WO2022133612A1 - Parenteral cannabinoid formulations and uses thereof - Google Patents

Parenteral cannabinoid formulations and uses thereof Download PDF

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Publication number
WO2022133612A1
WO2022133612A1 PCT/CA2021/051882 CA2021051882W WO2022133612A1 WO 2022133612 A1 WO2022133612 A1 WO 2022133612A1 CA 2021051882 W CA2021051882 W CA 2021051882W WO 2022133612 A1 WO2022133612 A1 WO 2022133612A1
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Prior art keywords
pharmaceutical composition
ontemabez
emulsifier
solvent
nanoemulsion
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PCT/CA2021/051882
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French (fr)
Inventor
Harpreet Kaur
Hashim FAROOQ
Subhagini THIRUNAVUKARASU
Dawaye GEORGEWILL
Jixing Wang
Di XU
Omar Sharif
Melanie KELLY
Graham Wood
Charles Campbell
Erin BASSETT
Orlando Hung
Christian Lehmann
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Tetra Bio-Pharma Inc.
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Publication of WO2022133612A1 publication Critical patent/WO2022133612A1/en

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    • 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
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • 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
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to formulations of cannabinoid compounds, particularly formulations for parenteral delivery.
  • Systemic inflammatory responses can be triggered by a variety of infectious and non- infectious stimuli such as bacterial, viral and fungal infections, trauma, and in response to certain drugs.
  • Systemic inflammatory responses involving organ dysfunction are often seen in diseases such as sepsis/severe sepsis/septic shock, systemic inflammatory response syndrome (SIRS), cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (MODS) (see for example, https://wwwZncbi.nlm.nih.gov/books/NBK5477669).
  • Sepsis is a clinical syndrome that arises due to infection, trauma or non-infectious triggers. Sepsis is often characterized by physiological and pathological abnormalities caused by a dysregulated host immune response to infection. Sepsis is normally classified on a continuum of severity progressing from systemic inflammatory response to sepsis, followed by severe sepsis, and the most severe and refractory state of septic shock. Severe sepsis and septic shock are associated with organ dysfunction, which may progress to MODS, and a high mortality. This mortality is particularly high (40-75%) in patients with sepsis-associated ARDS and MODS.
  • the endocannabinoid system regulates many critical physiological processes including those underlying the functions of the immune system.
  • the endocannabinoid system comprises at least two receptors: cannabinoid 1 (CB1) and cannabinoid 2 (CB2), and several endogenous ligands (endocannabinoids).
  • Immune cells may express both CB1 and CB2 receptors and may metabolize, produce and respond to both exogenous and endogenous cannabinoids.
  • Cannabinoids of natural or synthetic origin, have been shown to be potent inhibitors of certain immune system components and act as anti-inflammatory agents in vitro and in vivo through four main mechanisms: induction of apoptosis, inhibition of cell proliferation, inhibition of cytokines and chemokine production, and via the induction of regulatory T cells.
  • Cannabinoids that act at the CB2 receptor which is highly expressed on some immune cells, have shown promise to reduce immune system dysregulation. CB2 is upregulated following infection, trauma and inflammation, and activation of CB2 by cannabinoids is associated with inhibition of immune cells, particularly those cells involved in the innate immune response, and pro-inflammatory mediators. Development of potent and selective CB2 receptor cannabinoid ligands affords the opportunity to activate CB2 receptor independent of CB1 receptor behavioral effects.
  • HU308 (also known by International Nonproprietary Name (INN) as ontemabez; [(lR,2R,5R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4- bicyclo[3.1.1]hept-3-enyl]methanol) is a synthetically derived analog of cannabidiol and an example of a potent, selective agonist for the CB2 receptor.
  • INN International Nonproprietary Name
  • HU308 delivered via various routes of delivery has shown efficacy in reducing inflammation and immune system hyperactivation in experimental models of sepsis and septic lung injury as well as in organspecific models of inflammatory disease, highlighting the potential of HU308 and selective CB2 ligands for the treatment of systemic inflammatory responses and sepsis.
  • cannabinoids such as HU308 typically have poor drug like properties and are generally very lipophilic small molecules with tow kinetic solubility ( ⁇ 1 pg/ml) making preparation of a formulation suitable for delivery to animals and humans difficult. For these reasons and others, cannabinoids are often considered poor candidates for parenteral formulation to patients.
  • Parenteral formulations are sterile preparations used to deliver drugs, for example, but not limited to intravenously, subcutaneously, intraperitoneally or intramuscularly, and include solutions, suspensions or emulsions for injection or infusion.
  • Parenteral formulations afford advantages over enteral routes of delivery in that parenteral drug formulations can be used in conscious or unconscious patients providing rapid drug delivery with predictable pharmacokinetics largely avoiding the first-pass metabolism and unpredictable bioavailability seen with some enteral drugs (e.g. oral, oro-mucosal).
  • Some of the issues that needed to be addressed in the development of a parenteral cannabinoid formulation include finding a solvent that lipophilic cannabinoids can be dissolved in at high enough concentration for therapeutic efficacy but that is also safe to use in intravenous formulations, testing a number of emulsifiers that can form stable nanoparticles that are under 200 nanometers which permits terminal filter sterilization and finally determining the ideal antioxidants and other stabilizers so that the formulation will have sufficient stability.
  • the present invention relates to pharmaceutical compositions comprising cannabinoids suitable for parenteral delivery.
  • the present invention also relates to methods of production and uses of the compositions.
  • a pharmaceutical composition comprising ontemabez, a first solvent, and a first emulsifier.
  • the composition comprises water. In further embodiments, the composition is a nanoemulsion. [0016] In some embodiments, the pharmaceutical composition is sterilized. In further embodiments, the pharmaceutical composition is sterilized by fdtration through a filter. In even further embodiments, the filter has a pore size of about 0.2 pm or about 0.22 pm.
  • the nanoemulsion is a suspension of nanoparticles in water.
  • a mean diameter of the nanoparticles is about 100-400 nanometers (nm).
  • the mean diameter may be about 120-250 nm, for example about 200 nm. In a further embodiment, a mean diameter below about 200 nm is preferred.
  • the pharmaceutical composition comprises about 0.1-about 3% w/w ontemabez, such as, but not limited to, about 1 %w/w ontemabez or about 2 %w/w ontemabez.
  • the first solvent is soybean oil, or medium-chain triglyceride (MCT) oil.
  • MCT oil may comprise Miglyol 812N.
  • the pharmaceutical composition further comprises a second solvent.
  • the second solvent may be soybean oil, or medium-chain triglyceride (MCT) oil.
  • MCT medium-chain triglyceride
  • a total solvent content of the pharmaceutical composition is about 5-25 %w/w.
  • the total solvent content may be about 16.1 %w/w or about 20 %w/w, for example.
  • the first solvent is soybean oil and a total solvent content is about 20 %w/w.
  • the first solvent is soybean oil
  • the second solvent is MCT oil and a total solvent content is about 16.1 %w/w.
  • the pharmaceutical composition comprises about 15 %w/w of soybean oil and about 1.1 %w/w MCT oil.
  • the first emulsifier is egg lecithin or polyoxyl 15- hydroxystearate. In further embodiments, the first emulsifier is egg lecithin. In still further embodiments, the compositions further comprise a co-emulsifier, such as, but not limited to, sodium oleate. In some embodiments, the first emulsifier is polyoxyl 15-hydroxystearate and the co-emulsifier is sodium oleate. In further embodiments, the first emulsifier is egg lecithin at a concentration of about 1.2 %w/w.
  • the first emulsifier is polyoxyl 15 -hydroxy stearate at a concentration of about 4.4 %w/w
  • the co-emulsifier is sodium oleate at a concentration of about 0.48 %w/w.
  • compositions as described herein may further comprise a first preservative.
  • the first preservative may be EDTA, benzyl alcohol or sodium benzoate.
  • Some embodiments may further comprise a second preservative.
  • the second preservative may be EDTA, benzyl alcohol or sodium benzoate, wherein the first preservative and the second preservative are different.
  • the first preservative is EDTA at a concentration of about 0.01 %w/w.
  • the first preservative is benzyl alcohol at a concentration of about 0.15 %w/w
  • the second preservative is sodium benzoate at about 0.10 %w/w.
  • compositions may further comprise alpha-tocopherol.
  • the compositions comprise about 0.03 %w/w alpha-tocopherol, although other concentrations are contemplated.
  • compositions further comprise glycerol.
  • the compositions may comprise about 2.25 %w/w glycerol.
  • compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w soybean oil; about 4.40 %w/w polyoxyl 15-hydroxystearate; about 0.48 %w/w sodium oleate; about 2.25 %w/w glycerol; about 0.03 %w/w alphatocopherol; about 0.01 %w/w disodium EDTA; and about 70.84 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
  • compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w soybean oil; about 4.40 %w/w polyoxyl 15-hydroxystearate; about 0.48 %w/w sodium oleate; about 2.25 %w/w Glycerol; about 0.03 %w/w alphatocopherol; about 0.15 %w/w benzyl alcohol; about 0.10 %w/w sodium benzoate; and about 70.59 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
  • compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w soybean oil; about 1.20 %w/w egg lecithin; about 2.25 %w/w glycerol; about 0.03 %w/w alpha-tocopherol; about 0.01 %w/w disodium EDTA; and about 74.52 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
  • compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w egg lecithin; about 2.25 %w/w glycerol; about 0.03 %w/w alpha-tocopherol; about 0.15 %w/w benzyl alcohol; about 0.10 %w/w sodium benzoate; and about 74.27 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
  • Methods for preparation of nanoemulsions comprising nanoparticles of ontemabez, a first solvent, a first emulsifier and water, are disclosed herein.
  • the methods comprise: mixing the ontemabez, the first solvent, the first emulsifier and water to make a crude emulsion, homogenizing the cmde emulsion under high pressure; and repeating the homogenizing step at least about five times.
  • a diameter of the nanoparticles is about 100-400 nanometers (nm). In some cases, the diameter is about 150-250 nm, such as 200 nm.
  • high pressure is about 700-2000 bar.
  • the high pressure may be about 750-1500 bar, such as, but without wishing to be limiting, about 750 bar or about 1500 bar.
  • repeating comprises undertaking the homogenizing step at least ten times. In some embodiments, repeating comprises undertaking the homogenizing step up to at least twenty times.
  • Some embodiments further comprise cooling the crude emulsion after homogenizing. Cooling may comprise cooling with an ice-water bath. In further embodiments, repeating comprises undertaking the homogenizing and cooling steps. In some embodiments, repeating comprises undertaking the homogenizing and cooling steps at least ten times.
  • the methods further comprise adjusting the pH prior to homogenization.
  • adjusting may comprise adjusting the pH to 7.5.
  • the methods comprise premixing ontemabez, the first solvent, and the first emulsifier, prior to the mixing step. Some such embodiments further comprise heating the premixed components and/or heating the water, prior to the mixing step. Heating may comprise heating to about 70°C, but other temperatures are also contemplated.
  • Uses of the pharmaceutical compositions are also disclosed herein. For example, use of any of the compositions for parenteral administration is contemplated. Use of the compositions for the treatment of systemic inflammation responses is contemplated herein.
  • the systemic inflammation response is one or more of: systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), or multiple organ dysfunction syndrome (MODS).
  • SIRS systemic inflammatory response syndrome
  • CRS cytokine release syndrome
  • CSS cytokine storm syndrome
  • ARDS acute respiratory distress syndrome
  • MODS multiple organ dysfunction syndrome
  • FIGURES 1A-F show the appearance of the nanoemulsion preparations and microscopic views of the crude primary emulsion, and nanoemulsions obtained at 750 bar and 1500 bar high pressure homogenizations.
  • Fig. 1 A depicts appearance of the lecithinbased reference nanoemulsion and priority placebo DOE (Design of Experiments) Formulation #1 nanoemulsion after high pressure homogenization.
  • Fig. IB is a microscopic image (X400 magnification) of a crude primary emulsion of priority placebo DOE Formulation #1 after high speed mixing at 10,000 rpm for 2 minutes.
  • Fig. 1C is a first microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed at 750 bar for 10 cycles.
  • Fig. ID is a second microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed at 750 bar for 10 cycles.
  • Fig. IE is a first microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed further at 1500 bar for 10 cycles.
  • Fig. IF is a second microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed further at 1500 bar for 10 cycles.
  • FIGURES 2A-D are microscopic images of the reference nanoemulsion batch.
  • Fig. 2A is a first microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 750 bar for 10 cycles.
  • Fig. 2B is a second microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 750 bar for 10 cycles.
  • Fig. 2C is a first microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 1500 bar for 10 cycles.
  • Fig. 2D is a second microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 1500 bar for 10 cycles.
  • FIGURES 3A-B are particle size analyses for the reference nanoemulsion batch processed at 750 bar for 10 cycles (Fig. 3A) and the priority placebo DOE Formulation #1 processed at 750 bar for 10 cycles (Fig. 3B).
  • FIGURE 4 is a graph depicting the effect of formulation composition and high pressure homogenization (HPH) cycle on DOE nanoemulsion formulation droplet size.
  • FIGURE 5 shows appearance of priority ontemabez DOE nanoemulsion batches #1, 3, 6 and 8 (left to right) processed at 1500 bar high pressure homogenization for 20 cycles.
  • FIGURES 6A-D show microscopic appearances of ontemabez DOE nanoemulsion batches at 200X magnification.
  • Fig. 6A shows microscopic appearance of ontemabez DOE Formulation #3, nanoemulsion batch at X200 magnification.
  • Fig. 6B shows microscopic appearance of ontemabez DOE Formulation #6, nanoemulsion batch at X200 magnification.
  • Fig. 6C shows microscopic appearance of ontemabez DOE Formulation #1, nanoemulsion batch at X200 magnification.
  • Fig. 6D shows microscopic appearance of ontemabez DOE Formulation #8, nanoemulsion batch at X200 magnification.
  • FIGURES 7A-C show results of IL-6 production (Fig 7A), Ashcroft score (Fig 7B) and hydroxyproline content (Fig 7C) for various test groups treated with vehicle or HU308, respectively.
  • FIGURE 15 shows results of BHK-21 cells infected with sequential dilutions of hCoV-OC43 produced by infected cells cotreated with HU308 at various concentrations.
  • Top horizontal axis depicts hCoV-OC43 dilution factor; left vertical axis depicts conditions that virus-producing cells were exposed to; right vertical axis depicts viral titer relative to control conditions.
  • FIGURE 16 shows results of BHK-21 cells infected with sequential dilutions of hCoV-OC43 produced by infected cells cotreated with HU308 at various concentrations.
  • Top horizontal axis depicts hCoV-OC43 dilution factor; left vertical axis depicts conditions that virus-producing cells were exposed to; right vertical axis depicts viral titer relative to control conditions.
  • FIGURE 17 shows results of health score analysis at Day 6 post-infection with SARS in groups treated daily with vehicle (VEH) or HU308 (HU; 3 or 10 mg/kg/day).
  • VH vehicle
  • HU308 HU; 3 or 10 mg/kg/day.
  • N 6 - 9 mice/group. ***, P ⁇ 0.0005; ****, P0.0001.
  • FIGURES 18A-C show results of sera analysis allowing quantification of monocyte chemoattractant protein-1 (MCP-1) (Fig 18A), inflammatory interferon gamma (INFg) (Fig 18B), and Interleukin 6 (Fig 18C) at the point of infection (Day 0), as well as Day 3 and Day 6 post-infection in groups treated daily with either parenteral (i.p.) vehicle (Control), Remdesivir (REM; 10 mg/kg/day) or HU308 (HU; 3 or 10 mg/kg/day). ** PO.Ol.
  • MCP-1 monocyte chemoattractant protein-1
  • IFNg inflammatory interferon gamma
  • Fig 18C Interleukin 6
  • FIGURES 19A-D show results of BALF analysis allowing quantification of tumor necrosis factor-alpha (TNFa or TNF-a) (Fig 19A), interferon gamma (IFNg or IFNy) (Fig 19B), interleukin 6 (IL-6) (FIG 19C), or interleukin 10 (IL-10) (FIG 19D) at Day 3 and Day 6 post-infection in groups treated daily with either parenteral (i.p.) vehicle (VEH), Remdesivir (REM, 10 mg/kg/day) or HU308 (HU; 3 or 10 mg/kg/day).
  • TNFa or TNF-a tumor necrosis factor-alpha
  • IFNg or IFNy interferon gamma
  • IL-6 interleukin 6
  • IL-10) interleukin 10
  • FIGURE 29 shows results of Interleukin- 1 -alpha levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats.
  • Saline IVT intravitreal
  • n 6, 200 ng LPS IVT
  • n 7, 3 mg/kg HU308 IV
  • n 7.
  • FIGURE 30 shows results of Interferon-gamma levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats.
  • Saline IVT intravitreal
  • n 6, 200 ng LPS IVT
  • n 7, 3 mg/kg HU308 IV
  • n 7.
  • FIGURE 31 shows results of Vascular Endothelial Growth Factor levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats.
  • Saline IVT intravitreal
  • FIGURE 32 shows results of Interleukin-4 levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats.
  • Saline IVT Intravitreal
  • FIGURE 33 shows results of Tumor Necrosis Factor- alpha levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats.
  • Saline IVT intravitreal
  • n 6, 200 ng LPS IVT
  • n 7, 3 mg/kg HU308 IV
  • n 7.
  • FIGURE 36 shows results of intestinal intravital microscopy allowing quantification of leukocytes per square millimeter of vascular endothelium in VI venules at 6-hours following disease induction.
  • (*) represents P ⁇ 0.05
  • (***) represents P ⁇ 0.001.
  • FIGURE 37 shows results of histopathologic scoring of lung tissue collected at 6- hours following disease induction. Increasing score indicates more severe disease. (**) represents P ⁇ 0.01.
  • FIGURE 38 shows results of levels of the pro-inflammatory mediator, Interleukin-6, in peripherally obtained plasma at 6-hours following pulmonary administration of LPS.
  • CON Control
  • LPS lipopolysaccharide
  • HU HU308.
  • (*) represents P ⁇ 0.05.
  • FIGURE 39 shows results of the levels of the pro-inflammatory mediator, CXCL2, in peripherally obtained plasma at 6-hours following pulmonary administration of LPS. CON; Control, LPS; lipopolysaccharide, HU; HU308. (*) represents P ⁇ 0.05.
  • FIGURE 40 shows results of the levels of the pro-inflammatory mediator, TNF- alpha, in peripherally obtained plasma at 6-hours following pulmonary administration of LPS. CON; Control, LPS; lipopolysaccharide, HU; HU308. (**) represents P ⁇ 0.01, (***) represents P ⁇ 0.001.
  • compositions, formulations, methods of production and uses relating to injectable formulations of cannabinoids are described by way of example. Described herein are compositions, formulations, methods of production and uses relating to injectable formulations of cannabinoids. It will be appreciated that embodiments and examples are provided for illustrative purposes intended for those skilled in the art, and are not meant to be limiting in any way. All references to embodiments, examples, aspects, formulas, compounds, compositions, solutions, kits and the like is intended to be illustrative and non-limiting.
  • HU308 and ontemabez are used interchangeably herein.
  • Described herein is the development and manufacture of a sterile nanoemulsion of ontemabez for parenteral administration to a subject, for example, a mammalian subject, more preferably a human subject.
  • a subject for example, a mammalian subject, more preferably a human subject.
  • Embodiments of the formulations described herein may be employed, for example, but not limited to, for parenteral delivery in the treatment of systemic inflammation responses such as SIRS, sepsis, severe sepsis, septic shock, CRS & CSS, ARDS and MODS.
  • Embodiments of the formulations may comprise at least one solvent, such as soybean oil, an emulsifier, such as egg lecithin or polyoxyl 15-HS, and sodium EDTA as a preservative and homogenized with the required pressure and cycles to achieve stable nanoparticles that allow sterilization by filtration.
  • solvent such as soybean oil
  • an emulsifier such as egg lecithin or polyoxyl 15-HS
  • sodium EDTA as a preservative and homogenized with the required pressure and cycles to achieve stable nanoparticles that allow sterilization by filtration.
  • cannabinoid may refer to C21 or C22 terpenophenolic compounds, or carboxylic acids, analogs and transformation products thereof. Cannabinoid may also refer to endogenous or artificial compounds that bind to cannabinoid receptors, such as CB1 and/or CB2. An example of a suitable cannabinoid is HU308 or ontemabez.
  • solvent may refer to aqueous or organic fluids that dissolve or otherwise solvate one or more desired compounds, such as a cannabinoid.
  • the solvent may be in a non-liquid form, such as in frozen, dried, dehydrated or lyophilized compositions. Such non-liquid solvents may be rehydrated or melted prior to use.
  • Emulsion as used herein may be understood as a fine dispersion of droplets of one or more non-soluble or immiscible liquids. Emulsions are made through the mixing of a two-phase system (such as oil and water) to form a single phase. In some cases, emulsifying agents are used. Emulsions may be categorized by the size of the droplets within the emulsion. For example, a typical oil-in-water emulsion or “macroemulsion” may have a lipid droplet size greater than 1 micrometer (1 pm). The term “nanoemulsion” may refer to emulsions with a lipid droplet size generally between 10 and 1000 nanometers (nm).
  • emulsifier may refer to an additive that acts to stabilize an emulsion.
  • mean diameter as used herein may be understood as the mean value of a population of particles, such as the lipid droplets in a nanoemulsion.
  • Mean diameter may be measured by a suitable technique, such as dynamic light scattering (DLS).
  • the mean diameter may be measured as the mean value of a particle size distribution of the population of nanoparticles.
  • sterile or “sterilized” as used herein may refer to compositions that are substantially free of viable or reproducible microorganisms or harmful biological agents.
  • microorganisms include fungi, bacteria, viruses, spores, protozoa and others.
  • harmful biological agents includes prions, protein-based toxins and others.
  • %w/w will be understood by a person of skill in the art as a “weight concentration”.
  • the weight concentration is calculated by [weight of the component (g)/total weight of the solution (g)] x 100%.
  • the remaining balance comprises water (unless otherwise specified, such as in lyophilized embodiments).
  • %w/v will be understood by a person of skill in the art as a “weight per volume concentration”.
  • the weight per volume concentration is calculated by [weight of the component (g)/total volume of the solution (ml)] x 100%.
  • compositions and formulations for parenteral delivery of pharmaceuticals that exhibit poor solubility in aqueous solvent, such as water.
  • Embodiments of the compositions comprise: ontemabez, a first solvent, and a first emulsifier.
  • the pharmaceutical compositions described herein may be suitable for parenteral administration.
  • Some embodiments of the pharmaceutical compositions are formulated as a nanoemulsion.
  • the compositions also comprise an aqueous phase, such as water.
  • the first solvent may act as the non-aqueous phase of the emulsion and form droplets/nanoparticles.
  • the compositions comprise water. Unless otherwise specified, water may comprise the majority of the composition by weight. In some cases, 50 %w/w or greater of the compositions is water. In some cases, about 0.01- ⁇ 100 %w/w or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as about 70 to less than 100 %w/w of water may be used.
  • the pharmaceutical compositions are dried such that there is no appreciable aqueous phase present.
  • the pharmaceutical compositions may be freeze dried, lyophilized or the aqueous phase is removed via vacuum.
  • nanoemulsions described herein may be understood as a suspension of nanoparticles in water.
  • the nanoparticles may be droplets of solvent/oil that dissolve lipophilic molecules, such as ontemabez, and are suspended in an immiscible solvent, such as water.
  • the nanoparticles may have a suitable mean diameter, for example about 100-400 nanometers (nm).
  • the mean diameter may be about 220 nm or less, such as about 200 nm or less.
  • the mean diameter is about 150-250 nm, such as about 200 nm.
  • a range of about 0. 1-500 nm or any value therebetween (optionally rounded to the nearest 0. 1), or any sub-range spanning between any two of these values, such as 0.1-220 nm, may be suitable.
  • the pharmaceutical composition may be sterilized by a suitable method.
  • sterilization occurs after the nanoemulsion is formed.
  • Sterilization methods include heat treatment via a suitable method such as an autoclave or dry heating, filter sterilization, radiation, and others.
  • the formulations are filter sterilized through a suitable filter.
  • the filter may have a pore size that is sized to allow for the passage of nanoparticles and prevent the passage of harmful biological agents. For example, the filter pore size is about 0.2 pm or about 0.22 pm.
  • a range of 0.1-200 nm or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values is also contemplated, for example, pore sizes of 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30 pm and others are considered.
  • the pharmaceutical compositions described herein comprise at least one cannabinoid.
  • the cannabinoid is ontemabez.
  • Other compounds with suitable solubility profiles may be used without departing from the invention, such as other cannabinoids.
  • Suitable concentrations of cannabinoids in the compositions may be about 1-3 %w/w. In some cases, a range of 0.01-10 %w/w or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 0.1-3 %w/w may be used.
  • the pharmaceutical compositions described herein comprise a first solvent.
  • the first solvent is an oil suitable for dissolving a lipophilic molecule.
  • the first solvent may be soybean oil, or medium-chain triglyceride (MCT) oil.
  • MCT oil may comprise one or more medium-chain triglycerides, such as Miglyol 812N.
  • the first solvent/oil may be refined, such that they are suitable for parenteral delivery.
  • Other solvents may be used, such as glycerin, propylene glycol, ethanol, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, castor oil, polysorbate, polyoxyl 40 stearate, PEG 400 or others.
  • the first solvent may be suitable and safe for parenteral delivery.
  • soybean oil may be a preferred first solvent.
  • the second solvent may be soybean oil, or medium-chain triglyceride (MCT) oil, with the first solvent being different from the second solvent.
  • MCT medium-chain triglyceride
  • the second solvent may be miscible with the first solvent to provide a continuous phase.
  • the MCT oil may comprise one or more mediumchain triglycerides, such as Miglyol 812N.
  • the second solvent/oil may be refined, such that it is suitable for parenteral delivery.
  • Other solvents may be used, such as glycerin, Propylene glycol, Ethanol, Polyoxyl 35 castor oil, Polyoxyl 40 hydrogenated castor oil, Castor oil, Polysorbate, Polyoxyl 40 stearate, PEG 400 or others.
  • MCT oil may be a preferred second solvent.
  • the pharmaceutical compositions described herein may have a total solvent content.
  • the total solvent content may be varied to accommodate different concentrations of solute, such as ontemabez.
  • the total oil content may be increased to dissolve a higher amount of solute.
  • the total solvent content of the pharmaceutical composition is about 5-25 %w/w, such as, without limitation, about 16.1 %w/w or about 20 %w/w.
  • about 0.1-50 %w/w or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning any two of these values, such as 5-25 %w/w total solvent content may be used.
  • the first solvent is soybean oil and a total solvent content is about 20 %w/w.
  • the first solvent is soybean oil, the second solvent is MCT oil and a total solvent content is about 16.1 %w/w, such as about 15 %w/w of soybean oil and about 1.1 %w/w MCT oil.
  • the pharmaceutical compositions described herein comprise a suitable first emulsifier.
  • a suitable first emulsifier may be any emulsifier that acts to stabilize the emulsion that forms from mixing the one or more solvents with water.
  • the first emulsifier may be egg lecithin, polyoxyl 15 -hydroxy stearate or sodium oleate. In one embodiment, the first emulsifier is egg lecithin.
  • the pharmaceutical compositions described herein may comprise more than one emulsifier.
  • the second emulsifier is egg lecithin, polyoxyl 15- hydroxystearate or sodium oleate, and the second emulsifier is different from the first emulsifier.
  • the first emulsifier is polyoxyl 15 -hydroxy stearate and the second emulsifier is sodium oleate.
  • the first and second emulsifier may have a suitable concentration or range of concentrations.
  • the concentration of the first or second emulsifier is about 0.1-10 %w/w, such as about 1.2 %w/w, 4.4 %w/w or about 0.48 %w/w. In some cases, about 0. 1-50 %w/w or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as 0.1-15 %w/w may be used.
  • the first emulsifier is egg lecithin at a concentration of 1.2 %w/w. In another embodiment, the first emulsifier is polyoxyl 15-hydroxystearate at a concentration of 4.4 %w/w, and the coemulsifier is sodium oleate at a concentration of 0.48 %w/w.
  • the pharmaceutical compositions described herein may comprise one or more preservatives.
  • a preservative may be understood as a component that extends the shelf-life of the composition, such as by protecting the active ingredient from damage caused by oxidation, radiation or other sources.
  • the first preservative may be EDTA, benzyl alcohol, sodium benzoate, sorbic acid or others. Preservatives may also have antimicrobial properties to prevent spoilage. Suitable preservatives known in the art may be used, for example those found in Meyer et al., Antimicrobial preservative use in parenteral products: past and present. J Pharm Sci. 2007, 96(12):3155-67, herein incorporated by reference.
  • a second preservative is used. Suitable second preservatives include EDTA, benzyl alcohol or sodium benzoate.
  • the first and second preservative may have a suitable concentration or concentration range.
  • the concentration of the first or second preservative is about 0.1-10 %w/w, such as about 0.01 %w/w, 0.15 %w/w or about 0.1 %w/w.
  • about 0.1-50 %w/w or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as about 0.1-15 %w/w may be used.
  • the first preservative is EDTA at a concentration of about 0.01 %w/w.
  • the first preservative is benzyl alcohol at a concentration of about 0.15 %w/w
  • the second preservative is sodium benzoate at about 0.10 %w/w.
  • compositions described herein may comprise additional components.
  • a suitable antioxidant such as alpha-tocopherol. Suitable antioxidants may prevent the oxidation of lipids or other ingredients in the compositions.
  • the compositions comprise an isotonic agent, such as glycerol.
  • the components may be at a suitable concentration or concentration range. In some cases, about 0.1-5 %w/w or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as 0.1-5 %w/w may be used.
  • the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil as a first solvent; about 4.40 %w/w Polyoxyl 15 -Hydroxy stearate as a first emulsifier; about 0.48 %w/w Sodium Oleate as a second emulsifier; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.01 %w/w Disodium EDTA; and about 70.84 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
  • the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 4.40 %w/w Polyoxyl 15 -Hydroxy stearate; about 0.48 %w/w Sodium Oleate; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.15 %w/w Benzyl Alcohol; about 0.10 %w/w Sodium Benzoate; and about 70.59 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
  • the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w Egg Lecithin; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.01 %w/w Disodium EDTA; and about 74.52 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
  • the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w Egg Lecithin; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.15 %w/w Benzyl Alcohol; about 0.10 %w/w Sodium Benzoate; and about 74.27 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
  • the pharmaceutical composition described herein may optionally further comprise a pharmaceutically acceptable excipient, diluent, or carrier.
  • a pharmaceutically acceptable excipient examples of such pharmaceutically acceptable excipients, diluents, and carriers may be found in Remington: The Science and Practice of Pharmacy (2012).
  • examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999, each of which are herein incorporated by reference in their entireties.
  • a pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art.
  • suitable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch.
  • pharmaceutically acceptable excipients may include suitable fdlers, binders, lubricants, buffers, glidants, and disintegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2012)).
  • Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • Cannabinoid compounds such as ontemabez, may be prepared using synthetic methodology known in the art, for example US6903137, which is herein incorporated by reference.
  • the initial components are mixed to form a crude emulsion.
  • Mixing may comprise simple mixing via a mechanical stirrer or magnetic stir bar.
  • This may also be known as macro-emulsification.
  • Methods of macro-emulsification known in the art may be used, for example: Goodarzi, F. and Zendehboudi, S. (2019), A Comprehensive Review on Emulsions and Emulsion Stability in Chemical and Energy Industries. Can. J. Chem. Eng., 97: 281-309, which is herein incorporated by reference.
  • two or more components of the compositions are pre-mixed prior to the mixing step (macro-emulsification).
  • the first solvent and the alpha-tocopherol are mixed together prior to macro-emulsification.
  • all of the non-aqueous components are pre-mixed prior to the macroemulsification step.
  • one or both of the premixed components and the water are heated.
  • the water and premixed components may be heated to a suitable temperature, for example 70°C.
  • Suitable heating temperatures include a range of 20-100°C or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 60-75°C. For example, temperatures of 20, 20.5, 21, 21.5, 22,
  • the crude emulsion is homogenized using a high pressure homogenizer (HPH).
  • HPH high pressure homogenizer
  • High pressure homogenization techniques are known in the art, for example in Yadav, K.S., Kale, K. High Pressure Homogenizer in Pharmaceuticals: Understanding Its Critical Processing Parameters and Applications. J Pharm Innov 15, 690-701 (2020), which is herein incorporated by reference.
  • Homogenization or high pressure homogenization may be conducted at various pressures. For example, a range of 700-2000 bar or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 750-1500 bar. Homogenization at other pressures is also contemplated, for example, but not limited to 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,
  • the homogenizing steps may be repeated until the desired mean diameter of the nanoparticles is achieved.
  • the homogenizing steps may be repeated at least five times, such as ten, fifteen or twenty times.
  • the homogenizing step may be repeated at least 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,
  • repeating comprises undertaking the homogenizing step at least ten times.
  • the crude emulsion may be cooled after the homogenizing step. Cooling the macroemulsion may occur when the temperature of said emulsion rises too high during high- pressure homogenization.
  • the crude emulsion may be cooled using conventional techniques, such as cooling in a cooling solution, for example an ice-water bath. Cooling may be used if the temperature rises above 45°C, for example 50°C.
  • repeating comprises undertaking both of the homogenizing and cooling steps. For example, the homogenizing and cooling steps may be repeated at least five times, such as ten, fifteen or twenty times.
  • the homogenizing and cooling step may be repeated at least 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,
  • repeating comprises undertaking the homogenizing step and cooling step at least ten times.
  • the pH of the crude emulsion may be adjusted prior to or after homogenization.
  • pH is adjusted after homogenization.
  • the pH may be adjusted to 7.5.
  • the pH may be adjusted using conventional techniques, such as the addition of acid or base.
  • a pH range of 5-8 or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 7-7.5 may be used.
  • a pH of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or others may be suitable.
  • compositions as described herein may be used for parenteral administration.
  • the compositions may be formulated as a nanoemulsion in water with a nanoparticle mean diameter sufficient for filter sterilization prior to administration.
  • the compositions may be used to treat systemic inflammation responses.
  • ontemabez may act as a selective agonist for the CB2 receptor, which has been linked to inhibition of immune cell activity, particularly those cells involved in the innate immune response, and pro-inflammatory mediators.
  • the compositions as described herein may be used as anti-inflammatory agents.
  • compositions described herein may be used for the treatment or prevention of systemic inflammation responses.
  • suitable systemic inflammation responses include: systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), or multiple organ dysfunction syndrome (MODS).
  • SIRS systemic inflammatory response syndrome
  • CRS cytokine release syndrome
  • CSS cytokine storm syndrome
  • ARDS acute respiratory distress syndrome
  • MODS multiple organ dysfunction syndrome
  • the composition may be used for treating a bacterial infection or a viral infection.
  • Ontemabez solubility was first evaluated in aqueous solvents. Solubility of ontemabez in aqueous buffers ranged from 0.000032 mg/mL to 0.000775 mg/mL (Table 1 A). These results indicated that a more complex formulation is required to achieve a target concentration of 10-20 mg/mL.
  • Table 1A Solubility of ontemabez in various aqueous buffers
  • Example 2 Preliminary nanoemulsion composition and process for placebo nanoemulsions
  • Implementation of the nanoemulsion procedures known in the art involves the preparation of a primary emulsion using conventional macro emulsification technique followed by 2-stage high pressure homogenization.
  • the primary emulsification process the Lipid Phase Components (LPC) and the Aqueous Phase Components (APC) are combined to form the primary emulsion using convention mixing with an overhead stirrer and propeller blade.
  • the primary emulsion is subsequently subjected to high pressure homogenization for further processing to generate the nanoemulsion.
  • Table 2 List of excipient raw materials for an injectable nanoemulsion formulation of ontemabez:
  • Example 2.2 Reference nanoemulsion batch preparation
  • the nanoemulsion formulation composition comprises 5%w/v soybean oil, 5%w/v MCT, 1.2%w/v egg lecithin, 2.5%w/v glycerol, and QS with water to 100%.
  • a crude emulsion was prepared using a Pro Scientific PRO250 mixer fitted with a 30 mm x 200 mm generator probe. The crude emulsion was subjected to high pressure homogenization at 750 bar for 10 cycles. No cooling of the batch between the HPH cycles was required at this pressure setting. A milky white, uniform and opaque nanoemulsion was obtained (Fig. 1 A).
  • a portion of the nanoemulsion was removed and set aside for microscopic and droplet size analysis. The remaining material was then processed at 1500 bar for an additional 10 cycles. The batch required cooling using an ice-water bath as the temperature rose up to 51 °C after each cycle of homogenization. The nanoemulsion remained the original milky white appearance.
  • Example 2.3 Preparation of priority placebo DOE nanoemulsion formulation
  • Priority DOE nanoemulsion formulation #1 whose composition consists of 20%w/v soybean oil, 1.2%w/v egg lecithin, 2.25%w/v glycerol, and QS with water to 100% (Table 3), was used.
  • the crude emulsion was prepared as before and was subjected to high pressure homogenization at 750 bar for 10 cycles. A portion of the processed material was set aside for microscopic examination (Figs. 1C-1D) and droplet size analysis (Fig. 3B). The temperature of the batch increased to a maximum of 44°C during the 10 cycles of processing and did not require cooling. Another portion of the crude emulsion was also processed at 1500 bar for 20 cycles.
  • the nanoemulsion obtained was milky white and opaque in appearance (Fig. 1A).
  • nanoemulsion was then processed at 1500 bar for an additional 10 cycles.
  • the batch required cooling using an ice-water bath as the temperature rose to 51 °C after each cycle of homogenization.
  • the nanoemulsion remained milky white and opaque at the end of the processing at 1500 bar. Samples of the nanoemulsions were subjected to bright field microscopic and droplet size evaluations.
  • Placebo Runs #5,8, 7,10 Comparison of cycle numbers and formulation composition [00150] Based on Run #1 results, four additional priority placebo DOE nanoemulsion formulation batches, Runs #5, 7, 8 and 10, were prepared by following the composition and a modified procedure outlined in Table 3. Briefly, the crude emulsions were prepared essentially as for Run#l, except the high-speed mixing was performed at 18,000 rpm to further reduce the droplet size before HPH. The HPH was performed at 1500 bar for up to 20 cycles and samples were collected for droplet size analysis at 0, 5, 10, 15 and 20 cycles. These four additional formulation batches were set up to test the performance of the composition of the oil fraction and the emulsifier(s) used for dispersion (Table 4).
  • Table 4 Priority placebo DOE nanoemulsion trials (Runs #5, 7, 8, 10)
  • DOE Run #5 (contains 20% soybean oil and Polyoxyl 15-hydroxystearate/sodium oleate) and DOE Run #8 (contains 15% Soybean Oil, 1.1% Miglyol 812N and 1.2% Egg Lecithin) could be filtered with the PES membrane filter. Subsequent DLS results confirming no significant changes in droplet size following PES membrane filtration of DOE Runs #5 and #8 are presented in Table 6.
  • Example 3 Formulation of priority developmental DOE batch nanoemulsions containing ontemabez
  • the four priority API DOE nanoemulsions were prepared following the same procedure as for priority placebo DOE batches to obtain preliminary information on drug loading capacity, droplet size and filterability. Crude emulsion preparation and HPH were performed as with the priority placebo DOE batches. Samples were taken at the same intermediate steps during HPH processing (1, 5, 10, 15 and 20 cycles) for comparability and tested for droplet size and ontemabez concentration by HPLC.
  • Fig. 6A shows microscopic appearance of ontemabez DOE Formulation #3, nanoemulsion batch at X200 magnification.
  • Fig. 6B shows microscopic appearance of ontemabez DOE Formulation #6, nanoemulsion batch at X200 magnification.
  • Fig. 6C shows microscopic appearance of ontemabez DOE Formulation #1, nanoemulsion batch at X200 magnification.
  • Fig. 6D shows microscopic appearance of ontemabez DOE Formulation #8, nanoemulsion batch at X200 magnification.
  • Results from ontemabez DOE nanoemulsion show that the smallest droplet size and only formulation consistently meeting the preestablished size criterion ( ⁇ 200 nm) was achieved with the formulation containing the polyoxyl 15-hydroxystearate+sodium oleate emulsifier combination (Table 8).
  • the HPLC assay results for all batches (Table 8) demonstrated good incorporation of ontemabez in the nanoemulsion.
  • Table 9 Pre- and post-PES membrane filtration HPLC assay and droplet size analysis for placebo and ontemabez DOE nanoemulsion batches a Pre- or post-filtration droplet diameter after high pressure homogenization at 1500 bar for 20 cycles
  • Prototypes 1A, IB, 2A and 2B were prepared by following the composition and procedure outlined in Table 10 below and tested for droplet size distribution, filterability and stability over a 3 week period. Results are shown in Table 11. [00167] Table 10: Composition of Prototype 1A, IB, 2 A and 2B formulations, and processing parameters for HPH, filtration and stability testing
  • A Change between values at Time 0 (TO) compared to 12 months (12mo), calculated as T0-12mo.
  • the particle size of formulation containing 2% ontemabez/20% soybean oil could remain well below 200 nm with the emulsification system of Polyoxyl HS- 15 + sodium oleate. (See priority API DOE Run #6 in Table 8 and Prototypes 1A and IB in Table 10).
  • Example 5 HU3Q8/Ontemabez as a therapeutic candidate for the treatment of inflammatory disorders.
  • IL-6 Interleukin 6
  • TGF-a tumor necrosis factor alpha
  • TGF-B transforming growth factor beta
  • IL-6 is an inflammatory cytokine which can induce a variety of proteins involved in the acute phase response to injury, infection, or other sources of inflammation.
  • a histological analysis was performed using formalin-buffered and paraffin-embedded lung tissue samples acquired from the HU308 or vehicle-treated mice.
  • the histological analysis used a fibrosis grading scale ranging from 0 (“normal lung”) to 8 (“total fibrous obliteration of the field”).
  • This grading scheme known as the Ashcroft score, is a standard used to measure the severity and extent of fibrotic damage.
  • animals treated with HU308 every three days throughout the 21 -day post-BLM period displayed significantly lower fibrosis grades than those treated with a vehicle on the same schedule ( Figure 7).
  • these findings confirm that the previously identified HU308 mediated attenuation of inflammatory responses to chemical lung insult consequently reduce tissue damage in a significant manner.
  • HU308 appears to offer significant anti-inflammatory protection against chemical insult modeling IPF when administered intravenously over a variety of schedules. Further refinements to the administration regimen may further attenuate inflammatory signaling and resultant tissue damage.
  • parenteral formulations used in IV delivery of HU308 are notably deficient from a safety standpoint for application in human subjects and indications, necessitating development and refinement of drug products with key quality attributes in keeping with current regulatory requirements.
  • key quality attributes for an acceptable clinical formulation include (but are not limited to) an acceptable nonclinical safety profile; excipients at levels accepted by regulatory agencies for IV administration; desirable stability profiles; and compatibility with terminal sterilization techniques (including by filtration).
  • Example 6 HU3Q8/Ontemabez as an anti-viral and anti-inflammatory therapeutic.
  • Study 1 HU308 in vitro antiviral efficacy against infection with Influenza, Respiratory Syncytial Virus, Adenovirus Type-5, a-coronavirus, and -coronavirus.
  • Study 1 was conducted to determine the EC50 & CC50 of test compounds in an in vitro model of infection against 5 viral strains (Influenza, Respiratory Syncytial Virus, Adenovirus Type-5, a-coronavirus, and [3-coronavirus).
  • a dilution series of each test compound (8-point, 2-fold dose titration) was added to cells at various time-points.
  • Vehicle and positive control wells were set up to control for any influence of the compounds alone on cell viability.
  • Cells were visually inspected for the appearance of any cytopathic effects (CPE).
  • a cell viability assay was performed once CPE was complete.
  • EC50 is the concentration which results in 50% viral inhibition, following the addition of compounds.
  • CC50 is the concentration which results in 50% cell viability, following the addition of compounds.
  • Table 14 Cell and viral strains used in anti-infectivity studies.
  • Table 15 Test compounds used in anti-infectivity studies with respective concentration ranges. Text in parentheses indicates target viral strain for positive controls.
  • CBD cannabidiol
  • HU308 elicited inhibition of viral growth
  • Table 16 Summary of EC50 (pM) and CC50 (pM) values of CBD, HU308, or positive controls in the indicated viral infection models. In conditions where antiviral effects were observed but high drug concentration produced a decrease in cell viability that prohibited the calculation of an EC50, EC50 is listed as >CC50.
  • Study 2 HU308 antiviral and anti-inflammatory efficacy against infection with HIV-1 in human neurospheres.
  • Study 2 was conducted to address the potential of HU308 to alter viral and cytokine RNA expression in primary neurospheres infected with HIV-1 and treated with combined antiretroviral therapy (cART).
  • NPCs Normal Human Neural Progenitor Cells (ATCC® ACS-5003TM) were expanded per the manufacturer’s recommendations.
  • NPCs were seeded at high density in ultra-low attachment, U-shaped bottom 96-well plates in Neural Progenitor Medium. After approximately 48 hours, the medium was replaced with prodifferentiation medium to induce differentiation and replaced every 2 to 3 days for a period of 2 weeks.
  • Neurospheres were cultured in the presence of HIV-1 dual tropic 89.6
  • Combined antiretroviral therapy (cART) treatment was simulated by incubation of infected neurospheres for one week with a cocktail consisting of equal parts lamivudine, tenofovir disoproxil fumarate, emtricitabine, and indinavir at a concentration of 45pM.
  • a subset of cART-treated neurospheres were additionally treated with HU308 EE: 98.3%;
  • Exosomes were isolated from HIV-1 viral particles in neurosphere supernatants by sequential centrifugation, precipitation, and fdtration.
  • RNA was isolated utilizing TRIzol Reagent according to manufacturer’s instructions and subjected to RT-qPCR using TaqMan primers specific for TAR and HIV-1 genomic RNA. Copy number was quantified using serial dilutions of DNA from 8E5 positive control cells as quantitative standards and significance was determined using Student’s t-test.
  • Study 3 was conducted to address the antiviral potential of HU308 in cell lines infected with [3-coronavirus (OC43).
  • HU308 Hamster kidney fibroblasts (BHK-21) were expanded per the manufacturer’s recommendations.
  • HU308 (Tocris) was dissolved in DMSO to a stock concentration of 40 mM and stored at -80°C.
  • BHK-21 cells were infected with OC43 with an MOI of 0.1 for 1 hour. After the infection, HU308 was added to the cell culture medium and incubated for 23 h. At 24 h post infection cells and supernatant were collected and freeze-thawed to help liberate cell- associated virus into the medium. Viral stocks were titred on BHK-21 cells using a Median Tissue Culture Infectious Dose (TCID50) assay. Virus stocks were subjected to 5-fold dilutions and used to infect the cells.
  • TCID50 Median Tissue Culture Infectious Dose
  • Antiviral activity of HU308 was measured as % viral inhibition via comparison of viral titre (TCID50 units/mL), calculated by observing the transition of wells from those containing cells (visualized by crystal violet staining) to those that have been cleared due to infection-mediated lysing of the cells. Two independent experiments were performed in triplicate.
  • HU308 exhibited positive antiviral effects on various in vitro models of viral infection against several strains of human virus.
  • HU308 treatment elicited viral inhibition in cells infected with Influenza A/Panama/2007/99 (H3N2); Respiratory Syncytial Virus A2; Adenovirus Type 5; a-coronavirus (229E), and [3-coronavirus (OC43, in two separate models) (Studies 1 and 3); and elicited inhibition of viral RNA and cytokine production in human neurospheres infected with HIV-1 in combination with cART therapy (Study 2).
  • This data suggests that HU308 treatment may be beneficial in viral-mediated inflammatory indications both as an anti-inflammatory and as an anti-viral agent.
  • Example 7 HU308/Qntemabez as therapeutic against SARS.
  • SARS-CoV-2 (Italy strain) viral infection was induced in mice (K18-ACE2, n
  • mice were administered either parenteral HU308 (3 or 6 mg/kg/day), a vehicle control, or a standard of care comparator (Remdesivir; 10 mg/kg/day). Mice were monitored daily for disease symptoms, including changes in body temperature or weight, and analyzed using a clinical health scoring scale. Additionally, peripheral whole blood was collected at multiple time points for processing to sera and analyzed for quantity of various inflammatory cytokines or mediators using a Meso Quickplex SQ 120 system. Bronchoalveolar lavage fluid (BALF) was similarly collected at multiple time points and for quantity of various inflammatory cytokines or mediators.
  • BALF Bronchoalveolar lavage fluid
  • mice The clinical health of mice was monitored daily via an established clinical Health Score scale by trained observers.
  • MCP-1 monocyte chemoattractant protein- 1
  • MIP-la macrophage inflammatory protein la
  • TNF-alpha tumor necrosis factor alpha
  • IFN-g interleukin gamma
  • mice Following infection with SARS-CoV-2 (day 0), exposed mice exhibited symptoms corresponding with disease progression over the course of the following 6 days. These symptoms were quantified using an established clinical health scoring scale (Figure 17; increased Health Score indicates poor clinical health). Mice exposed to the virus and treated daily with a parenterally administered (intraperitoneal, i.p.) vehicle control exhibited significantly elevated Health Scores as compared to mice who were not exposed to the virus (No Infection). Mice treated with HU308 (3 or 6 mg/kg/day) on the same schedule (1 day prior to viral exposure, then once daily from day 0 to day 6), exhibited significantly lower Health Score ratings, indicating decreased disease severity.
  • Figure 17 increased Health Score indicates poor clinical health.
  • bronchoalveolar lavage fluid indicated decreased concentrations of several inflammatory cytokines in mice treated with parenteral (i.p.) HU308 (3 or 10 mg/kg) when tested either 3- or 6-days post-infection.
  • concentrations of TNF-a were lower in HU308-treated mice as compared to those treated either with vehicle or with the standard of care for COVID-19, Remdesivir (10 mg/kg/day).
  • decreased levels of the inflammatory mediators IL-10, INF-g, and IL- 6 were found in HU308-treated mice as compared to either vehicle- or Remdesivir-treated mice when BALF was analyzed 6 days following viral exposure.
  • mice K18-ACE2
  • SARS-CoV-2 SARS-CoV-2
  • HU308 reduced systemic cytokine release (primary study outcome parameter) in blood sera collected from mice (K18-ACE2) infected with SARS-CoV-2, a model of the COVID-19 disease.
  • Blood sera and BALF measures were collected immediately following viral exposure, as well as 3- and 6-days post-infection from mice treated daily (including one day prior to viral exposure) with HU308; 3 or 6 mg/kg/day, a vehicle control, or the current standard of care, Remdesivir (10 mg/kg/day).
  • Clinical health scoring was significantly improved by both injected dosages of HU308.
  • analyses of blood sera and bronchoalveolar lavage fluid (BALF) demonstrated a protective effect ofHU308 against infection-induced elevations in several inflammatory cytokines at either 3- or 6-days post-infection.
  • Example 8 HU3Q8/Ontemabez as therapeutic against pan-ocular inflammation.
  • EIU Endotoxin-induced uveitis
  • uveitis was in induced in one eye via a single intravitreal injection of LPS.
  • Control animals received an intravitreal injection of saline at equivalent volume.
  • rats received a single intravenous bolus of HU308 (3 mg/kg).
  • rats were euthanized and the uveitis eye was enucleated and flash frozen in liquid nitrogen. Eyes were subsequently thawed, mechanically homogenized, and total protein levels were quantified. Eye homogenate was analyzed via Luminex, and analyte levels were normalized against total protein concentrations.
  • IV catheter Under local anesthesia with subcutaneous 1% lidocaine, a 24G intravenous (IV) catheter was inserted into the marginal ear vein. Subsequently, animals were placed under deep sedation via a single bolus of 5 mg/kg propofol followed by a propofol infusion from 0.7 - 1.4 mg/kg/min, titrated to effect (deep sedation without respiratory or cardiovascular compromise). Following a baseline ocular examination (described below in d), the ocular surface was anesthetized with proparacaine (2 drops), and the pars plana was marked using an indentation from a Castroviejo caliper.
  • Asepsis was performed by applying 1% povidone iodine to the ocular surface, before EIU was induced via an intravitreal injection of LPS, administered to one eye via the location indented at the pars plana, using a 0.5-inch 28-gauge needle.
  • the total inj ectate volume was 50 pL, which contained 200 ng LPS (isolated from Escherichia coli) in saline. Control animals received an injection with 50 pL saline.
  • HU308 (Tocris Bioscience) was prepared in a vehicle suitable for parenteral administration, yielding a working concentration of 3 mg/mL. Rabbits received a twice daily intravenous bolus of 1.5 mg/kg HU308, with the initial dose 10 minutes following discontinuation of anesthesia (for baseline examination and intravitreal LPS administration). Subsequent doses were administered at 12-, 24-, and 36-hours (total of 4 doses).
  • the rabbit was then mounted in a table slit lamp biomicroscope and anterior chamber cells and flare were quantified as per the Standardization of Uveitis Nomenclature (SUN) scoring system - using a 1 mm x 2 mm slit beam at maximum brightness intensity and at a ⁇ 90-degree angle between incident beam and examiner ocular lenses. Two drops of 1 % tropicamide were applied to each eye, and once dilated, direct ophthalmoscopy was performed (Volk InView). Two images were collected, one of the fundus, and one of the peripheral retinal vasculature. At the same time, SUN scoring was utilized to assign scores for vitreous haze. At the 48-hour timepoint, following final ocular examination/ data collection, animals were euthanized via barbiturate overdose (150 mg/kg sodium pentobarbital IV push).
  • SUN Uveitis Nomenclature
  • Rats were placed under general anesthesia using isoflurane.
  • an intravitreal injection of LPS was administered to one eye via the pars plana, under a dissecting microscope and using a Hamilton microliter syringe fitted with a 30-gauge needle.
  • the total injectate volume was 5 pL, which contained 200 ng LPS (isolated from Escherichia coli) in normal saline.
  • the puncture site was sealed with 3M Vetbond Tissue Adhesive. The animal was allowed to recover and was observed for complications.
  • HU308 (Tocris Bioscience) was prepared by dissolving in a vehicle suitable for parenteral administration, yielding a working concentration of 10 mg/mL. Rats received a single intravenous bolus of 3 mg/kg HU308, 15-minutes after induction of EIU.
  • Luminex Assay A magnetic bead-based multiplex Luminex® assay (Research & Development Systems) was used to assess for presence of: IL-l-alpha, IL-l-beta, IL-2, IL-4, IL-6, IL-10, IL-13, ICAM-1, IFN-gamma, TNF-alpha, and GM-CSF. The whole eye homogenate was diluted 1:4 using the diluent provided by the Luminex® kit. Samples were run in duplicate. Each n value represents one eye. All samples were run on a Bio-Rad 200 instrument with Bio-Plex software, according to the manufacturer's instructions. Standard curves for each analyte were generated based on reference compounds provided by the Luminex® kit.
  • Table 17 Summary of inflammatory mediator results in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Mean (picograms/mL) +/- SEM.
  • Example 9 HU3Q8/Ontemabez as therapeutic agent for interstitial cystitis
  • Interstitial cystitis was induced in mice via systemic administration of lipopolysaccharide (LPS; endotoxin).
  • Intravital microscopy was used to quantify leukocytes adhering to the endothelium of submucosal venules in the bladder 1.5-hours following parenteral treatment with HU308.
  • IVAM Intravital microscopy
  • FCD Functional capillary density
  • FCD was used to assess the microcirculatory function in bladder tissue. Following LPS administration, FCD levels decreased significantly, and HU308 treatment restored levels to those in control mice receiving a saline injection instead of LPS.
  • mice under general anesthesia IC was induced either via systemic administration of LPS (20 mg/kg; single intraperitoneal [IP] injection). Treatment with parenteral HU308 (5 mg/kg; single IP injection) was administered 30-minutes following administration of LPS.
  • a fluorescent dye Rhodamine 6G and fluorescein isothiocyanate; FITC
  • IV intravenously
  • mice were secured in a stereotaxic frame, the bladder was covered with a coverslip to enhance optics, and IVM was performed 2-hours following LPS administration. Offline video analysis was then performed, allowing quantification of adherent leukocytes and FCD scoring.
  • mice Female CD-I mice (30 ⁇ 3 g) were used for IC experiments. IC was induced via an intraperitoneal (IP) injection of LPS (20 mg/kg) from Escherichia coli (serotype 026:B6, Sigma-Aldrich, Oakville, ON, Canada) dissolved in 50 pL normal saline (Hospira, Montreal, Canada). LPS was administered 15-minutes following induction of anesthesia. Once 30-minutes had elapsed following LPS administration, a total volume of 50 pL of drug treatment was administered parenterally.
  • IP intraperitoneal
  • mice were anesthetized via IP injection of sodium pentobarbital (65 mg/kg, Ceva Sante Animate, Montreal, QC, Canada). Following induction of anesthesia, mice were placed on a heating pad to maintain a body temperature of 37 °C, which was monitored with a rectal temperature probe. Anesthesia was maintained with repeated IP administration of 5 mg/kg sodium pentobarbital, while the depth of anesthesia was monitored by clinical examination (return of pedal withdrawal reflex). Once the animals achieved a surgical depth of anesthesia, a Maylard incision of the lower abdomen was performed using surgical scissors. The abdominal muscle layer was lifted using forceps, and an incision made along the linea alba to expose the bladder. Using saline-soaked cotton-tipped applicators, the bladder was gently exteriorized. C rede’s maneuver was then performed to manually void urine from the bladder. IVM was performed 2-hours following LPS administration.
  • HU308 (Tocris Bioscience) was prepared by dissolving in a vehicle suitable for parenteral administration. Mice received a single injection of 5 mg/kg HU308 (IP), 30- minutes after LPS administration.
  • a tail vein (IV) injection of two fluorochrome dyes was performed 15-minutes prior to the start of IVM.
  • the fluorochrome dye mixture consisted of Rhodamine 6G (1.5 ml/kg, 0.75 mg/kg body weight, Sigma-Aldrich, ON, Canada) and fluorescein isothiocyanate (FITC)- albumin (1 ml/kg, 50 mg/kg, Sigma-Aldrich, ON, Canada).
  • the fluorochrome dyes were injected into the tail vein.
  • Rhodamine 6G allows for visualization of leukocytes
  • FITC-albumin was used to facilitate evaluation of functional capillary density by providing enhanced illumination of the bladder capillary beds. All tail vein injections were carried out in minimum light to minimize the photobleaching of fluorochromes.
  • a small clean glass cover slip was positioned on top of the bladder, and the animal positioned under the microscope.
  • a metal arm was used to apply gentle pressure to the upper abdominal area. Any areas of the abdomen that were not subject to experimentation were covered in gauze that was saturated in saline solution maintained at physiological temperature as to avoid dehydration and exposure to ambient air.
  • Intravital fluorescent video microscopy was performed using the following technical devices: an epifluorescence microscope (Leica DMLM, Wetzlar, Germany, Filterset: 13, green light filter), light source (LEG EBQ 100, Jena, Germany), and a black and white monitor (Speco Technologies, Texas, US).
  • the images were transferred to a Windows desktop computer and recorded using WinDV software (version 1.2.3, Czech Republic).
  • the leukocytes within the microcirculation of the bladder were visible under the 20x objective with green light.
  • Five to seven randomly selected visual fields containing bladder venules were recorded for 30 seconds.
  • the filter was then changed for examination with FITC-albumin under blue light, allowing for the examination of capillary blood flow.
  • Five to seven randomly selected visual fields with capillaries were recorded again for 30 seconds.
  • FCD When analyzing FCD, a single rectangular field covering the maximum possible area was drawn for each video segment, and capillaries containing FITC-albumin marked plasma found in the rectangular field were chosen. The length of perfused capillaries was measured by manually drawing a line within the lumen of the capillary. Capillaries with absent and/or intermittent flow were characterized as dysfunctional capillaries and not accounted for, whereas capillaries with continuous flow, regardless of the speed of the flowing cells, were characterized as functional capillaries. Summing the length of all corresponding capillaries and dividing the sum length with the measured area of the rectangular field calculated the FCD. For the initial pilot segment of this study, FCD was expressed as a percentage relative to the FCD of the control group. At the 2- hour timepoint following video collection for IVM/FCD, animals were euthanized via barbiturate overdose (150 mg/kg sodium pentobarbital, IP).
  • Example 10 HU3Q8/Ontemabez as therapeutic agent for treatment of systemic cytokine release and sepsis.
  • Peripheral whole blood was collected at 6-hours via cardiac puncture and centrifuged to isolate plasma for cytokine/chemokine analysis (CXCL1, CXCL2, ICAM, IL-1, IL-10, IL-6, P-selectin, IFN-gamma, TNF-alpha, and LIX) using a Luminex® assay.
  • Mouse lungs were collected for analysis using a histopathological scoring system established by the American Thoracic Society.
  • mice 10 mice per condition
  • LPS working solution was prepared by diluting LPS, purified from Pseudomonas aeruginosa, in sterile saline to yield a final concentration of 10 mg/mL.
  • LPS working solution was brought to physiological temperature prior to administration.
  • a pipette was used to administer LPS solution via the left nares at a rate of 1 droplet every 7-10 seconds, up to the calculated dose drawn in the pipette tip. Note a maximum of 20 pL per mouse was delivered. Following administration, the nares was flushed with 10 pL sterile saline and the animal was recovered and observed for complications.
  • HU308 (Tocris Bioscience) was prepared in a vehicle suitable for parenteral administration, yielding a working concentration of 10 mg/mL. Mice received a single intravenous bolus of 3 mg/mL HU308 at time 0, immediately following induction of pulmonary injury.
  • mice received a single intravenous bolus injection of fluorescent dyes (Rhodamine-6G and FITC) in order to non-specifically stain leukocytes and visualize blood flow, respectively.
  • Laparotomy was performed via a vertical midline incision, followed by dissection to allow identification and isolation of the small intestine.
  • a loop of terminal ileum, proximal to the ileocecal valve, was selected for imaging and placed on a specifically designed stage attached to the intravital microscope. Throughout the period of imaging, the intestine was superfused with normal saline at physiologic temperature to prevent desiccation. Videos were collected for offline analysis. Intravital microscopy was performed 6 hours after pulmonary delivery of LPS.
  • the trachea and lungs were harvested and a 23G blunt-tipped needle connected to a 10 mL syringe containing 10% formalin was gently threaded into the trachea.
  • the trachea was then clasped with forceps in order to hold the needle in place, and the lungs were gently inflated with formalin until fully expanded, but not over-inflated.
  • the whole lung lobes and trachea were then placed in ajar filled with formalin, allowing fixation of tissues for a minimum of 24 hours. Following fixation, the lungs were embedded in paraffin and subsequently sectioned at 5 pm. Tissues sections were then stained with hematoxylin and eosin and cover-slipped with non-aqueous mounting media.
  • Scores were assigned by assessing multiple parameters, including the presence/magnitude of: neutrophils in the alveolar space, neutrophils in the interstitial space, proteinaceous debris filling the airspaces, alveolar septal thickening, and hyaline membranes - based on a grading system for experimental acute lung injury in animals published by the American Thoracic Society. Scores for each section were generated from the score assigned to 10 unique histological fields, selected at random using 20 X magnification. Fields selected were areas that are not close proximity to large pulmonary arteries/veins or bronchioles, and further were not selected when the vessels present encompass more than 50% of the field of view.
  • Each aforementioned parameter was assigned a score of 0 (indicating ‘none’), 1 (indicating 1-5), or 2 (indicating >5). The sum of these scores yielded a total score per field, which ranged from 0 - 6. All 10 scores were averaged to yield a final histopathologic score per animal. Histopathologic scoring for all animals was performed primarily by one individual, and each animal was scored by one individual. All those assigning scores were blinded to treatment groups.
  • a magnetic bead based multiplex Luminex assay (Research & Development Systems) was used to assess the presence of: CXCL1, CXCL2, ICAM, IL-1, IL-10, IL-6, P- selectin, IFN-gamma, TNF-alpha, and LIX in heparinized plasma samples.
  • Plasma was diluted (1:2) using the 10-Plex Mouse Cytokine ELISA Kit diluent. Samples were run in duplicate on a Bio-Rad 200 instrument with Bio-Plex software, according to the manufacturer's instructions. Standard curves for each analyte were generated based on reference compounds provided by the kits.
  • Cannabinoid receptor type 2 a possible target in SARS- CoV-2 (CoV-19) infection. Int. J. Mol. Sci., 21, in press,'

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Abstract

Pharmaceutical compositions for parenteral delivery of cannabinoids are disclosed herein. The pharmaceutical compositions comprise onternabez, a first solvent, and a first emulsifier. Uses thereof and methods of producing the compositions are also disclosed.

Description

PARENTERAL CANNABINOID FORMULATIONS AND USES THEREOF
FIELD OF INVENTION
[0001] The present invention relates to formulations of cannabinoid compounds, particularly formulations for parenteral delivery.
BACKGROUND OF THE INVENTION
[0002] Systemic inflammatory responses can be triggered by a variety of infectious and non- infectious stimuli such as bacterial, viral and fungal infections, trauma, and in response to certain drugs. Systemic inflammatory responses involving organ dysfunction are often seen in diseases such as sepsis/severe sepsis/septic shock, systemic inflammatory response syndrome (SIRS), cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (MODS) (see for example, https://wwwZncbi.nlm.nih.gov/books/NBK5477669).
[0003] Sepsis is a clinical syndrome that arises due to infection, trauma or non-infectious triggers. Sepsis is often characterized by physiological and pathological abnormalities caused by a dysregulated host immune response to infection. Sepsis is normally classified on a continuum of severity progressing from systemic inflammatory response to sepsis, followed by severe sepsis, and the most severe and refractory state of septic shock. Severe sepsis and septic shock are associated with organ dysfunction, which may progress to MODS, and a high mortality. This mortality is particularly high (40-75%) in patients with sepsis-associated ARDS and MODS. Estimates of the global disease burden in 2017 cites sepsis as accounting for greater than 48 million cases and 11 million sepsis-related deaths. The magnitude of this is further represented by the fact that sepsis deaths represent 20% of all global deaths. There is a lack of sepsis-specific treatments despite the disease burden. Accordingly, there remains an ongoing need to identify new approaches for treating patients with systemic immune system dysregulation and sepsis.
[0004] While inflammation is normally an essential host response, dysregulation of this response can trigger a cataclysmic sequela of events that can progress to widespread tissue and organ damage. Dysregulation is sometimes typified by increases in both pro- inflammatory and anti-inflammatory mediators. In some cases, this dysregulation results in progression to a state of immunosuppression involving immune cell paralysis. The majority of sepsis patients die during this latter immunosuppressant “second phase”.
[0005] The endocannabinoid system regulates many critical physiological processes including those underlying the functions of the immune system. The endocannabinoid system comprises at least two receptors: cannabinoid 1 (CB1) and cannabinoid 2 (CB2), and several endogenous ligands (endocannabinoids). Immune cells may express both CB1 and CB2 receptors and may metabolize, produce and respond to both exogenous and endogenous cannabinoids.
[0006] Cannabinoids, of natural or synthetic origin, have been shown to be potent inhibitors of certain immune system components and act as anti-inflammatory agents in vitro and in vivo through four main mechanisms: induction of apoptosis, inhibition of cell proliferation, inhibition of cytokines and chemokine production, and via the induction of regulatory T cells.
[0007] Cannabinoids that act at the CB2 receptor, which is highly expressed on some immune cells, have shown promise to reduce immune system dysregulation. CB2 is upregulated following infection, trauma and inflammation, and activation of CB2 by cannabinoids is associated with inhibition of immune cells, particularly those cells involved in the innate immune response, and pro-inflammatory mediators. Development of potent and selective CB2 receptor cannabinoid ligands affords the opportunity to activate CB2 receptor independent of CB1 receptor behavioral effects.
[0008] HU308 (also known by International Nonproprietary Name (INN) as ontemabez; [(lR,2R,5R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4- bicyclo[3.1.1]hept-3-enyl]methanol) is a synthetically derived analog of cannabidiol and an example of a potent, selective agonist for the CB2 receptor. HU308 delivered via various routes of delivery has shown efficacy in reducing inflammation and immune system hyperactivation in experimental models of sepsis and septic lung injury as well as in organspecific models of inflammatory disease, highlighting the potential of HU308 and selective CB2 ligands for the treatment of systemic inflammatory responses and sepsis.
[0009] However, cannabinoids such as HU308 typically have poor drug like properties and are generally very lipophilic small molecules with tow kinetic solubility (<1 pg/ml) making preparation of a formulation suitable for delivery to animals and humans difficult. For these reasons and others, cannabinoids are often considered poor candidates for parenteral formulation to patients.
[0010] Parenteral formulations are sterile preparations used to deliver drugs, for example, but not limited to intravenously, subcutaneously, intraperitoneally or intramuscularly, and include solutions, suspensions or emulsions for injection or infusion. Parenteral formulations afford advantages over enteral routes of delivery in that parenteral drug formulations can be used in conscious or unconscious patients providing rapid drug delivery with predictable pharmacokinetics largely avoiding the first-pass metabolism and unpredictable bioavailability seen with some enteral drugs (e.g. oral, oro-mucosal).
[0011] While several cannabinoid drugs have been approved for delivery via the oral and oro-mucosal routes for various indications, currently there are no approved parenteral cannabinoid formulations, reflecting the challenges of developing injectable for highly lipophilic cannabinoid drugs for human clinical use. Some of the issues that needed to be addressed in the development of a parenteral cannabinoid formulation include finding a solvent that lipophilic cannabinoids can be dissolved in at high enough concentration for therapeutic efficacy but that is also safe to use in intravenous formulations, testing a number of emulsifiers that can form stable nanoparticles that are under 200 nanometers which permits terminal filter sterilization and finally determining the ideal antioxidants and other stabilizers so that the formulation will have sufficient stability.
[0012] There exists a need in the art for parenteral formulations of cannabinoid compounds, particularly in the treatment of systemic inflammation responses.
SUMMARY OF THE INVENTION
[0013] The present invention relates to pharmaceutical compositions comprising cannabinoids suitable for parenteral delivery. The present invention also relates to methods of production and uses of the compositions.
[0014] According to the present invention, there is provided a pharmaceutical composition comprising ontemabez, a first solvent, and a first emulsifier.
[0015] In some embodiments, the composition comprises water. In further embodiments, the composition is a nanoemulsion. [0016] In some embodiments, the pharmaceutical composition is sterilized. In further embodiments, the pharmaceutical composition is sterilized by fdtration through a filter. In even further embodiments, the filter has a pore size of about 0.2 pm or about 0.22 pm.
[0017] In some embodiments, the nanoemulsion is a suspension of nanoparticles in water. In further embodiments, a mean diameter of the nanoparticles is about 100-400 nanometers (nm). The mean diameter may be about 120-250 nm, for example about 200 nm. In a further embodiment, a mean diameter below about 200 nm is preferred.
[0018] In some embodiments, the pharmaceutical composition comprises about 0.1-about 3% w/w ontemabez, such as, but not limited to, about 1 %w/w ontemabez or about 2 %w/w ontemabez.
[0019] In some embodiments of the compositions, the first solvent is soybean oil, or medium-chain triglyceride (MCT) oil. Without limitation, the MCT oil may comprise Miglyol 812N.
[0020] In some embodiments, the pharmaceutical composition further comprises a second solvent. The second solvent may be soybean oil, or medium-chain triglyceride (MCT) oil. Generally, the first solvent and the second solvent are not the same.
[0021] In some embodiments, a total solvent content of the pharmaceutical composition is about 5-25 %w/w. The total solvent content may be about 16.1 %w/w or about 20 %w/w, for example. In some embodiments, the first solvent is soybean oil and a total solvent content is about 20 %w/w. In other embodiments, the first solvent is soybean oil, the second solvent is MCT oil and a total solvent content is about 16.1 %w/w. In further embodiments, the pharmaceutical composition comprises about 15 %w/w of soybean oil and about 1.1 %w/w MCT oil.
[0022] In some embodiments, the first emulsifier is egg lecithin or polyoxyl 15- hydroxystearate. In further embodiments, the first emulsifier is egg lecithin. In still further embodiments, the compositions further comprise a co-emulsifier, such as, but not limited to, sodium oleate. In some embodiments, the first emulsifier is polyoxyl 15-hydroxystearate and the co-emulsifier is sodium oleate. In further embodiments, the first emulsifier is egg lecithin at a concentration of about 1.2 %w/w. In yet further embodiments, the first emulsifier is polyoxyl 15 -hydroxy stearate at a concentration of about 4.4 %w/w, and the co-emulsifier is sodium oleate at a concentration of about 0.48 %w/w.
[0023] Compositions as described herein may further comprise a first preservative. The first preservative may be EDTA, benzyl alcohol or sodium benzoate. Some embodiments may further comprise a second preservative. The second preservative may be EDTA, benzyl alcohol or sodium benzoate, wherein the first preservative and the second preservative are different. In some embodiments, the first preservative is EDTA at a concentration of about 0.01 %w/w. In further embodiments, the first preservative is benzyl alcohol at a concentration of about 0.15 %w/w, and the second preservative is sodium benzoate at about 0.10 %w/w.
[0024] Some embodiments may further comprise alpha-tocopherol. In some cases, the compositions comprise about 0.03 %w/w alpha-tocopherol, although other concentrations are contemplated.
[0025] Some embodiments further comprise glycerol. For example, but not wishing to be limiting, the compositions may comprise about 2.25 %w/w glycerol.
[0026] Some embodiments of the pharmaceutical compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w soybean oil; about 4.40 %w/w polyoxyl 15-hydroxystearate; about 0.48 %w/w sodium oleate; about 2.25 %w/w glycerol; about 0.03 %w/w alphatocopherol; about 0.01 %w/w disodium EDTA; and about 70.84 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
[0027] Other embodiments of the pharmaceutical compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w soybean oil; about 4.40 %w/w polyoxyl 15-hydroxystearate; about 0.48 %w/w sodium oleate; about 2.25 %w/w Glycerol; about 0.03 %w/w alphatocopherol; about 0.15 %w/w benzyl alcohol; about 0.10 %w/w sodium benzoate; and about 70.59 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
[0028] Further embodiments of the pharmaceutical compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w soybean oil; about 1.20 %w/w egg lecithin; about 2.25 %w/w glycerol; about 0.03 %w/w alpha-tocopherol; about 0.01 %w/w disodium EDTA; and about 74.52 %w/w water, wherein the pharmaceutical composition is a nanoemulsion. [0029] Yet further embodiments of the pharmaceutical compositions comprise: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w egg lecithin; about 2.25 %w/w glycerol; about 0.03 %w/w alpha-tocopherol; about 0.15 %w/w benzyl alcohol; about 0.10 %w/w sodium benzoate; and about 74.27 %w/w water, wherein the pharmaceutical composition is a nanoemulsion.
[0030] Methods for preparation of nanoemulsions comprising nanoparticles of ontemabez, a first solvent, a first emulsifier and water, are disclosed herein. The methods comprise: mixing the ontemabez, the first solvent, the first emulsifier and water to make a crude emulsion, homogenizing the cmde emulsion under high pressure; and repeating the homogenizing step at least about five times.
[0031] In some embodiments, a diameter of the nanoparticles is about 100-400 nanometers (nm). In some cases, the diameter is about 150-250 nm, such as 200 nm.
[0032] In some embodiments, high pressure is about 700-2000 bar. The high pressure may be about 750-1500 bar, such as, but without wishing to be limiting, about 750 bar or about 1500 bar.
[0033] In some embodiments, repeating comprises undertaking the homogenizing step at least ten times. In some embodiments, repeating comprises undertaking the homogenizing step up to at least twenty times.
[0034] Some embodiments further comprise cooling the crude emulsion after homogenizing. Cooling may comprise cooling with an ice-water bath. In further embodiments, repeating comprises undertaking the homogenizing and cooling steps. In some embodiments, repeating comprises undertaking the homogenizing and cooling steps at least ten times.
[0035] In further embodiments, the methods further comprise adjusting the pH prior to homogenization. Without wishing to be limiting in any manner, adjusting may comprise adjusting the pH to 7.5.
[0036] In yet further embodiments, the methods comprise premixing ontemabez, the first solvent, and the first emulsifier, prior to the mixing step. Some such embodiments further comprise heating the premixed components and/or heating the water, prior to the mixing step. Heating may comprise heating to about 70°C, but other temperatures are also contemplated. [0037] Uses of the pharmaceutical compositions are also disclosed herein. For example, use of any of the compositions for parenteral administration is contemplated. Use of the compositions for the treatment of systemic inflammation responses is contemplated herein. In some embodiments, the systemic inflammation response is one or more of: systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), or multiple organ dysfunction syndrome (MODS). Further uses of the compositions as described herein and throughout are as antibacterials, antivirals or both. This summary of the invention does not necessarily describe all features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
[0039] FIGURES 1A-F show the appearance of the nanoemulsion preparations and microscopic views of the crude primary emulsion, and nanoemulsions obtained at 750 bar and 1500 bar high pressure homogenizations. Fig. 1 A depicts appearance of the lecithinbased reference nanoemulsion and priority placebo DOE (Design of Experiments) Formulation #1 nanoemulsion after high pressure homogenization. Fig. IB is a microscopic image (X400 magnification) of a crude primary emulsion of priority placebo DOE Formulation #1 after high speed mixing at 10,000 rpm for 2 minutes. Fig. 1C is a first microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed at 750 bar for 10 cycles. Fig. ID is a second microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed at 750 bar for 10 cycles. Fig. IE is a first microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed further at 1500 bar for 10 cycles. Fig. IF is a second microscopic image (X400 magnification) of priority placebo DOE Formulation #1 nanoemulsion processed further at 1500 bar for 10 cycles.
[0040] FIGURES 2A-D are microscopic images of the reference nanoemulsion batch. Fig. 2A is a first microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 750 bar for 10 cycles. Fig. 2B is a second microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 750 bar for 10 cycles. Fig. 2C is a first microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 1500 bar for 10 cycles. Fig. 2D is a second microscopic image (X400 magnification) of trial lecithin-based reference nanoemulsion processed at 1500 bar for 10 cycles.
[0041] FIGURES 3A-B are particle size analyses for the reference nanoemulsion batch processed at 750 bar for 10 cycles (Fig. 3A) and the priority placebo DOE Formulation #1 processed at 750 bar for 10 cycles (Fig. 3B).
[0042] FIGURE 4 is a graph depicting the effect of formulation composition and high pressure homogenization (HPH) cycle on DOE nanoemulsion formulation droplet size.
[0043] FIGURE 5 shows appearance of priority ontemabez DOE nanoemulsion batches #1, 3, 6 and 8 (left to right) processed at 1500 bar high pressure homogenization for 20 cycles.
[0044] FIGURES 6A-D show microscopic appearances of ontemabez DOE nanoemulsion batches at 200X magnification. Fig. 6A shows microscopic appearance of ontemabez DOE Formulation #3, nanoemulsion batch at X200 magnification. Fig. 6B shows microscopic appearance of ontemabez DOE Formulation #6, nanoemulsion batch at X200 magnification. Fig. 6C shows microscopic appearance of ontemabez DOE Formulation #1, nanoemulsion batch at X200 magnification. Fig. 6D shows microscopic appearance of ontemabez DOE Formulation #8, nanoemulsion batch at X200 magnification.
[0045] FIGURES 7A-C show results of IL-6 production (Fig 7A), Ashcroft score (Fig 7B) and hydroxyproline content (Fig 7C) for various test groups treated with vehicle or HU308, respectively.
[0046] FIGURES 8A-B show the effect of test compounds on influenza A/Panama/2007/99 (Fig 8A). Data are presented as mean percentage inhibition of viral infection ±SEM (n=3). N.B. Cytotoxic concentrations have been removed from the analysis. Fig 8B. Effect of test compounds on MDCK cells. Data are presented as mean percentage cell viability ±SEM (n=3).
[0047] FIGURES 9A-B show the effect of test compounds on RSV (A2) (Fig 9A). Data are presented as mean percentage inhibition of viral infection ±SEM (n=3). N.B. Cytotoxic concentrations have been removed from the analysis. Fig 9B. Effect of test compounds on HeLa cells. Data are presented as mean percentage cell viability ±SEM (n=3). [0048] FIGURES 10-B show the effect of test compounds on adenovirus (type 5) (Fig 10A). Data are presented as mean percentage inhibition of viral infection ±SEM (n=3). N.B. Cytotoxic concentrations have been removed from the analysis. Fig 10B. Effect of test compounds on HeLa cells. Data are presented as mean percentage cell viability ±SEM (n=3).
[0049] FIGURES 11A-B show the effect of test compounds on a-coronavirus (229E) (Fig. 11 A). Data are presented as mean percentage inhibition of viral infection ±SEM (n=3). N.B. Cytotoxic concentrations have been removed from the analysis. Figure 11B. Effect of test compounds on 16HBE cells. Data are presented as mean percentage cell viability ±SEM (n=3).
[0050] FIGURES 12A-B show the effect of test compounds on [3-coronavirus (OC43) (Fig 12A). Data are presented as mean percentage inhibition of viral infection ±SEM (n=3). N.B. Cytotoxic concentrations have been removed from the analysis. Fig 12B. Effect of test compounds on 16HBE cells. Data are presented as mean percentage cell viability ±SEM (n=3).
[0051] FIGURES 13A-B show results of TAR (Fig 13A) and TAR-gag/env RNA (Fig 13B) expression in control untreated human neurospheres +/- HIV-1 infection (columns 1, 2) vs. human neurospheres infected with HIV-1 and treated with cART +/- HU308 (columns 3, 4). Bars indicate an average and ± S.D. for each technical triplicate. Two-tailed Student’s t-test: p < 0.01 = **; p < 0.001 = ***.
[0052] FIGURES 14A-B show results of IL-1 p (Fig 14A) and TNF-a (Fig 14B) proinflammatory cytokine RNA expression in control untreated human neurospheres +/- HIV-1 infection vs. human neurospheres infected with HIV-1 and treated with cART +/- HU308. Bars indicate an average and ± S.D. for each technical triplicate. Two-tailed Student’s t-test: p < 0.01 = **; p < 0.001 = ***.
[0053] FIGURE 15 shows results of BHK-21 cells infected with sequential dilutions of hCoV-OC43 produced by infected cells cotreated with HU308 at various concentrations. Top horizontal axis depicts hCoV-OC43 dilution factor; left vertical axis depicts conditions that virus-producing cells were exposed to; right vertical axis depicts viral titer relative to control conditions. [0054] FIGURE 16 shows results of BHK-21 cells infected with sequential dilutions of hCoV-OC43 produced by infected cells cotreated with HU308 at various concentrations. Top horizontal axis depicts hCoV-OC43 dilution factor; left vertical axis depicts conditions that virus-producing cells were exposed to; right vertical axis depicts viral titer relative to control conditions.
[0055] FIGURE 17 shows results of health score analysis at Day 6 post-infection with SARS in groups treated daily with vehicle (VEH) or HU308 (HU; 3 or 10 mg/kg/day). N = 6 - 9 mice/group. ***, P<0.0005; ****, P0.0001.
[0056] FIGURES 18A-C show results of sera analysis allowing quantification of monocyte chemoattractant protein-1 (MCP-1) (Fig 18A), inflammatory interferon gamma (INFg) (Fig 18B), and Interleukin 6 (Fig 18C) at the point of infection (Day 0), as well as Day 3 and Day 6 post-infection in groups treated daily with either parenteral (i.p.) vehicle (Control), Remdesivir (REM; 10 mg/kg/day) or HU308 (HU; 3 or 10 mg/kg/day). ** PO.Ol.
[0057] FIGURES 19A-D show results of BALF analysis allowing quantification of tumor necrosis factor-alpha (TNFa or TNF-a) (Fig 19A), interferon gamma (IFNg or IFNy) (Fig 19B), interleukin 6 (IL-6) (FIG 19C), or interleukin 10 (IL-10) (FIG 19D) at Day 3 and Day 6 post-infection in groups treated daily with either parenteral (i.p.) vehicle (VEH), Remdesivir (REM, 10 mg/kg/day) or HU308 (HU; 3 or 10 mg/kg/day).
[0058] FIGURE 20 shows results of eyelid edema score prior to induction of uveitis (0- hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0059] FIGURE 21 shows results of iris inflammation score prior to induction of uveitis (0- hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0060] FIGURE 22 shows results of chemosis score prior to induction of uveitis (0-hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM. [0061] FIGURE 23 shows results of vitreous haze SUN score prior to induction of uveitis (0-hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0062] FIGURE 24 shows results of ciliary injection score prior to induction of uveitis (0- hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0063] FIGURE 25 shows results of anterior chamber flare SUN score prior to induction of uveitis (0-hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0064] FIGURE 26 shows results of nterior chamber cells score prior to induction of uveitis (0-hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0065] FIGURE 27 shows results of palpebral conjunctival injection score prior to induction of uveitis (0-hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0066] FIGURE 28 shows results of nictitating membrane irritation score prior to induction of uveitis (0-hours), and at 24- and 48-hours following treatment with 1.5 mg/kg HU308 IV administered twice daily in test rabbit (n=l) vs. vehicle (n=4). Mean +/- SEM.
[0067] FIGURE 29 shows results of Interleukin- 1 -alpha levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Saline IVT (intravitreal); n=6, 200 ng LPS IVT; n=7, 3 mg/kg HU308 IV; n=7. Mean +/- SEM.
[0068] FIGURE 30 shows results of Interferon-gamma levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Saline IVT (intravitreal); n=6, 200 ng LPS IVT; n=7, 3 mg/kg HU308 IV; n=7. Mean +/- SEM.
[0069] FIGURE 31 shows results of Vascular Endothelial Growth Factor levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Saline IVT (intravitreal); n=6, 200 ng LPS IVT; n=7, 3 mg/kg HU308 IV; n=7. Mean +/- SEM. [0070] FIGURE 32 shows results of Interleukin-4 levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Saline IVT (intravitreal); n=6, 200 ng LPS IVT; n=7, 3 mg/kg HU308 IV; n=7. Mean +/- SEM.
[0071] FIGURE 33 shows results of Tumor Necrosis Factor- alpha levels (picograms/mL) in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Saline IVT (intravitreal); n=6, 200 ng LPS IVT; n=7, 3 mg/kg HU308 IV; n=7. Mean +/- SEM.
[0072] FIGURE 34 shows results of leukocyte adhesion in submucosal bladder venules of female CD-I mice for the following experimental groups: control (CON; n=9), untreated LPS-induced IC (LPS; n=9), LPS-induced IC treated with HU308 (5mg/kg, LPS+HU308; n=4). Saline, LPS, and HU308 were parenterally administered via intraperitoneal injection. Data presented as mean ± SD. * p < 0.05 vs. CON. # p < 0.05 vs LPS.
[0073] FIGURE 35 shows results of capillary perfusion quantified through FCD within the bladder microcirculation of female CD-I mice for the following experimental groups: control (CON; n=9), untreated LPS-induced IC (LPS; n=9), LPS-induced IC treated with HU308 (5mg/kg, LPS+HU308; n=4). Saline, LPS, and HU308 were parenterally administered via intraperitoneal injection. Data are presented as percentage relative to the control group. * p < 0.05 vs. CON. # p < 0.05 vs LPS.
[0074] FIGURE 36 shows results of intestinal intravital microscopy allowing quantification of leukocytes per square millimeter of vascular endothelium in VI venules at 6-hours following disease induction. (*) represents P < 0.05, (***) represents P < 0.001.
[0075] FIGURE 37 shows results of histopathologic scoring of lung tissue collected at 6- hours following disease induction. Increasing score indicates more severe disease. (**) represents P < 0.01.
[0076] FIGURE 38 shows results of levels of the pro-inflammatory mediator, Interleukin-6, in peripherally obtained plasma at 6-hours following pulmonary administration of LPS. CON; Control, LPS; lipopolysaccharide, HU; HU308. (*) represents P < 0.05.
[0077] FIGURE 39 shows results of the levels of the pro-inflammatory mediator, CXCL2, in peripherally obtained plasma at 6-hours following pulmonary administration of LPS. CON; Control, LPS; lipopolysaccharide, HU; HU308. (*) represents P < 0.05. [0078] FIGURE 40 shows results of the levels of the pro-inflammatory mediator, TNF- alpha, in peripherally obtained plasma at 6-hours following pulmonary administration of LPS. CON; Control, LPS; lipopolysaccharide, HU; HU308. (**) represents P < 0.01, (***) represents P < 0.001.
DETAILED DESCRIPTION
[0079] One or more illustrative embodiments have been described by way of example. Described herein are compositions, formulations, methods of production and uses relating to injectable formulations of cannabinoids. It will be appreciated that embodiments and examples are provided for illustrative purposes intended for those skilled in the art, and are not meant to be limiting in any way. All references to embodiments, examples, aspects, formulas, compounds, compositions, solutions, kits and the like is intended to be illustrative and non-limiting.
[0080] HU308 and ontemabez are used interchangeably herein.
[0081] Described herein is the development and manufacture of a sterile nanoemulsion of ontemabez for parenteral administration to a subject, for example, a mammalian subject, more preferably a human subject. Embodiments of the formulations described herein may be employed, for example, but not limited to, for parenteral delivery in the treatment of systemic inflammation responses such as SIRS, sepsis, severe sepsis, septic shock, CRS & CSS, ARDS and MODS. Embodiments of the formulations may comprise at least one solvent, such as soybean oil, an emulsifier, such as egg lecithin or polyoxyl 15-HS, and sodium EDTA as a preservative and homogenized with the required pressure and cycles to achieve stable nanoparticles that allow sterilization by filtration.
[0082] Definitions
[0083] The term “cannabinoid” as used herein, may refer to C21 or C22 terpenophenolic compounds, or carboxylic acids, analogs and transformation products thereof. Cannabinoid may also refer to endogenous or artificial compounds that bind to cannabinoid receptors, such as CB1 and/or CB2. An example of a suitable cannabinoid is HU308 or ontemabez.
[0084] The term “solvent” as used herein, may refer to aqueous or organic fluids that dissolve or otherwise solvate one or more desired compounds, such as a cannabinoid. In some cases, the solvent may be in a non-liquid form, such as in frozen, dried, dehydrated or lyophilized compositions. Such non-liquid solvents may be rehydrated or melted prior to use.
[0085] The term “emulsion” as used herein may be understood as a fine dispersion of droplets of one or more non-soluble or immiscible liquids. Emulsions are made through the mixing of a two-phase system (such as oil and water) to form a single phase. In some cases, emulsifying agents are used. Emulsions may be categorized by the size of the droplets within the emulsion. For example, a typical oil-in-water emulsion or “macroemulsion” may have a lipid droplet size greater than 1 micrometer (1 pm). The term “nanoemulsion” may refer to emulsions with a lipid droplet size generally between 10 and 1000 nanometers (nm).
[0086] The term “emulsifier” as used herein may refer to an additive that acts to stabilize an emulsion.
[0087] The term “mean diameter” as used herein may be understood as the mean value of a population of particles, such as the lipid droplets in a nanoemulsion. Mean diameter may be measured by a suitable technique, such as dynamic light scattering (DLS). The mean diameter may be measured as the mean value of a particle size distribution of the population of nanoparticles.
[0088] The term “sterile” or “sterilized” as used herein may refer to compositions that are substantially free of viable or reproducible microorganisms or harmful biological agents. Examples of microorganisms include fungi, bacteria, viruses, spores, protozoa and others. Suitable examples of harmful biological agents includes prions, protein-based toxins and others.
[0089] The term “%w/w” will be understood by a person of skill in the art as a “weight concentration”. The weight concentration is calculated by [weight of the component (g)/total weight of the solution (g)] x 100%. A person of skill in the art will understand that where the total number of components listed does not add up to 100%, the remaining balance comprises water (unless otherwise specified, such as in lyophilized embodiments). Similarly, the term “%w/v” will be understood by a person of skill in the art as a “weight per volume concentration”. The weight per volume concentration is calculated by [weight of the component (g)/total volume of the solution (ml)] x 100%. A person of skill in the art will understand that where the total number of components listed does not add up to 100%, the remaining balance comprises water (unless otherwise specified). [0090] The term “about” as used herein will be understood as meaning +/- 10% of the following value, unless otherwise indicated. For example, “about 200 nm” may be interpreted as a range of 180-220 nm.
[0091] Pharmaceutical Compositions
[0092] Described herein are pharmaceutical compositions and formulations for parenteral delivery of pharmaceuticals that exhibit poor solubility in aqueous solvent, such as water. Embodiments of the compositions comprise: ontemabez, a first solvent, and a first emulsifier. The pharmaceutical compositions described herein may be suitable for parenteral administration. Some embodiments of the pharmaceutical compositions are formulated as a nanoemulsion. In such embodiments, the compositions also comprise an aqueous phase, such as water. The first solvent may act as the non-aqueous phase of the emulsion and form droplets/nanoparticles.
[0093] In some embodiments, the compositions comprise water. Unless otherwise specified, water may comprise the majority of the composition by weight. In some cases, 50 %w/w or greater of the compositions is water. In some cases, about 0.01-<100 %w/w or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as about 70 to less than 100 %w/w of water may be used. For example, percentages of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 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 %w/w and others are considered and contemplated for use herein.
[0094] In some embodiments, the pharmaceutical compositions are dried such that there is no appreciable aqueous phase present. For example, the pharmaceutical compositions may be freeze dried, lyophilized or the aqueous phase is removed via vacuum. In some embodiments, there is about 5 %w/w or less water present. A range of about 0-10% or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as about 0.1-1%, may be suitable. For example, percentages of 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 %w/w and others are considered. Water may be added to the water-free embodiments to reconstitute to a biphasic or emulsified form prior to use.
[0095] As discussed herein, preferred embodiments of the pharmaceutical composition are formulated as a nanoemulsion. The nanoemulsions described herein may be understood as a suspension of nanoparticles in water. The nanoparticles may be droplets of solvent/oil that dissolve lipophilic molecules, such as ontemabez, and are suspended in an immiscible solvent, such as water.
[0096] The nanoparticles may have a suitable mean diameter, for example about 100-400 nanometers (nm). In embodiments that are sterilized by filtration, the mean diameter may be about 220 nm or less, such as about 200 nm or less. In some embodiments, the mean diameter is about 150-250 nm, such as about 200 nm. A range of about 0. 1-500 nm or any value therebetween (optionally rounded to the nearest 0. 1), or any sub-range spanning between any two of these values, such as 0.1-220 nm, may be suitable. For example, diameters of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,
205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 nm and others are considered.
[0097] The pharmaceutical composition may be sterilized by a suitable method. In some cases, sterilization occurs after the nanoemulsion is formed. Sterilization methods include heat treatment via a suitable method such as an autoclave or dry heating, filter sterilization, radiation, and others. In such embodiments, the formulations are filter sterilized through a suitable filter. The filter may have a pore size that is sized to allow for the passage of nanoparticles and prevent the passage of harmful biological agents. For example, the filter pore size is about 0.2 pm or about 0.22 pm. A range of 0.1-200 nm or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values is also contemplated, for example, pore sizes of 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30 pm and others are considered.
[0098] The pharmaceutical compositions described herein comprise at least one cannabinoid. In a preferred embodiment, the cannabinoid is ontemabez. Other compounds with suitable solubility profiles may be used without departing from the invention, such as other cannabinoids. Suitable concentrations of cannabinoids in the compositions may be about 1-3 %w/w. In some cases, a range of 0.01-10 %w/w or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 0.1-3 %w/w may be used. For example, percentages of 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35,
1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3,
2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25,
3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4, 4.5, 5, 5.5, 6, 6.5, 7,
7.5, 8, 8.5, 9, 9.5, 10 %w/w and others are considered.
[0099] The pharmaceutical compositions described herein comprise a first solvent. In some embodiments, the first solvent is an oil suitable for dissolving a lipophilic molecule. For example, the first solvent may be soybean oil, or medium-chain triglyceride (MCT) oil. The MCT oil may comprise one or more medium-chain triglycerides, such as Miglyol 812N. The first solvent/oil may be refined, such that they are suitable for parenteral delivery. Other solvents may be used, such as glycerin, propylene glycol, ethanol, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, castor oil, polysorbate, polyoxyl 40 stearate, PEG 400 or others. As will be understood by a person of skill in the art, the first solvent may be suitable and safe for parenteral delivery. In embodiments where ontemabez is used, soybean oil may be a preferred first solvent.
[00100] In some cases, more than one solvent may be used. In such embodiments, the second solvent may be soybean oil, or medium-chain triglyceride (MCT) oil, with the first solvent being different from the second solvent. The second solvent may be miscible with the first solvent to provide a continuous phase. The MCT oil may comprise one or more mediumchain triglycerides, such as Miglyol 812N. The second solvent/oil may be refined, such that it is suitable for parenteral delivery. Other solvents may be used, such as glycerin, Propylene glycol, Ethanol, Polyoxyl 35 castor oil, Polyoxyl 40 hydrogenated castor oil, Castor oil, Polysorbate, Polyoxyl 40 stearate, PEG 400 or others. In embodiments where ontemabez is used, MCT oil may be a preferred second solvent. Although the embodiments described herein have a first and second solvent, more than two solvents may be used.
[00101] The pharmaceutical compositions described herein may have a total solvent content. A person of skill in the art will understand that the total solvent content may be varied to accommodate different concentrations of solute, such as ontemabez. For example, the total oil content may be increased to dissolve a higher amount of solute. In some embodiments, the total solvent content of the pharmaceutical composition is about 5-25 %w/w, such as, without limitation, about 16.1 %w/w or about 20 %w/w. In some cases, about 0.1-50 %w/w or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning any two of these values, such as 5-25 %w/w total solvent content may be used. For example, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,
10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,
20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30,
30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40,
40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50
%w/w and others are considered. In one embodiment, the first solvent is soybean oil and a total solvent content is about 20 %w/w. In another embodiment, the first solvent is soybean oil, the second solvent is MCT oil and a total solvent content is about 16.1 %w/w, such as about 15 %w/w of soybean oil and about 1.1 %w/w MCT oil.
[00102] The pharmaceutical compositions described herein comprise a suitable first emulsifier. A suitable first emulsifier may be any emulsifier that acts to stabilize the emulsion that forms from mixing the one or more solvents with water. The first emulsifier may be egg lecithin, polyoxyl 15 -hydroxy stearate or sodium oleate. In one embodiment, the first emulsifier is egg lecithin.
[00103] The pharmaceutical compositions described herein may comprise more than one emulsifier. In some embodiments, the second emulsifier is egg lecithin, polyoxyl 15- hydroxystearate or sodium oleate, and the second emulsifier is different from the first emulsifier. In one embodiment, the first emulsifier is polyoxyl 15 -hydroxy stearate and the second emulsifier is sodium oleate.
[00104] The first and second emulsifier may have a suitable concentration or range of concentrations. In some embodiments, the concentration of the first or second emulsifier is about 0.1-10 %w/w, such as about 1.2 %w/w, 4.4 %w/w or about 0.48 %w/w. In some cases, about 0. 1-50 %w/w or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as 0.1-15 %w/w may be used. For example, 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4, 4.05, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35, 4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95, 5, 6, 7, 8, 9, and 10 %w/w are considered. In one embodiment, the first emulsifier is egg lecithin at a concentration of 1.2 %w/w. In another embodiment, the first emulsifier is polyoxyl 15-hydroxystearate at a concentration of 4.4 %w/w, and the coemulsifier is sodium oleate at a concentration of 0.48 %w/w.
[00105] The pharmaceutical compositions described herein may comprise one or more preservatives. A preservative may be understood as a component that extends the shelf-life of the composition, such as by protecting the active ingredient from damage caused by oxidation, radiation or other sources. The first preservative may be EDTA, benzyl alcohol, sodium benzoate, sorbic acid or others. Preservatives may also have antimicrobial properties to prevent spoilage. Suitable preservatives known in the art may be used, for example those found in Meyer et al., Antimicrobial preservative use in parenteral products: past and present. J Pharm Sci. 2007, 96(12):3155-67, herein incorporated by reference. In some embodiments, a second preservative is used. Suitable second preservatives include EDTA, benzyl alcohol or sodium benzoate.
[00106] The first and second preservative may have a suitable concentration or concentration range. In some embodiments, the concentration of the first or second preservative is about 0.1-10 %w/w, such as about 0.01 %w/w, 0.15 %w/w or about 0.1 %w/w. In some cases, about 0.1-50 %w/w or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as about 0.1-15 %w/w may be used. For example, 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5,
1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45,
2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4,
3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4, 4.05, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35,
4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95, and 5 %w/w are considered. In one embodiment, the first preservative is EDTA at a concentration of about 0.01 %w/w. In another embodiment, the first preservative is benzyl alcohol at a concentration of about 0.15 %w/w, and the second preservative is sodium benzoate at about 0.10 %w/w.
[00107] The pharmaceutical compositions described herein may comprise additional components. For example, some embodiments may comprise a suitable antioxidant, such as alpha-tocopherol. Suitable antioxidants may prevent the oxidation of lipids or other ingredients in the compositions. In some embodiments, the compositions comprise an isotonic agent, such as glycerol. The components may be at a suitable concentration or concentration range. In some cases, about 0.1-5 %w/w or any value therebetween (optionally rounded to the nearest 0. 1), or any subrange spanning between any two of these values, such as 0.1-5 %w/w may be used. For example, 0.01, 0.015, 0.02, 0.025, 0.03, 0.036, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4, 4.05, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35, 4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95, and 5 %w/w are considered. In one embodiment, the compositions comprise 0.03 %w/w alpha-tocopherol. In some embodiments, the compositions comprise 2.25 %w/w glycerol.
[00108] In one embodiment, the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil as a first solvent; about 4.40 %w/w Polyoxyl 15 -Hydroxy stearate as a first emulsifier; about 0.48 %w/w Sodium Oleate as a second emulsifier; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.01 %w/w Disodium EDTA; and about 70.84 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
[00109] In another embodiment, the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 4.40 %w/w Polyoxyl 15 -Hydroxy stearate; about 0.48 %w/w Sodium Oleate; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.15 %w/w Benzyl Alcohol; about 0.10 %w/w Sodium Benzoate; and about 70.59 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
[00110] In yet another embodiment, the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w Egg Lecithin; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.01 %w/w Disodium EDTA; and about 74.52 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
[00111] In another embodiment, the pharmaceutical composition comprises: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w Egg Lecithin; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.15 %w/w Benzyl Alcohol; about 0.10 %w/w Sodium Benzoate; and about 74.27 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
[00112] In some embodiments, the pharmaceutical composition described herein may optionally further comprise a pharmaceutically acceptable excipient, diluent, or carrier. Examples of such pharmaceutically acceptable excipients, diluents, and carriers may be found in Remington: The Science and Practice of Pharmacy (2012). As well, examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999, each of which are herein incorporated by reference in their entireties. In certain embodiments, a pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art. Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch. The person of skill in the art will recognize that pharmaceutically acceptable excipients may include suitable fdlers, binders, lubricants, buffers, glidants, and disintegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2012)). Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
[00113] Methods of production
[00114] Cannabinoid compounds, such as ontemabez, may be prepared using synthetic methodology known in the art, for example US6903137, which is herein incorporated by reference.
[00115] Disclosed herein are methods for preparing nanoemulsions comprising ontemabez, a first solvent, a first emulsifier and water, the method comprising: mixing the ontemabez, the first solvent, the first emulsifier and water to make a cmde emulsion; homogenizing the crude emulsion under high pressure; and repeating the homogenizing step at least five times.
[00116] As described herein, the initial components are mixed to form a crude emulsion. Mixing may comprise simple mixing via a mechanical stirrer or magnetic stir bar. This may also be known as macro-emulsification. Methods of macro-emulsification known in the art may be used, for example: Goodarzi, F. and Zendehboudi, S. (2019), A Comprehensive Review on Emulsions and Emulsion Stability in Chemical and Energy Industries. Can. J. Chem. Eng., 97: 281-309, which is herein incorporated by reference.
[00117] In some cases, two or more components of the compositions are pre-mixed prior to the mixing step (macro-emulsification). For example, ontemabez, the first solvent and the alpha-tocopherol are mixed together prior to macro-emulsification. In some embodiments, all of the non-aqueous components are pre-mixed prior to the macroemulsification step. In further embodiments, one or both of the premixed components and the water are heated. In some cases, the water and premixed components may be heated to a suitable temperature, for example 70°C.
[00118] Suitable heating temperatures include a range of 20-100°C or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 60-75°C. For example, temperatures of 20, 20.5, 21, 21.5, 22,
22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32,
32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42,
42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 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°C and others are considered.
[00119] As described herein, the crude emulsion (macroemulsion) is homogenized using a high pressure homogenizer (HPH). High pressure homogenization techniques are known in the art, for example in Yadav, K.S., Kale, K. High Pressure Homogenizer in Pharmaceuticals: Understanding Its Critical Processing Parameters and Applications. J Pharm Innov 15, 690-701 (2020), which is herein incorporated by reference.
[00120] Homogenization or high pressure homogenization may be conducted at various pressures. For example, a range of 700-2000 bar or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 750-1500 bar. Homogenization at other pressures is also contemplated, for example, but not limited to 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, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 205, 210, 215, 220,
225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320,
330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,
510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000,
1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500 bar and others are considered.
[00121] The homogenizing steps may be repeated until the desired mean diameter of the nanoparticles is achieved. For example, the homogenizing steps may be repeated at least five times, such as ten, fifteen or twenty times. The homogenizing step may be repeated at least 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, or 100 times. In one embodiment, repeating comprises undertaking the homogenizing step at least ten times.
[00122] The crude emulsion may be cooled after the homogenizing step. Cooling the macroemulsion may occur when the temperature of said emulsion rises too high during high- pressure homogenization. The crude emulsion may be cooled using conventional techniques, such as cooling in a cooling solution, for example an ice-water bath. Cooling may be used if the temperature rises above 45°C, for example 50°C. In some embodiments, repeating comprises undertaking both of the homogenizing and cooling steps. For example, the homogenizing and cooling steps may be repeated at least five times, such as ten, fifteen or twenty times. The homogenizing and cooling step may be repeated at least 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, or 100 times. In one embodiment, repeating comprises undertaking the homogenizing step and cooling step at least ten times.
[00123] The pH of the crude emulsion may be adjusted prior to or after homogenization. In a preferred embodiment, pH is adjusted after homogenization. For example, the pH may be adjusted to 7.5. The pH may be adjusted using conventional techniques, such as the addition of acid or base. In some cases, a pH range of 5-8 or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, such as 7-7.5 may be used. For example, a pH of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or others may be suitable.
[00124] Uses of the pharmaceutical compositions
[00125] The pharmaceutical compositions as described herein may be used for parenteral administration. The compositions may be formulated as a nanoemulsion in water with a nanoparticle mean diameter sufficient for filter sterilization prior to administration. The compositions may be used to treat systemic inflammation responses. Without wishing to be bound by theory, ontemabez may act as a selective agonist for the CB2 receptor, which has been linked to inhibition of immune cell activity, particularly those cells involved in the innate immune response, and pro-inflammatory mediators. As such, the compositions as described herein may be used as anti-inflammatory agents.
[00126] The compositions described herein may be used for the treatment or prevention of systemic inflammation responses. Some examples of suitable systemic inflammation responses include: systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), or multiple organ dysfunction syndrome (MODS). In a further embodiment, which is not meant to be limiting, the composition may be used for treating a bacterial infection or a viral infection. [00127] The present invention will be further illustrated in the following examples.
Examples
[00128] Example 1 : Solubility studies of ontemabez formulations
[00129] The solubility of ontemabez was evaluated in various buffers and solvents prior to formulation development. Cannabinoid compounds, including ontemabez, typically have poor aqueous solubility profiles which present significant challenges to their development as drugs. The solvent system should be safe for parenteral injection and dissolve sufficient quantities of ontemabez.
[00130] Ontemabez solubility was first evaluated in aqueous solvents. Solubility of ontemabez in aqueous buffers ranged from 0.000032 mg/mL to 0.000775 mg/mL (Table 1 A). These results indicated that a more complex formulation is required to achieve a target concentration of 10-20 mg/mL.
[00131] Results from solubilization experiments determined that ontemabez has high solubility in Cremophor EL, Tween 80, Castor oil, soybean oil, mineral oil and PEG 400 (Table IB). However, based on the limits in the FDA’s Inactive Ingredient Guide, soybean oil will have a good carrying capacity for a parenteral formulation that can be injected intravenously. Therefore, soybean oil was chosen as a starting point for a solvent.
[00132] Table 1A: Solubility of ontemabez in various aqueous buffers
Figure imgf000028_0001
Figure imgf000029_0002
[00133] Table IB: Solubility of ontemabez in various solvents
Figure imgf000029_0001
Maximal dissolution in solvent was not reached and not deemed necessary due to low IIG limit of the solvent in drug formulations.
[00134] Example 2: Preliminary nanoemulsion composition and process for placebo nanoemulsions
[00135] Example 2.1: Nanoemulsion process for developmental placebo batches
[00136] After solubility testing of the API demonstrated greater ontemabez solubility in lipophilic solvents, the efficacy of producing nanoemulsions with candidate solvents was evaluated using developmental placebo batches (no API).
[00137] Implementation of the nanoemulsion procedures known in the art involves the preparation of a primary emulsion using conventional macro emulsification technique followed by 2-stage high pressure homogenization. In the primary emulsification process the Lipid Phase Components (LPC) and the Aqueous Phase Components (APC) are combined to form the primary emulsion using convention mixing with an overhead stirrer and propeller blade. The primary emulsion is subsequently subjected to high pressure homogenization for further processing to generate the nanoemulsion.
[00138] To determine the operating parameters to be employed for the primary emulsion and high-pressure homogenization steps, a preliminary set of placebo nanoemulsion batches were processed using a fixed composition of the key formulation excipients. A list of excipients and their function in the formulation is provided in Table 2.
[00139] Table 2: List of excipient raw materials for an injectable nanoemulsion formulation of ontemabez:
Figure imgf000030_0001
Figure imgf000031_0001
[00140] Example 2.2: Reference nanoemulsion batch preparation
[00141] To establish the high-pressure homogenizer equipment parameters, a trial reference batch of a known nanoemulsion was prepared using the GEA Niro Soavi Panda PLUS 2000. The formulation composition and processing were taken from a report on lecithin-based nanoemulsion obtained by spontaneous emulsification or high pressure homogenization (Schuh, et. al. 2014). Specifically, the nanoemulsion formulation composition comprises 5%w/v soybean oil, 5%w/v MCT, 1.2%w/v egg lecithin, 2.5%w/v glycerol, and QS with water to 100%. A crude emulsion was prepared using a Pro Scientific PRO250 mixer fitted with a 30 mm x 200 mm generator probe. The crude emulsion was subjected to high pressure homogenization at 750 bar for 10 cycles. No cooling of the batch between the HPH cycles was required at this pressure setting. A milky white, uniform and opaque nanoemulsion was obtained (Fig. 1 A).
[00142] A portion of the nanoemulsion was removed and set aside for microscopic and droplet size analysis. The remaining material was then processed at 1500 bar for an additional 10 cycles. The batch required cooling using an ice-water bath as the temperature rose up to 51 °C after each cycle of homogenization. The nanoemulsion remained the original milky white appearance.
[00143] Samples of the nanoemulsions were subjected to bright field microscopic evaluation and were also analyzed for droplet size distribution using a Malvern Zetasizer. The results indicate that the mean peak of the distribution is about 200 nm and the mean droplet size diameter is about 180 nm. Two microscopic views each of the nanoemulsions obtained at 750 bar and 1500 bar of high-pressure homogenizations are shown in Figs. 2A-2D. The droplet-size analysis report for the Reference nanoemulsion processed at 750 bar is shown in Fig. 3A.
[00144] The batches used herein were used as proof of concept and would be less suitable for use with the API due to the low solvent/oil content (~5%). The lower oil amount may be less effective for dissolving the desired amount of ontemabez (10-20 mg/mL).
[00145] Example 2.3: Preparation of priority placebo DOE nanoemulsion formulation
[00146] Following establishment of process parameters using the trial reference nanoemulsion batch, a number of priority design of experiment (DOE) placebo batches were made for droplet size evaluation and filterability to identify the optimal manufacturing process settings and formulation composition before including the API in the batch. The amount of oil was increased in these formulations to allow the loading of ontemabez into the nanoemulsion to reach the 10-20 mg/mL concentration target. Priority placebo DOE formulations composition and processing parameters are listed in Table 3.
Table 3: Composition and processing parameters of priority placebo DOE formulations
Figure imgf000032_0001
Placebo Run #1: Comparison of pressure settings
[00147] Priority DOE nanoemulsion formulation #1 whose composition consists of 20%w/v soybean oil, 1.2%w/v egg lecithin, 2.25%w/v glycerol, and QS with water to 100% (Table 3), was used. The crude emulsion was prepared as before and was subjected to high pressure homogenization at 750 bar for 10 cycles. A portion of the processed material was set aside for microscopic examination (Figs. 1C-1D) and droplet size analysis (Fig. 3B). The temperature of the batch increased to a maximum of 44°C during the 10 cycles of processing and did not require cooling. Another portion of the crude emulsion was also processed at 1500 bar for 20 cycles. The nanoemulsion obtained was milky white and opaque in appearance (Fig. 1A).
[00148] The remaining nanoemulsion was then processed at 1500 bar for an additional 10 cycles. The batch required cooling using an ice-water bath as the temperature rose to 51 °C after each cycle of homogenization. The nanoemulsion remained milky white and opaque at the end of the processing at 1500 bar. Samples of the nanoemulsions were subjected to bright field microscopic and droplet size evaluations.
[00149] Samples were also analyzed for droplet size distribution using Malvern Zetasizer. For Priority DOE Formulation #1 nanoemulsion processed at 750 bar, the results indicate that the mean peak of the distribution is about 480 nm (Fig. 3B) and the mean droplet diameter is about 350 nm (Table 5). Sample analysis demonstrated that homogenization at 1500 bar further reduced the nanoemulsion mean droplet diameter to about 300 nm (Table 5), which is still larger than the target of < 200 nm that would enable filtration. As shown earlier, the mean peak of the distribution is about 200 nm for the reference batch, which contains only 10% of oil phase. The high oil content (equal to or greater than 10%) was used to get the water insoluble ontemabez into solution but was causing the droplet size to be too large for filtration.
Placebo Runs #5,8, 7,10: Comparison of cycle numbers and formulation composition [00150] Based on Run #1 results, four additional priority placebo DOE nanoemulsion formulation batches, Runs #5, 7, 8 and 10, were prepared by following the composition and a modified procedure outlined in Table 3. Briefly, the crude emulsions were prepared essentially as for Run#l, except the high-speed mixing was performed at 18,000 rpm to further reduce the droplet size before HPH. The HPH was performed at 1500 bar for up to 20 cycles and samples were collected for droplet size analysis at 0, 5, 10, 15 and 20 cycles. These four additional formulation batches were set up to test the performance of the composition of the oil fraction and the emulsifier(s) used for dispersion (Table 4).
[00151] Table 4: Priority placebo DOE nanoemulsion trials (Runs #5, 7, 8, 10)
Figure imgf000034_0001
[00152] The priority placebo DOE nanoemulsion formulation analysis results are summarized in Table 5 and the plots of the effect of high-pressure homogenization cycle on droplet size reduction is presented in Figure 4. The results suggest that the droplet size obtained is affected by the formulation composition and the number of cycles of homogenization. The two batches with the smallest droplets, Runs #5 and #10, were both emulsified with polyoxyl 15-hydroxystearate+sodium oleate, suggesting that this emulsifier combination is preferable to egg lecithin. From the plot of mean droplet size as a function of HPH cycles (Figure 4), it is observed that with the right emulsifiers, a minimum number of 10 cycles of HPH is required to obtain the target mean droplet size.
[00153] Table 5: Droplet size analysis results of placebo DOE nanoemulsion formulations
Filtration of priority placebo DOE formulations
[00154] Trial liltrations of the reference batch and priority placebo DOE
Figure imgf000035_0001
nanoemulsions Run #1, Run #5 and Run #8 were performed using 0.2 micron (pm) syringe filters to explore filterability of the nanoemulsions and to facilitate the identification of a desirable filter membrane for filter sterilization of the formulation. The preliminary qualitative data obtained with three types of filter membrane are presented in Table 6. Only the reference nanoemulsion formulation (contains a 10% total oil content) could be filtered with polyvinylidene fluoride (PVDF), Mixed Cellulose Esters (MCE), and polyethersulfone (PES) membrane filters. Of the three DOE formulations tested, DOE Run 1, containing 20% soybean oil and egg lecithin could not be filtered with any of the filters tried. DOE Run #5 (contains 20% soybean oil and Polyoxyl 15-hydroxystearate/sodium oleate) and DOE Run #8 (contains 15% Soybean Oil, 1.1% Miglyol 812N and 1.2% Egg Lecithin) could be filtered with the PES membrane filter. Subsequent DLS results confirming no significant changes in droplet size following PES membrane filtration of DOE Runs #5 and #8 are presented in Table 6.
[00155] Table 6: Filterability Summary of Reference and Priority Placebo DOE
Nanoemulsion Formulations
Figure imgf000036_0001
[00156] Example 3: Formulation of priority developmental DOE batch nanoemulsions containing ontemabez
Ontemabez Runs #3, 6, 1, 8: Formulation and processing of API-containing nanoemulsion batches
After placebo batches were completed and analyzed for droplet size distribution, four DOE nanoemulsion batches including ontemabez were undertaken. Priority API DOE formulations composition and processing parameters are listed in Table 7.
Table 7 Composition and processing parameters of API DOE Nanoemulsion Formulation
Figure imgf000037_0001
[00157] The four priority API DOE nanoemulsions were prepared following the same procedure as for priority placebo DOE batches to obtain preliminary information on drug loading capacity, droplet size and filterability. Crude emulsion preparation and HPH were performed as with the priority placebo DOE batches. Samples were taken at the same intermediate steps during HPH processing (1, 5, 10, 15 and 20 cycles) for comparability and tested for droplet size and ontemabez concentration by HPLC.
[00158] Pictures of the visual and microscopic appearance of the batches are presented in Figs. 5 and 6A-D, respectively.
[00159] Fig. 6A shows microscopic appearance of ontemabez DOE Formulation #3, nanoemulsion batch at X200 magnification. Fig. 6B shows microscopic appearance of ontemabez DOE Formulation #6, nanoemulsion batch at X200 magnification. Fig. 6C shows microscopic appearance of ontemabez DOE Formulation #1, nanoemulsion batch at X200 magnification. Fig. 6D shows microscopic appearance of ontemabez DOE Formulation #8, nanoemulsion batch at X200 magnification.
[00160] Results from ontemabez DOE nanoemulsion show that the smallest droplet size and only formulation consistently meeting the preestablished size criterion (< 200 nm) was achieved with the formulation containing the polyoxyl 15-hydroxystearate+sodium oleate emulsifier combination (Table 8). The HPLC assay results for all batches (Table 8) demonstrated good incorporation of ontemabez in the nanoemulsion.
[00161] Table 8: Assay and droplet size analysis results of priority API DOE formulations
Figure imgf000038_0001
Figure imgf000039_0001
[00162] Trial liltrations of ontemabez DOE nanoemulsion batches using 0.2 gm PES membrane sterilizing filter were performed. Samples of both unfiltered and the filtrate were submitted for droplet size testing and HPLC assay for ontemabez. For clarity, filtration results are presented in Table 9. As shown, little to no API loss/adsorption was observed for these formulations and mean droplet diameter was similar pre- and post-filtration, indicating feasibility for sterile filtration.
[00163] A plot of the droplet size as a function of the number of high-pressure homogenization cycle at 1500 bar for the completed priority placebo and API DOE batches was prepared to determine the minimum number of cycles required to achieve a droplet size below 200 nm (see Figure 4). Ten cycles were found to be adequate to generate the required droplet size for the formulation of interest, as exemplified by priority API DOE nanoemulsion Run #6. Polyoxyl 15 HS/Na Oleate (P15-HS/Na Oleate) is a suitable emulsifier combination that yielded the smallest mean droplet size (Fig. 4). Several formulations showed feasibility for sterile filtration with filter adsorption of API (Table 9).
[00164] Table 9: Pre- and post-PES membrane filtration HPLC assay and droplet size analysis for placebo and ontemabez DOE nanoemulsion batches
Figure imgf000040_0001
a Pre- or post-filtration droplet diameter after high pressure homogenization at 1500 bar for 20 cycles
[00165] Example 4: Stability studies of prototype formulations
[00166] Four candidate formulation batches, Prototypes 1A, IB, 2A and 2B, were prepared by following the composition and procedure outlined in Table 10 below and tested for droplet size distribution, filterability and stability over a 3 week period. Results are shown in Table 11. [00167] Table 10: Composition of Prototype 1A, IB, 2 A and 2B formulations, and processing parameters for HPH, filtration and stability testing
Figure imgf000041_0001
[00168] Table 11 : Summary of analytical testing results for prototype batches
Figure imgf000042_0001
* A = Change between values at W3 compared to WO, calculated as W3-W0.
[00169] The results of the droplet size analysis (Table 11) confirmed the droplet sizes remained similar to the priority placebo and API DOE batches.
[00170] Stability testing at 25°C and 40°C for up to three weeks (Table 11) showed that mean droplet size and pH did not change substantially over the three weeks, but the concentration of ontemabez decreased in all samples and was more pronounced in accelerated storage conditions (40°C).
[00171] Two formulations, 1A and 3A, were prepared following the same process as the first prototype batches and tested for stability, at 5°C, 25°C and 40°C for three weeks. Their respective compositions are listed in Table 10 and stability testing results show that decreases in ontemabez concentration over time are mitigated at 5°C (Table 11).
[00172] In an early placebo trial with 20% soybean oil and egg lecithin only as the emulsifier, mean droplet diameter was >300 nm even at HPH pressure of 1500 bar for 10 cycles (see placebo DOE Run #1 in Table 5).
[00173] Adding co-emulsifier sodium oleate to the above placebo formulation decreased droplet size to a mean diameter of 155 nm after 10 cycles at 1500 bar (see placebo DOE Run # 7 in Table 5). Development batch 3A, which was prepared using an egg lecithin + sodium oleate emulsification system with addition of API (2% ontemabez), showed that mean droplet size was >200 nm (Table 11). Therefore, adding sodium oleate does not improve mean droplet size in lecithin-based formulations as demonstrated by comparing results from prototype batches 2A and 3A that are identical in composition, except for the absence or presence of sodium oleate, respectively.
[00174] Additional analytical data on the batches of unfiltered active (2% ontemabez) and placebo Prototype 1 A nanoemulsion used in nonclinical studies demonstrated stability of ontemabez concentration, pH, droplet size, osmolality, and viscosity over a period of one year when stored refrigerated (2 to 8 °C; see Table 12). [00175] Table 12: Stability of Prototype 1A (Unfiltered) Under Refrigerated (2 to 8°C) Storage
Figure imgf000044_0001
ND, not detected; LC, label claim
* A = Change between values at Time 0 (TO) compared to 12 months (12mo), calculated as T0-12mo.
[00176] Ultimately, the particle size of formulation containing 2% ontemabez/20% soybean oil could remain well below 200 nm with the emulsification system of Polyoxyl HS- 15 + sodium oleate. (See priority API DOE Run #6 in Table 8 and Prototypes 1A and IB in Table 10).
[00177] Example 5: HU3Q8/Ontemabez as a therapeutic candidate for the treatment of inflammatory disorders.
[00178] In order to assess the efficacy of HU308/Ontemabez as a therapeutic candidate for the treatment of inflammatory disorders, a model of idiopathic pulmonary fibrosis (IPF) was used. In this study, bleomycin (BLM), a cytotoxic antibiotic drug frequently employed as a chemotherapeutic agent, was administered to mice (C57BL/6) via a single intratracheal application in saline (3 mg/kg). For comparison of certain measures, an additional group (sham) was administered intratracheal saline alone. BLM administration is a well-established model of IPF which induces both acute inflammation and associated tissue fibrosis. Previous studies have employed this model to elucidate the mechanisms underlying the development of interstitial lung fibrosis following inflammatory insult and identified critical inflammatory signaling molecules involved in the induction of fibrotic development including Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), and transforming growth factor beta (TGF-B).
[00179] Following BLM administration, four different groups received HU308/ontemabez via parenteral intravenous (IV) injection at 0 (vehicle control) or 3 mg/kg, according to the study design in Table 13 below. Analyses were conducted on bronchoalveolar lavage fluid (BALF) and lung tissue following animal sacrifice.
[00180] Table 13 - Treatment and analysis schedule
Figure imgf000045_0001
Day 0 - BLM administration
[00181] First, levels of the cytokines IL-6, TNF-a, and TGF-B in BALF from HU308 or vehicle-treated mice were quantified using commercial ELISA kits. Compared to vehicle- treated mice, there was a significant decrease in levels of IL-6 detected in BALF collected from mice treated with HU308 at day 0 and tested at day 3 during the inflammatory phase of the model (Figure 1). No such difference was detected in the levels of TGF-B or TNF-a. IL-6 is an inflammatory cytokine which can induce a variety of proteins involved in the acute phase response to injury, infection, or other sources of inflammation. Thus, decreasing IL-6 with acute treatment by HU308 immediately following inflammatory insult is a promising approach to dampening downstream inflammatory signaling and consequences.
[00182] Next, a quantification of lung hydroxyproline content was performed using frozen lung samples acquired from the HU308 or vehicle-treated mice. Hydroxyproline is a standard quantitative biomarker for IPF. In mice who received two administrations of HU308 (at day 0 and day 3), there was a significant decrease in lung hydroxyproline content when tested at day 6 as compared to vehicle-administered mice tested at day 21 (Figure 7). When mice who received the same treatment of HU308 (day 0 and day 3) were tested at day 21, no difference in hydroxyproline content of the lungs was detected as compared to vehicle-treated mice tested at day 21. Thus, the data suggest that HU308 acutely reduces inflammation in this model of lung injury, but prolonged treatment may be necessary to effectively reduce fibrotic lung damage.
[00183] Additionally, a histological analysis was performed using formalin-buffered and paraffin-embedded lung tissue samples acquired from the HU308 or vehicle-treated mice. The histological analysis used a fibrosis grading scale ranging from 0 (“normal lung”) to 8 (“total fibrous obliteration of the field”). This grading scheme, known as the Ashcroft score, is a standard used to measure the severity and extent of fibrotic damage. On this grading scale, animals treated with HU308 every three days throughout the 21 -day post-BLM period displayed significantly lower fibrosis grades than those treated with a vehicle on the same schedule (Figure 7). Importantly, these findings confirm that the previously identified HU308 mediated attenuation of inflammatory responses to chemical lung insult consequently reduce tissue damage in a significant manner.
[00184] Thus, intravenous HU308 treatment in a variety of timing protocols has demonstrated significant anti-inflammatory effect in the mouse model of IPF. Compared to control animals, decreased levels of the inflammatory mediator, IL-6, were found following a single administration of HU308 at the day of chemical insult. Additionally, when treated twice with HU308 over the first three days following insult, levels of the IPF marker, hydroxyproline, were comparatively reduced when the mice were tested at day 6. These findings were further supported by a reduction in the Ashcroft score, a histological scoring scale for fibrotic damage, when the mice were treated with HU308 every three days during the three weeks after BLM. Thus, HU308 appears to offer significant anti-inflammatory protection against chemical insult modeling IPF when administered intravenously over a variety of schedules. Further refinements to the administration regimen may further attenuate inflammatory signaling and resultant tissue damage. In addition, while promising, parenteral formulations used in IV delivery of HU308 are notably deficient from a safety standpoint for application in human subjects and indications, necessitating development and refinement of drug products with key quality attributes in keeping with current regulatory requirements. Notably, key quality attributes for an acceptable clinical formulation include (but are not limited to) an acceptable nonclinical safety profile; excipients at levels accepted by regulatory agencies for IV administration; desirable stability profiles; and compatibility with terminal sterilization techniques (including by filtration).
[00185] Example 6: HU3Q8/Ontemabez as an anti-viral and anti-inflammatory therapeutic.
[00186] Study 1: HU308 in vitro antiviral efficacy against infection with Influenza, Respiratory Syncytial Virus, Adenovirus Type-5, a-coronavirus, and -coronavirus.
[00187] Study 1 was conducted to determine the EC50 & CC50 of test compounds in an in vitro model of infection against 5 viral strains (Influenza, Respiratory Syncytial Virus, Adenovirus Type-5, a-coronavirus, and [3-coronavirus). A dilution series of each test compound (8-point, 2-fold dose titration) was added to cells at various time-points. Vehicle and positive control wells were set up to control for any influence of the compounds alone on cell viability. Cells were visually inspected for the appearance of any cytopathic effects (CPE). A cell viability assay was performed once CPE was complete. EC50 is the concentration which results in 50% viral inhibition, following the addition of compounds. CC50 is the concentration which results in 50% cell viability, following the addition of compounds.
[00188] Methods:
[00189] Cells permissible to infection, indicated in the Conditions Table, were grown and seeded into 96-well plates to a confluency of 80-90%. The test compounds were serially diluted half-log into 8 concentrations in total and added to cells for 1 hour at 37°C and 5% CO2. HU308/ontemabez; EE: 97.68%; 98.84% HU308/ontemabez : 1.16% HU433. After 1 hour, the virus was added to the cells for Ih at lOOx TCID50. A mock infection of blank media was added to the uninfected controls. After infection, the virus/media was removed, and an overlay medium was added with equivalent concentrations of the test compounds. Cells were incubated until extensive cytopathic effects (CPE) were seen in the infected control wells. A cell viability assay was performed on all conditions, with cells receiving the test treatment compared to the vehicle treated and uninfected controls. Calculations are as follows: % Inhibition = [(A-B)Z (C-B)] x 100, where: A: mean optical density of test, B: mean optical density of virus controls, C: mean optical density of cell controls. Negative values occur when A<B, due either to natural variation or compound toxicity. Selectivity index (SI) = CC50 / EC50. Higher numbers indicate a more selective compound. Additional details of the study are outlined in Tables 14 and 15 below:
[00190] Table 14: Cell and viral strains used in anti-infectivity studies.
Figure imgf000047_0001
[00191] Table 15: Test compounds used in anti-infectivity studies with respective concentration ranges. Text in parentheses indicates target viral strain for positive controls.
Figure imgf000048_0001
[00192] Results:
[00193] For all viruses tested, cannabidiol (CBD) and HU308 elicited inhibition of viral growth (Figures 8-12 summarized in Table 15 above and Table 16 below).
[00194] Table 16: Summary of EC50 (pM) and CC50 (pM) values of CBD, HU308, or positive controls in the indicated viral infection models. In conditions where antiviral effects were observed but high drug concentration produced a decrease in cell viability that prohibited the calculation of an EC50, EC50 is listed as >CC50.
Figure imgf000048_0002
[00195] Study 2: HU308 antiviral and anti-inflammatory efficacy against infection with HIV-1 in human neurospheres.
[00196] Study 2 was conducted to address the potential of HU308 to alter viral and cytokine RNA expression in primary neurospheres infected with HIV-1 and treated with combined antiretroviral therapy (cART).
[00197] Methods:
[00198] Neurosphere generation and infection:
[00199] Normal Human Neural Progenitor Cells (ATCC® ACS-5003™) were expanded per the manufacturer’s recommendations. For neurosphere generation, NPCs were seeded at high density in ultra-low attachment, U-shaped bottom 96-well plates in Neural Progenitor Medium. After approximately 48 hours, the medium was replaced with prodifferentiation medium to induce differentiation and replaced every 2 to 3 days for a period of 2 weeks.
[00200] Infection and treatment of infected neurospheres:
[00201] Neurospheres were cultured in the presence of HIV-1 dual tropic 89.6
(MOI: 10). Combined antiretroviral therapy (cART) treatment was simulated by incubation of infected neurospheres for one week with a cocktail consisting of equal parts lamivudine, tenofovir disoproxil fumarate, emtricitabine, and indinavir at a concentration of 45pM. A subset of cART-treated neurospheres were additionally treated with HU308 EE: 98.3%;
99.15% HU308Z 0.85% HU433) at a concentration of IpM. Neurospheres and supernatants were collected 7-days post-treatment for downstream analysis.
[00202] Isolation of exosomes and analysis of RNA expression:
[00203] Exosomes were isolated from HIV-1 viral particles in neurosphere supernatants by sequential centrifugation, precipitation, and fdtration. RNA was isolated utilizing TRIzol Reagent according to manufacturer’s instructions and subjected to RT-qPCR using TaqMan primers specific for TAR and HIV-1 genomic RNA. Copy number was quantified using serial dilutions of DNA from 8E5 positive control cells as quantitative standards and significance was determined using Student’s t-test.
[00204] Results: [00205] cART-HU308 combination treatment appeared to increase reduction of TAR and viral genomic RNA expression in neurospheres infected with HIV-1 (Figure 13) vs. cART treatment alone, while reduction in pro-inflammatory cytokine TNF-a RNA expression was maintained (Figure 14).
[00206] Study 3: HU308 in vitro antiviral efficacy against infection with human f>- coronavirus
[00207] Study 3 was conducted to address the antiviral potential of HU308 in cell lines infected with [3-coronavirus (OC43).
[00208] Methods:
[00209] Cell culture, HU308 handling, and target dose determination
[00210] Hamster kidney fibroblasts (BHK-21) were expanded per the manufacturer’s recommendations. HU308 (Tocris) was dissolved in DMSO to a stock concentration of 40 mM and stored at -80°C. CC50 of HU308 was determined in BHK-21 cells (n=3) using alamarBlue (Thermo, DAL 1025). CC50 was determined to be 6.1 pM, which served as the basis for determining the target doses (3, 1, and 0.3 pM) of HU308 to use in the antiviral assay.
[00211] Antiviral activity assay:
[00212] BHK-21 cells were infected with OC43 with an MOI of 0.1 for 1 hour. After the infection, HU308 was added to the cell culture medium and incubated for 23 h. At 24 h post infection cells and supernatant were collected and freeze-thawed to help liberate cell- associated virus into the medium. Viral stocks were titred on BHK-21 cells using a Median Tissue Culture Infectious Dose (TCID50) assay. Virus stocks were subjected to 5-fold dilutions and used to infect the cells. Antiviral activity of HU308 was measured as % viral inhibition via comparison of viral titre (TCID50 units/mL), calculated by observing the transition of wells from those containing cells (visualized by crystal violet staining) to those that have been cleared due to infection-mediated lysing of the cells. Two independent experiments were performed in triplicate.
[00213] Results:
[00214] HU308 displayed a positive dose-dependent effect on viral inhibition (Figures
15 and 16) in BHK-21 cells infected with [3-coronavirus (OC43), with % inhibition (relative to DMSO control) values of up to 41% (Figure 15) and 88% (Figure 16) in independent experiments.
[00215] Summary:
[00216] HU308 exhibited positive antiviral effects on various in vitro models of viral infection against several strains of human virus. HU308 treatment elicited viral inhibition in cells infected with Influenza A/Panama/2007/99 (H3N2); Respiratory Syncytial Virus A2; Adenovirus Type 5; a-coronavirus (229E), and [3-coronavirus (OC43, in two separate models) (Studies 1 and 3); and elicited inhibition of viral RNA and cytokine production in human neurospheres infected with HIV-1 in combination with cART therapy (Study 2). This data suggests that HU308 treatment may be beneficial in viral-mediated inflammatory indications both as an anti-inflammatory and as an anti-viral agent.
[00217] Example 7: HU308/Qntemabez as therapeutic against SARS.
[00218] SARS-CoV-2 (Italy strain) viral infection was induced in mice (K18-ACE2, n
= 3 - 9 per group) via intranasal administration in an established model of COVID-19 disease. On the day prior to, and for every day following infection, mice were administered either parenteral HU308 (3 or 6 mg/kg/day), a vehicle control, or a standard of care comparator (Remdesivir; 10 mg/kg/day). Mice were monitored daily for disease symptoms, including changes in body temperature or weight, and analyzed using a clinical health scoring scale. Additionally, peripheral whole blood was collected at multiple time points for processing to sera and analyzed for quantity of various inflammatory cytokines or mediators using a Meso Quickplex SQ 120 system. Bronchoalveolar lavage fluid (BALF) was similarly collected at multiple time points and for quantity of various inflammatory cytokines or mediators.
[00219] Methods:
[00220] Induction of SARS-CoV-2 Infection Model
[00221] Mice (K18-ACE2; n = 6/group) were administered either inactivated or replication competent virus (SARS-CoV-2, Italy strain) via intranasal challenge (54 pfu/mouse). Animals were expected to exhibit symptoms of the disease, including change in body weight and temperature, followed by mortality, within 7 - 9 days post-infection in the absence of therapeutic intervention. [00222] Treatment with Parenteral HU308
[00223] HU308 (HU308; EE: 97.68%; 98.84% HU308Z 1.16% HU433) was prepared by dissolving in a vehicle suitable for parenteral administration. Mice (K18-ACE2; n = 6 - 9/group) were administered intraperitoneal HU308, 3 or 10 mg/kg one day prior to viral exposure. Following viral exposure, animals were administered HU308 once daily until sacrifice for analysis.
[00224] Clinical Health Score
[00225] The clinical health of mice was monitored daily via an established clinical Health Score scale by trained observers.
[00226] Sera and BALF Collection and Processing
[00227] Prior to euthanasia, blood was collected on day 0, day 3, or day 6 postinfection via submandibular puncture. Plasma was extracted and stored at -80C until further analyzed. BAL fluid was collected from terminally anesthetized mice and stored at -80C until further analyzed.
[00228] Multiplex Assay
[00229] Blood sera was analyzed using a multiplex assay (Quickplex 120; Meso Scale Diagnostics, LLC) to assess the presence and quantity of: monocyte chemoattractant protein- 1 (MCP-1), macrophage inflammatory protein la (MIP-la), tumor necrosis factor alpha (TNF-alpha), interleukin (IL-) lb, IL-10, IL-6, and interferon gamma (IFN-g).
[00230] Statistical Analysis
[00231] All data are expressed as means ± standard deviation (SD). Statistical analyses were conducted in GraphPad Prism 9 (GraphPad Software, LLC, La Jolla, CA, USA). After confirmation of normal distribution by Kolmogorov-Smimov testing, differences between groups were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test for group wise comparisons. The significance level was considered at p < 0.05.
[00232] Results:
[00233] Health Score
[00234] Following infection with SARS-CoV-2 (day 0), exposed mice exhibited symptoms corresponding with disease progression over the course of the following 6 days. These symptoms were quantified using an established clinical health scoring scale (Figure 17; increased Health Score indicates poor clinical health). Mice exposed to the virus and treated daily with a parenterally administered (intraperitoneal, i.p.) vehicle control exhibited significantly elevated Health Scores as compared to mice who were not exposed to the virus (No Infection). Mice treated with HU308 (3 or 6 mg/kg/day) on the same schedule (1 day prior to viral exposure, then once daily from day 0 to day 6), exhibited significantly lower Health Score ratings, indicating decreased disease severity.
[00235] Blood sera
[00236] Analyses of blood sera indicated decreased concentrations of several inflammatory cytokines in mice treated with parenteral (i.p.) HU308 (3 or 10 mg/kg) when tested either on the day of viral exposure (day 0), or 3- or 6-days post-infection. Specifically, concentrations of MCP-1 (Figure 18) were lower in HU308-treated mice as compared to those treated either with vehicle or with the standard of care for COVID-19, Remdesivir (10 mg/kg/day). Similarly, comparatively decreased concentrations of the cytokines INF-g and IL-6 were found in mice treated with ARDS -003 as compared to either control- or Remdesivir-treated mice.
[00237] Bronchoalveolar lavage fluid
[00238] Analyses of bronchoalveolar lavage fluid (BALF) indicated decreased concentrations of several inflammatory cytokines in mice treated with parenteral (i.p.) HU308 (3 or 10 mg/kg) when tested either 3- or 6-days post-infection. Specifically, concentrations of TNF-a (Figure 19) were lower in HU308-treated mice as compared to those treated either with vehicle or with the standard of care for COVID-19, Remdesivir (10 mg/kg/day). Similarly, decreased levels of the inflammatory mediators IL-10, INF-g, and IL- 6 were found in HU308-treated mice as compared to either vehicle- or Remdesivir-treated mice when BALF was analyzed 6 days following viral exposure.
[00239] The results provided demonstrate parenteral treatment with HU308 reduced systemic cytokine release (primary study outcome parameter) in blood sera collected from mice (K18-ACE2) infected with SARS-CoV-2, a model of the COVID-19 disease. Blood sera and BALF measures were collected immediately following viral exposure, as well as 3- and 6-days post-infection from mice treated daily (including one day prior to viral exposure) with HU308; 3 or 6 mg/kg/day, a vehicle control, or the current standard of care, Remdesivir (10 mg/kg/day). Clinical health scoring was significantly improved by both injected dosages of HU308. Further, analyses of blood sera and bronchoalveolar lavage fluid (BALF) demonstrated a protective effect ofHU308 against infection-induced elevations in several inflammatory cytokines at either 3- or 6-days post-infection.
[00240] Example 8: HU3Q8/Ontemabez as therapeutic against pan-ocular inflammation.
[00241] Endotoxin-induced uveitis (EIU), a model of pan-ocular inflammation, was established in rabbits and rats, via a single intravitreal injection of lipopolysaccharide (LPS; isolated from Escherichia coli). In a case-study rabbit, parenteral administration (intravenous) treatment with HU308 (1.5 mg/kg) led to a trend towards decreased scores in multiple clinical parameters of ocular irritation and inflammation as assessed at 24- and 48-hours. In rats, intravenous treatment with the active pharmaceutical ingredient in ARDS-003 (HU308, 3 mg/kg) led to decreased levels of several key pro-inflammatory mediators 8-hours following induction of EIU.
[00242] Rabbit Uveitis
[00243] In sedated rabbits, a comprehensive ocular examination was performed as a baseline measure. This included slit lamp biomicroscopy with fluorescein staining, retinal examination via direct ophthalmoscopy, and a clinical uveitis score (Standardization of Uveitis Nomenclature ‘SUN’ scoring system). Uveitis was induced in one eye via a single intravitreal injection of LPS. Control animals received an intravitreal injection of saline, at equal volume. Ten minutes following discontinuation of sedation, rabbits received a single intravenous bolus of HU308 (3 mg/kg), or vehicle, with subsequent doses at 12-, 24-, and 36- hours (total 4 doses). Ocular examinations were repeated at 24- and 48-hours.
[00244] Rat Uveitis
[00245] In rats under general anesthesia, uveitis was in induced in one eye via a single intravitreal injection of LPS. Control animals received an intravitreal injection of saline at equivalent volume. Fifteen minutes following LPS administration, rats received a single intravenous bolus of HU308 (3 mg/kg). After 8-hours, rats were euthanized and the uveitis eye was enucleated and flash frozen in liquid nitrogen. Eyes were subsequently thawed, mechanically homogenized, and total protein levels were quantified. Eye homogenate was analyzed via Luminex, and analyte levels were normalized against total protein concentrations.
[00246] Animal Care - Rabbits
[00247] Male New Zealand White Rabbits (2.8 - 4.5 kg) were kept on a light/dark cycle (07:00-19:00) and fed ad libitum. Ethics approval was obtained for all procedures.
[00248] Anesthesia & Endotoxin-Induced Uveitis
[00249] Under local anesthesia with subcutaneous 1% lidocaine, a 24G intravenous (IV) catheter was inserted into the marginal ear vein. Subsequently, animals were placed under deep sedation via a single bolus of 5 mg/kg propofol followed by a propofol infusion from 0.7 - 1.4 mg/kg/min, titrated to effect (deep sedation without respiratory or cardiovascular compromise). Following a baseline ocular examination (described below in d), the ocular surface was anesthetized with proparacaine (2 drops), and the pars plana was marked using an indentation from a Castroviejo caliper. Asepsis was performed by applying 1% povidone iodine to the ocular surface, before EIU was induced via an intravitreal injection of LPS, administered to one eye via the location indented at the pars plana, using a 0.5-inch 28-gauge needle. The total inj ectate volume was 50 pL, which contained 200 ng LPS (isolated from Escherichia coli) in saline. Control animals received an injection with 50 pL saline.
[00250] Treatment with Intravenous HU308
[00251] HU308 (Tocris Bioscience) was prepared in a vehicle suitable for parenteral administration, yielding a working concentration of 3 mg/mL. Rabbits received a twice daily intravenous bolus of 1.5 mg/kg HU308, with the initial dose 10 minutes following discontinuation of anesthesia (for baseline examination and intravitreal LPS administration). Subsequent doses were administered at 12-, 24-, and 36-hours (total of 4 doses).
[00252] Clinical Ocular Examination
[00253] At 0-, 24-, and 48-hours, a comprehensive ocular examination was performed using a portable slit lamp biomicroscope (Keeler PSL). One drop of 2% sodium fluorescein was applied to the ocular surface of each eye, followed by examination for comeal/conjunctival epithelial compromise (indicating abrasions, ulceration etc.) under cobalt blue light. For parameters of ocular irritation, a clinical score of 0 - 3 was assigned - 0: not present, 1: trace/mild, 2: moderate, 3: extensive/severe. The rabbit was then mounted in a table slit lamp biomicroscope and anterior chamber cells and flare were quantified as per the Standardization of Uveitis Nomenclature (SUN) scoring system - using a 1 mm x 2 mm slit beam at maximum brightness intensity and at a ~90-degree angle between incident beam and examiner ocular lenses. Two drops of 1 % tropicamide were applied to each eye, and once dilated, direct ophthalmoscopy was performed (Volk InView). Two images were collected, one of the fundus, and one of the peripheral retinal vasculature. At the same time, SUN scoring was utilized to assign scores for vitreous haze. At the 48-hour timepoint, following final ocular examination/ data collection, animals were euthanized via barbiturate overdose (150 mg/kg sodium pentobarbital IV push).
[00254] Animal Care -Rats
[00255] Male Lewis Rats (250 - 350g) were kept on a light/dark cycle (07:00-19:00) and fed ad libitum. Ethics approval was obtained for all procedures.
[00256] Endotoxin-Induced Uveitis
[00257] Rats were placed under general anesthesia using isoflurane. To induce uveitis, an intravitreal injection of LPS was administered to one eye via the pars plana, under a dissecting microscope and using a Hamilton microliter syringe fitted with a 30-gauge needle. The total injectate volume was 5 pL, which contained 200 ng LPS (isolated from Escherichia coli) in normal saline. Upon completion of the injection, the puncture site was sealed with 3M Vetbond Tissue Adhesive. The animal was allowed to recover and was observed for complications.
[00258] Treatment with Intravenous HU308
[00259] HU308 (Tocris Bioscience) was prepared by dissolving in a vehicle suitable for parenteral administration, yielding a working concentration of 10 mg/mL. Rats received a single intravenous bolus of 3 mg/kg HU308, 15-minutes after induction of EIU.
[00260] Euthanasia, Eye Tissue Collection & Homogenization
[00261] Animals were euthanized via barbiturate overdose (150 mg/kg sodium pentobarbital IV push), eyes were immediately enucleated and flash-frozen in liquid nitrogen, before being stored at -80°C.
[00262] Luminex Assay [00263] A magnetic bead-based multiplex Luminex® assay (Research & Development Systems) was used to assess for presence of: IL-l-alpha, IL-l-beta, IL-2, IL-4, IL-6, IL-10, IL-13, ICAM-1, IFN-gamma, TNF-alpha, and GM-CSF. The whole eye homogenate was diluted 1:4 using the diluent provided by the Luminex® kit. Samples were run in duplicate. Each n value represents one eye. All samples were run on a Bio-Rad 200 instrument with Bio-Plex software, according to the manufacturer's instructions. Standard curves for each analyte were generated based on reference compounds provided by the Luminex® kit.
Analyte levels were normalized against total protein concentrations for each eye homogenate.
[00264] Results: Rabbit Uveitis
[00265] Treatment with 1.5 mg/kg HU308 IV twice daily led to a trend towards lower scores in 8 parameters of ocular inflammation and disease-specific uveitis scores at 24- and/or 48-hours following disease induction, as displayed in Figures 20-26 and Figures 27- 28. For anterior chamber cells (SUN score), no difference was observed between the HU308- treated rabbit and vehicle-treated rabbits at 24- and 48-hours, as displayed in Figure 26.
[00266] Results: Rat Uveitis
[00267] Treatment with a single dose of 3 mg/kg HU308 IV led to decreased levels of five inflammatory mediators in whole-eye tissue 8-hours following induction of EIU, as compared to animals with EIU that did not receive HU308 (Figures 29-33) and Table 17.
[00268] Table 17. Summary of inflammatory mediator results in whole eye homogenate at 8-hours following induction of EIU in Lewis rats. Mean (picograms/mL) +/- SEM.
IL-la Pro- 254.0±l 87.1 / Increased 1795±301.1 / Decreased Inflammatory 1795±301.1 1421il59.1
IFN-Y Pro- 661.6±458.9 / Increased 2098il217 / Decreased Inflammatory 2098±1217 O.OOiO.OO
VEGF Pro- Angiogenic 147.7i29.06 / Unchanged 133.2il5.73 / Decreased & Pro- 133.2±15.73 93.94±7.92 Inflammatory
IL-4 Pro- 32.72il0.27 / Increased 48.29il7.54 / Decreased
Inflammatory 48.29il7.54 17.82±6.68
TNF-a Pro- 125.3±27.5 / Increased 321.0i92.96 / Decreased
Inflammatory 321.0i92.96 180.7±35.59
[00269] Example 9: HU3Q8/Ontemabez as therapeutic agent for interstitial cystitis [00270] Interstitial cystitis (IC) was induced in mice via systemic administration of lipopolysaccharide (LPS; endotoxin). Intravital microscopy (IVM) was used to quantify leukocytes adhering to the endothelium of submucosal venules in the bladder 1.5-hours following parenteral treatment with HU308. Treatment with HU308 led to a significant decrease in levels of adherent leukocytes. Functional capillary density (FCD) was used to assess the microcirculatory function in bladder tissue. Following LPS administration, FCD levels decreased significantly, and HU308 treatment restored levels to those in control mice receiving a saline injection instead of LPS.
[00271] Methods:
[00272] In mice under general anesthesia, IC was induced either via systemic administration of LPS (20 mg/kg; single intraperitoneal [IP] injection). Treatment with parenteral HU308 (5 mg/kg; single IP injection) was administered 30-minutes following administration of LPS. At 2-hours post-LPS administration, mice were placed under general anesthesia and a fluorescent dye (Rhodamine 6G and fluorescein isothiocyanate; FITC) was administered intravenously (IV) in order to non-specifically stain leukocytes and provide video contrast of vessels. The bladder was then surgically isolated and gently exteriorized. C rede’s maneuver was performed to manually void urine from the bladder. Mice were secured in a stereotaxic frame, the bladder was covered with a coverslip to enhance optics, and IVM was performed 2-hours following LPS administration. Offline video analysis was then performed, allowing quantification of adherent leukocytes and FCD scoring.
[00273] Systemic LPS-Induced IC & Animal Groups
[00274] Female CD-I mice (30 ± 3 g) were used for IC experiments. IC was induced via an intraperitoneal (IP) injection of LPS (20 mg/kg) from Escherichia coli (serotype 026:B6, Sigma-Aldrich, Oakville, ON, Canada) dissolved in 50 pL normal saline (Hospira, Montreal, Canada). LPS was administered 15-minutes following induction of anesthesia. Once 30-minutes had elapsed following LPS administration, a total volume of 50 pL of drug treatment was administered parenterally.
[00275] The experimental groups for this model were as follows: (1) healthy control animals (CON; 50 pL normal saline IP, treated with normal saline IP, n = 9), (2) untreated IC animals (LPS; 50 pL LPS IP, treated with normal saline IP, n = 9), (3) IC treated with HU308 (LPS + HU308; 50 pL LPS IP, treated with 5 mg/kg HU308 dissolved in 30% DMSO, n = 4).
[00276] Animals were anesthetized via IP injection of sodium pentobarbital (65 mg/kg, Ceva Sante Animate, Montreal, QC, Canada). Following induction of anesthesia, mice were placed on a heating pad to maintain a body temperature of 37 °C, which was monitored with a rectal temperature probe. Anesthesia was maintained with repeated IP administration of 5 mg/kg sodium pentobarbital, while the depth of anesthesia was monitored by clinical examination (return of pedal withdrawal reflex). Once the animals achieved a surgical depth of anesthesia, a Maylard incision of the lower abdomen was performed using surgical scissors. The abdominal muscle layer was lifted using forceps, and an incision made along the linea alba to expose the bladder. Using saline-soaked cotton-tipped applicators, the bladder was gently exteriorized. C rede’s maneuver was then performed to manually void urine from the bladder. IVM was performed 2-hours following LPS administration.
[00277] Drug and Vehicle Preparation & Treatment
[00278] HU308 (Tocris Bioscience) was prepared by dissolving in a vehicle suitable for parenteral administration. Mice received a single injection of 5 mg/kg HU308 (IP), 30- minutes after LPS administration.
[00279] Intravital Microscopy
[00280] After confirming the depth of anesthesia via the absence of a toe-pinch reflex, a tail vein (IV) injection of two fluorochrome dyes was performed 15-minutes prior to the start of IVM. The fluorochrome dye mixture consisted of Rhodamine 6G (1.5 ml/kg, 0.75 mg/kg body weight, Sigma-Aldrich, ON, Canada) and fluorescein isothiocyanate (FITC)- albumin (1 ml/kg, 50 mg/kg, Sigma-Aldrich, ON, Canada). With the needle bevel of a 29G x !4 inch 0.5ml U-100 insulin syringe (BD Canada) facing upwards parallel to the vein, the fluorochrome dyes were injected into the tail vein. Rhodamine 6G allows for visualization of leukocytes, while FITC-albumin was used to facilitate evaluation of functional capillary density by providing enhanced illumination of the bladder capillary beds. All tail vein injections were carried out in minimum light to minimize the photobleaching of fluorochromes.
[00281] After the 15-minute period, a small clean glass cover slip was positioned on top of the bladder, and the animal positioned under the microscope. To avoid movement of the bladder as a result of diaphragm activity, a metal arm was used to apply gentle pressure to the upper abdominal area. Any areas of the abdomen that were not subject to experimentation were covered in gauze that was saturated in saline solution maintained at physiological temperature as to avoid dehydration and exposure to ambient air. Intravital fluorescent video microscopy was performed using the following technical devices: an epifluorescence microscope (Leica DMLM, Wetzlar, Germany, Filterset: 13, green light filter), light source (LEG EBQ 100, Jena, Germany), and a black and white monitor (Speco Technologies, Texas, US). The images were transferred to a Windows desktop computer and recorded using WinDV software (version 1.2.3, Czech Republic). The leukocytes within the microcirculation of the bladder were visible under the 20x objective with green light. Five to seven randomly selected visual fields containing bladder venules were recorded for 30 seconds. The filter was then changed for examination with FITC-albumin under blue light, allowing for the examination of capillary blood flow. Five to seven randomly selected visual fields with capillaries were recorded again for 30 seconds.
[00282] Video Analysis: Adherent Leukocytes & Functional Capillary Density
[00283] Experimental microcirculatory parameters were assessed via off-line analysis of the captured IVM videos using ImageJ V 1.50f software (National Institute of Health, USA). Specific parameters that were analyzed included leukocyte adhesion and functional capillary density (FCD), a microcirculatory perfusion parameter. When examining leukocyte adhesion, the length and diameter of the venule in the field of view was used to calculate the endothelial surface area. Adhering leukocytes were defined as white blood cells that stayed immobile on the endothelial surface for the complete observation period of 30 seconds. The number of adherent leukocytes in a randomly selected vessel was reported as number of cells per square millimeter of endothelial surface area. When analyzing FCD, a single rectangular field covering the maximum possible area was drawn for each video segment, and capillaries containing FITC-albumin marked plasma found in the rectangular field were chosen. The length of perfused capillaries was measured by manually drawing a line within the lumen of the capillary. Capillaries with absent and/or intermittent flow were characterized as dysfunctional capillaries and not accounted for, whereas capillaries with continuous flow, regardless of the speed of the flowing cells, were characterized as functional capillaries. Summing the length of all corresponding capillaries and dividing the sum length with the measured area of the rectangular field calculated the FCD. For the initial pilot segment of this study, FCD was expressed as a percentage relative to the FCD of the control group. At the 2- hour timepoint following video collection for IVM/FCD, animals were euthanized via barbiturate overdose (150 mg/kg sodium pentobarbital, IP).
[00284] Statistical Analysis
[00285] All data are expressed as means ± standard deviation (SD). Statistical analyses of the results were performed using the software GraphPad Prism 6.0 (GraphPad Software Inc, La Jolla, CA, USA). After confirmation of normal distribution by Kolmogorov-Smirnov testing, differences between groups were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test for group wise comparisons. The significance level was considered at p < 0.05.
[00286] Results:
[00287] Systemically administered LPS significantly increased the number of leukocytes adhering to the endothelium of submucosal bladder venules. Parenteral treatment with HU308 (5 mg/kg) significantly reduced the number of adherent leukocytes in LPS challenged animals (Figure 34).
[00288] The functional capillary density of the bladder microcirculation was significantly reduced following LPS administration. Treatment with HU308 restored capillary perfusion to levels observed in healthy control animals (Figure 35).
[00289] Example 10: HU3Q8/Ontemabez as therapeutic agent for treatment of systemic cytokine release and sepsis.
[00290] Parenteral (Intravenous) treatment with the active pharmaceutical ingredient of ARDS-003 (HU308; 3 mg/kg) significantly reduced systemic cytokine release (primary study outcome parameter) in acute lung injury -induced systemic inflammation - a model of experimental sepsis. In addition to a reduction in pro-inflammatory cytokines, at six hours following disease induction by intranasal pathogen administration, both attenuation of histological lung damage as well as a reduction of extrapulmonary leukocyte activation using intravital imaging of the intestinal microcirculation were observed.
[00291] Methods:
[00292] Pulmonary and systemic inflammation were induced in anesthetized mice via intranasal administration of the endotoxin, lipopolysaccharide (LPS; 5 mg/kg). Immediately following LPS administration, mice received a single intravenous bolus of HU308 (3 mg/kg). Mice were allowed to recover following which, at 6 hours, and under general anesthesia, mice received an intravenous injection of fluorescent dyes before a segment of small intestine was exposed via laparotomy for intravital microscopy (IVM). Videos were collected for offline quantification of leukocytes adhering to the intestinal vascular endothelium. Peripheral whole blood was collected at 6-hours via cardiac puncture and centrifuged to isolate plasma for cytokine/chemokine analysis (CXCL1, CXCL2, ICAM, IL-1, IL-10, IL-6, P-selectin, IFN-gamma, TNF-alpha, and LIX) using a Luminex® assay. Mouse lungs were collected for analysis using a histopathological scoring system established by the American Thoracic Society.
[00293] Induction of Pulmonary Injury via Intranasal LPS Administration
[00294] Mice (n = 10 mice per condition) were anesthetized using isoflurane. LPS working solution was prepared by diluting LPS, purified from Pseudomonas aeruginosa, in sterile saline to yield a final concentration of 10 mg/mL. LPS working solution was brought to physiological temperature prior to administration. To administer LPS at 5 mg/kg, the mouse was held vertically, and the tongue was swept to one side and gently held in place. A pipette was used to administer LPS solution via the left nares at a rate of 1 droplet every 7-10 seconds, up to the calculated dose drawn in the pipette tip. Note a maximum of 20 pL per mouse was delivered. Following administration, the nares was flushed with 10 pL sterile saline and the animal was recovered and observed for complications.
[00295] Treatment with Intravenous HU308
[00296] HU308 (Tocris Bioscience) was prepared in a vehicle suitable for parenteral administration, yielding a working concentration of 10 mg/mL. Mice received a single intravenous bolus of 3 mg/mL HU308 at time 0, immediately following induction of pulmonary injury.
[00297] Peripheral Blood Collection & Processing
[00298] Blood was collected from anesthetized mice via cardiac puncture. For cardiac puncture, a 25G heparinized needle was inserted into the heart at the apex and blood was slowly withdrawn into a syringe, before being transferred to heparinized Eppendorf tubes. Blood samples were processed via centrifugation (15 minutes at 1500g) to yield plasma and samples were stored at -80°C. [00299] Intestinal IVM & Analysis
[00300] Under general anesthesia, mice received a single intravenous bolus injection of fluorescent dyes (Rhodamine-6G and FITC) in order to non-specifically stain leukocytes and visualize blood flow, respectively. Laparotomy was performed via a vertical midline incision, followed by dissection to allow identification and isolation of the small intestine. A loop of terminal ileum, proximal to the ileocecal valve, was selected for imaging and placed on a specifically designed stage attached to the intravital microscope. Throughout the period of imaging, the intestine was superfused with normal saline at physiologic temperature to prevent desiccation. Videos were collected for offline analysis. Intravital microscopy was performed 6 hours after pulmonary delivery of LPS.
[00301] Analysis was performed using ImageJ 32-bit version software (National Institutes of Health, USA). A VI vessel (>50 pm diameter) segment was selected, and its surface area calculated. The number of leukocytes adhering to the endothelium over the full video course of 30 seconds were counted as ‘adherent leukocytes.’ The final data output in adherent leukocytes per square millimeter of endothelium was subsequently calculated for each analyzed vessel. Quantification of leukocytes in the intestine is representative of the levels of systemic inflammation. Adherence is mediated by levels of cytokines/chemokines and cellular adhesion molecules in the vasculature of a distant organ not associated with the route of delivery of LPS.
[00302] Lung Tissue Collection & Histologic Preparation
[00303] The trachea and lungs were harvested and a 23G blunt-tipped needle connected to a 10 mL syringe containing 10% formalin was gently threaded into the trachea. The trachea was then clasped with forceps in order to hold the needle in place, and the lungs were gently inflated with formalin until fully expanded, but not over-inflated. The whole lung lobes and trachea were then placed in ajar filled with formalin, allowing fixation of tissues for a minimum of 24 hours. Following fixation, the lungs were embedded in paraffin and subsequently sectioned at 5 pm. Tissues sections were then stained with hematoxylin and eosin and cover-slipped with non-aqueous mounting media.
[00304] Histopathologic Scoring
[00305] Scores were assigned by assessing multiple parameters, including the presence/magnitude of: neutrophils in the alveolar space, neutrophils in the interstitial space, proteinaceous debris filling the airspaces, alveolar septal thickening, and hyaline membranes - based on a grading system for experimental acute lung injury in animals published by the American Thoracic Society. Scores for each section were generated from the score assigned to 10 unique histological fields, selected at random using 20 X magnification. Fields selected were areas that are not close proximity to large pulmonary arteries/veins or bronchioles, and further were not selected when the vessels present encompass more than 50% of the field of view. Each aforementioned parameter was assigned a score of 0 (indicating ‘none’), 1 (indicating 1-5), or 2 (indicating >5). The sum of these scores yielded a total score per field, which ranged from 0 - 6. All 10 scores were averaged to yield a final histopathologic score per animal. Histopathologic scoring for all animals was performed primarily by one individual, and each animal was scored by one individual. All those assigning scores were blinded to treatment groups.
[00306] Luminex Assay
[00307] A magnetic bead based multiplex Luminex assay (Research & Development Systems) was used to assess the presence of: CXCL1, CXCL2, ICAM, IL-1, IL-10, IL-6, P- selectin, IFN-gamma, TNF-alpha, and LIX in heparinized plasma samples. Plasma was diluted (1:2) using the 10-Plex Mouse Cytokine ELISA Kit diluent. Samples were run in duplicate on a Bio-Rad 200 instrument with Bio-Plex software, according to the manufacturer's instructions. Standard curves for each analyte were generated based on reference compounds provided by the kits.
[00308] Statistical Analysis
[00309] Data were analyzed via one-way analysis of variance (ANOVA). Animal numbers per experimental group: n=10.
[00310] Results:
[00311] At 6-hours following pulmonary installation of LPS, significant local and systemic inflammation was observed as evidenced by pulmonary histopathologic scores and leukocyte adhesion to the endothelium of intestinal vessels (Figures 36-40). Further, LPS administration led to a systemic inflammatory response at 6-hours, evidenced by increased levels of Interleukin-6 (IL-6), C-X-C Motif Chemokine Ligand 2 (CXCL2), and tumor necrosis factor alpha (TNF-a) in peripheral whole blood. [00312] Treatment with a single intravenous dose of HU308 at time of disease induction led to a significant (P<0.05) reduction in the number of adherent leukocytes in intestinal vessels as demonstrated by IVM (Figure 36). Similarly, HU308 treatment led to a significant (P<0.01) reduction in lung histopathological scores (Figure 37). Levels of CXCL2 and TNF-a in plasma obtained from peripheral whole blood were also significantly decreased following HU308 treatment. Further, there was no significant difference in analyte levels between control and HU308 treatment group animals for IL-6, CXCL2, and TNF-a.
[00313] The following are references that provide background for aspects of the invention described herein and throughout.
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[00314] All citations are hereby incorporated by reference. In the event of conflicting information with statements between any reference to or incorporated herein, and the present disclosure, the present disclosure will act as the guiding authority.
[00315] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

- 69 - WHAT IS CLAIMED IS:
1. A pharmaceutical composition comprising: ontemabez; a first solvent; and a first emulsifier.
2. The pharmaceutical composition of claim 1, further comprising water.
3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is a nanoemulsion.
4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is sterilized.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is sterilized by filtration through a filter.
6. The pharmaceutical composition of claim 5, wherein the filter has a pore size of about 0.2 pm or about 0.22 pm.
7. The pharmaceutical composition of any one of claims 3-6, wherein the nanoemulsion is a suspension of nanoparticles in water. - 70 -
8. The pharmaceutical composition of claim 7, wherein a mean diameter of the nanoparticles is about 100-400 nanometers (nm).
9. The pharmaceutical composition of claim 8, wherein the mean diameter is about 150- 250 nm.
10. The pharmaceutical composition of claim 9, wherein the mean diameter is about 200 nm or less.
11. The pharmaceutical composition of any one of claims 1-10, wherein the pharmaceutical composition comprises about 0.1-3% w/w ontemabez.
12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition comprises about 1 % w/w ontemabez.
13. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition comprises about 2 % w/w ontemabez.
14. The pharmaceutical composition of any one of claim 1-13, further comprising HU433, the enantiomer of ontemabez, desmethyl ontemabez, didesmethyl ontemabez pivalate, ontemabez pivalate or a combination thereof.
15. The pharmaceutical composition of claim 14, wherein the weight ratio of ontemabez to HU433 is between 100-97.5% ontemabez: 0-2.5% HU433. - 71 -
16. The pharmaceutical composition of any one of claims 1-15, wherein the first solvent is soybean oil, or medium-chain triglyceride (MCT) oil.
17. The pharmaceutical composition of claim 16, wherein the MCT oil comprises Miglyol 812N.
18. The pharmaceutical composition of any one of claims 1-17, further comprising a second solvent.
19. The pharmaceutical composition of claim 18, wherein the second solvent is soybean oil, or medium-chain triglyceride (MCT) oil, and wherein the first solvent and the second solvent are different.
20. The pharmaceutical composition of any one of claims 1-19, wherein a total solvent content of the pharmaceutical composition is about 5-25 %w/w.
21. The pharmaceutical composition of claim 20, wherein the total solvent content is about 16.1 %w/w.
22. The pharmaceutical composition of claim 20, wherein the total solvent content is about 20 %w/w.
23. The pharmaceutical composition of any one of claims 20, wherein the first solvent is soybean oil and a total solvent content is about 20 %w/w. - 72 -
24. The pharmaceutical composition of claim 18, wherein the first solvent is soybean oil, the second solvent is MCT oil and a total solvent content is about 16.1 %w/w.
25. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition comprises about 15 %w/w of soybean oil and about 1.1 %w/w MCT oil.
26. The pharmaceutical composition of any one of claims 1-25, wherein the first emulsifier is egg lecithin, polyoxyl 15 -hydroxy stearate or sodium oleate.
27. The pharmaceutical composition of claim 26, wherein the first emulsifier is egg lecithin.
28. The pharmaceutical composition of any one of claims 1-27, further comprising a second emulsifier.
29. The pharmaceutical composition of claim 28, wherein the second emulsifier is egg lecithin, polyoxyl 15 -hydroxy stearate or sodium oleate, and the first emulsifier and the second emulsifier are different.
30. The pharmaceutical composition of claim 29, wherein the first emulsifier is polyoxyl 15 -hydroxy stearate and the second emulsifier is sodium oleate.
31. The pharmaceutical composition of claim 30, wherein the polyoxyl 15- hydroxystearate is present in an amount greater than sodium oleate by an amount of between - 73 - about 5 and about 20 times by weight, preferably between about 7 and about 15 times by weight, more preferably about 9.2 times by weight.
32. The pharmaceutical composition of any one of claims 1-31, wherein the first emulsifier is egg lecithin at a concentration of 1.2 %w/w.
33. The pharmaceutical composition of claim 31 wherein the first emulsifier is polyoxyl 15 -hydroxy stearate at a concentration of 4.4 %w/w, and the second emulsifier is sodium oleate at a concentration of 0.48 %w/w.
34. The pharmaceutical composition of any one of claims 1 to 33, comprising ontemabez and soybean oil, wherein the weight ratio of ontemabez to soybean oil is 1:5 to 1:20, preferably 1:10.
35. The pharmaceutical composition of any one of claims 1 to 34, comprising ontemabez and kolliphor HS-15 (polyoxyl 15 -hydroxy stearate), wherein the weight ratio or ontemabez to kolliphor HS-15 (polyoxyl 15 -hydroxy stearate) is 1: 1 to 1:3, preferably 1:2.2.
36. The pharmaceutical composition of any one of claims 1-35, further comprising a first preservative.
37. The pharmaceutical composition of claim 36, wherein the first preservative is EDTA, benzyl alcohol or sodium benzoate. - 74 -
38. The pharmaceutical composition of any one of claims 36-37, further comprising a second preservative.
39. The pharmaceutical composition of claim 38, wherein the second preservative is EDTA, benzyl alcohol or sodium benzoate, and wherein the first and second preservatives are different.
40. The pharmaceutical composition of any one of claims 36-39, wherein the first preservative is EDTA at a concentration of 0.01 %w/w.
41. The pharmaceutical composition of any one of claims 36-40, wherein the first preservative is benzyl alcohol at a concentration of 0.15 %w/w, and the second preservative is sodium benzoate at 0.10 %w/w.
42. The pharmaceutical composition of any one of claims 1-41, further comprising alphatocopherol.
43. The pharmaceutical composition of claim 42, comprising 0.03 %w/w alphatocopherol.
44. The pharmaceutical composition of any one of claims 1-43, further comprising glycerol.
45. The pharmaceutical composition of claim 44, comprising 2.25 %w/w glycerol.
46. The pharmaceutical composition of any one of claims 1 to 45, comprising ontemabez, Soybean Oil, Polyoxyl 15-Hydroxystearate, Sodium Oleate, Glycerol, and water.
47. The pharmaceutical composition of claim 46, comprising one or more of the following characteristics: a) suitable for parenteral administration in a subject; b) an oil in water nanoemulsion with a mean diameter of particles less than 200 nm, preferably less than about 160nm, more preferably less than about 150nm, still more preferable about 145 nm; c) an osmolality between about 400 mOsmol/kg to about 500 mOsm/kg, preferably about 430 to 475 mOsmol/kg; d) stability for at least about 3 weeks, more preferably about 7 months at conditions of about 5, about 25 and about 40 degrees in relative humidities of about 60%, about 60% and about 70%, respectively, wherein stability is defined as the ontemabez degrading less than about 5% over time, preferably less than about 2%, more preferably less than about 1.6% over time as measured by liquid chromatography; e) a viscosity of between about 3.7 to 4.4 Cps, preferably between about 3.9 to about 4.3; f) pH of about 7-8, more preferably about 7.2-7.8, more preferably about 7.5.
48. The pharmaceutical composition of any one of claims 1-47 prepared by high pressure homogenization at about 1500 bar.
49. The pharmaceutical composition of claim 48, prepared by at least 5 cycles of homogenization.
50. A pharmaceutical composition comprising: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 4.40 %w/w Polyoxyl 15 -Hydroxy stearate; about 0.48 %w/w Sodium Oleate; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.01 %w/w Disodium EDTA; and about 70.84 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
51. A pharmaceutical composition comprising: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 4.40 %w/w Polyoxyl 15 -Hydroxy stearate; about 0.48 %w/w Sodium Oleate; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.15 %w/w Benzyl Alcohol; about 0.10 %w/w Sodium Benzoate; and about 70.59 %w/w Water, - 77 - wherein the pharmaceutical composition is a nanoemulsion.
52. A pharmaceutical composition comprising: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w Egg Lecithin; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.01 %w/w Disodium EDTA; and about 74.52 %w/w Water, wherein the pharmaceutical composition is a nanoemulsion.
53. A pharmaceutical composition comprising: about 2.00 %w/w ontemabez; about 20.00 %w/w Soybean Oil; about 1.20 %w/w Egg Lecithin; about 2.25 %w/w Glycerol; about 0.03 %w/w Alpha-Tocopherol; about 0.15 %w/w Benzyl Alcohol; about 0.10 %w/w Sodium Benzoate; and about 74.27 %w/w Water, - 78 - wherein the pharmaceutical composition is a nanoemulsion.
54. A method for preparation of a nanoemulsion comprising nanoparticles of ontemabez, a first solvent, a first emulsifier and water, the method comprising: mixing the ontemabez, the first solvent, the first emulsifier and water to make a crude emulsion; homogenizing the crude emulsion under high pressure; and repeating the homogenizing step at least five times.
55. The method of claim 54, wherein a diameter of the nanoparticles is about 100-400 nanometers (nm).
56. The method of claim 55, wherein the diameter is about 150-250 nm.
57. The method of claim 56, wherein the diameter is under about 200 nm.
58. The method of any one of claims 54-57, wherein high pressure is about 700-2000 bar.
59. The method of claim 58, wherein the high pressure is about 750-1500 bar.
60. The method of claim 59, wherein the high pressure is about 750 bar.
61. The method of claim 59, wherein the high pressure is about 1500 bar. - 79 -
62. The method of any one of claims 54-61, wherein repeating comprises repeating the homogenizing step at least 5, 6, 7, 8, 9, 10 times or more.
63. The method of any one of claims 54-62, further comprising cooling the crude emulsion after homogenizing.
64. The method of claim 63, wherein cooling comprises cooling with an ice-water bath.
65. The method of any one of claims 62-65, wherein repeating comprises repeating the homogenizing and cooling steps.
66. The method of claim 65, wherein repeating comprises repeating at least 5, 6, 7, 8, 9, 10 or more times.
67. The method of any one of claims 54-66, further comprising adjusting the pH prior to homogenization.
68. The method of claim 67, wherein adjusting comprises adjusting the pH to 7.5.
69. The method of any one of claims 54-68, further comprising premixing ontemabez, the first solvent, and the first emulsifier, prior to the mixing step. - 80 -
70. The method of claim 69, further comprising heating the premixed components and heating the water, prior to the mixing step.
71. The method of claim 70, wherein heating comprises heating to about 70°C.
72. Use of the pharmaceutical composition of any one of claims 1-53 for parenteral administration.
73. Use of the pharmaceutical composition of any one of claims 1-53 for treatment of inflammation.
74. Use of the pharmaceutical composition of any one of claims 1-53 for treatment of viral infection or one or more effects or symptoms associated with a viral infection.
75. Use of claim 73 wherein the inflammation is systemic inflammation.
76. The use of claim 73, wherein the systemic inflammation is one or more of, or caused by one or more of: systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, cytokine release syndrome (CRS), cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), or multiple organ dysfunction syndrome (MODS).
PCT/CA2021/051882 2020-12-24 2021-12-23 Parenteral cannabinoid formulations and uses thereof WO2022133612A1 (en)

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