CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional application no. 62/929,576, filed Nov. 1, 2019 and entitled Self-Emulsifying drug delivery system and uses thereof, the entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present disclosure relates generally to delivery systems, and more particularly to self- emulsifying drug delivery systems and uses thereof.

INTRODUCTION

[0003] Self-emulsifying drug delivery systems (SEDDS) are complex lipid drug delivery systems comprising a mixture of oils, co-solvents, surfactants, and/or co-surfactants that can spontaneously self-assemble into oil-in-water (O/W) or water-in-oil (W/O) nano (-100-400 nm in diameter) or micro (<100 nm in diameter) emulsions. Surfactants delay droplet coalescence by reducing the surface free energy associated with the oil-water interface creating a rigid, viscous mono-layer at the interface. Self-emulsifying microemulsions require ultra-low interfacial tension that may be achievable, to some extent, with the use of co-surfactants (generally polyols like glycerol and propylene glycol or short chain alcohols like ethanol).

[0004] The use of polyols and short chain alcohols in microemulsions have been reported to “tune” two critical interfacial parameters. First, the amphiphilic nature of polyols can lead to their spontaneous self-assembly at the oil-water interface where the polar portion of the polyol aligns towards the aqueous phase and the apolar aliphatic chain aligns toward the oil phase, which collectively modifies film curvature. Second, the self-assembly of polyols at the interface can increase the elasticity of the interfacial film (referred to as the bending moduli).

[0005] Microemulsions can have low energy requirements (spontaneous formation), thermodynamic stability, low viscosity, and high solubilization capacity, which may make microemulsions a useful delivery-vehicle for cosmetic, food, and pharmaceutical formulations. However, translation of microemulsions into practice has been limited due to the high surfactant concentrations typically required, the need for co-surfactants (exceeding 10%), and low drug loading efficiency. [0006] Despite the advances made to date in the development of microemulsion delivery vehicles, there is room for improvement to address the above-mentioned problems and shortcomings.

[0007] It is an object of the present disclosure to obviate or mitigate at least one of the above- mentioned disadvantages, and to provide a novel SEDDS and uses thereof.

SUMMARY OF THE INVENTION

[0008] The teachings described herein may, in one broad aspect, relate to a self-emulsifying drug delivery system (SEDDS) comprising an avocado polyol, a surfactant, and an oil.

[0009] Other aspects of the teachings described herein, which may be used in combination with any other aspect, including the broad aspect listed above, may include that the avocado polyol comprises at least one of avocadene, avocadyne, and a mixture thereof.

[0010] The surfactant may include a non-ionic surfactant.

[0011] The surfactant may include a polysorbate.

[0012] The surfactant may include polysorbate 80.

[0013] The surfactant may include CREMOPHOR EL™.

[0014] The oil may include a medium chain triglyceride oil.

[0015] The oil may include at least one of coconut oil and NEOBEE® M-5.

[0016] The surfactant and oil may be in a ratio of 1:1 (w/w).

[0017] The surfactant may include polysorbate 80 and the oil comprises NEOBEE® M-5.

[0018] The oil and surfactant may be diluted at least 10 fold in an aqueous buffer.

[0019] The aqueous buffer is phosphate buffered saline (PBS).

[0020] The SEDDS may include about 1-2% w/w avocadene.

[0021] The SEDDS may include about 1.5% w/w avocadene. [0022] The SEDDS may include about 1-2% w/w avocadyne.

[0023] The avocado polyol may include a mixture of avocadene and avocadyne in a 1 : 1 w/w ratio (avocatin B).

[0024] The avocado polyol may include a mixture of avocadene and avocadyne in a 3:1 w/w ratio. [0025] The avocado polyol may include a mixture of avocadene and avocadyne in a 3:2 w/w ratio. [0026] The SEDDS may include about 1-2% w/w avocatin B.

[0027] In accordance with another broad aspect of the teachings described herein, an in vitro method of treating acute myeloid leukemia cells, may include treating the cells with a therapeutically effective amount of the SEDDS.

[0028] The concentration of the avocado polyol in the SEDDS may be about 20 mg/ml_ (2% w/w).

[0029] In accordance with another broad aspect of the teachings described herein, a method of treating a disease or condition may include administering to a subject in need thereof a therapeutically effective amount of the SEDDS.

[0030] The SEDDS may include about 20 mg/ml_ avocatin B.

[0031] The SEDDS may be administered in an oral dosage form.

[0032] The SEDDS may be administered in at least one daily dose.

[0033] The at least one daily dose may include from about 25 mg to about 150 mg of avocado polyol, the avocado polyol may include a mixture of avocadene and avocadyne.

[0034] The at least one daily dose may include from about 25 mg to about 150 mg of avocado polyol, the avocado polyol may include a mixture of avocadene and avocadyne in a ratio of about 3:2 w/w.

[0035] The at least one daily dose may include from about 25 mg to about 150 mg of avocado polyol, the avocado polyol may include a mixture of avocadene and avocadyne in a ratio of about 3:1 w/w. [0036] The at least one daily dose com may include prises from about 25 mg to about 150 mg of avocatin B.

[0037] The at least one daily dose may include about 50 mg of avocado polyol, the avocado polyol may include a mixture of avocadene and avocadyne.

[0038] The at least one daily dose may include about 50 mg of avocado polyol, the avocado polyol may include a mixture of avocadene and avocadyne in a ratio of about 3:2 w/w.

[0039] The at least one daily dose may include about 50 mg of avocado polyol, the avocado polyol may include a mixture of avocadene and avocadyne in a ratio of about 3:1 w/w.

[0040] The at least one daily dose may include about 50 mg of avocatin B.

[0041] The disease may be acute myeloid leukemia.

[0042] The disease or condition may be diet-induced obesity.

[0043] The disease or condition may be obesity-associated lipotoxicity.

[0044] The disease or condition may be characterized by dysregulation of glucose-stimulated insulin secretion (GSIS).

[0045] The disease or condition may be characterized by insulin resistance.

[0046] The disease or condition may be characterized by reduced insulin sensitivity.

[0047] Another broad aspect of the teachings described herein may relate to the use of the SEDDS to encapsulate a pharmaceutical compound.

[0048] The pharmaceutical compound may be poorly-water soluble.

[0049] The SEDDS may be adsorbed onto a solid carrier.

[0050] The solid carrier may include NEUSILIN™.

[0051] The solid carrier may include Aeroperl® 300 Pharma.

[0052] The solid carrier may include Aeroperl® 3375/20. [0053] The solid carrier may include Aeroperl® 300/30.

[0054] The pharmaceutical compound may include naproxen.

[0055] The concentration of naproxen may be about 3 mg/ml_.

[0056] The pharmaceutical compound may include curcumin.

[0057] The concentration of curcumin may be about 5 mg/ml_.

[0058] Another broad aspect of the teachings described herein relates to a pharmaceutical composition that may include an active agent and the SEDDS.

[0059] Thus, the present inventors have developed a novel SEDDS comprising one or more bioactive avocado polyols, a surfactant, and an oil. The present SEDDS can form oil-in-water nano- and microemulsions and may be used to deliver bioactive avocado polyols as well as improve encapsulation of poorly water-soluble drugs. The present SEDDS may also provide an increase in potency and bioactivity compared to conventional cell culture delivery systems and, when delivered orally, may accumulate in blood and in key target tissues in vivo. The present therapeutic SEDDS and uses thereof may provide a pharmaceutical delivery vehicle with improved cellular delivery and enhanced bioactivity as compared to previously reported delivery vehicles.

[0060] Other advantages of the present disclosure will become apparent to those of skill in the art upon reviewing the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Embodiments of the present disclosure will be described with reference to the accompanying drawings, wherein like reference numerals denote like parts, and in which:

Figure 1 illustrates avocado polyol analytical characterization, showing ¹ H NMR spectra of avocado seed polyols. (A) avocadyne. (B) avocadene. (C) avocatin B. (D) 3:1 w/w avocadene- avocadyne. (E) 3:2 w/w avocadene-avocadyne.

Figure 2 illustrates LC-MS chromatographs of (A) avocatin B, (B) 3:1 avocadene- avocadyne, (C) 3:2 avocadene-avocadyne. Avocadyne and avocadene [M+H] ⁺ and [M+H-FhO] ^* mass fragments illustrated by employing an extraction window of 10 mDa. Figure 3 illustrates eutectic phase behaviour of avocatin B. (A) Chemical structures of avocadene and avocadyne. (B) Representative DSC melting thermograms for avocadene, avocadyne and their mixtures. Plots have been nudged for illustration. (C) Melting temperatures (left Y-axis) and melting enthalpies (right Y-axis) as a function of avocadene and avocadyne composition. (D) Domain size (left Y-axis) and melting entropy (right Y-axis), AS _m, as a function of avocadene and avocadyne composition. For (C-D) values are means ± SD from three independent experiments.

Figure 4 illustrates small and wide-angle powder X-ray diffraction spectra for (A) avocadyne, (B) avocadene, (C) avocatin B, (D) 3:1 avocadene-avocadyne, (E) 3:2 avocadene- avocadyne.

Figure 5 illustrates small and wide-angle powder X-ray diffraction spectra and corresponding domain sizes as obtained from Williamson Hull plots for (A) 1-heptadecanol, (B) heptadecanoic acid, (C) 16-heptadecynoic acid.

Figure 6 illustrates droplet size and PDI for varying amounts of NeoBee®M5 MCT oil tested for self-emulsifying properties when combined with varying amounts of Tween 80 or Cremophor EL. All SEDDS presented here were diluted 10 fold in PBS. Data represents mean ± SEM of two independent experiments.

Figure 7 illustrates in vitro cytotoxicity measurements of SEDDS vehicles in AML and non- AML cell lines. In vitro cytotoxicity of 1 :1 NeoBee®M5-Tween 80 or NeoBee®M5-CrEL blank SEDDS in (A) AML cell line OCI-AML-2 and non-AML cell lines, (B) INS-1 (832/13), (C) Caco-2, and (D) HepG2. Cell lines were incubated with varying concentrations of blank SEDDS. For OCI- AML-2 cells, cell viability was measured by the MTS assay after 72 h, whereas for non-AML cells viability was measured after 24 h treatments. All data represents mean ± SEM from two independent experiments performed in triplicate.

Figure 8 illustrates that avocatin B reduces the droplet size of an MCT oil-polysorbate 80 based SEDDS. (A) SEDDS method of preparation. (B) Effect of avocatin B concentration on average hydrodynamic diameter (Z-average) of NeoBee ^® M5 — Tween 80 SEDDS overtime. Inset: visual appearance of control (blank SEDDS) and avocatin B containing SEDDS on day 0. (C) Polydispersity index of SEDDS described in (B). Values are means ± SEM of three independent experiments. (D) Cryo-TEM images of control (blank SEDDS) and 20 mg/mL avocatin B containing SEDDs. Scale bar represents 100 nm. Figure 9 illustrates the effect of avocatin B on droplet size of coconut oil-Tween 80 and NeoBee®M5-CrEL SEDDS. (A) Effect of AVO concentration on Z-average of coconut oil-Tween 80 SEDDS over time. Inset: visual appearance of control (blank SEDDS) and AVO containing SEDDS on day 0. (B) Polydispersity index of SEDDS described in (A). (C) Effect of AVO concentration on Z-average of NeoBee®M5-CrEL SEDDS over time. Inset: visual appearance of control SEDDS and AVO containing SEDDS on day 0. (D) Polydispersity index of SEDDs described in (C). For A-D, values are means ± SEM of three independent experiments; *p<0.05, **p<0.01 , ***p<0.001, ****p<0.0001 compared to control, two-way ANOVA, Dunnett’s post hoc test.

Figure 10 illustrates SEDDS droplet size and stability when avocadene and avocadyne at varying ratios are incorporated into SEDDS in increasing concentrations. (A) Effect of 3:1 avocadene-avocadyne concentration on average hydrodynamic diameter (Z-average) of NeoBee ^® M5 ween 80 SEDDS over time. Inset: visual appearance of control (blank SEDDS) and 3:1 avocadene-avocadyne containing SEDDS on day 0. (B) Polydispersity index of SEDDS described in (A). (C) Effect of 3:2 avocadene-avocadyne concentration on Z-average of NeoBee ^® M5-Tween 80 SEDDS overtime. Inset: visual appearance of control (blank SEDDS) and 3:2 avocadene-avocadyne containing SEDDs on day 0. (D) Polydispersity index of SEDDS described in (C). For A-D, values are means ± SEM of three independent experiments. The size and polydispersity data for both 3:1 and 3:2 avocadene-avocadyne at a concentration of 20 mg/ml_ were excluded from the results starting on day 3 and onwards due, at least in part, to emulsion destabilization. Embodiments of the teachings herein that utilize these ratios at this concentration may therefore be best utilized in applications in which they can be used/consumed within less than 3 days from preparation.

Figure 11 illustrates the variable behavior of avocadene and avocadyne in SEDDS. (A) Effect of avocadene concentration on average hydrodynamic diameter (Z-average) of NeoBee ^® M5-Tween 80 SEDDS over time. Inset: visual appearance of control (blank SEDDS) and avocadene containing SEDDS on day 0. (B) Polydispersity index of SEDDS described in (A). (C) Effect of avocadyne concentration on Z-average of NeoBee ^® M5-Tween 80 SEDDS when final emulsions are heated for 3-5 min and droplet size is measured at 37 °C. Inset: visual appearance of control (blank SEDDS) and avocadyne containing SEDDS before and after application of heat (emulsion formation). (D) Polydispersity index of SEDDS described in (C). For A-D, values are means ± SEM of three independent experiments. (A-B) or one-way ANOVA (C-D), Dunnett’s post hoc test. The size and polydispersity data for avocadene at a concentration of 20 mg/ml_ is missing on day 7 and onwards due, at least in part, to emulsion destabilization. Embodiments of the teachings herein that utilize avocadene at this concentration may therefore be best utilized in applications in which they can be used/consumed within less than 7 days from preparation.

Figure 12 illustrates melting point, enthalpy of fusion, and melting entropy data for avocadene and avocadyne incorporated, at varying weight ratios, into 1:1 NeoBee®M5 — Tween 80 oil phase only. Avocado polyols were mixed in 1:1 NeoBee®M5-Tween 80 oil phase at a concentration of 200 mg/ml_ and analyzed via DSC. (A) DSC melting temperatures (left Y-axis) and melting enthalpies (right Y-axis) as a function of avocadene and avocadyne composition. (B) Melting entropy, AS _m as a function of avocadene and avocadyne composition, calculated using experimental enthalpies of fusion and melting temperatures. Values for (A-B) are means ± SD from two independent experiments.

Figure 13 illustrates mean particle size distribution (PSD) over four weeks at ambient temperature storage for (A) 1% (w/w) AVO emulsion, (B) 1.5% (w/w) AVO emulsion, and (C) 2% (w/w) AVO emulsion. (D) Evidence of thermodynamic stability for fresh 20 mg/ml_ avocatin B SEDDS during centrifugation test. (E) Evidence of thermodynamic stability for fresh 20 mg/ml_ avocatin B SEDDS during free-thaw test. (F) Normal light photomicrograph of crystals in a six month aged and destabilized 2% (w/w) AVO SEDDS (left) re-emulsifying into fine droplets upon brief application of heat (right). For A-C data represents mean PSD from three independent experiments.

Figure 14 illustrates mean particle size distribution (PSD) over four weeks at ambient temperature storage for (A) 1% (w/w) avocadene SEDDS, (B) 1.5% (w/w) avocadene SEDDS, (C) 1% (w/w) 3:1 avocadene-avocadyne SEDDS, (D) 1.5% (w/w) 3:1 avocadene-avocadyne SEDDS, (E) 1% (w/w) 3:2 avocadene-avocadyne SEDDS, and (F) 1.5% (w/w) 3:2 avocadene- avocadyne SEDDS. Data represents mean PSD from three independent experiments.

Figure 15 illustrates the optical isotropy of SEDDS assessed by normal and polarized light microscopy. (A) Normal light photomicrograph of control SEDDS (1:1 NeoBee®M5-Tween 80) (left) and when viewed between crossed polarizers (right). (B) Normal light photomicrograph of 20 mg/ml_ avocatin B SEDDS (left) and when viewed between crossed polarizers (right). All images were captured at 20x magnification.

Figure 16 illustrates in vitro activity of avocado polyol SEDDS evaluated in AML cell lines OCI-AML-2 and TEX. Cells were incubated with varying concentrations of avocado polyols dissolved in DMSO or as SEDDS. After 72 hours, cell viability was measured by the MTS assay. For avocadyne (A), avocadene (B), avocatin B (C), and 3:1 avocadyne-avocadene (D) 20 mg/mL (2% w/w) SEDDS were freshly prepared and used for serial dilution whereas for 3:2 avocadene- avocadyne (E), 15 mg/ml_ (1.5% w/w) SEDDS were freshly prepared and used. Data represent logarithmic transformation of avocado polyol concentrations (pg/mL) and cell viability data that was fit to a nonlinear four-parameter logistic curve (log(inhibitor) vs. response-variable slope) to determine inhibitory concentration 50 (IC50). All data represents mean ± SEM from three independent experiments performed in triplicate.

Figure 17 illustrates in vitro toxicity data of avocado polyols in non-AML cell lines. In vitro cytotoxicity of avocado polyols was evaluated in non-AML cell lines (Caco-2, INS-1 (832/13), C2C12 myotubes and HepG2). Cell lines were incubated with varying concentrations of avocado polyols dissolved in DMSO or as SEDDS. After 24 hours, cell viability was measured by the MTS assay. For avocadyne (A), avocadene (B), avocatin B (C) and 3:1 avocadyne-avocadene (D) 20 mg/mL (2% w/w) SEDDS were freshly prepared and used whereas for 3:2 avocadene-avocadyne (E) 15 mg/m L (1.5% w/w) SEDDS were freshly prepared and used. Data fit to nonlinear regression curve as described for Figure 16. All data represents mean ± SEM from three independent experiments performed in triplicate.

Figure 18 illustrates the bioaccessibility of avocadene and avocadyne during 360-min dynamic in vitro TIM-1 digestion of avocatin B emulsion. 100 mg avocatin B powder was emulsified in 1:1 NeoBee®M5-polysorbate 80 SEDDS and administered to TIM-1. (A) Mean particle size distribution (PSD) of TIM-1 start solution and (B) start solution spiked with AVO emulsion (20 mg/mL) to administer 100 mg to TIM-1. Cumulative bioaccessibility of avocadene and avocadyne in (C) jejunum, (D) ileum, and (E) the sum of jejunum and ileum. Non-cumulative, absolute concentrations of avocadene and avocadyne over time in (F) jejunum, (G) ileum, (H) the sum of jejunum and ileum. For (A-B) data represents mean PSD (±SEM) from three independent experiments. For (C-E) data represents mean (±SEM) % cumulative bioaccessibility relative to input amount. For (F-H) data represents mean (±SEM) concentration. For (A-F) *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 for avocadene vs. avocadyne, two-way AN OVA, Sidak’s post hoc test, n=3.

Figure 19 illustrates the bioavailability and biodistribution of avocatin B SEDDS in an in vivo pharmacokinetic study. Avocatin B SEDDS (2% w/w) was delivered via gavage (100 mg/kg body weight (b.w.)) to 6-8 week old female C57BL/6J mice. (A) 50-100 pL blood was collected via tail-snips at 2 hr and 6 hr and at endpoint (24 hr) and avocadene and avocadyne were quantified in blood using a validated LC-MS bio-analytical method. (B) Tissues (bone marrow, heart, pancreas, liver, gonadal fat pad, inguinal fat pad, and brain) were collected at endpoint for avocadene and avocadyne quantification. Data are shown as mean ± S.D., N=3 in each group.

Figure 20 illustrates (A) a method of solid-SEDDS preparation and characterization and (B) droplet size distribution for control or blank solid SEDDS and AVO solid SEDDS. Data represents mean droplet size distribution from three independent experiments

Figure 21 illustrates encapsulation of poorly water soluble drugs. (A) Histograms represent Z-average (left Y-axis) of naproxen (0.5% (w/w)) encapsulated in NeoBee ^® -Tween 80 SEDDS (control SEDDS) and AVO (1% (w/w)) SEDDS. Symbol represents PDI (right Y-axis). (B) Visual appearance of SEDDS described in (A). (C) Z-average and PDI of curcumin (0.5% (w/w)) in control or AVO (1% (w/w)) SEDDS. (D) Visual appearance of SEDDS described in (C). (E) In vitro cytotoxicity of curcumin formulated in control SEDDS or AVO (1% (w/w)) SEDDS was evaluated in OCI-AML-2 cells. Data fit to nonlinear regression curve described for Figure 17. For A and C, data represents mean ± SEM of two independent experiments. For (E), data represents mean ± SEM from three independent experiments performed in triplicate.

DETAILED DESCRIPTION

[0062] Various uses or methods will be described below to provide examples of embodiments of the claimed invention. No embodiment described below limits any claimed invention and any claimed invention may cover uses or methods that differ from those described below. The claimed inventions are not limited to uses or methods having all of the features of any one use or method described below or to features common to multiple or all of the uses or methods described below. It is possible that a use or method described below is not an embodiment of any claimed invention. Any invention disclosed in a use or method described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicants, inventors or owners do not intend to abandon, disclaim, or dedicate to the public any such invention by its disclosure in this document.

[0063] Self-emulsifying drug delivery systems (SEDDS) are complex lipid drug delivery systems comprised of a mixture of oils, co-solvents, surfactants, and/or co-surfactants. The present SEDDS comprises an avocado polyol, a surfactant, and an oil. [0064] Avocados ( Persea americana Mill.; Lauraceae) are a source of long carbon chain (C ₁₇ to C ₂₁ ) polyhydroxylated fatty alcohols or polyols. For example, avocados are a source of the 17- carbon polyols, avocadyne and avocadene. Avocatin B (AVO) is a 1:1 mixture of avocadyne and avocadene.

[0065] Avocado polyols can be extracted from avocado seeds and purified. For example, Figure 1 illustrates an analytical characterization of avocado polyols extracted and purified from Hass avocado seeds.

[0066] In the illustrated example, Avocatin B was extracted from Hass avocado seeds, as originally described by Kashman and colleagues with some modifications. Seeds were air dried, crushed and placed in glass bottles with ethyl acetate in a 2:1 solvent to seed ratio. The bottles were sealed and allowed to rotate on a bench top roller (120 VAC Wheaton Mini Bench Top Roller) for 24 hours and extracts were gravity filtered and the solvent was evaporated using a rotary evaporator. This process was repeated twice, and all extracts were combined. The crude extract was purified using flash silica gel chromatography using ethyl acetate as the mobile phase Column fractions were analyzed for purity using thin layer chromatography (eluent: ethyl acetate, Rf = 0.30) and visualized using p-anisaldehyde stain. Additionally, different weight ratios of avocadene and avocadyne were blended using commercially available avocadene and avocadyne powders (Microsource Discovery Systems Inc. (CT, USA)) in a 3:2 or 3:1 (w/w) avocadene: avocadyne ratio in glass scintillation vials.

[0067] All purified samples were characterized by ¹ H NMR. Samples were dissolved to 2 mg/ml_ in deuterated chloroform (CDCI ₃ ) and transferred to an NMR tube. Samples were analyzed using an Avance 400 MHz spectrometer (Bruker, Canada) and recorded as parts per million (ppm) using CDCb as internal standard (CDC : d 7.24 ppm ( ¹ H at 400 MHz)). Figure 1 shows chemical shifts (d (ppm)) and coupling constant ( J (hertz)) for avocadyne, avocadene, avocatin B, 3:2 avocadene — avocadyne, and 3:1 avocadene — avocadyne. Avocadyne: ¹ H NMR (400 MHz, CDCb) d: 3.93-3.88 (m, 2H), 3.64-3.60 (m, 1 H), 3.48-3.44 (m, 2H), 2.49 (br. s, 1 H), 2.15 (td, J = 7.1 and 2.6 Hz, 2H), 2.06-1.98 (m, 1H), 1.91 (t, J = 2.6 Hz), 1.57-1.44 (m, 7H), 1.37-1.24 (m, 20H). Avocadene: Ή NMR (400 MHz, CDCb) d: 5.83-5.73 (m, 1 H), 4.98-4.87 (m, 2H), 3.96-3.85 (m, 2H), 3.61 (dd, J = 11 and 3.5 Hz, 1 H), 3.45 (dd, J = 11 and 6.4 Hz), 1.99-1.97 (m, 2H), 1.56- 1.14 (m, 43H). [0068] Purity was also validated using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Commercially available avocatin B (Microsource Discovery Systems Inc.) was used to generate standard curves for avocadene and avocadyne and then used to quantify and determine avocadene and avocadyne ratios in the extracted and purified seed samples. All test samples were dissolved in 1:1 methanol-acetonitrile at a concentration of 5.71 mg/ml_ and then diluted to 0.006 mg/ml_ using the initial composition of the LC mobile phase (60% water-40% acetonitrile + 0.1% FA). All samples were prepared in duplicate and 10 pLwas injected into the UHPLC-MS. Figure 2 shows representative LC-MS chromatographs.

[0069] Differential scanning calorimeter (DSC) was used to determine the melting point and transition enthalpies for avocado polyol powders using Q2000 DSC (TA Instruments, Mississauga, ON, Canada). Four to five mg of solid material was measured and placed and sealed in aluminum pans (Mettler Toledo, 51119871, ON, Canada) and heated from 20 °C to 90 °C at 5 °C/min, under a nitrogen purge (gas flow of 18 ml/min). Peak melting temperatures (°C) and enthalpies (J/g) were determined by integrating the endothermic transition using the linear peak integration method in Universal Analysis 2000 (TA Instruments, New Castle, DE) software. Tangent skim method was also utilized to determine melting onset temperature. Melting temperatures and enthalpies were also determined for avocado polyol powders mixed in oil/surfactant (1:1 NeoBee®M5-Tween 80) at a concentration of 200 mg/mL (final amount loaded was 20-30 pL) using the same DSC parameters as described above.

[0070] The crystal structure and polymorphic forms of avocado polyols were assayed by X-ray powder diffraction (Multiflex Powder XRD spectrometer, Rigaku, Tokyo, Japan). The copper X- ray tube (wavelength of 1.54 A) was run at 40 kV and 44 mA. The measurement scan rate was set at 0.5 min in the range 2Q = 1-30° at 22-23°C. Peak positions (d-spacings) calculated from Bragg’s law were determined by MDI Jade 9 (MDI, Livermore, CA, USA) software. Domain sizes (thickness of the nano-crystal) in angstroms (A) were obtained using Williamson Hull equation as outlined below: where k is the Scherrer’s constant (0.94) which depends on the shape of the crystal, l is wavelength of the x-ray radiation (0.15406 nm), FWHM is the full width of half-maximum intensity expressed in radians, and Q is the diffraction (Bragg) angle in radians. Williamson Hull plots of FWHMcos(0) versus sin(0) (for 20 = 1-30°) were plotted for all samples. The slope was used to determine crystal strain and the y-intercept was used to calculate domain size.

[0071] In this example, purification of avocado seed ethyl acetate extracts yielded a fraction pure in avocadene and avocadyne (Figure 3A) at a 1:1 (w:w) ratio (avocatin B). Commercially available standards of pure avocadene and avocadyne (purchased from Microsource Discovery Systems Inc. (CT, USA)) were used for physical characterization studies and also used to generate samples with a 3:1 or 3:2 weight ratio of avocadene:avocadyne. DSC showed avocadyne had a significantly higher melting point than avocadene, (Figure 3B). Avocatin B had the lowest peak melting point suggesting that it may be a eutectic composition of avocadene and avocadyne (Figure 3C). A eutectic composition refers to a binary mixture of two structurally distinct solids that exhibit a depressed melting point relative to either pure component. These trends were unaltered when melting onset temperatures for all avocado polyol samples were assessed. Furthermore, avocatin B exhibited a lower value for AS _m (the ratio of melting enthalpy (AH _m ) to the melting point (T _m ) referred to as entropy of melting) compared to pure avocadyne or 3:1 or 3:2 mixtures, suggesting a more disordered or liquid-like crystal (Figure 3D). Eutectic mixtures identified in other research areas also have low melting points and entropies which is explained by significant differences in intermolecular interactions that introduce greater molecular mobility or disorder.

[0072] Analysis of powder X-ray diffraction patterns for all polyols in the small angle region (SAXS region) demonstrated that all crystals displayed a lamellar arrangement of similar size with characteristic reflections associated with the [001] molecular plane and its higher orders, i.e., [002], [003], [004], etc., evident at the expected ratio of spacings (1:1/2: 1/3: 1/4:1, and so on) (Figure 4A-E). However, avocadyne lamellae were generally larger and two species of 31 and 40 Angstroms were evident in the [001] plane suggesting the presence of two polymorphs or two geometric isomers. The wide angle patterns (WAXS) for avocadene and mixtures of avocadene and avocadyne were very similar, suggesting that the long hydrocarbon chains of the fatty alcohols pack in similar fashion or in the same polymorphic form. Avocadyne however, displayed different WAXS patterns than the rest of the samples, suggesting a different unit cell structure. Indexing and unit cell identification were not attempted due to concerns over crystal purity. The observed eutectic behaviour of avocatin B was attributable to its domain size (i.e., the persistence length, and the physical thickness of the nanocrystals in the [001] crystal planes’ direction). This analysis showed that the domain size of avocatin B was noticeably smaller compared to all other avocado polyol samples whereas avocadyne exhibited the largest domain size (Figure 3D). The relationship between small domain sizes and lower melting points has been reported for both eutectic and monotectic systems. XRD analysis on structurally related odd-numbered carbon lipids (i.e., 1-heptadecanol, heptadecanoic acid and 16-heptadecynoic acid) showed completely different diffraction patterns and domain sizes compared to avocado polyols (Figure 5A-C).

[0073] In some embodiments of the present disclosure, the avocado polyol comprises at least one of avocadyne, avocadene, or a mixture thereof. In some embodiments, the avocado polyol comprises about 1% to about 1.5% w/w avocadene. In some embodiments, the avocado polyol comprises about 1.5% w/w avocadene. In some embodiments, the avocado polyol comprises from about 1% to about 2% avocadyne. In some embodiments, the avocado polyol comprises avocatin B. In some embodiments, the SEDDS may comprise from about 1% to about 2% w/w avocatin B.

[0074] The surfactant according to the present disclosure may be non-ionic, anionic, cationic, or amphoteric. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant is a polysorbate, such as polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 85. In other embodiments, the surfactant comprises polyoxyl castor oil, such as CREMOPHOR EL™, KOLLIPHOR EL™, ACRYSOL EL- 135™, ACRYSOL K-140™ or CRODURET 50™, or ETOCAS 35™.

[0075] The oil of the present SEDDS is preferably a medium chain triglyceride oil. For example, the oil may be coconut oil, NEOBEE M5™, CAPTEX 8000™, CAPTEX 300 EP/NF™, CAPTEX 355 EP/NF™, MIGLYOL 808™, MIGLYOL 810N™, MIGLYOL 812N™, NEOBEE 1053™, LABRAFAC LIPOPHILE WL1349™, KOLLISOL MCT 70™, or CRODAMOL GTCC™.

[0076] In some embodiments, the surfactant is polysorbate 80 and the oil is NEOBEE M-5™.

[0077] The amount of oil in the present SEDDS may vary with the amount of surfactant. In some embodiments, the ratio of oil to surfactant is 1:1.

[0078] The oil and surfactant may be diluted with an aqueous buffer suitable for in vitro, in vivo, and clinical formulations, such as phosphate buffered saline (PBS), PIPES (piperazine-A/./V- jb/s(2-ethanesulfonic acid)), MOPS (3-(/\/-morpholino)propanesulfonic acid), or HEPES (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the aqueous buffer is PBS. In some embodiments, the oil and surfactant are diluted 10-fold or more with the aqueous buffer. [0079] Table 1 summarizes examples of SEDDS formulations that were tested for self emulsification. The tested formulations consisting of either long or medium chain triglycerides and surfactants/co-surfactants with varying HLB values, at various oil-to-surfactant weight ratios (9:1, 5:1 , 1:1) assessed for SEDDS characteristics.

[0080] The following materials were used: polysorbate (tween) 20, 60 and 80, span 65 and 80, Cremophor EL (Kolliphor® EL), polyethylene glycol, polyethylene glycol monolaurate, soybean oil, PBS, 1-heptadecanol, heptadecanoic acid, palmitic acid, oleic acid, penicillin/streptomycin, trypsin solution, p-anisaldehyde stain and formic acid (FA) purchased from Sigma-Aldrich. Neobee®M-5 was a provided as gift from Stepan Company (Northfield, IL). Capryol 90, Labrafil M 1944 CS, Labrasol, Transcutol, HP, and Maisine CC were received as a gift from Gattefosse (St-Priest, France).

Table 1. Formulations tested forSEDDS properties.

Oil (% v/v) Surfactant Co-surfactant Self-Emulsifying Z- Polydispersity (% v/v) (% v/v or w/w if Formulation after average Index (PDI) specified) 1:10 dilution in (d.nm)

_ PBS (Y/N)

LONG CHAIN TRIGLYCERIDE OILS

Olive oil 50% Tween 80 50% _ N

Vegetable oil 50% Tween 80 50% N

Soybean oil 50% Tween 80 50% _ N

Soybean oil 50% Tween 20 50% _ N

Castor oil 50% Tween80 50% N

Avocado oil 50% Tween 80 50% N

MEDIUM CHAIN TRIGLYCERIDE OILS

NeoBee M5 50% Tween 80 50% Y 189 0.241

NeoBee M5 50% CrEL 50% Ϋ 82 0.345

NeoBee M5 50% Labrasol 50% N

NeoBee M5 50% Tween 20 50% N

NeoBee M5 50% Span 80 50% N

NeoBee M5 53% Tween 80 35% Maisine 10% Y 119 0.240

NeoBee M5 45% Tween 80 45% Maisine 10% Y 100 0.370 NeoBee M5 42% Tween 80 42% Maisine 16% _ Y 103 0.300 NeoBee M5 38% Tween 80 38% Maisine 14% Y 100 0.294

NeoBee M5 47% Tween 80 47% Span80 6% Y 157 0.263

NeoBee M5 45% Tween 80 45% Span80 10% Y ΪΪ 2 0.350

NeoBee M5 43% Tween 80 43% Span80 14% Y ~98~ 0.350 NeoBee M5 42% Tween 80 42% Span80 16% Y 87 0.320 NeoBee M5 40% Tween 80 40% Span80 20% Y 180 0.197

NeoBee M5 38% Tween 80 38% Span80 24% Y 280 0.300

NeoBee M5 50% Tween 80 15% Labrasol 35% Y 260 0.155

NeoBee M5 50% Tween 80 25% Labrasol 25% Y 253 0.190

NeoBee M5 50% Tween 80 35% Labrasol 15% Y 280 0.280

NeoBee M5 50% Tween 80 35% Span 65 19% Y T73 0.260

_ (w/w) _

NeoBee M5 50% Tween 80 35% Span 65 42% Y 310 0.540

_ (w/w) _

NeoBee M5 45% Tween 80 45% Transcutol 10% Y 238.8 0.238

Coconut oil 50% Tween 80 50% _ Y 195 0.219

Coconut oil 50% CrEL 50% _ Y 92 0.254

Coconut oil 60% Labrasol 40% _ N

Coconut oil 50% Span 80 50% _ N

Coconut oil 45% Tween 80 45% Transcutol 10% Y 233 0.230

Coconut oil 45% Tween 80 45% Maisine 10% Y Ϊ93 0.225

Coconut oil 42% Tween 80 42% Maisine 16% _ Y Too 0.294

Coconut oil 38% Tween 80 38% Maisine 24% _ Y 333 0.363

Coconut oil 47% Tween 80 47% Span80 6% Y 130 0.270

Coconut oil 45% Tween 80 45% Span80 10% Y To9 0.280

Coconut oil 43% Tween 80 43% Span80 14% Y 0.350

Coconut oil 41% Tween 80 41% Span80 18% Y ^" 75 ^" 0.360

Coconut oil 40% Tween 80 40% Span80 20% Y T98 0.225

Coconut oil 38% Tween 80 38% Span80 24% Y Tq5 0.291

Coconut oil 50% Tween 80 15% Labrasol 35% Y 268 0.215

Coconut oil 50% Tween 80 25% Labrasol 25% Y 278 0.218

Coconut oil 50% Tween 80 35% Labrasol 15% Y 310 0.280

Labrafil 20% Labrasol 80% N

Labrafil 47% Labrasol 47% Capryol 90 6% N

Labrafil 47% Labrasol 47% Transcutol 6% N

Labrafil 15% Labrasol 80% Capryol 5% N

Labrafil 10% _ Labrasol 80% Transcutol 10% _ N

Labrafil 5% Labrasol 65% Transcutol 30% N Capryol 90 47% Tween 80 47% Transcutol HP N

6%

[0081] As summarized in Table 1 , SEDDS comprising Neobee®M5 or coconut oil in combination with Tween 80 or CrEL, at an oil/surfactant ratio of 1 :1 , were suitable SEDDS, when diluted 10 folds in PBS (Table 1 and Figure 6). The cytotoxicity of 1:1 Neobee®M5-CrEL SEDDS (mean droplet diameter of ~80 nm) compared to Neobee®M5-Tween 80 SEDDS (mean droplet diameter of -200 nm) was significantly greater in AML (OCI-AML-2) and non-AML cell lines (INS-1 (832/13), Caco-2, and HepG2) (Figure 7). The 1:1 Neobee®M5-Tween 80 was further studied as the SEDDS system in the illustrated example described herein.

[0082] To characterize the illustrated example of the present SEDDS (i.e. , the 1 :1 Neobee®M5- Tween 80 SEDDS), formulations were prepared in 2 ml polypropylene centrifuge tubes, then vortexed and heated at 75 °C for 5 minutes. The heated oil was then diluted 10-fold with immediate addition of water phase (PBS) and vortexed for 30 sec. All formulations were visually analyzed for macroscopic appearance (characterized as clear, opalescent or milky appearance). The mean droplet size (Z-average in nm), polydispersity index (PDI), and zeta potential for each formulation was determined using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Malvern, U.K.). A refractive index of 1.33 was used for the aqueous phase, and refractive indices for each oil/surfactant mix were determined using a table top refractometer (Zeiss Abbe, NY, USA). Droplet size was measured on three independently prepared formulations and averaged from three readings per formulation, whereas zeta potential was analyzed on two independent formulations, with each measurement averaged from four readings per formulation. All measurements were carried out at 25 °C except for avocadyne SEDDS where the zetasizer compartment temperature was set to 37 °C to prevent destabilization.

[0083] As shown in Figure 8, avocado polyols self-assembled at the O/W interface of the illustrated SEDDS example. Avocado polyols (1-20 mg) were pre-dissolved in 100 pL of the oil/surfactant phase by heating to 75 °C for 2 hr (or until a clear oily solution formed), after which 900 pL water phase (PBS) was added directly to the oil phase and vortexed for 30 sec (Figure 8A). This method of preparation produced consistent emulsion droplet sizes; another method where hot oil phase was added to PBS under constant stirring did not produce consistent results.

[0084] When 1-2% (w/w) avocatin B was incorporated into SEDDS, significant reduction in turbidity (as characterized by an increase in transparency) and mean droplet size were observed where 2% (w/w) avocatin B reduced SEDDS droplet diameter below 25 nm (Figure 8B). Ambient temperature polydispersity and droplet size measurements increased for 1.5-2% (w/w) avocatin B containing SEDDS over four weeks compared to control SEDDS (Figure 8C).

[0085] Cryo-transmission electron microscopy (TEM) images of the control and 2% (w/w) avocatin B emulsions highlighted the drastic reduction in mean droplet size caused by avocatin B (Figure 8D). Similarly, avocatin B significantly reduced droplet diameter of 1 :1 coconut oil-Tween 80 SEDDS (Figure 9A and B) and 1 :1 Neobee®M5-CrEL SEDDS (Figure 9C and D) in a concentration dependent manner. Hydrophilic co-surfactants like glycerol, propylene glycol and ethanol have also been reported to penetrate the interfacial film and decrease droplet size of O/W emulsions; however, no such effects were observed in the illustrated example SEDDS system.

[0086] Choice of raw material (avocado pulp or seed from different cultivars) and differences in extraction methods can result in the purification of variable weight ratios of avocadene and avocadyne. Since avocatin B is a 1:1 weight ratio of avocadene and avocadyne, 3:1 and 3:2 ratios were generated using pure avocadene and avocadyne and incorporated into the SEDDS example to determine their self-emulsifying properties. Similar to avocatin B, 3:1 avocadene-avocadyne SEDDS formed mean droplet sizes ~ 25 nm at 2% (w/w); however, this emulsion destabilized after 24 hr (Figure 10A and B). In contrast, 3:2 avocadene-avocadyne SEDDS exhibited larger droplet sizes at concentrations between 0.1-0.15 % (w/w) compared to avocatin B or 3:1 avocadene-avocadyne SEDDS (Figure 10C and D). This highlights the importance of avocadene: avocadyne ratios for optimal incorporation into SEDDS.

[0087] Next, pure avocadene or avocadyne were incorporated into the illustrated SEDDS example to examine their individual effects. Avocadene in SEDDS behaved similarly to avocatin B where 0.5-2 % (w/w) avocadene caused a significant reduction in droplet diameter compared to control (Figure 11A and B). Avocadyne did not exhibit the same behaviour as avocatin B or avocadene in the illustrated SEDDS example and microemulsions did not form when water phase was added to hot oil phase containing 1-20 mg of avocadyne (Figure 11C and D). However, fine transparent microemulsions for 1-2% (w/w) avocadyne were observed after application of heat (75 °C for 3-5 minutes) and keeping samples at 37 °C.

[0088] DSC was also used to evaluate the melting temperatures of avocado polyols when incorporated in only the oil/surfactant phase (at a concentration of 200 mg/ml_) of the SEDDS (1 :1 Neobee®M5-Tween 80). This analysis demonstrated that avocadyne exhibited larger thermal and entropic parameters compared to other polyol samples tested (Figure 12A and B). Collectively, differences in crystal structure and packing as well as solubility of avocado polyols in the chosen oil phase may be integral factors that impact self-assembly.

[0089] Formulations chosen for further study were selected based on their self-emulsification properties, small droplet size (below 300 nm), low PDI (below 0.4), and no co-surfactants. Formulations of avocado polyol-SEDDS contained 1-20 mg powder of each polyol added to 100 pl_ of a 1 : 1 mixture of oil and surfactant, which was heated to 75 °C for 2 hr in an incubator (MaxQ 4450, Barnstead/Lab-Line, IL, USA). After dissolution of polyols in the oil phase, 900 mI_ water phase (PBS) was added to the oil phase and vortexed for 30 s. Visual appearance of fresh polyol emulsions were noted followed by droplet size, PDI and zeta potential measurements on day zero (immediate measurement on fresh emulsion), day 3 and weekly for 4 weeks.

[0090] To obtain physical images of the illustrated SEDDS, cryo-transmission electron microscopy was conducted. Transmission electron microscopy (TEM) was conducted at the Advanced Imaging Center in the University of Guelph. Approximately 5 mI of emulsion was placed on a carbon grid with a perforated carbon film (Canemco & Marivac, Quebec, Canada). Filter paper was used to blot the excess liquid. The grid was then immediately dipped into liquid ethane and transferred to a cryo-holder for direct observation at -176°C on a FEI Tecnai G2 F20 energy- filtered Cryo-TEM (FEI Corp., Hillsboro, Oregon, USA) operated at 200 kV in low dose mode, equipped with a Gatan 4k CCD camera (Gatan Inc., Pleasanton, California, USA).

[0091] Optical and polarized light microscopy was also performed on the illustrated SEDDS using an optical microscope model BX60 (Olympus Optical Co., Tokyo, Japan). Images were captured (using 20* objective lens) with a model DP71 digital camera (Olympus Optical Co., Tokyo, Japan) using the cellSens (version 1.0) software. 20-50 mI_ of emulsion samples were placed on a microscope slide and micrographs were obtained using either brightfield or polarized light. Polarized light microscopy was performed on emulsions to confirm absence of crystalline phases (i.e. , confirm optical isotropy).

[0092] Emulsion thermodynamic stability was assessed by centrifugation at 21.1 x g for 15 min and phase separation, creaming or flocculation was observed. The effect of temperature on SEDDS was also evaluated using freeze-thaw tests where formulations were subject to six freezing-heating cycles (-20°C for 24 hr followed by 37°C for 24 hr) after which visual observations and droplet size measurements were performed. [0093] Table 2 illustrates results demonstrating that a combination of Ostwald ripening and coalescence took place as determined by regression analysis on the cube of mean droplet radius (r ³ ) (Ostwald ripening model) and inverse of the square of mean droplet radius (1/r ² ) (coalescence model).

Table 2. Experimentally determined Ostwald ripening rate and coalescence rate for blank SEDDs (control) and avocado polyol SEDDS (between 1-2% (w/w)) at room temperature.

SEDDS Description Ostwald ripening Correlation Coalescence Correlation rate (r ² ) rate (nm ² IT ¹ ) (r ² )

(nm ³ IT ¹ )

Control 227.5 0.5235 1.97 x 10- ⁸ 0.4977

AVO 1% w/w 73.78 0.9526 2.31 x 10 ⁷ 0.8894

AVO 1 .5% w/w 14.93 0.9652 6.27 x 10 ^s 0.9151

AVO 2% w/w 74.56 0.9327 1.07 x 10 ^s 0.8195

Avocadene 1 % w/w 80.61 0.9253 3.15 x 10 ⁷ 0.8251

Avocadene 1 .5% w/w 39.14 0.8544 7.89 x 10 ^s 0.9583

3:1 Avocadene-Avocadyne

46.58 0.9539 2.57 x 10 ⁷ 0.9156 1 % w/w

3:1 Avocadene-Avocadyne

67.16 0.934 2.43 x 10 ^s 0.9532 1 .5% w/w

3:2 Avocadene-Avocadyne

31.59 0.8808 6.72 x 10 ^s 0.8654 1 % w/w

3:2 Avocadene-Avocadyne

86.94 0.9867 5.43 x 10 ⁷ 0.97 1 .5% w/w

[0094] Particle size distribution (PSD) analysis over time provided additional insight into the kinetics of the two destabilization mechanisms. Both PSD peak broadening (suggestive of coalescence occurring at a faster rate) and sharpening (suggestive of Ostwald ripening following first order kinetics) were observed over time in a concentration independent manner for all polyol SEDDS examined (Figure 13A-C and Figure 14A-F). In contrast, further dilution of the described avocado polyol SEDDS (up to 1000 folds in PBS) slowed droplet size growth significantly (data not shown); a finding widely reported for micro and nano self-emulsifying systems. PBS was used as the aqueous phase for all SEDDS characterization studies described herein to ensure no changes to osmotic and pH balance in vitro and in vivo. Zeta potential values for all SEDDS prepared in PBS at 10-1000 fold dilutions were not significantly different (i.e., < -6 mV) (data not shown). [0095] Thermodynamic stability was assessed for freshly prepared SEDDS using centrifugation and freeze-thaw tests. Avocatin B SEDDS (up to 2% (w/w)) did not flocculate or cream after ultracentrifugation (Figure 13D). Avocatin B SEDDS froze at -20 °C, but an isotropic emulsion was reformed after thawing at 37 °C (Figure 13E). Brightfield microscopy of a six-month aged 2% (w/w) avocatin B SEDDS showed large crystals (a combination of avocatin B, oil, and surfactant), which disappeared after heating (45 °C) for 3-5 min (Figure 13F). While not wishing to be bound by any particular theory or mode of action, these results may suggest that a destabilized polyol microemulsion can undergo self-emulsification with heating. Polarized light microscopy confirmed that the illustrated SEDDS examples were isotropic colloidal dispersions void of lamellar liquid crystals (Figure 15), as no birefringence was observed between crossed polarizing plates. These properties were observed for all freshly prepared avocado polyol SEDDS.

In vitro Cytotoxicity

[0096] The present SEDDS may be used in vitro to treat acute myeloid leukemia cells, wherein the cells are treated with a therapeutically effective amount of the SEDDS. In some embodiments, the concentration of the avocado polyol in the in vitro SEDDS treatment may be about 10 to about 25 mg/ml_. In some embodiments, the concentration of the avocado polyol in the in vitro SEDDS treatment may be about 20 mg/ml_.

[0097] Figures 16-17 and Table 3 set out the results from an in vitro cytotoxicity study using the illustrated SEDDS example. Cells were cultured at 37°C with 5% CO2. OCI-AML2 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% penicillin/streptomycin (100 U/mL of streptomycin and 100 mg/mL of penicillin; Sigma). TEX cells were similarly cultured except with 15% FBS, 2 mmol/L (mM) L-glutamine (Sigma), 20 ng/mL stem cell factor, and 2 ng/mL interleukin 3 (IL3; Peprotech). AML-2 and TEX cells between passages 5-25 were used for all experiments.

[0098] Non-AML cell lines included INS-1 (832/13) rat pancreatic b-cell line, C2C12 mouse skeletal myoblast cell line, Caco-2 human epithelial colorectal adenocarcinoma cells, and HepG2 human hepatocellular carcinoma cells. INS-1 (832/13) cells were cultured in RPMI 1640 medium containing 11.1 mM glucose, supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L- glutamine, 1 mM sodium pyruvate, and 50 mM b-mercaptoethanol. INS-1 (832/13) cells between passages 75-100 were used for all experiments. C2C12 myoblast cells were cultured in growth media consisting of low-glucose (5.5 mM) Dulbecco’s Modified Eagles Medium (DMEM; Hyclone, ThermoFisher) supplemented with 10% FBS and 1% penicillin/streptomycin. Differentiation of C2C12 myoblasts into myotubes was induced by switching 90% confluent cells to differentiation media consisting of low-glucose DMEM supplemented with 2% horse-serum and 1% penicillin/streptomycin. Differentiation media was changed every 24 hr for up to 5 days prior to all experimental treatments. C2C12 cells between passages 5-20 were used for all experiments. Caco-2 and HepG2 cells were cultured in DMEM medium containing 25 mM glucose, 10% FBS and 1% penicillin/streptomycin. Caco-2 and HepG2 cells between passages 10-40 were used for all experiments. All cell lines were maintained at 37 °C with 5% C02 and 95% humidity.

[0099] The cytotoxicity of avocado polyols was tested in AML cell lines OCI-AML-2 and TEX. Avocado polyols were delivered in either dimethyl sulfoxide (DMSO) or as SEDDS. For DMSO delivery, all avocado polyols were dissolved in DMSO at a concentration of 11.4 mg/mL, which was then diluted to 0.6 mg/mL in cell culture media as a working stock to treat cells at varying concentrations from 1-29 pg/mL. DMSO vehicle control cells were treated with no more than 0.03% DMSO. For SEDDS delivery, a 2% (w/w) (or 20 mg/mL) SEDDS stock of avocadyne, avocadene, avocatin B, and 3:1 avocadene — avocadyne was diluted to 0.6 mg/mL in cell culture media and cells were treated with the same concentrations as mentioned above. For 3:2 avocadene — avocadyne SEDDS delivery, a 1.5% (w/w) (or 15 mg/mL) stock was used due to the higher stability of these emulsion. SEDDS vehicle control cells were treated with no more than 0.3% control SEDDS (1:1 NeoBee®M5 — Tween 80 diluted 10 folds in PBS)

[0100] For OCI-AML-2 and TEX cells, 1.25x10 ^s cells/ml were seeded in 96-well plates and treated with test compounds for 72 hours. Cells were then incubated with 20 pL of 3-(4, 5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz olium salt (MTS; Promega; Madison, Wl) for two hours at 37 °C and 5% CO2. Metabolically active cells express extracellular enzymes which can cleave MTS into a coloured formazan product. The formazan product was then quantified measuring absorbance at 490 nm using a Biotek Synergy HT spectrophotometer (Biotek; Winooski, VT). For non-AML cell lines INS-1 (832/13), Caco-2, and HepG2, 1x10 ^s cells/ml were seeded in 96 well plates, allowed to attach for 24 hr after which they were treated with test compounds for 24 hr before addition of MTS reagent. C2C12 myoblast cells were seeded in 12- well plates and day 5 differentiated myotubes were treated for 24 hr before addition of MTS reagent. Results were statistically analyzed in GraphPad 6.0 prism software and represent logarithmic transformation of avocado polyol concentrations (pg/mL) and cell viability (% MTS reduction relative to vehicle control) that was fit to a nonlinear regression curve (log(agonist) vs. response-variable slope (four parameters)) to determine inhibitory concentration 50 (IC50). All data represented by mean ± SEM from three independent experiments performed in triplicate.

[0101] Previously, it has been reported that avocatin B induces mitochondria-mediated death in AML cells, where avocatin B was delivered using a conventional cell culture vehicle (DMSO). Solubility of long aliphatic chain avocado polyols in DMSO is likely a limiting factor for in vitro cellular delivery, thus we directly compared the cytotoxicity of avocado polyols in AML cells when delivered using DMSO orSEDDS (Figure 16A-E; Table 3). All avocado polyol delivered in SEDDS lowered AML cell viability with doubled potency compared to DMSO delivery.

Table 3. IC50 values of avocado polyols delivered either in DMSO or SEDDS to OCI-AML-2 or TEX cells. IC50 was calculated using non-linear regression model (logarithmic inhibitor vs. normalized response-variable slope).

Different superscript letters within each column for each cell line indicates significant differences (P < 0.0001) between DMSO and SEDDS delivery, two-way ANOVA with Sidak’s post hoc test (n=3).

[0102] Avocado polyols selectively exert cytotoxicity in AML cells while sparing non-AML cells.

Figure 17A-E illustrates the cytotoxicity of avocado polyols (delivered in DMSO or SEDDS) in non-AML, adherent, cells such as INS-1 (832/13), C2C12 myotubes, Caco-2, and HepG2. All avocado polyols delivered in DMSO showed no activity in any of the non-AML cell lines even at high concentration ranges between 15-29 pg/mL. However, avocado polyols delivered in SEDDS at the supraphysiological concentration of 29 pg/mL (> 50 pM) showed some reductions in viability in INS-1 (932/13), C2C12 myotubes and HepG2 cells, which may suggest enhanced delivery.

Bioaccessibility

[0103] To assess the bioaccessibility of the illustrated SEDDS example, a TIM-1 protocol was used. TIM-1 simulates digestion in the upper gastrointestinal tract of an adult human. TIM-1 includes four compartments: stomach, duodenum, jejunum, and ileum. Prior to introducing the meal into the TIM-1 , a small intestinal electrolyte solution (SIES: 132 mM NaCI, 5.28 mM KCI, 0.75 mM CaCh), a 7% (w/v) pancreatin solution, a duodenal start residue (15 g SIES, 30 g fresh 20% porcine bile solution, 2 mg trypsin, 15 g of 7% pancreatin solution), a jejunal start residue (35 g SIES, 70 g fresh 20% porcine bile solution, 35 g of 7% pancreatin solution), and an ileal start residue (140 g SIES) were injected into their respective compartments and the system was heated to 37 °C. The initial amount of gastric juice was simulated by loading the gastric compartment with 15 g (fasted protocol) of gastric enzyme solution (4800 U/mL pepsin, 20 U/mL lipase, and 47 U/mL amylase in gastric electrolyte solution: 150 mM NaCI, 20 mM KCI, 1 mM CaCh _, 10 mM sodium acetate buffer) and 15 g of 0.4% HPMC/0.04% bile solution. The rate of secretion of digestive juices, peristaltic movements, nutrient and water absorption, gastric emptying, pH, and transit times in each compartment was set in accordance to manufacturer’s protocol (Triskelion/TIM021 and 040) for fed state-lipid digestion or a fasted state-lipid digestion. Simulated digestive fluids were prepared fresh on the day of the experiment, and enzyme solutions were stored between 0-5 °C before use. For fasted state digestion the pH of the stomach was pre-set as follows: 3.0 at 0 min, 2.2 at 10 min, 1.8 at 30 min, and 1.7 between 60-360 min. The pH in all other compartments was kept constant (5.9, 6.5, and 7.4 for the duodenum, jejunum and ileum, respectively). Meals were administered to TIM-1 and samples were collected from the jejunal filtrates, ileal filtrates and ileal efflux (effluent) at 60, 120, 180, 240, 300 and 360 min after which the TIM runs were terminated, and samples were solvent extracted the same day. A water control was also subjected to TIM-1 digestion (fed protocol) where dialysates were sampled at the same time points and used to generate matrix matched calibration curves and perform analyte recovery experiments for LC-MS method validation and analysis.

[0104] For sample extraction and validation, 100 pL of TIM-1 dialysates from jejunum, ileum, and effluent were extracted and samples prepared with the same procedures described for mouse blood. Blank TIM-1 dialysates (water administration to TIM-1 in fed state protocol) from 1 and 6 h samples were also extracted the same way for recovery experiments and the generation of matrix- matched AVO standard curves. All TIM-1 digestion experiments were completed in triplicate.

[0105] An emulsion containing 2% (w/w) avocatin B was prepared in deionized water as described above and administered to TIM-1 for digestion using the fasted state-lipid digestion protocol. Avocatin B emulsion was added to 270 g of start solution (251 g gastric electrolyte solution, 18.9 g HPMC, 11 mg amylase) such that a total of 100 mg of avocatin B was added to the stomach compartment. The particle size of the emulsion in the start solution was measured using dynamic light scattering, as described above, prior to administration to TIM-1.

[0106] Cumulative bioaccessibility was the accumulation of avocadene or avocadyne quantified during each time interval in the jejunum, ileum and the sum of the two filtrates, expressed as a percent of the input amount (absolute fatty alcohol content). The amounts quantified in the effluent were also expressed as a percent of the input amount but excluded from the bioaccessibility analysis. Non-cumulative, absolute concentrations of avocadene and avocadyne was plotted against time to perform area under curve (AUC) analysis on GraphPad Prism 6.0 software (CA, USA).

[0107] Mathematical modelling was also applied on all TIM-1 cumulative data which displayed a sigmoidal pattern (initial lag phase followed by a steep increase in fatty alcohol bioaccessibility that approaches a plateau). A three-parameter shifted logistic model commonly used to characterize free fatty acids generated as a result of lipolytic activity in the gastrointestinal tract as a function of time ( t ):

[0108] Here, C _asymp is the total amount of fatty alcohol released (asymptotic level at the plateau region), k is the rate of fatty alcohol released per unit time, and tc is the critical time (induction time) at which half the total amount of fatty alcohol released is achieved. Nonlinear analysis was performed in Graphpad Prism 6.0 software for each compartment on the three bioaccessibility parameters from the equation above (Casymp, k, and tc).

[0109] Dynamic in vitro digestion model, TIM-1, was utilized to assess the in vitro bioaccessability of 2% (w/w) avocatin B SEDDS prepared in deionized water and spiked into 270 g of TIM-1 starting solution (a mixture of gastric electrolyte solution, HPMC and amylase) such that 100 mg of avocatin B would be administered to the TIM. Particle size distribution analysis of the TIM-1 starting solution revealed a mean droplet diameter of 410.8 ± 0.1 nm (PDI: 0.4 ± 0.1) (Figure 18A), whereas the mean droplet diameter of the TIM-1 starting solution with the spiked avocatin B emulsion was 25.3 ± 0.01 nm (PDI: 0.1 ± 0.01) (Figure 18B). Spiking avocatin B emulsion into TIM-1 starting solution resulted in a transparent mixture void of any signs of creaming or coalescence. Cumulative TIM-1 bioaccessibility analysis of avocatin B emulsion demonstrated that avocadene had significantly greater bioaccessibility than avocadyne in both ileum and jejunum as early as two hours into digestion (Figure 18C-E). In the jejunum, the cumulative bioaccessibility for avocadene and avocadyne was 60.3 ± 0.5% and 47.1 ± 0.7%, respectively. In the ileum, cumulative bioaccessibility for avocadene and avocadyne was 12.0 ± 0.4% and 15.2 ± 0.4%, respectively. In the effluent, less than 8% of both avocadene and avocadyne was quantified (data not shown). Collectively, the cumulative bioaccessibility (sum of jejunum and ileum compartments) for avocadene and avocadyne delivered via SEDDS was determined to be 72.2 ± 0.9% and 62.3 ± 1.0%, respectively. Interestingly, these cumulative values are only moderately higher than what was previously observed for avocado pulp powder digestion (see, for example PCT/CA2020/051220, Avocatin B for the treatment of diseases and conditions, filed Sept. 10, 2020 which is incorporated herein in its entirety by reference), suggesting some level of equivalency between the two delivery systems of avocadene and avocadyne.

[0110] Figure 18F-H illustrate a non-cumulative, absolute concentration-time analysis, which showed a slight lag in avocadene and avocadyne bioaccessibility in the first hour followed by a rapid increase until two hours. Between one and four hours, avocadene had significantly higher bioaccessibility than avocadyne in the jejunum; however, the opposite trend (statistically insignificant) was observed in the ileum where avocadyne bioaccessibility was elevated above that of avocadene until 6 hours. A plateau was seen between two and three hours after which amounts of avocadene and avocadyne rapidly declined towards baseline. AUC analysis on the concentration-time curves revealed almost 1.2 folds higher bioaccessibility of avocadene compared to avocadyne (T able 4). While not wishing to be bound by any particular theory or mode of action, these results suggest that physicochemical differences as well stoichiometric ratios of avocadene and avocadyne may play a role in bioaccessibility.

Table 4. Area under curve analysis on non-cumulative concentration — time graphs for jejunum, ileum, and jejunum+ileum for AVO emulsion administered to TIM-1. _

Jejunum Ileum Jejunum+ileum

Avocadene 116.4 ± 1.8 ^a 24.4 ± 2.0 ^a 140.8 ± 3.8 ^a

(pg*h/mL) _

Avocadyne 85.9 ± 3.1 ^b 28.8 ± 3.0 ^a 114.7 ± 6.1 ^b

(pg*h/mL) _

Different superscript letters within each column indicates significant differences (P < 0.0001) between avocadene and avocadyne, two-way ANOVA with Sidak’s post hoc test (n=3).

[0111] A three-parameter shifted logistical model for lipolytic release of free fatty acids was adopted and used to characterize free fatty alcohol release in each digestive compartment as a function of time. Table 5 highlights the jejunal bioaccessibility (Casymp) of both avocadene and avocadyne compared to the ileum, as supported by the observed lower jejunal induction times ( t _c ) and higher jejunal rate constants (k) for both avocadene and avocadyne.

Table 5. Fitted parameter logistical model applied on cumulative data showing PFA bioaccessibility, induction time and rate of bioaccessibility in jejunum, ileum, and jejunum+ileum for AVO SEDDS administered to TIM-1.

Avocadene Avocadyne

Jejunum

PFA Bioaccessibility ( C _asy mp ) (%) 61 .5 ± 0.6 ^a _’ ^# 48.8 ± 0.6 ^{a #}

Induction time ( t _c ) (min) 132.2 ± 1.4 ^{a #} 129.5 ± 1.8 ^{a #}

Rate constant ( k ) (%/m in) 0.0270 ± 0.0011 ^a 0.0247 ± 0.0011 ^a

Ileum

PFA Bioaccessibility ( C _asy mp ) (%) 12.3 ± 0.3 ^b _’ ^# 15.7 ± 0.4 ^{b #}

Induction time ( t _c ) (min) 176.1 ± 3.7 ^{b #} 182.4 ± 3.7 ^{b #}

Rate constant (k) (%/m in) 0.0218 ± 0.0018 ^b 0.0206 ± 0.0016 ^b

Jejunum+lleum

PFA Bioaccessibility ( Casymp ) (%) 73.8 ± 0.9 ^# 64.5 ± 1 1 ^#

Induction time (t _c ) (min) 138.2 ± 1.8 140.3 ± 2.2

Rate constant (k) (%/min) 0.0254 ± 0.0012 0.0227 ± 0.0013

Different superscript letters ( ^a or ^b ) within each column indicate significant difference for each fitted parameter between jejunum and ileum (p < 0.05). Two-way ANOVA with Tukey’s post hoc test (n=3). # within each row indicates significant difference (p < 0.05) between avocadene and avocadyne. Two-way ANOVA with Tukey’s post hoc test (n=3).

[0112] Statistically significant differences between avocadene and avocadyne were observed for C _asymp , t _c , or k in jejunum and ileum but not overall (jejunum+ileum), where all fitted parameters were higher for avocadene (jejunum and overall) as initially observed in the cumulative and non- cumulative bioaccessibility analysis (Figure 18). While not wishing to be bound by any particular theory or mode of action, the mathematical model applied here suggests that avocadene and avocadyne bioaccessibility from avocatin B SEDDS may be generally higher than previously reported delivery systems of lyophilized avocado pulp powder (see, for example PCT/CA2020/051220, Avocatin B for the treatment of diseases and conditions, filed Sept. 10, 2020 which is incorporated herein in its entirety by reference). In vivo Pharmacokinetics

[0113] The present SEDDS may also be used to treat a disease or condition by administering the SEDDS in a therapeutically effective amount to a subject in need thereof. The SEDDS may be administered in an oral or intravenous dosage form and may be administered at least once daily.

[0114] The amount of avocado polyol, surfactant, and oil in the SEDDS may vary based on the disease or condition being treated. For example, in some embodiments, the SEDDS may be administered in an at least once daily dose that comprises from about 25 mg to about 150 mg of avocado polyol. In some embodiments, the avocado polyol may be avocatin B (i.e., avocadene and avocadyne in a 1 :1 w/w ratio). In some embodiments, the avocado polyol may comprise avocadene and avocadyne in a 3:2 w/w ratio, or in a 3:1 w/w ratio, or in a 1 :1 w/w ratio. In some embodiments, the treatment comprises administering SEDDS at least once daily, wherein the SEDDS comprises about 50 mg of avocatin B.

[0115] The diseases or conditions that may be treated with the present SEDDS may include, but are not limited to, acute myeloid leukemia, diet-induced obesity, and obesity-associated lipotoxicity. The disease or condition may be characterized by a dysregulation of glucose- stimulated insulin secretion (GSIS), insulin resistance, or reduced insulin sensitivity.

[0116] Figure 19 and Tables 6-7 illustrate results from a pharmacokinetic study using the illustrated SEDDS example. Ten-week-old, female, C57BL/6J mice were purchased (Jackson Laboratory, Bar Harbor, ME) and allowed to acclimatize for 1 week. After acclimatization, mice were randomly assigned (n=3 per treatment group) to receive an oral bolus dose of 100 mg/kg body weight (b.w.) avocatin B (formulated as 1% w/w SEDDS) or vehicle control (control SEDDS). The gavage volume was 5 mL/kg b.w. After 2 hr and 6 hr post gavage, up to 100 pL of whole blood was drawn per animal via tail bleed and collected in K2EDTA coated tubes (Sarstedt, Canada). At endpoint (24 hr post gavage), animals were euthanized via CO2 followed by exsanguination from which 500-800 pL of whole blood was collected and stored as described above.

[0117] For whole blood extraction, 100 pL of whole blood was extracted using a modified Folch protocol. Briefly, 100 pL of whole blood was macerated and mixed in 3 mL of 2:1 chloroform- methanol (v/v) at room temperature after which 0.5 mL of 0.2 M sodium-phosphate (NaHPCL) buffer in ddH ₂ 0 (pH 4.4) was added to induce layer separation. After inversion, samples were centrifuged for 5 min at 1500 ref. The total lipid containing organic layer was collected and an additional 2 ml_ chloroform was added to the aqueous layer as a wash step and as an additional round of extraction. The second organic layer was combined with the first and the buffer layer was discarded. Chloroform extracts were dried under a gentle stream of nitrogen and stored at 4 °C until sample preparation was required for LC-MS analysis. Blank whole blood (blood from non- treated or control mice) was also extracted the same way for recovery experiments and the generation of matrix- matched AVO standard curves.

[0118] Modified Folch protocol was also utilized for tissue extraction where 75 mg of flash frozen and pulverized tissue was macerated in 3 ml_ of 2:1 chloroform-methanol (v/v) and homogenized 40 times with a tissue grinder. 0.5 ml_ of NaHPC was then added to induce layer separation and double extraction of organic layer was completed as described before. Tissue chloroform extracts were dried under a gentle stream of nitrogen and stored at 4 °C until sample preparation was required for LC-MS analysis. Tissue from non-treated or control mice were also extracted the same way for recovery experiments and the generation of matrix-matched AVO standard curves. On analysis day, all dried blood and tissue samples were reconstituted in 250 pL of LC starting gradient (60% water-40% acetonitrile + 0.1% FA) and 10 pL of each was injected into the LC- SIM-MS method.

[0119] Tissue (inguinal fat pad, gonadal fat pad, liver, pancreas, heart, femur (to obtain bone marrow), and brain) was harvested and flash frozen in liquid N2. All blood and tissue samples were then maintained at -80 °C until extraction was performed for the analytical determination of avocadene and avocadyne in whole blood and tissues. Results for avocadene or avocadyne quantitation are presented as mean ± S.D., in ng/ml for whole blood or in ng/g wet tissue for tissues.

[0120] A previously developed LC-MS method for the quantitation of avocadene and avocadyne in avocado seed and pulp was further validated for mouse whole blood and tissue as well as TIM- 1 jejunum, ileum, and effluent dialysates. All chromatography and mass spectrometer parameters utilized were the same as previously described except all quantitative analysis was performed in high-resolution selected-ion monitoring (SIM) mode, which enabled avocadene and avocadyne fragmentation ions ([M+H-FhO]*) to be detected at low detection limits compared to the LC-MS method.

[0121] Pharmacokinetic parameters were calculated using non-compartmental analysis with the PK Functions add-in for Microsoft® Excel (Joel I. Usansky, PhD, Atul Desai, MS and Diane Tang- Liu, PhD, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA 92606, USA). The total area under the curve (AUCO-t) was determined with the linear trapezoidal rule from the time of dosing while AUCO-inf was extrapolated to infinity. The elimination rate constant (kel) and plasma concentration half life (t ½) were determined by regression of the two terminal data points on the semi-logarithmic concentration versus time plot. The maximal concentration (Cmax) and time at maximal (Tmax) were obtained directly from the concentration versus time plot.

[0122] The pharmacokinetics of a 100 mg/kg (body weight) oral bolus dose containing 20 mg/mL avocatin B SEDDS was examined in the mice. Non-compartmental pharmacokinetic analysis showed that the maximum concentration (Cmax) of avocadyne and avocadene in whole blood was 1687.90 and 1544.83 ng/mL, respectively (Table 6).

Table 6. Avocatin B non-compartmental pilot pharmacokinetic analysis. Data are shown as mean ± S.D., N=3 in each group.

Avocadyne Avocadene

Mean S.D. Mean S.D.

Cmax (ng/mL) 1687.90 79.08 1544.83 216.37

Tmax (h) 2.00 0.00 2.00 0.00

Kel (IT ¹ ) 0.18 0.06 0.20 0.03 ti/ ₂ (h) 4.37 1.88 3.55 0.50

AUCo t (ng/ml*h) 9499.48 1243.22 13032.37 838.88

AUCojn _f (ng/mL*h) 9071.26 3480.83 9868.07 2253.16

Cmax denotes maximum concentration of bioactive in blood; Tmax denotes time at which Cmax occurs; Kel denotes elimination rate constant; ti denotes half-life of bioactive in blood; AUCo_t denotes area-under-the-curve (AUC) from time 0-24 hr; AUCoj _nf denotes AUC from time 0 to infinity.

[0123] Despite similar maximal plasma concentrations (Figure 19A), exposure to avocadyne was greater than avocadene due to its higher half-life (t ½) of 4.37 hours compared to 3.55 hours for avocadene. Furthermore, avocadyne and avocadene were detectable in various metabolically active tissues, accumulating most in the liver and pancreas (Figure 19B). This pharmacokinetic study also allowed for the development and validation of a bioanalytical method that has selectivity, linearity, extraction recovery, accuracy, and precision for mouse whole blood and tissue matrices (Table 7). [0124] Intra- and inter-day accuracy and precision of the developed method were determined by assaying three concentrations of quality control (QC) samples (for blood matrix: LLOQ = 2 ng/mL, low = 100 ng/mL, and high QC = 1800 ng/mL; for tissue matrix: see Table 7) in duplicates on two different analytical days. Precision was reported as percent coefficient of variation (%CV) of replicates within one sample run (intra-assay) or between sample runs (inter-assay). Intra- and inter-assay accuracy was reported as percent relative error (% RE) or the percent deviation of QC replicates from nominal concentration. The acceptance limit for accuracy and precision, at low and high QC concentration levels, were set to 15% RE and 15% CV, respectively. For LLOQ, accuracy and precision acceptance limits were set to below 20% RE and 20% CV, respectively. See Table 7 for accuracy and precision parameters for whole blood, tissue, and TIM-1 dialysates.

[0125] Recovery of avocadene and avocadyne from mouse whole blood, tissue, and TIM-1 dialysates was determined by comparing the peak areas of extracted QC samples (at LLOQ, low and high concentrations as highlighted in Table 7) with the peak areas of post-extraction blood, tissue or TIM-1 dialysate blanks spiked at corresponding concentrations. The matrix effect of the different samples on the ionization of avocadene and avocadyne was evaluated by comparing the peak areas of post-extraction blank biological or TIM-1 samples spiked at concentrations of QC samples with the areas obtained by QC samples prepared in solvent (LC starting gradient). This analysis was performed for biological replicates.

Table 7. AVO bioanalytical method validation parameters

Sample Linear Range, QC Accuracy (% error) Precision (% CV) Recovery (%) Matrix Effect (%) concentrations

Solid Form and Encapsulation of Pharmaceutical Compounds

[0126] The present SEDDS may also be used to encapsulate a pharmaceutical compound. In some embodiments, the pharmaceutical compound may be poorly water-soluble. In some embodiments, the SEDDS may be adsorbed onto a solid carrier, such as but not limited to NEUSILIN™, AEROPERL® 300 PHARMA™, AEROPERL 3375/20™ or AEROPERL® 300/30™. In some embodiments, the solid carrier may be NEUSILIN™.

[0127] In some embodiments, the pharmaceutical compound may comprise naproxen. In some embodiments, the concentration of naproxen may be about 1 mg/mL to about 3 mg/mL. In some embodiments, the concentration of naproxen may be about 3 mg/mL.

[0128] In some embodiments, the pharmaceutical compound may comprise curcumin. In some embodiments, the concentration of curcumin may be about 1 mg/mL to about 5 mg/mL. In some embodiments, the concentration of curcumin may be about 5 mg/mL.

[0129] In some embodiments, there may be a pharmaceutical composition comprising an active agent and the SEDDS.

[0130] Figure 20A illustrates an example in which a solid preparation of the present SEDDS was prepared. The liquid SEDDS were converted to a solid form (solid-SEDDS) using the solid carrier NEUSILIN™ (an amorphous form of magnesium aluminometasilicate which has adsorption capacity and flow enhancing properties; purchased from Fuji Chemical Industries Inc (NJ, USA)). Avocatin B was heated in the oil/surfactant phase at 200 mg/mL, which was then adsorbed to 200mg NEUSILIN™ carrier by mixing and sonication for 30 min. The powder was then reconstituted in 4 ml double-distilled water and mean droplet size was measured using dynamic light scattering as described above.

[0131] The oil phase for both control and avocatin B SEDDS adsorbed easily onto the solid carrier NEUSILIN™. These solid-SEDDS self-assembled into expected droplet size distributions upon dissolution in water (Figure 20B), suggesting the described SEDDS may be scalable and suitable for common commercial dosage forms.

[0132] The ability of avocatin B to enhance encapsulation of two poorly water soluble compounds, naproxen and curcumin (both purchased from Cayman Chemicals (Ml, USA)), is illustrated in Figure 21. Naproxen or curcumin were added to the oil phase (1:1 NeoBee®M5-Tween 80) at a concentration of 50 mg/mL with or without 100 mg/mL avocatin B and heated overnight at 75 °C and then the oil phase was diluted 10 fold with PBS and vortexed for 30 s. Droplet size and PDI were then measured as described above.

[0133] Here, 5 mg/ml_ naproxen encapsulated in avocatin B-SEDDS had a mean droplet size of 32 nm compared to naproxen in control SEDDS which was 229 nm (Figure 21A-B). Similarly, 5 mg/ml_ curcumin encapsulated in avocatin B-SEDDS showed a mean droplet size of 52 nm compared to 297 nm in control SEDDS (Figure 21C-D). Both naproxen and curcumin control and avocatin B SEDDS destabilized over the course of 3 days; however, higher dilutions (above 1 : 100) slowed this effect (data not shown).

[0134] Curcumin has an established in vitro bioactivity profile in AML cells ; thus, curcumin was delivered to AML-2 cells in a DMSO or SEDDS formulation (Figure 21 E). Curcumin in avocatin B- SEDDS had greatest potency compared to curcumin/avocatin B in DMSO or curcumin in control SEDDS (Figure 21 E and Table 5).

Table 5. IC50 values of curcumin delivered via DMSO or in SEDDS to OCI-AML-2 cells. IC50 was calculated using non-linear regression model (logarithmic inhibitor vs. normalized response- variable slope).

DMSO SEDDS

(pg/mL) (pg/mL)

Curcumin _ 8.55 ± 0.1Q ^a _ 5.83 ± 0.19 ^b _

Curcumin + AVO _ 3.75 ± 0.18 ^a _ 1.08 ± 0.13 ^b _

Different superscript letters within each row indicate significant differences (P < 0.0001) between treatments, two-way ANOVA with Sidak’s post hoc test (n=3).

[0135] While not wishing to be bound by any particular theory or mode of action, the eutectic phase behaviour of avocatin B may be advantageous for its incorporation into simple, low-energy, bio-compatible SEDDS. Eutectic mixtures have been reported to improve drug dissolution/solubility and bioavailability; however, these systems typically employ structurally unrelated molecules (e.g. drug and excipients) to form eutectics. In contrast, avocadene and avocadyne are two structurally similar natural compounds that form a eutectic mixture at a molar ratio of 1:1, which may translate to their efficient incorporation into a microemulsion-based delivery system. While not wishing to be bound by any particular theory or mode of action, the XRD data discussed herein suggest that molecular size and potentially polymorphic state contribute to the eutectic formation seen in avocatin B.

[0136] The incorporation of small amounts of avocatin B into SEDDS resulted in reductions in droplet diameter without adding energy or a co-surfactant. While not wishing to be bound by any particular theory or mode of action, this may be indicative of avocatin B self-assembling at the oil- water interface altering the interface curvature and film flexibility. The co-surfactant effects of avocado polyols in MCT oil-high HLB surfactant SEDDS was found to be correlated with their physical properties (notably melting temperatures, enthalpies and entropy as well as crystal structure and thickness).

[0137] The tested SEDDS having concentrations greater than or equal to about 2% (w/w) avocado polyol showed at least some signs of destabilization on extended storage. One explanation for this considered by the inventors may be that relatively higher polyol concentrations may penetrate the interfacial film in such a way that pushes the spontaneous curvature of the surfactant film past the hydrophilic — lipophilic balance point.

[0138] While this invention has been described with reference to illustrative embodiments and examples, the description is not intended to be construed in a limiting sense. Thus, various modifications of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to this description. It is therefore contemplated that the appended claims will cover any such modifications or embodiments.

[0139] All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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