneal administration of OEA attenuates hepatic steatosis in obese Zucker rats (Fu et al., 2005) and in a rat model of nonalcoholic fatty liver disease (Li et al., 2015). Furthermore, intraperitoneal OEA administration stimulates fatty acid oxidation in isolated rat hepatocytes and promotes ketogenesis in live rats (Guzman et al., 2004). Finally, subchronic intraperitoneal OEA administration reduces lipid accumulation, inflammatory responses, and fibrosis in the liver of diet-induced obese mice (Lin et al., 2022; PMID: 35691287).

[0005] Although intraperitoneal OEA administration is clearly beneficial in rodent models of hepatic steatosis, the clinical application of OEA as an oral therapeutic for NAFLD is hindered by two important factors: (1) after oral administration, OEA is rapidly degraded in vivo, catalyzed by fatty acid amide hydrolase and other lipid amidases (Piomelli, 2013; PMID: 23567058); and (2) OEA is a lipophilic molecule with limited drug-like properties.

[0006] There remains a need for means to deliver OEA in a manner that makes it bioavailable, and in particular, in a manner that makes OEA effective and available to the liver. Summary of the Invention

[0007] The compositions and methods described herein provide a means to deliver OEA in a bioavailable formulation that reduces lipid accumulation in the liver of a subject. Described herein is a novel OEA formulation that allows the transport of the compound to the liver parenchyma, where this natural lipid amide exerts an anti-steatotic effect. This formulation has significantly higher oral bioavailability than either unformulated ("neat") OEA or a different formulation of OEA (Levagen®-OEA). Furthermore, oral administration of the novel OEA formulation attenuates hepatic steatosis occurring in a mouse model of human metabolic syndrome. The data described herein support the use of the composition in the treatment of human NAFLD, including NASH.

[0008] Described herein are compositions comprising 5-15% by weight OEA and 85-95% self-emulsifying drug delivery system (SEDDS). In some embodiments, the SEDDS comprises a carrier oil comprising a medium chain triglyceride, a citrus oil, and lecithin. In some embodiments, the composition promotes at least a two-fold increase in bioavailability of OEA. In some embodiments, the composition promotes at least a three-fold increase in bioavailability of OEA. In some embodiments, the SEDDS comprises AquaCelle®.

[0009] SEDDS, commercially available as AquaCelle®, is described in U.S. Patent Publication No. 20190374494, published December 12, 2019. See also Bremmell, KE;et al, A self-emulsifying Omega-3 ethyl ester formulation (AquaCelle) significantly improves eicosapentaenoic and docosahexaenoic acid bioavailability in healthy adults. Eur. J. Nutr.2019,59, 2729-2737. Further information regarding delivery methods for dietary supplements can be found in Subramanian, P. Lipid-Based Nanocarrier System for the Effective Delivery of Nutraceuticals. Molecules 2021, 26, 5510 https: / / doi.org / 10.3390 / molecules26185510.

[0010] In some embodiments, the composition comprises 10-12% OEA and 88-90% AquaCelle®. In some embodiments, the composition comprises 11% OEA and 89% AquaCelle®. In some embodiments, the carrier oil comprises up to about 11% by weight, the citrus oil comprises up to about 10% by weight, and the lecithin comprises up to about 11% by weight.

[0011] In some embodiments, the composition percentage of SEDDS is such that when the composition is dispersed in an aqueous environment, it forms a population of micelles having an average diameter of about 1-20 micrometers. In some embodiments, the composition forms a population of micelles having an average diameter of about 5-15 micrometers. In some embodiments, the composition forms a population of micelles having an average diameter of about 12-13 micrometers.

[0012] In some embodiments, the composition further comprises an antioxidant. Exemplary antioxidants include, but are not limited to, lecithin, ascorbyl palmitate, d-alpha-tocopherol, dl-alpha-tocopherol, d-alpha-tocopheryl acetate, dl-alpha-tocopheryl acetate, d-alpha-tocopheryl succinate, dl-alpha-tocopheryl succinate, vitamin E and its derivatives, olive polyphenols, algal polyphenols, and mixtures thereof. In some embodiments, the antioxidant is present at a concentration of 0-0.5% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.01% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.02% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.1% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.5% by weight.

[0013] In some embodiments, the composition further comprises an excipient. Representative excipients include, but are not limited to, colloidal silica, corn starch, hydroxypropyl methylcellulose (HPMC), maltodextrin, magnesium stearate, magnesium hydroxide, microcrystalline cellulose, dextrin, sorbitol, mannitol, and trehalose. In some embodiments, the excipient is present at a concentration of about 30% to about 90% by weight. In some embodiments, the excipient is present at a concentration of about 30% to about 90% by weight.

[0014] Also described herein is a method for delivering oleoylethanolamide (OEA) to the liver of a subject in need thereof.In some embodiments, the method comprises oral administration of the composition described herein.Also described is a method for reducing liver steatosis in a subject, as well as a method for treating non-alcoholic fatty liver disease (NAFLD) in a subject, and a method for treating non-alcoholic steatohepatitis (NASH) in a subject.In some embodiments, each of these methods comprises oral administration of the composition described herein. [Brief description of the drawings]

[0015] [Figure 1] Effect of HFD exposure on fasting-induced histamine signaling in the portal circulation and liver. (A,B) Histamine identification in portal plasma. (A) Representative LC / MS-MS trace showing elution of authentic [2H4]-histamine and native histamine in portal plasma. (B) Mass spectra of native and [2H4]-histamine showing the presence of diagnostic ions [M-NH3]+ (m / z=95.1 and m / z=99.1) in portal plasma. (C-E) Effects of ad libitum feeding (circles), food deprivation (squares), and food deprivation / refeeding (triangles) in chow-fed (open symbols) and HFD-fed (filled symbols) mice. (C) Histamine concentration in portal plasma. (D) Transcription of histamine H1 receptor (H1r) in liver. (E) Transcription of histamine H2 receptor (H2r) in liver. (F) Transcription of histamine H4 receptor (H4r) in liver. Data are presented as mean ± SEM (n = 4–6) and analyzed using two-way ANOVA followed by Sidak's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Diagram 2] Figure 1 shows the effect of HFD exposure on fasting-induced OEA signaling in liver tissue. (A) Representative LC / MS-MS traces showing the presence of OEA and its local isomer VEA in liver. (B-F) Effects of ad libitum feeding (circles), food deprivation (squares), and food deprivation / refeeding (triangles) in chow-fed (open symbols) and HFD-fed (filled symbols) mice. (B) OEA content in liver. (C) AEA content in liver. (D) PEA content in liver. (E) NAPE-PLD (Napepld) transcription in liver. (F) FAAH (Faah) transcription in liver. Data are presented as mean ± SEM (n = 4-9) and analyzed using two-way ANOVA followed by Sidak's multiple comparison test. * or #p<0.05, ** or ##p<0.01, *** or ###p<0.001. VEA, vaccenoylethanolamide. [Figure 3A]Effects of VEH or OEA administration on the liver of HFD-fed mice. Representative images of in situ liver (left) and liver sections stained with Oil Red O (second from left), BODIPY (third from left), and PSR (right). [Figure 3B] Figure 1 shows the effect of VEH or OEA administration on the liver of HFD-fed mice. Quantification of BODIPY fluorescence in the liver of VEH-treated (open symbols) or OEA-treated (filled symbols) mice exposed to HFD. Data are presented as mean ± SEM (n = 4–6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3C] Figure 1 shows the effect of VEH or OEA administration on the liver of HFD-fed mice. Quantification of PSR fluorescence in the liver of VEH-treated (open symbols) or OEA-treated (filled symbols) mice exposed to HFD. Data are presented as mean ± SEM (n = 4–6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3D] Effect of VEH or OEA administration on liver of HFD-fed mice. Total triglyceride content in liver of VEH-treated (open symbols) or OEA-treated (filled symbols) mice exposed to HFD. Data are presented as mean ± SEM (n = 4–6) and analyzed using unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3E] Effect of VEH or OEA administration on the liver of HFD-fed mice. Interleukin IL6 protein is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3F]Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Il1b is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3G] Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Ccl2 is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3H] Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Col1a1 is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3I] Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Hmox1 is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3J] Figure 1 shows the effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Nrf1 is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3K] Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Tnfa is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3L]Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Tgfb1 is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 3M] Effect of VEH or OEA administration on the liver of HFD-fed mice. Hepatic transcription of Nqo1 is shown. Data are presented as mean ± SEM (n = 4-6) and analyzed using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. VEH, vehicle. [Figure 4] Male C57Bl / 6J mice exposed to HFD develop obesity, hyperglycemia, and hepatic steatosis. (A) Time course of weight gain in mice exposed to a typical chow (open symbols) or HFD (filled symbols). (B) Fasting glucose concentration in cardiac plasma of chow-fed or HFD-fed mice. (C) Total triglyceride content in liver of chow-fed or HFD-fed mice. (D) Representative images of liver tissue sections from chow-fed (left) or HFD-fed (right) mice stained with Oil Red O. Data are presented as mean ± SEM (A, n = 20–26; B–C, n = 4–7) and analyzed using two-way ANOVA followed by Sidak's multiple comparison test (A) or unpaired two-tailed Student's t test (B, C). *P<0.05, **P<0.01. ***P<0.001. [Diagram 5] Effect of OEA administration on body weight gain in HFD-fed mice. (A) Body weight trajectory before the start of OEA treatment is shown. (B) Body weight trajectory after the start of OEA treatment is shown. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Sidak's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Figure 6] H&E staining of liver sections showing the effect of vehicle (VEH) or OEA on liver histology in diet-induced obese mice. [Figure 7A]Figure 1 shows the pharmacokinetic (PK) profiles of unformulated OEA (neat-OEA), Levagen®-OEA, and Aquacelle®-OEA in mice. Dose was equivalent to 30 mg / kg free OEA. Inset shows area under the curve (AUC) values. Comparison of PK profiles of neat OEA vs. Levagen®-OEA in plasma. Data are presented as mean ± SEM (n=9-11) and analyzed using 2-way ANOVA followed by Sidak's multiple comparison test or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 7B] Figure 1 shows the pharmacokinetic (PK) profiles of unformulated OEA (neat-OEA), Levagen®-OEA, and Aquacelle®-OEA in mice. Dose was equivalent to 30 mg / kg free OEA. Inset shows area under the curve (AUC) values. Comparison of PK profiles of neat OEA vs. Levagen®-OEA in liver. Data are presented as mean ± SEM (n=9-11) and analyzed using 2-way ANOVA followed by Sidak's multiple comparison test or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 7C] Figure 1 shows the pharmacokinetic (PK) profiles of unformulated OEA (neat-OEA), Levagen®-OEA, and Aquacelle®-OEA in mice. Dose was equivalent to 30 mg / kg free OEA. Inset shows area under the curve (AUC) values. Comparative PK profiles of neat OEA vs. Aquacelle®-OEA in plasma. Data are presented as mean ± SEM (n=9-11) and analyzed using 2-way ANOVA followed by Sidak's multiple comparison test or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 7D]Figure 1 shows the pharmacokinetic (PK) profiles of unformulated OEA (neat-OEA), Levagen®-OEA, and Aquacelle®-OEA in mice. Dose was equivalent to 30 mg / kg free OEA. Inset shows area under the curve (AUC) values. Comparative PK profiles of neat OEA vs. Aquacelle®-OEA in liver. Data are presented as mean ± SEM (n=9-11) and analyzed using 2-way ANOVA followed by Sidak's multiple comparison test or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 8] (A) A scheme summarizing the study design is shown. (B) The weights of all animal subjects included in the study before randomization into control (AquaCelle® alone, circles) and OEA treatment groups (AquaCelle®-OEA, triangles) after 3 weeks of exposure to a high-fat diet (HFD). (C) Weight gain of mice during treatment with AquaCelle® alone and AquaCelle®-OEA is shown. Data are presented as mean ± SEM (n=9 / group) and analyzed using unpaired t-tests or 2-way ANOVA followed by Sidak's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Figure 9A] Figure 1 shows the effect of oral administration of AquaCelle® alone or AquaCelle®-OEA in diet-induced obese mice with hepatic steatosis. Representative images of excised livers (left), liver sections stained with BODIPY (middle), and liver sections stained with Oil Red O (right). Data are presented as mean ± SEM and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 9B]Figure 1 shows the effect of oral administration of AquaCelle® alone or AquaCelle®-OEA in diet-induced obese mice with hepatic steatosis. Mean liver weights (n=8 / 9) of mice treated with AquaCelle® alone (circles) or AquaCelle®-OEA (triangles). Data are presented as mean ± SEM and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 9C] Figure 1 shows the effect of oral administration of AquaCelle® alone or AquaCelle®-OEA in diet-induced obese mice with hepatic steatosis. Quantification of BODIPY fluorescence in the liver of mice treated with AquaCelle® alone or AquaCelle®-OEA (n=4 / group) is shown. Data are presented as mean ± SEM and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 9D] Figure 1 shows the effect of oral administration of AquaCelle® alone or AquaCelle®-OEA in diet-induced obese mice with hepatic steatosis. Figure 2 shows droplet size distribution in liver sections of mice treated with AquaCelle® alone or AquaCelle®-OEA. Data are presented as mean ± SEM and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10A] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. Triglycerides (TAG) are shown. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10B]Plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. AST, aspartate transaminase. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10C] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. ALT, alanine transaminase. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10D] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. Cholesterol is shown. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10E] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. Glucose is shown for vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10F]Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. Albumin is shown. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10G] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. Globulins are shown. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10H] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. Amylase is shown. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. [Figure 10I] Figure 1 shows plasma blood profiles of diet-induced obese mice with hepatic steatosis treated with AquaCelle® alone or AquaCelle®-OEA. CPK, creatine kinase. Vehicle, AquaCelle® alone, OEA, AquaCelle®-OEA. Data are expressed as mean ± SEM (n=6 / 7) and analyzed using unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The compositions and methods described herein provide a novel OEA formulation that allows the transport of compounds to the liver parenchyma, where the natural lipid amide exerts its anti-steatotic effect.The formulation has significantly higher oral bioavailability than either unformulated OEA or Levagen®-OEA.Furthermore, oral administration of the novel OEA formulation attenuates hepatic steatosis occurring in a mouse model of human metabolic syndrome.The data described herein support the use of the composition in the treatment of non-alcoholic hepatic steatosis in humans.

[0017] definition All scientific and technical terms used in this application have the meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified:

[0018] As used herein, "control" or "reference" sample refers to a sample that represents the normal measurement value of each marker, as obtained from a normal, healthy control subject, or a baseline amount of the marker that can be used for comparison. Typically, the baseline will be a measurement value obtained from the same subject or patient. The sample can be the actual sample used for testing, or a reference level or range based on the known normal measurement value of the corresponding marker.

[0019] As used herein, "significant difference" refers to a difference that can be detected by a method that is considered reliable by those skilled in the art, such as a statistically significant difference, or a difference of a magnitude that can be detected with a reasonable level of reliability under the circumstances.In one example, a 10% increase or decrease compared to a reference sample is a significant difference.In another example, a 20%, 30%, 40%, or 50% increase or decrease compared to a reference sample is considered a significant difference.In yet another example, a 2-fold increase compared to a reference sample is considered significant.

[0020] As used herein, "pharmaceutical acceptable carrier" or "excipient" includes any material that, when combined with an active ingredient, retains the biological activity of the ingredient and does not react with the subject's immune system. Compositions that include such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0021] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In an exemplary embodiment, the subject is a human.

[0022] As used herein, "a" or "an" means at least one, unless clearly indicated otherwise.

[0023] composition Described herein are compositions comprising 5-15% by weight OEA and 85-95% self-emulsifying drug delivery system (SEDDS). In some embodiments, the SEDDS comprises a carrier oil comprising a medium chain triglyceride, a citrus oil, and lecithin. In some embodiments, the composition, when administered to a subject, promotes at least a two-fold increase in bioavailability of OEA compared to administration of neat OEA. In some embodiments, the composition promotes at least a three-fold increase in bioavailability of OEA. In some embodiments, the SEDDS comprises AquaCelle®.

[0024] SEDDS, commercially available as AquaCelle®, is described in U.S. Patent Publication No. 20190374494, published December 12, 2019. See also Bremmell, KE;et al, A self-emulsifying Omega-3 ethyl ester formulation (AquaCelle®) significantly improves eicosapentaenoic and docosahexaenoic acid bioavailability in healthy adults. Eur. J. Nutr.2019,59, 2729-2737. Further information regarding delivery methods for dietary supplements can be found in Subramanian, P. Lipid-Based Nanocarrier System for the Effective Delivery of Nutraceuticals. Molecules 2021, 26, 5510 https: / / doi.org / 10.3390 / molecules26185510.

[0025] In some embodiments, the composition comprises 10-12% OEA and 88-90% AquaCelle®. In some embodiments, the composition comprises 11% OEA and 89% AquaCelle®. In some embodiments, the carrier oil comprises up to about 11% by weight, the citrus oil comprises up to about 10% by weight, and the lecithin comprises up to about 11% by weight.

[0026] In some embodiments, the composition percentage of SEDDS is such that when the composition is dispersed in an aqueous environment, it forms a population of micelles having an average diameter of about 1-20 micrometers. In some embodiments, the composition forms a population of micelles having an average diameter of about 5-15 micrometers. In some embodiments, the composition forms a population of micelles having an average diameter of about 12-13 micrometers.

[0027] In some embodiments, the composition is formulated for delivery with enteric coating, such as enteric coated capsule.Enteric coated capsule is designed to remain intact in stomach and release active substance in intestine.Enteric coating can be applied to solid dosage forms such as granules, pellets, capsules or tablets to prevent acid or gastric enzyme unstable drug from decomposing, thereby improving bioavailability.

[0028] In some embodiments, the composition further comprises an antioxidant. Exemplary antioxidants include, but are not limited to, lecithin, ascorbyl palmitate, d-alpha-tocopherol, dl-alpha-tocopherol, d-alpha-tocopheryl acetate, dl-alpha-tocopheryl acetate, d-alpha-tocopheryl succinate, dl-alpha-tocopheryl succinate, vitamin E and its derivatives, olive polyphenols, algal polyphenols, and mixtures thereof. In some embodiments, the antioxidant is present at a concentration of 0-0.5% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.01% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.02% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.1% by weight. In some embodiments, the antioxidant is present at a concentration of about 0.5% by weight.

[0029] In some embodiments, the composition further comprises an excipient. Representative excipients include, but are not limited to, colloidal silica, corn starch, hydroxypropyl methylcellulose (HPMC), maltodextrin, magnesium stearate, magnesium hydroxide, microcrystalline cellulose, dextrin, sorbitol, mannitol, and trehalose. In some embodiments, the excipient is present at a concentration of about 30% to about 90% by weight. In some embodiments, the excipient is present at a concentration of about 30% to about 90% by weight.

[0030] method Provided is a method for delivering OEA to the liver of a subject who needs to deliver OEA.In some embodiments, the method comprises oral administration of the composition described herein.Also described is a method for reducing liver steatosis in a subject, a method for treating NAFLD in a subject, and a method for treating NASH in a subject.In some embodiments, each of these methods comprises oral administration of the composition described herein.As shown in the following examples, administration of the composition leads to reduced lipid accumulation in the liver, reduced circulating triglycerides, and reduced levels of liver enzymes, namely AST and ALT.

[0031] In some embodiments, the dosage is 30-90 mg per administration. In some embodiments, the dosage is 90-300 mg. In yet other embodiments, the dosage is 300-600 mg. Administration includes providing the composition to the subject via a route known in the art, including, but not limited to, oral, buccal, rectal, or intragastric routes of administration. In certain embodiments, the oral route of administering the composition is preferred. The composition can be delivered alone or in combination with food or beverages. In some embodiments, the composition is administered in an enteric coating, such as an enteric coated capsule.

[0032] In some embodiments, the composition is administered daily. In some embodiments, administration is twice daily. In some embodiments, the composition is administered for 4-6 weeks. In some embodiments, the composition is administered for 5 weeks. In some embodiments, the composition is administered for 4-24 weeks. EXAMPLES

[0033] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same, and are not intended to otherwise limit the scope of the invention in any way.

[0034] Example 1: Diet-induced obesity disrupts histamine-dependent oleoylethanolamide signaling in mouse liver This example demonstrates that subchronic OEA administration reduces lipid accumulation and the transcription of proinflammatory and profibrotic genes in the liver of HFD-exposed mice. The data reported herein indicate that disruption of histamine-dependent OEA signaling in the liver may contribute to the pathology of obesity-associated nonalcoholic fatty liver disease.

[0035] Oleoylethanolamide (OEA) is an important lipid-derived regulator of energy balance [1]. In the small intestine, where its function has been extensively studied, OEA is generated postprandially from dietary oleic acid [2] and acts as a local messenger promoting satiety [3–5]. This effect is mediated by the ligand-activated transcription factor, peroxisome proliferator-activated receptor-α (PPAR-α), which binds OEA with high affinity [6, 7]. Food intake also regulates OEA mobilization in the liver, but in the opposite direction to that observed in the small intestine. Hepatic OEA content increases in the fasting state and decreases after feeding [8, 9]. The physiological significance of these changes has been investigated in recent studies

[10] . Results suggest that fasting stimulates extrahepatic mast cells to release histamine, which enters the liver via the portal circulation and activates H1-type receptors to enhance local OEA production. Interventions that disrupt this signaling process reduce fasting-induced ketogenesis by approximately 50%

[10] . This effect is consistent with the important role played by PPAR-α in controlling ketogenesis

[11] , and with data showing that OEA associates with PPAR-α to stimulate lipolysis, enhance fatty acid oxidation, and reduce weight gain and hepatic triacylglycerol content in obese rats and mice [12-15]. The findings summarized above detail a paracrine signaling process involving fasting-induced histamine release from mast cells into the portal circulation, stimulation of OEA formation in the liver, and enhancement of PPAR-α-mediated ketogenesis by OEA. Here, we raise the question of whether diet-induced obesity, known to disrupt feeding-dependent OEA production in the small intestine

[16] , might affect such a process.

[0036] Materials and Methods chemicals

[0037] OEA, palmitoylethanolamide (PEA), anandamide, and their deuterium-containing analogs were purchased from Cayman Chemicals (Ann Arbor, MI). 11) were prepared as described in

[16] . Histamine and [ 2 H4]-histamine was from Toronto Research Chemicals Canada (Toronto, CA), ethanolamine and Oil Red O were from Sigma Aldrich (St. Louis, MO, USA), and ProLong™ Gold antifade mounting medium with BODIPY and DAPI (4',6-diamidino-2-phenylindole) was from Thermo Fisher Scientific (Waltham, MA, USA). All solvents and reagents were of the highest grade available.

[0038] animal Male C57Bl / 6J mice (17 weeks) were purchased from Charles River (Wilmington, MA). Unless otherwise stated, they were group-housed (4-5 / cage) in ventilated cages with free access to food and water. Mice were maintained at a controlled temperature (22 ± 1 o The animals were maintained at 25 °C (55 ± 10%) and relative humidity (55 ± 10%) under a 12-h light / dark cycle (lights on at 6:30 am) and handled for 1 week prior to experimentation. Housing, animal maintenance, and all procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

[0039] Diet-induced obesity Mice at 18 weeks of age were randomized into two groups; one group received a standard chow (6.5% kcal fat, Envigo 2020X, Livermore, CA) and the other group received a high-fat diet (HFD, 60% kcal fat, D12492, Research Diets, New Brunswick, NJ). Both were allowed to eat ad libitum for 11 weeks. Body weight and food intake were recorded three times per week. Animals from both groups were randomly assigned to three cohorts: a) ad libitum feeding, b) fasting (6 pm to 12 pm the following day), c) fasting / refeding (access to food 2 hours after fasting). Mice assigned to the fasting and fasting / refed groups were housed in cages with wire attached to the bottom to prevent defecation.

[0040] OEA preparation OEA was dissolved in a vehicle of 95% saline / 5% Tween 80 (v / v) immediately prior to the experiment and administered by intraperitoneal (ip) injection at 5 mg / kg in a volume of 10 mL / kg.

[0041] Tissue collection

[0042] Portal blood was collected from anesthetized mice using a 1 mL syringe fitted with a 23G×1 needle (0.6 mm×25 mm) coated with potassium-EDTA (K2-EDTA). Samples were transferred to K2-EDTA-coated 0.5 mL polypropylene tubes and placed on ice. The tubes were centrifuged at 490×g for 15 min at 4° C., and the plasma layer was collected and stored at −80° C. o The animals were euthanized by decapitation, and the livers were extracted and flash frozen on dry ice and stored at -80 °C until analysis. o Saved in C.

[0043] Liver triglyceride measurement Hepatic triglycerides were quantified using a colorimetric assay kit (Cayman Chemicals). Briefly, approximately 30 mg of the right liver lobe was mixed with 0.2 mL of assay buffer and processed according to the manufacturer's instructions.

[0044] Blood glucose measurement Groups of chow-fed and HFD mice (n = 7–8) were placed in wire-bottomed cages and fasted overnight. Tail glucose levels were measured using an Accu-Chek Aviva meter and test strips (Roche Diabetes Care, Indianapolis, IN).

[0045] Other biochemical analyses A comprehensive blood chemistry panel was performed at Antech Diagnostics (Irvin, Calif.).

[0046] Histological analysis Oil Red O staining: Liver samples were embedded in molds and cut into 7 μm thick sections on a cryostat, which were fixed in 4% paraformaldehyde (PFA) for 15 min. After fixation, sections were immersed in phosphate-buffered saline (PBS) for 2 min, placed in isopropanol (60%) for 2 min, and incubated in Oil Red O (60% in isopropanol) for 30 min. Finally, sections were immersed in 60% isopropanol 40% water and mounted on glass slides with 50% glycerol. Images were taken at 20x magnification.

[0047] BODIPY staining: Liver sections (7 μm thick) were immersed in PBS for 5 min, fixed with 4% PFA for 15 min, and washed with PBS. Each specimen was circled with a hydrophobic pen and stained with BODIPY (boron dipyrromethene; 1 / 1000 dilution) for 30 min. The specimens were immersed in PBS and mounted with antifade mounting medium with DAPI (CAT:P36931). Images were captured at 20x magnification.

[0048] lipid analysis Analyte extraction

[0049] Frozen liver samples (approximately 30-40 mg) were transferred into 2 mL Precellys® soft tissue vials and diluted with an internal standard ([ 2 H4]-OEA, 2 H4]-PEA and [ 2H4]-anandamide, 100 nM each). Samples were homogenized using a Bertin homogenizer for 1 min at 4°C for 15 s / cycle with 2 cycles with a 20 s pause between cycles. The homogenates were centrifuged at 490 x g for 15 min at 4°C, and the supernatants were transferred into 8 mL glass vials and dried under N2. Each sample was diluted with chloroform / methanol / water (2 mL / 1 mL / 1 mL, by volume), vortexed, and centrifuged at 490 x g for 15 min at 4°C. The organic phase was collected and dried under N2. The pellets were reconstituted in acetonitrile (100 μL), transferred to a deactivated glass insert, and placed in an amber glass vial for liquid chromatography mass spectrometry (LC / MS-MS) analysis. For histamine extraction, portal vein plasma samples (50 μL) were transferred to 1.5 mL plastic vials and [ 2 Proteins were precipitated by adding ice-cold acetonitrile (0.4 mL) containing [H4]-histamine (50 μL, 0.5 μg / mL). 2 [H4]-histamine was prepared in a solution of distilled water and EDTA to prevent binding to glass

[17] . Samples were vortexed for 30 s and centrifuged at 490 × g for 10 min at 4 °C. The supernatant was transferred to a deactivated glass insert and placed in an amber glass vial for LC / MS-MS analysis.

[0050] LC / MS-MS analysis Quantification of OEA: Fatty acylethanolamides were fractionated using a 1260 series LC system (Agilent Technologies, Santa Clara, CA). Step gradient separation was performed on a Poroshell 120 column (1.9 μm, 2.1x100 mm; Agilent Technologies, Wilmington, DE) using a mobile phase consisting of 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B. A linear gradient was used: 80% B from 0.0 to 9.5 min; 95% B from 9.51 to 11.0 min; and 55% B from 11.1 to 15.50 min. The column temperature was maintained at 40°C and the autosampler temperature at 9°C. The injection volume was 2 μl, the flow rate was 0.3 ml / min, and the total analysis time was 15.5 min. The injection needle was washed at the autosampler port for 20 s using a cleaning solution consisting of 10% acetone in water / methanol / isopropanol / acetonitrile (1:1:1:1, by volume) before each injection. The mass spectrometer was operated in positive electrospray ionization mode. Analytes were quantified by multiple reaction monitoring using the acquisition parameters shown in Table 1. The capillary and nozzle voltages were 3500 V and 300-500 V, respectively. The drying gas temperature was 300 °C and the flow rate was 10 L / min. The sheath gas temperature was 300 °C and the flow rate was 10 L / h. The nebulizer pressure was set at 40 psi. MassHunter software (Agilent Technologies) was used for instrument control, data acquisition, and analysis.

[0051] [Table 1]

[0052] Histamine quantification: A BEH Amide column (1.7 μm, 2.1 × 50 mm, Waters Corporation, Milford, MA) was used. The mobile phase consisted of 40 mM ammonium formate adjusted to pH 3.0 with formic acid as solvent A, and 0.1% formic acid in acetonitrile as solvent B. A linear gradient was used: 75% B from 0.0 to 1.5 min; 65% B from 1.51 to 2.5 min; and held at 75% B from 2.51 to 5.50 min. The flow rate was 0.5 ml / min. The column temperature was maintained at 40 °C and the autosampler temperature at 9 °C. The injection volume was 2 μL. The mass spectrometer was operated in positive mode. Quantification was performed using the MRM transitions reported in Table 1. The capillary voltage was 2.8 kV. The source parameters were as follows: drying gas temperature was 230° C. and flow rate was 9 L / min; nebulizer pressure was set at 30 psi; sheath gas temperature was 300° C.; flow rate was 12 L / min; capillary voltage was set at 2000 V.

[0053] Real-time PCR Total RNA was extracted from liver using TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MS) and purified with the PureLink™ RNA Mini Kit (Invitrogen, Waltham, MS) according to the supplier's instructions. Prior to purification, samples were passed through gDNA Eliminator spin columns (Qiagen, Germantown, CA). RNA was quantified using a NanoDrop 2000 / 2000-c spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from 2 mg of total RNA using the High Capacity cDNA RT Kit with RNase Inhibitor (Applied BioSystems, Foster City, CA) according to the manufacturer's instructions. First strand cDNA was amplified using TaqMan™ Universal PCR Master Mixture (Thermo Fisher Scientific). Real-time PCR reactions were performed in 96-well plates using a CFX96™ Real-time System (Bio-Rad, Hercules, CA). The thermal cycling conditions were as follows: initial denaturation was set at 95°C for 10 min, followed by 45 cycles, each cycle performed at 95°C for 30 s followed by 55°C for 60 s. ΔCt values ​​were calculated using the geometric mean of three different housekeeping genes, and the relative fold change to the control group (no feed fed) was calculated as 2 -ΔΔCtThe ratios were calculated using the method

[18] . Real-time PCR primers and fluorogenic probes were purchased from Applied Biosystems (TaqMan® Gene Expression Assays, Foster City, CA). Mouse Actb (Mm00607939_s1), Hprt (Mm00446968_m1), Gapdh (Mm99999915_g1), Tnfa (Mm00443258_m1), Il1b (Mm00434228_m1), Tgfb1 (Mm_01178820_m1), Ccl2 (Mm_00441242_m1), and β-actin (Mm_00441242_m1) were used. TaqMan gene expression assays were used for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 122, 123, 124, 130, 135, 140, 141, 152

[0054] statistical analysis Data were analyzed using GraphPad Prism version 8.0 for Windows (GraphPad Software, San Diego, CA). Statistical significance was determined using two-tailed Student's t-tests, one-way or two-way analysis of variance (ANOVA), as well as Bonferroni post-hoc tests for multiple comparisons, as appropriate. Differences between groups were considered statistically significant at a value of p<0.05. Results are expressed as mean ± SEM.

[0055] result A high-fat diet interferes with fasting-induced histamine release into the hepatic portal system.

[0056] Fasting stimulates visceral mast cells to release histamine into the portal circulation

[10] . To determine whether diet-induced obesity influences this response, we exposed mice to a HFD or a typical diet for 12 weeks (Fig. 4) and measured histamine concentrations in portal plasma using an ad hoc developed LC-MS / MS assay (Fig. 1a, b). Confirming previous results

[10] , in chow-fed mice, food deprivation significantly increased portal histamine concentrations, which were 4-fold higher in fasted mice than in free-feeding mice, returning to baseline after 2 h of refeeding (Fig. 1c). No such increase occurred in HFD-fed animals, whose free-feeding histamine levels were approximately 3-fold higher than chow-fed controls and did not change in response to fasting (Fig. 1c). We also examined the effect of food deprivation on the transcription of H1 and H2 histamine receptors in the liver of chow-fed and HFD-fed mice. As shown in Figures 1d and 1e, fasting increased H1 and H2 receptor mRNA levels twofold in chow-fed controls but had no effect in HFD-fed mice. These results suggest that exposure to an HFD interferes with fasting-induced release of histamine into the portal system as well as fasting-induced expression of histamine receptors in mouse liver.

[0057] A high-fat diet blocks fasting-induced hepatic OEA production Mast cell-derived histamine stimulates hepatic OEA biosynthesis via activation of H1 receptors

[10] . To assess the effect of diet-induced obesity on this response, we administered OEA (18:1Δ 9 ) with its local isomer, vaccenoylethanolamide (VEA, 18:1Δ 11We measured OEA content in the livers of chow-fed and HFD-fed mice using an LC-MS / MS assay that can separate OEA from chow-fed mice (Fig. 2a) [16, 19]. As previously reported [8, 11], fasting significantly increased hepatic OEA content in chow-fed mice, which returned to baseline after refeeding (Fig. 2b). No such response was observed in HFD-fed mice (Fig. 3b). Similarly, fasting increased the levels of the hepatic endocannabinoid anandamide in chow-fed mice, but not in HFD-fed mice (Fig. 2b). The content of a third bioactive lipid amide, PEA, was not affected by food deprivation and was slightly decreased by HFD exposure (Fig. 2d). RT-PCR analysis revealed that the transcription of NAPE-PLD (encoded by the Napepld gene in mice), which catalyzes OEA formation

[20] , was slightly increased by HFD, whereas the transcription of FAAH (Faah), which catalyzes OEA degradation

[20] , remained unchanged (Fig. 2e, f). Collectively, these findings indicate that HFD exposure abolishes fasting-induced OEA production in mouse liver.

[0058] Exogenous OEA corrects lipid accumulation in the liver of obese mice.

[0059] Diet-induced obesity is associated with the development of hepatic steatosis and fibrosis

[21] . We investigated whether OEA supplementation could correct these changes in HFD-fed mice, which have defective endogenous OEA production. Mice were exposed to a HFD for 5 weeks, then randomized into two groups and treated with either OEA (5 mg / kg, once daily) or vehicle for an additional 6 weeks of HFD exposure. As expected [2, 12, 22–26], OEA administration reduced body weight gain and lowered circulating total cholesterol and hepatic transaminase levels (Table 2, Figure 5). These effects were accompanied by an improvement in the hepatic lipid profile (Figure 3). Oil Red O and BODIPY staining of liver sections showed that lipid content was reduced in OEA-treated mice compared to control mice (Figure 3a, b). Consistent with these results, hepatic triglyceride content was lower in OEA-treated mice than in control mice (Figure 3c). Further supporting the protective effect of OEA, the transcription of genes involved in liver inflammation and fibrosis, such as CC motif chemokine ligand 2 (Ccl2)

[27] , interleukin 1β (Il1b)

[28] , and collagen type I alpha 1 (Col1a1)

[29] , was attenuated in OEA-treated mice compared to control mice (Fig. 3d-f), whereas the transcription of the protective factor heme oxygenase 1 (Hmox1) was enhanced (Fig. 3g). The transcription of other related genes (Nrf1, Tnfa, Tgf1b, and Nqo1) was not affected (Fig. 3h-j).

[0060] Table 2. Effects of vehicle (VEH) or OEA administration on weight gain and blood chemistry in HFD-fed mice. Abbreviations: AST: aspartate transaminase, ALT: alanine transaminase, SGOT: serum glutamic oxaloacetic transaminase, SGPT: serum glutamic pyruvic transaminase. Data are presented as mean ± SEM and analyzed using unpaired two-tailed Student's t-test. *P<0.05. [Table 2]

[0061] Consideration In fasted mice, histamine secreted from visceral mast cells into the portal vein is secreted into the hepatic G q Histamine-dependent OEA signaling activates the binding H1 receptor and stimulates local OEA biosynthesis

[10] . Genetic or pharmacological manipulations that abrogate this process, such as mast cell ablation, H1 receptor blockade, and deletion of histamine or OEA-generating enzymes, reduce fasting-induced ketogenesis by approximately 50%. These findings identify histamine-dependent OEA signaling as a significant contributor to ketogenesis in mouse liver

[10] . Herein, we report that diet-induced obesity disrupts this paracrine signaling mechanism, such that fasting no longer induces histamine release and is unable to induce OEA biosynthesis in the liver. Furthermore, we provide new evidence confirming that OEA administration reduces hepatic steatosis in obese rodents [13, 14, 22-24]. Similar results have been reported in humans [25, 26]. Taken together, the findings suggest that HFD-induced histamine release and deficient hepatic OEA mobilization may contribute to the pathology of nonalcoholic fatty liver disease (NAFLD), a condition that affects approximately 25% of adults worldwide

[30] .

[0062] The results show that mouse obesity is accompanied by two notable changes in portal histamine (Fig. 1c). First, under ad libitum feeding conditions, histamine levels were higher in the portal blood of HFD-fed animals than in chow-fed animals. Second, fasting-induced histamine secretion [31, 32] - a process that may be mediated by vagal input - was suppressed in HFD-exposed animals. The mechanism behind these changes is unclear but likely involves the proinflammatory environment generated in the intestine by exposure to HFD. Studies using lymphatic fistula rat models have demonstrated that intestinal absorption of a high-fat diet is accompanied by activation of mucosal mast cells, resulting in the release of histamine and prostaglandin D2 [33–35]. Degranulation of intestinal mast cells has also been reported in diet-induced obese mice

[36] . Thus, it is plausible, but remains to be determined, that the elevated histamine release in the portal blood of obese ad libitum-fed mice is due to abnormal mast cell activity in inflamed mucosal tissue. Inflammation-related changes in mast cell function may also explain the failure of the cells to respond appropriately to fasting.

[0063] In addition to suppressing histamine release from mast cells, HFD blunts fasting-induced transcription of H1 and H2 receptors in the liver (Fig. 1d, e), which may further impair the organ's ability to produce OEA. Guinea pig and rat hepatocytes express H1 and H2 receptors, and their activation promotes glycogenolysis, gluconeogenesis, and ureogenesis in vitro [37, 38]. Consistent with these data, studies have shown that chronic treatment with H1 receptor antagonists and genetic deletion of H2 receptors exacerbate hepatic steatosis in mice [39, 40].

[0064] Confirming previous studies [13, 14], we found that subchronic treatment with OEA reduced hepatic steatosis. In humans, obesity is a major risk factor for the development of NAFLD, which can progress to pathologies such as nonalcoholic steatohepatitis (NASH) and cirrhosis

[41] . Interventions that reduce obesity slow this progression

[42] , but no pharmacological therapy is currently approved for either NAFLD or NASH

[43] . Previous studies have suggested that OEA supplementation may improve the prognosis of patients with NAFLD [24, 25] by activating PPAR-α in adipose organs and liver, promoting lipolysis [12, 44], possibly enhancing fatty acid oxidation and ketogenesis [12, 44], and attenuating local inflammation [45-47]. Our results are consistent with that proposal and further identify histamine-dependent OEA signaling in the liver as a potential pathomechanism and target for therapeutic action.

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[0066] Example 2: AquaCelle®-OEA is more orally bioavailable than Levagen®-OEA This example shows that not all formulations designed to increase lipid bioavailability can be used interchangeably. As shown in this example, AquaCelle®-OEA is significantly more orally bioavailable than either unformulated ("neat") OEA or a different formulation of OEA (Levagen®-OEA).

[0067] Materials and Methods chemicals Authentic oleoylethanolamide (OEA) and its deuterium-containing analog [2H4]-OEA were obtained from Cayman Chemicals (Ann Arbor, MI). Three OEA formulations were tested: (1) unformulated OEA ("neat-OEA"); (2) OEA formulated using Levogen-Plus® technology ("Levogen-OEA"); and (3) OEA formulated using AquaCelle® technology ("AquaCelle-OEA"). All formulations were suspended in sterile saline / Tween-80 (95% / 5%, vol / vol) immediately prior to the experiment and administered by oral gavage in a total volume of 10 mL / kg. Ethanolamine and Oil Red O were obtained from Sigma Aldrich (St. Louis, MO), ProLong™ Gold antifade mounting medium with BODIPY and DAPI (4',6-diamidino-2-phenylindole) was obtained from Thermo Fisher Scientific (Waltham, MA). All other reagents and analytical solvents were of the highest grade available.

[0068] animal Male C57Bl / 6J mice (13 weeks old) were purchased from Charles River (Wilmington, MA). Unless otherwise stated, mice were group-housed in ventilated cages (4–5 per cage) and provided with diet (standard chow, 6.5% kcal fat, Envigo 2020X, Livermore, CA) and water ad libitum. Mice were maintained under a 12-h light / dark cycle (lights on at 6:30 AM) at controlled temperature (22 ± 1°C) and relative humidity (55 ± 10%) and were handled for 1 week prior to experimentation. Housing, animal maintenance, and all other procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

[0069] Tissue collection Blood was collected by cardiac puncture into syringes rinsed with ethylenediamine delta-tetraacetic acid (EDTA) and transferred to 1 mL polypropylene tubes containing spray-coated potassium-EDTA (K2-EDTA). Plasma was prepared by centrifugation at 1450 x g for 15 min at 4 °C, transferred to polypropylene tubes, immediately frozen, and stored at -80 °C. Animals were euthanized by decapitation, their livers were rapidly harvested, and the right lobe of each liver was separated from the other lobes, frozen on dry ice, and stored at -80 °C until analysis.

[0070] Pharmacokinetics of OEA formulations Eighteen-week-old mice were orally gavaged with neat-OEA, Levagen-OEA, or AquaCelle-OEA at a dose of 30 mg / kg. At various time points post-dosing, animals were anesthetized with isoflurane (min = 0, 15, 30, 45, 60, 120, 240, 360; n = 9–11 mice per time point), and blood and liver were processed as described above.

[0071] blood chemistry A comprehensive blood chemistry panel was performed at Antech Diagnostics (Irvin, Calif.).

[0072] Histological analysis Oil Red O staining: Liver samples (right lobe) were embedded in molds and cut into 7 μm thick sections on a cryostat, and the sections were fixed in 4% paraformaldehyde (PFA) for 15 min. After fixation, the sections were immersed in phosphate-buffered saline (PBS) for 2 min, placed in isopropanol (60%) for 2 min, and incubated in Oil Red O (60% in isopropanol) for 30 min. Finally, the sections were immersed in 60% isopropanol 40% water and mounted on glass slides with 50% glycerol. Images were taken at 20x magnification [Lin et al. 2022].

[0073] BODIPY staining: Liver sections (7 μm thick) were immersed in PBS for 5 min, fixed with 4% PFA for 15 min, and washed with PBS. Each specimen was circled with a hydrophobic pen and stained with BODIPY (1 / 1000 dilution, vol / vol) for 30 min. The specimens were immersed in PBS and mounted with antifade mounting medium with DAPI (CAT:P36931). Images were captured at 20x magnification [Lin et al. 2022].

[0074] lipid extraction Frozen liver samples (approximately 30-40 mg) were transferred into 2 mL Precellys® soft tissue vials and diluted with ice-cold acetone (1 mL) containing an internal standard ([2H4]-OEA, 100 nM). Samples were homogenized using a Bertin homogenizer for 1 min at 4 °C for 15 s / cycle with 2 cycles with a 20 s pause between cycles. Homogenates were centrifuged at 490 × g for 15 min at 4 °C, and the supernatants were transferred into 8 mL glass vials and dried under N2. Each sample was diluted with chloroform / methanol / water (2 mL / 1 mL / 1 mL, vol / vol), vortexed, and centrifuged at 490 × g for 15 min at 4 °C. The organic phase was collected and dried under N2. Pellets were reconstituted in acetonitrile (100 μL), transferred to a deactivated glass insert, and placed in an amber glass vial for liquid chromatography mass spectrometry (LC / MS-MS) analysis.

[0075] OEA quantification Chromatographic separations were performed using a 1260 Series LC system (Agilent Technologies, Santa Clara, California) consisting of a binary pump, degasser, temperature-controlled autosampler, and column compartment, and connected to a 6460C triple quadrupole mass spectrometry detector equipped with a JetStream electrospray ionization (ESI) interface. Step gradient separations were performed on a Poroshell 120 column (1.9 μm, 2.1x100 mm; Agilent Technologies, Wilmington, DE) using a mobile phase consisting of 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B. A linear gradient was used: 80% B from 0.0 to 9.5 min; 95% B from 9.51 to 11.0 min; and 55% B from 11.1 to 15.50 min. The column temperature was maintained at 40 °C, and the autosampler temperature at 9 °C. The injection volume was 2 μl, the flow rate was 0.3 ml / min, and the total analysis time was 15.5 min. The injection needle was washed at the autosampler port for 20 s using a cleaning solution consisting of 10% acetone in water / methanol / isopropanol / acetonitrile (1:1:1:1, by volume) before each injection. The mass spectrometer was operated in positive electrospray ionization mode and the following MRM transitions (m / z) were monitored: OEA, 326.3>62.0; [2H4]-OEA, 330.3>66.0. The OEA fragmentation and collision voltages were set at 148 and 14, respectively, and for [2H4]-OEA, the OEA fragmentation and collision voltages were 143 and 14, respectively. The capillary and nozzle voltages were 3500 V and 500 V, respectively. The drying gas temperature was 300° C. and the flow rate was 10 L / min. The sheath gas temperature was 300°C and the flow rate was 10 L / h. The nebulizer pressure was set at 40 psi. The lower limit of quantification was 0.6 ng / mL (3.7 fmol / 2.0 μL injection). MassHunter software (Agilent Technologies, Santa Clara, CA) was used for instrument control, data acquisition, and analysis.

[0076] statistical analysis Data were analyzed using Graphpad Prism version 8.0 for Windows (GraphPad Software, San Diego, CA). Statistical significance was determined using two-tailed Student's t-tests, one-way or two-way analysis of variance (ANOVA), as well as Bonferroni post-hoc tests for multiple comparisons, as appropriate. Differences between groups were considered statistically significant at a value of p<0.05. Results are expressed as mean ± SEM.

[0077] result Pharmacokinetic Profile of AquaCelle®-OEA The pharmacokinetic properties of three different OEA preparations - unformulated ("neat") OEA, Levogen®-OEA, and AquaCelle®-OEA were compared after a single oral (gavage) dose (equivalent to 30 mg / kg free OEA) in male C57B16 mice (n=X per group). Plasma OEA profiles were similar in animals fed neat OEA and Levogen®-OEA (Figure 7A, inset shows area under the curve, AUC). Small differences were observed in OEA content in liver, which was significantly higher (P<0.05) at 60 min in mice receiving Levogen®-OEA compared to mice fed the unformulated compound (Figure 7B). Nevertheless, AUC values ​​were similar in the two groups (Figure 7B). In contrast, significant differences were observed between neat OEA and AquaCelle®-OEA, with the latter producing significantly higher OEA levels in both plasma (FIG. 7C) and liver (FIG. 7D). Indeed, the overall liver exposure (AUC) to OEA was approximately 3-fold higher in mice treated with AquaCelle®-OEA than in mice treated with the unformulated compound (FIG. 7D). These results indicate that the AquaCelle® formulation technology effectively transports orally administered OEA to the liver.

[0078] Example 3: Treatment of Hepatic Steatosis with a Bioavailable Oral Formulation of Oleoylethanolamide Systemic administration of OEA, an endogenous lipid-derived mediator, prevents steatosis in diet-induced obese rats (PMID:15910890) or mutant mice lacking the fatty acid binding protein L-FABP (PMID:22327204). The lipolytic properties of OEA were confirmed in the study described in Example 1, using a mouse model of diet-induced obesity. The main obstacle to applying these findings to treatment is the extremely poor bioavailability of OEA, which makes its use as an oral drug almost impossible. Gencor Pacific (https: / / www.gencoracific.com) has developed a self-emulsifying delivery system specifically designed to optimize the bioavailability of lipophilic molecules. This technology (AquaCelle®) achieves increased bioavailability by optimizing micelle formation and increasing the surface area of ​​the oil-water interface. It consists of lipids, surfactants, co-surfactants, and co-solvents that naturally form emulsions in digestive fluids and aid transport across the intestinal epithelium.

[0079] This example, in conjunction with Example 2, demonstrates that AquaCelle®-OEA formulations (consisting of 11% OEA and 89% AquaCelle®) increase the overall oral bioavailability of OEA, thereby enhancing its therapeutic efficacy. These results indicate that repeated oral administration of AquaCelle®-OEA significantly attenuates hepatic lipid accumulation in a mouse model of diet-induced obesity. These findings suggest that AquaCelle®-OEA is clinically applicable for the treatment of human nonalcoholic hepatic steatosis.

[0080] Diet-induced obesity Mice (18 weeks old) were exposed to a high-fat diet (HFD, 60% kcal fat, D12492, Research Diets, New Brunswick, NJ) for 3 weeks. Animals were randomized into two groups and gavaged twice daily with AquaCelle OEA (90 mg / kg) or its vehicle (AquaCelle without OEA) for 5 weeks while continuing the high-fat diet exposure. Individual weight gain and cage food intake were recorded 3 times weekly.

[0081] Effect of AquaCelle®-OEA on lipid accumulation in mouse liver A schematic diagram of the study protocol is shown in Figure 8A. Male C57B16 mice were exposed to a high-fat diet (HFD, 60% kcal from fat) for 3 weeks and then randomized into two groups (n=9 per group) of approximately equal weight (Figure 8B). With continued HFD exposure, animals were fed either AquaCelle®-OEA (equivalent to 90 mg free OEA per kg) or AquaCelle® alone (vehicle) by oral gavage twice daily for an additional 5 weeks. Mice were then euthanized and their liver and cardiac blood were collected for analysis.

[0082] Administration of AquaCelle®-OEA did not affect body weight gain, which was similar in the two groups (Figure 8C). Nevertheless, the average liver weight was significantly lower in mice treated with AquaCelle®-OEA than in mice treated with AquaCelle® alone (P<0.05, n=8=9) (Figure 9B). Furthermore, the outer surface of the organ appeared whiter in the latter group (Figure 9A, left). As both observations are consistent with the antisteatotic effect of AquaCelle®-OEA, lipid accumulation was measured in sections of liver parenchyma using BODIPY®, a strongly fluorescent lipophilic fluorophore. The results show that administration of AquaCelle®-OEA significantly (P<0.05; n=4) reduced BODIPY® fluorescence in liver sections compared to AquaCelle® alone (Figure 9A, center; Figure 9C). Frequency distribution analysis shown in Figure 9D further indicates that treatment with AquaCelle®-OEA reduced both the average size of lipid droplets and their number. Staining with the lipophilic dye Oil Red O confirmed these results (Figure 9C, right). Collectively, these findings indicate that subchronic oral administration of AquaCelle®-OEA prevents lipid accumulation in the liver of diet-induced obese mice without altering the trajectory of body weight. Consistent with the improvement of hepatic steatosis, treatment with AquaCelle®-OEA reduced circulating concentrations of triglycerides (P<0.01, n=7) (Figure 10A) and the hepatic enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Figures 10B, C). No statistically detectable effects were observed on circulating total cholesterol, glucose, albumin, globulin, amylase, and creatine phosphokinase (CPK) (Figures 10D-I).

[0083] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0084] Those skilled in the art will appreciate that the conception and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. 1. A composition comprising 5-15% by weight of oleoylethanolamide (OEA) and 85-95% of a self-emulsifying drug delivery system (SEDDS), wherein the SEDDS comprises a carrier oil comprising a medium chain triglyceride, a citrus oil, and lecithin, and wherein the composition promotes at least a two-fold increase in the bioavailability of the OEA.

2. 10. The composition of claim 1, wherein the SEDDS comprises AquaCelle®.

3. 3. The composition of claim 2, comprising 10-12% OEA and 88-90% AquaCelle®.

4. 3. The composition of claim 2, comprising 11% OEA and 89% AquaCelle®.

5. 10. Use of the composition of any one of claims 1 to 4 in the manufacture of a medicament for delivering oleoylethanolamide (OEA) to the liver of a subject in need thereof, wherein the composition is administered orally.

6. 10. Use of a composition according to any one of claims 1 to 4 in the manufacture of a medicament for reducing hepatic steatosis in a subject, wherein the composition is administered orally.

7. 10. Use of the composition of any one of claims 1 to 4 in the manufacture of a medicament for treating non-alcoholic fatty liver disease (NAFLD) in a subject, wherein the composition is administered orally.

8. 10. Use of the composition of any one of claims 1 to 4 in the manufacture of a medicament for treating non-alcoholic steatohepatitis (NASH) in a subject, wherein the composition is administered orally.

9. The use according to claim 5, wherein the subject is a human.

10. The use of claim 5, wherein the composition is administered in an enteric coated capsule.

11. 6. The use of claim 5, wherein the composition is administered once daily for 4 to 24 weeks.

12. 12. The use of claim 11, wherein the composition is administered twice daily for 4 to 24 weeks.

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