Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

• Nonalcoholic fatty liver disease is the most common cause of chronic liver disease in the Western world. It is projected to become a leading indication for liver transplantation, superseding hepatitis C. NAFLD is associated with obesity and may progress to nonalcoholic steatohepatitis (NASH) and end-stage liver disease. There are no approved drug treatments for NAFLD or NASH. Animal and human studies suggest that oleoylethanolamide (OEA) might find application in the treatment of NAFLD, but the clinical use of this naturally occurring molecule is greatly limited by its poor bioavailability, due to a combination of unfavorable physicochemical properties and rapid enzyme-mediated degradation.
• OEA OEA reduces food intake through a mechanism that involves recruitment of peripheral sensory afferents (Rodriguez de Fonseca et al., 2001) and activation of central pathways that utilize oxytocin and histamine as neurotransmitters (Gaetani et al., 2010, Provensi et al., 2014).
• OEA peroxisome proliferator-activated receptor-a
• OEA levels in small intestine from various vertebrate species - including fish, snakes and rodents - rise from ⁇ 50 nM in the fasting 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 that is compatible with PPAR-a activation (Fu et al., 2003, Astarita et al., 2006a).
• the available data indicate that OEA is a physiologically relevant endogenous agonist for small- intestinal PPAR-a, which participates in the control of satiety (DiPatrizio and Piomelli, 2015).
• intraperitoneal OEA administration stimulates fatty-acid oxidation in isolated rat hepatocytes and enhances ketone body production (ketogenesis) in live rats (Guzman et al., 2004).
• 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).
• OEA is a lipophilic molecule with limited drug-like properties.
• 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 compound vehiculation to the liver parenchyma, where this natural lipid amide exerts its anti-steatosis effects.
• This formulation provides a significantly greater oral bioavailability than either non-formulated (‘neat’) OEA or a different formulation of OEA (Levagen®-OEA).
• oral administration of the novel OEA formulation attenuates liver steatosis produced in a mouse model of human metabolic syndrome.
• the data described herein support use of the composition in the treatment of human NAFLD, including NASH.
• a composition comprising 5-15% by weight OEA and 85-95% self-emulsifying drug delivery system (SEDDS).
• the SEDDS comprises a carrier oil comprising medium chain triglycerides, a citrus oil, and lecithin.
• the composition promotes at least a twofold increase in bioavailability of the OEA.
• the composition promotes at least a threefold increase in the bioavailability of the OEA.
• the SEDDS comprises AquaCelle®.
• 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 lecithin comprises up to about 11% by weight.
• the percentage composition of the SEDDS is such that, when dispersed in an aqueous environment, the composition forms a population of micelles with a mean diameter of about 1 to 20 micrometers. In some embodiments, the composition forms a population of micelles with a mean diameter of about 5 to 15 micrometers. In some embodiments, the composition forms a population of micelles with a mean diameter of about 12 to 13 micrometers.
• the composition further comprises an antioxidant.
• 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 acid succinate, dl-alpha-tocopheryl acid succinate, Vitamin E and derivatives thereof, Olive polyphenols, Algal polyphenols, and mixtures thereof.
• the antioxidant is present at a concentration of 0-0.5% by weight.
• the antioxidant is present at a concentration of about 0.01% by weight.
• the antioxidant is present at a concentration of about 0.02% by weight.
• the antioxidant is present at a concentration of about 0.1 % by weight.
• the antioxidant is present at a concentration of about 0.5% by weight.
• the composition further comprises an excipient.
• excipients include, but are not limited to, colloidal silica, corn starch, hydroxypropylmethylcellulose (HPMC), maltodextrin, magnesium stearate, magnesium hydroxide, microcrystalline cellulose, dextrin, sorbitol, mannitol and trehalose.
• HPMC hydroxypropylmethylcellulose
• 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.
• a method of delivering oleoylethanolamide (OEA) to the liver of a subject in need thereof comprises oral administration of a composition as described herein.
• a method of reducing liver steatosis in a subject, as well as a method of treating non-alcoholic fatty liver disease (NAFLD) in a subject, and a method of treating non-alcoholic steatohepatitis (NASH) in a subject comprises oral administration of a composition as described herein.
• FIG. 1 Effects of HFD exposure on fasting-induced histamine signaling in portal circulation and the liver.
• A, B Histamine identification in portal plasma.
• A Representative LC/MS-MS tracing showing the elution of authentic [ 2 H4]-histamine and native histamine in portal plasma.
• C-E Effects of free feeding (circles), food deprivation (squares), and food deprivation/refeeding (triangles) in chow-fed (empty symbols) and HFD-fed (filled symbols) mice.
• C Histamine concentrations in portal plasma.
• D Histamine Hi receptor (/77r) transcription in the liver.
• E Histamine H2 receptor (H2 ) transcription in the liver.
• F Histamine H4 receptor (H4r) transcription in the liver.
• A Representative LC/MS-MS tracing showing the presence of OEA and its regional isomer VEA in the liver.
• B-F Effects of free feeding (circles), food deprivation (squares), and food deprivation/refeeding (triangles) in chow-fed (empty symbols) and HFD-fed (filled symbols) mice.
• B OEA content in the liver.
• C AEA content in the liver.
• D PEA content in the liver.
• E NAPE-PLD (Napepld) transcription in the liver.
• F FAAH (Faah) transcription in the liver.
• FIG. 3 Effects of VEH or OEA administration on liver of HFD-fed mice.
• A Representative images of the liver in situ (left) and liver sections stained with Oil Red O (2nd from the left), BODIPY (3rd from the left), and PSR (right).
• B Quantification of BODIPY fluorescence in livers from VEH-treated (empty symbols) or OEA-treated (filled symbols) mice exposed to HFD.
• C Quantification of PSR fluorescence in livers from VEH- treated (empty symbols) or OEA-treated (filled symbols) mice exposed to HFD.
• F-M Liver transcription of 111b (F), Cc/2 (G), Col1a1 (H), Hmoxl (I), Nrf1 (J), Tnfa (K), Tgfbl (L), and Nqo1 (M).
• VEH vehicle.
• FIG. 4 Male C57BI/6J mice exposed to HFD develop obesity, hyperglycemia, and liver steatosis.
• A Time-course of body weight gain in mice exposed to regular chow (empty symbols) or HFD (filled symbols).
• B Fasting glucose concentrations in cardiac plasma from 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.
• FIG. 5 Effect of OEA administration on body weight gain of HFD-fed mice.
• A Body weight trajectory
• B after the start of OEA treatment.
• Data are expressed as mean ⁇ S.E.M and were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons test. * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001.
• FIG. 6 H&E staining of liver sections showing the effects of vehicle (VEH) or OEA on liver histology in diet-induced obese mice.
• FIGS. 7A-7D Pharmacokinetic (PK) profiles of non-formulated OEA (neat-OEA), Levagen®-OEA, and Aquacelle®-OEA in mice. Dosing corresponded to 30 mg/kg free OEA. Insets show area under the curve (AUG) values.
• PK Pharmacokinetic
• 7A, 7B Comparative PK profiles of neat- OEA versus Levagen®-OEA in (7A) plasma and (7B) liver.
• (7C, 7D Comparative PK profiles of neat-OEA versus Aquacelle®-OEA in (7C) plasma and (7D) liver.
• FIG. 8. (A) Scheme summarizing the study design. (B) Body weight of all animal subjects included in the study after three weeks of high fat diet (HFD) exposure and before randomization into control (AquaCelle® alone, circles) and OEA-treated (AquaCelle®-OEA, triangles) groups. (C) Body weight gain in mice during treatment with AquaCelle® alone and AquaCelle®-OEA.
• FIGS. 9A-9D Effects of oral administration of AquaCelle® alone or AquaCelle®-OEA in diet-induced obese mice with liver steatosis.
• (9B) Average liver weight in mice treated with AquaCelle® alone (circles) or AquaCelle®-OEA (triangles) (n 8/9).
• (9C) Quantification of BODIPY fluorescence in livers from mice treated with AquaCelle® alone or AquaCelle®-OEA (n 4/group).
• FIGS. 10A-10I Plasma blood profile of diet-induced obese mice with liver steatosis treated with AquaCelle® alone or AquaCelle®-OEA.
• 10A Triglycerides (TAG), (10B) AST, aspartate transaminase, (10C) ALT, alanine transaminase, (10D) cholesterol, (10E) glucose, (10F) albumin, (10G) globulin, (10H) amylase, (101) CPK, creatine kinase.
• TAG Triglycerides
• 10B AST, aspartate transaminase
• 10C ALT
• 10D cholesterol
• 10E glucose
• 10F albumin
• 10G globulin
• 10H amylase
• CPK creatine kinase.
• Veh AquaCelle® alone
• OEA AquaCelle®-OEA
• compositions and methods described herein provide a novel OEA formulation that allows vehiculation of the compound to the liver parenchyma, where this natural lipid amide exerts its anti-steatosis effects.
• This formulation provides a significantly greater oral bioavailability than either non-formulated OEA or Levagen®-OEA.
• oral administration of the novel OEA formulation attenuates liver steatosis produced in a mouse model of human metabolic syndrome.
• the data described herein support use of the composition in the treatment of human non-alcoholic liver steatosis. Definitions
• a “control” or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects, or a baseline amount of marker to be used for comparison. Typically, a baseline will be a measurement taken from the same subject or patient. The sample can be an actual sample used for testing, or a reference level or range, based on known normal measurements of the corresponding marker.
• a “significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability.
• an increase or decrease of 10% relative to a reference sample is a significant difference.
• an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference.
• an increase of two-fold relative to a reference sample is considered significant.
• compositions comprising 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).
• 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.
• the subject is a human.
• a composition comprising 5-15% by weight OEA and 85-95% self-emulsifying drug delivery system (SEDDS).
• the SEDDS comprises a carrier oil comprising medium chain triglycerides, a citrus oil, and lecithin.
• the composition promotes at least a twofold increase in bioavailability of the OEA when administered to a subject relative to administration of neat OEA.
• the composition promotes at least a threefold increase in the bioavailability of the OEA.
• the SEDDS comprises AquaCelle®.
• 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 lecithin comprises up to about 11% by weight.
• the percentage composition of the SEDDS is such that, when dispersed in an aqueous environment, the composition forms a population of micelles with a mean diameter of about 1 to 20 micrometers. In some embodiments, the composition forms a population of micelles with a mean diameter of about 5 to 15 micrometers. In some embodiments, the composition forms a population of micelles with a mean diameter of about 12 to 13 micrometers.
• the composition is formulated for delivery in an enteric coating, such as, for example, an enteric-coated capsule.
• Enteric coated capsules are designed to remain intact in the stomach and then to release the active substance in the intestine.
• Enteric coating can be applied to solid dosage forms, such as granules, pellets, capsules, or tablet, to improve drug bioavailability by preventing degradation of acid or gastric enzyme labile drugs.
• the composition further comprises an antioxidant.
• 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 acid succinate, dl-alpha-tocopheryl acid succinate, Vitamin E and derivatives thereof, Olive polyphenols, Algal polyphenols, and mixtures thereof.
• 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.
• the composition further comprises an excipient.
• excipients include, but are not limited to, colloidal silica, corn starch, hydroxypropylmethylcellulose (HPMC), maltodextrin, magnesium stearate, magnesium hydroxide, microcrystalline cellulose, dextrin, sorbitol, mannitol and trehalose.
• HPMC hydroxypropylmethylcellulose
• 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.
• a method of delivering OEA to the liver of a subject in need thereof comprises oral administration of a composition as described herein.
• a method of reducing liver steatosis in a subject as well as a method of treating NAFLD in a subject, and a method of treating NASH in a subject.
• each of these methods comprises oral administration of a composition as described herein.
• administration of the composition results in reduced lipid accumulation in the liver, reduced circulating triglycerides, and reduced levels of the liver enzymes, AST and ALT.
• dosing is 30 to 90 mg per administration. In some embodiments, dosing is 90 to 300 mg. In yet other embodiments, dosing is 300 to 600 mg.
• the administering comprises providing a composition to a subject via a route known in the art, including but not limited to oral, buccal, rectal, or intragastric routes of administration. In certain embodiments, oral routes of administering a composition are preferred.
• the composition can be delivered alone or in combination with food or drink.
• the composition is administered in an enteric coating, such as an enteric- coated capsule.
• the composition is administered daily. In some embodiments, administration is twice daily. In some embodiments, the composition is administered for four to six weeks. In some embodiments, the composition is administered for five weeks. In some embodiments, the composition is administered for 4-24 weeks.
• This Example demonstrates that subchronic OEA administration reduces lipid accumulation and transcription of proinflammatory and profibrotic genes in liver of HFD- exposed mice.
• the data reported herein show that disruption of histamine-dependent OEA signaling in liver can contribute to pathology in obesity-associated non-alcoholic fatty liver disease.
• Oleoylethanolamide is an important lipid-derived regulator of energy balance [1], In the small intestine, where its functions have been studied extensively, OEA is generated postprandially from diet-derived oleic acid [2] and acts as a local messenger to promote satiety [3-5], This effect is mediated by the ligand-operated transcription factor, peroxisome prol iterator-activated receptor-a (PPAR-a), which binds OEA with high affinity [6,7], Food intake regulates OEA mobilization also in the liver, but in an opposite direction to that observed in 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 a recent study [10], Its 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.
• PPAR-a peroxisome prol iterator
• OEA palmitoylethanolamide
• PDA palmitoylethanolamide
• anandamide and their deuterium- containing analogs from Cayman Chemicals (Ann Arbor, Ml).
• Vaccenoylethanolamide (VEA, 18:1 A 11 ) was prepared as described [16], Histamine and [ 2 H4]-histamine were from Toronto Research Chemicals Canada (Toronto, CA), ethanolamine and Oil Red O from Sigma Aldrich (St. Louis, MO, USA) and BODIPY and ProLongTM Gold anti-fade mountant with DAPI (4',6-diamidino-2-phenylindole) from Thermo Fisher Scientific (Waltham, MA, USA). All solvents and reagents were of the highest available grade.
• mice Male C57BI/6J mice (17 weeks) were purchased from Charles River (Wilmington, MA). They were group housed (4-5/cage) in ventilated cages with free access to food and water, unless indicated otherwise. They 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 before experiments. Housing, animal maintenance and all procedures were conducted 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.
• mice 18-week-old mice were randomized into two groups; one received standard chow (6.5% kcal fat, Envigo 2020X, Livermore, CA) and the other a high-fat diet (HFD, 60% kcal fat, D12492, Research Diets, New Brunswick, NJ). Both were available 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) free feeding, b) fasting (from 6:00 pm to 12:00 pm on the following day) and c) fasting/refeeding (2 hr access to food after fasting). Mice assigned to the fasting and fasting/refeeding groups were housed in bottom-wired cages to prevent coprophagia.
• OEA was dissolved in a vehicle of 95% saline/5% Tween80 (v/v) shortly before experiments. It was administered by intraperitoneal (i.p.) injection at 5 mg/kg in a volume of 10 mL/kg.
• Liver triglyceride measurements [0057] Liver triglycerides were quantified using a colorimetric assay kit (Cayman Chemicals). Briefly, -30 mg of right liver lobe were mixed with 0.2 mL of assay buffer and processed following manufacturer’s instructions.
• Oil Red O Staining We embedded liver samples in a mold, cut them into 7-pm thick sections with a cryostat and fixed them with 4% paraformaldehyde (PFA) for 15 min. After fixation, we immersed the sections in phosphate-buffered saline (PBS) for 2 min, placed them in isopropanol (60%) for 2 min, and incubated them with Oil Red O (60% in isopropanol) for 30 min. Finally, we dipped the sections in 60% isopropanol 40% water and mounted them onto glass slides with 50% glycerol. Images were taken at 20x magnification.
• PFA paraformaldehyde
• BODIPY staining Liver sections (7-pm thick) were immersed in PBS for 5 min, fixed with 4% PFA for 15 min, and washed with PBS. Each sample was circled with a hydrophobic pen and stained with BODIPY (boron dipyrromethene; 1/1000 dilution) for 30 min. Samples were dipped in PBS and mounted with anti-fade mountant with DAPI (CAT: P36931). Images were captured at 20x magnification.
• Frozen liver samples (30-40 mg) were transferred into 2 mL Precellys® soft tissue vials and diluted with ice-cold acetone (1 mL) containing internal standards ([ 2 H4]-OEA, [ 2 H4]- PEA and [ 2 H4]-anandamide, 100 nM each).
• Samples were homogenized for 1 min using a Bertin homogenizer at 4°C in 15s/cycle for 2 cycles with 20 s pause between cycles. The homogenates were centrifuged for 15 min at 490 x g at 4°C and supernatants were transferred into 8-mL glass vials and dried under N2.
• OEA quantification Fatty acyl ethanolamides were fractionated using a 1260 series LC system (Agilent Technologies, Santa Clara, CA). A step gradient separation was performed on a Poroshell 120 column (1.9 pm, 2.1 x 100 mm; Agilent Technologies, Wilmington, DE) with 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: 0.0-9.5 min 80% B; 9.51-11.0 min 95% B; and 11.1-15.50 min maintained at 55% B. Column temperature was maintained at 40°C and autosampler temperature at 9°C.
• Injection volume was 2 pl, flow rate was 0.3 ml/min, and total analysis time was 15.5 min.
• the injection needle was washed in the autosampler port for 20 s before each injection, using a wash solution consisting of 10% acetone in water/methanol/isopropanol/acetonitrile (1 : 1 : 1 : 1 , vol).
• the mass spectrometer was operated in the positive electrospray ionization mode. Analytes were quantified by multiple reaction monitoring using acquisition parameters shown in Table 1. Capillary and nozzle voltages were 3500 V and 300-500 V, respectively. Drying gas temperature was 300°C with a flow of 10 L/min. Sheath gas temperature was 300°C with a flow of 10 L/hr. Nebulizer pressure was set at 40 psi.
• Table 1 Mass Spectrometry Acquisition Parameters
• Histamine quantification We used a BEH Amide column (1.7 pm, 2.1 x 50 mm, Waters Corporation, Milford, MA). The mobile phase consisted of 40 mM ammonium formate, pH adjusted to 3.0 with formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B. A linear gradient was used: 0.0-1.5 min 75% B 1.51-2.5 min 65% B; and 2.51- 5.50 min maintained at 75% B. Flow rate was 0.5 ml/min. Column temperature was maintained at 40°C and auto-sampler temperature at 9°C. Injection volume was 2 pL.
• the mass spectrometer was operated in the positive mode. Quantifications were performed using the MRM transitions reported in Table 1.
• the capillary voltage was 2.8 kV. Source parameters were as follows: drying gas temperature was 230 °C, with a flow of 9 L/min; nebulizer pressure was set at 30 psi; sheath gas temperature was 300°C with a flow of 12 L/min; capillary voltage was set at 2000 V.
• RNA was extracted total RNA from liver using TRIzolTM reagent (Thermo Fisher Scientific, Waltham, MS) and purified it with the PureLinkTM RNA Mini Kit (Invitrogen, Waltham, MS) as directed by the supplier. Prior to purification, samples were passed through a gDNA Eliminator spin column (Qiagen, Germantown, CA). We quantified RNA using 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) following manufacturer’s instructions.
• First-strand cDNA was amplified using TaqManTM Universal PCR Master Mixture (Thermo Fisher Scientific) Realtime PCR reactions were performed in 96-well plates using the CFX96TM Real-Time System (Bio-Rad, Hercules, CA). The thermal cycling conditions were as follows: initial denaturation set at 95°C for 10 min, followed by 45 cycles, where each cycle was performed at 95°C for 30 s followed by 55°C for 60 s.
• ACt values were calculated using the geometric mean of three different housekeeping genes, and the relative fold changes over control groups (chow-free feed) were calculated by the 2’ AACt method [18], Real-time PCR primers and fluorogenic probes were purchased from Applied Biosystems (TaqMan(R) Gene Expression Assays, Foster City, CA).
• Exogenous OEA corrects lipid accumulation in liver of obese mice.
• HFD In addition to curbing histamine release from mast cells, HFD also blunted fasting- induced transcription of Hi and H2 receptors in liver (Fig. 1 d, e), which may further impair the organ’s ability to produce OEA.
• Guinea pig and rat hepatocytes express Hi and H2 receptors and, in vitro, their activation enhances glycogenolysis, gluconeogenesis, and ureagenesis [37, 38], Consistent with those data, studies have shown that prolonged treatment with Hi receptor antagonists and genetic deletion of H2 receptors exacerbate hepatic steatosis in mice [39, 40], [0088] Confirming previous work [13, 14], we found that subchronic treatment with OEA alleviates liver steatosis.
• NAFLD non-alcoholic steatohepatitis
• OEA supplementation may improve prognosis in persons with NAFLD [24, 25] by activating PPAR-a in adipose organ and liver to enhance lipolysis [12, 44], possibly by enhancing fatty acid oxidation and ketogenesis [12, 44] and alleviating local inflammation [45-47],
• the present results are consistent with that proposal and further identify histamine-dependent OEA signaling in liver as a possible pathogenic mechanism and target for therapeutic action.
• Astarita G et al. J Pharmacol Exp Ther. 2006;318(2):563-70.
• This Example demonstrates that not all formulations designed to increase the bioavailability of lipids can be used interchangeably. As shown in this Example, AquaCelle®- OEA has a significantly greater oral bioavailability than either non-formulated (‘neat’) OEA or a different formulation of OEA (Levagen®-OEA).
• OEA oleoylethanolamide
• OEA deuterium-containing analog
• Cayman Chemicals Ann Arbor, Ml
• Three OEA formulations were tested: (1) non-formulated OEA (‘neat-OEA’); (2) OEA formulated with Levogen-Plus® technology (‘Levogen-OEA’); and (3) OEA formulated with AquaCelle® technology (‘AquaCelle-OEA’). All formulations were suspended in sterile saline/Tween-80 (95%/5% , vol/vol) shortly before experiments and were administered by gavage in a total volume of 10 mL/kg.
• Ethanolamine and Oil Red O were obtained from Sigma Aldrich (St. Louis, MO) and BODIPY and ProLongTM Gold anti-fade mountant with DAPI (4',6-diamidino-2-phenylindole) from Thermo Fisher Scientific (Waltham, MA). All other reagents and analytical solvents were of the highest available grade.
• mice Male C57BI/6J mice (13 weeks of age) were purchased from Charles River (Wilmington, MA). They were group housed in ventilated cages (4-5 per cage) with free access to food (standard chow, 6.5% kcal fat, Envigo 2020X, Livermore, CA) and water, unless indicated otherwise. They were maintained under a 12-h light/dark cycle (lights on at 6:30 am) at controlled temperature (22 ⁇ 1oC) and relative humidity (55 ⁇ 10%) and were handled for 1 week before experiments. Housing, animal maintenance, and all other procedures were conducted 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.
• EDTA ethylenediamine delta-tetra-acetic acid
• K2-EDTA spray- coated potassium-EDTA
• Oil Red O Staining We embedded liver samples (right lobe) in a mold, cut them with a cryostat into 7-pm thick sections, and fixed the sections with 4% paraformaldehyde (PFA) for 15 min. After fixation, we immersed the sections in phosphate-buffered saline (PBS) for 2 min, placed them in isopropanol (60%) for 2 min, and incubated them with Oil Red O (60% in isopropanol) for 30 min. Finally, we dipped the sections in 60% isopropanol 40% water and mounted them onto glass slides with 50% glycerol. Images were taken at 20x magnification [Lin et al. 2022],
• BODIPY staining Liver sections (7-pm thick) were immersed in PBS for 5 min, fixed with 4% PFA for 15 min, and washed with PBS. Each sample was circled with a hydrophobic pen and stained with BODIPY (1/1000 dilution, vol/vol) for 30 min. Samples were dipped in PBS and mounted with anti-fade mountant with DAPI (CAT: P36931). Images were captured at 20x magnification [Lin et al. 2022],
• Frozen liver samples (30-40 mg) were transferred into 2 mL Precellys® soft tissue vials and diluted with ice-cold acetone (1 mL) containing internal standard ([2H4]-OEA, 100 nM). Samples were homogenized for 1 min using a Bertin homogenizer at 4°C in 15s/cycle for 2 cycles with 20 s pause between cycles. The homogenates were centrifuged for 15 min at 490 x g at 4°C and supernatants were transferred into 8-mL glass vials and dried under N2.
• Chromatographic separations were carried out using a 1260 series LC system (Agilent Technologies, Santa Clara, California) consisting of a binary pump, degasser, temperature-controlled autosampler, and column compartment, coupled to a 6460C triple quadrupole mass spectrometric detector with JetStream electrospray ionization (ESI) interface.
• ESI JetStream electrospray ionization
• a step gradient separation was performed on a Poroshell 120 column (1.9 pm, 2.1 x 100 mm; Agilent Technologies, Wilmington, DE) with 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: 0.0-9.5 min 80% B; 9.51-11.0 min 95% B; and 11.1-15.50 min maintained at 55% B.
• Column temperature was maintained at 40°C and autosampler temperature at 9°C.
• Injection volume was 2 pl, flow rate was 0.3 ml/min, and total analysis time was 15.5 min.
• the injection needle was washed in the autosampler port for 20 s before each injection, using a wash solution consisting of 10% acetone in water/methanol/isopropanol/acetonitrile (1 :1:1:1, vol).
• the mass spectrometer was operated in the positive electrospray ionization mode, monitoring the following MRM transitions (m/z): OEA, 326.3 > 62.0; [2H4]-OEA, 330.3 > 66.0.
• OEA fragmentation and collision voltages were set at 148 and 14 respectively and for [2H4]-OEA they were 143 and 14 respectively.
• Capillary and nozzle voltages were 3500 V and 500 V, respectively.
• Drying gas temperature was 300°C with a flow of 10 L/min.
• Sheath gas temperature was 300°C with a flow of 10 L/hr.
• Nebulizer pressure was set at 40 psi. Lowest limit of quantification was 0.6 ng/mL (3.7 fmol/injection of 2.0 pL).
• We used the MassHunter software (Agilent Technologies, Santa Clara, CA) for instrument control, data acquisition and analysis.
• 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 spontaneously form an emulsion in digestive fluids, aiding transportation through the intestinal epithelium.
• Example 2 This Example, together with Example 2, demonstrates that an AquaCelle® ⁇ OEA formulation (consisting of 11% OEA and 89% AquaCelle®) increases the overall oral bioavailability of OEA and, by doing so, enhances its therapeutic effects.
• the results show that repeated oral administration of AquaCelle® ⁇ OEA significantly attenuates hepatic lipid accumulation in a mouse model of diet-induced obesity.
• the findings suggest that AquaCelle®- OEA has a clinical application in the treatment of human non-alcoholic liver steatosis.
• mice (18 weeks old) to a high-fat diet (HFD, 60% kcal fat, D12492, Research Diets, New Brunswick, NJ) for 3 weeks.
• HFD high-fat diet
• mice We randomized the animals into two groups and administered by gavage AquaCelle OEA (90 mg/kg) or its vehicle (AquaCelle without OEA) twice daily for 5 weeks, while continuing high-fat diet exposure.
• Individual body weight gain and cage food intake were recorded three times per week.
• FIG. 8A A schematic overview of the study protocol is shown in Figure 8A.
• HFD high-fat diet
• n 60% kcal from fat

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