DILUTABLE FORMULATIONS OF CANNABINOIDS AND PROCESSES FOR THEIR PREPARATION

Abstract

Provided are cannabinoid-loaded formulations, as well as processes for their preparation.

Claims

1.-49. (canceled)

50. A formulation comprising a microemulsion, said formulation comprising at least one oil mixture that comprises a mixture of oleic acid and linoleic acid, at least one hydrophilic surfactant, at least one co-surfactant, and at least 0.1 wt % of at least one cannabinoid, wherein the formulation comprises 10 wt % of water or less.

51. The formulation of claim 50, wherein the cannabinoid comprises CBD, CBD-A, THC, or a combination thereof.

52. The formulation of claim 51, wherein the cannabinoid comprises CBD.

53. The formulation of claim 50, wherein said at least one oil mixture comprises at least one oil selected from mineral oil, paraffinic oils, vegetable oils, glycerides, esters of fatty acids, liquid hydrocarbons and mixtures thereof.

54. The formulation of claim 50, wherein said at least one oil mixture comprises at least one oil selected from medium-chain triglycerides (MCT), olive oil, soybean oil, canola oil, cotton oil, palmolein, sunflower oil, corn oil, rapeseed oil, grape seeds oil, hemp oil, pomegranate oil, avocado oil, peppermint oil, tomato oil, isopropyl myristate, oleyl lactate, coco caprylocaprate, hexyl laurate, oleyl amine, oleyl alcohol, linoleyl alcohol, ethyl oleate, hexane, heptanes, nonane, decane, dodecane, D-limonene, neem oil, lavender oil, peppermint oil, anise oil, rosemary oil, sage oil, hibiscus oil, berries oil, menthol, capsaicin, grape seed oil, pumpkin oil, hemp oil and similar essential oils or triglycerides or esters of fatty acids and mixtures thereof.

55. The formulation of claim 50, wherein said formulation comprises between about 0.5 and 20 wt % of said at least one oil mixture.

56. The formulation of claim 50, wherein said at least one hydrophilic surfactant is selected from polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, and polyoxyethylene esters of saturated and unsaturated castor oil, ethoxylated monoglycerol esters, ethoxylated fatty acids and ethoxylated fatty acids of short and medium and long chain fatty acids.

57. The formulation of claim 50, wherein said formulation comprises between about 30 and 85 wt % of said at least one hydrophilic surfactant.

58. The formulation of claim 50, wherein said at least one co-surfactant is selected from polyols, diglycerides, and polyoxyethylenes.

59. The formulation of claim 50, wherein said formulation comprises between about 1 and 50 wt % of said at least one co-surfactant.

60. The formulation of claim 50, comprising between about 0.1 and 12 wt % of said cannabinoid.

61. The formulation of claim 50, comprising at least one oil droplet having an oil droplet size of between about 5 and about 30 nanometers.

62. The formulation of claim 50, wherein said cannabinoid is geometrically integrated, physically integrated, or both geometrically and physically integrated into the oil.

63. The formulation of claim 50, wherein said cannabinoid is geometrically integrated, physically integrated, or both geometrically and physically integrated into an interface between the oil and hydrophilic surfactant.

64. A pharmaceutical composition comprising the formulation of claim 50.

65. The pharmaceutical composition of claim 64, being in a form selected from a gel, a lotion, oil, soap, a spray, an emulsion, a cream, an ointment, capsules, soft-gel capsules, a patch, or a solution.

66. The pharmaceutical composition of claim 64, adapted for delivery of said cannabinoid topically, orally, by inhalation, nasally, transdermally, ocularly or parenterally into the circulatory system of a subject.

67. The pharmaceutical composition of claim 64, further comprising a pharmaceutically acceptable carrier.

68. The pharmaceutical composition of claim 64, further comprising a diluent.

69. A formulation comprising a microemulsion, said formulation comprising at least one oil mixture comprising a mixture of oleic acid and linoleic acid, polyglycerol ester, at least one hydrophilic surfactant different from polyglycerol esters, at least one co-surfactant, and at least 0.1 wt % of at least one cannabinoid, wherein the formulation comprises 10 wt % of water or less.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0116] FIG. 1 shows CBD-loaded 5CS formulation in various dilutions.

[0117] FIG. 2 shows electrical conductivity of empty and CBD-loaded 5CS formulation as a function of water (0.01M NaCl) content.

[0118] FIG. 3 shows viscosity of empty and CBD-loaded 5CS formulation as a function of the water content.

[0119] FIGS. 4A-4B show the diffusion coefficients (Dx) of the various components for unloaded and 1 wt % CBD-loaded formulation, respectively.

[0120] FIGS. 5A and 5B show long term stability of crystalline CBD solubilized in a concentration of 5 wt % in AX-1 and 5CS formulations, respectively.

[0121] FIGS. 6A-6C show LUMiFuge™ test results for CBD-loaded AX-1 concentrate, CBD-loaded AX-1 85wt % water diluted, and commercial ‘Plus CBD’ product, respectively.

[0122] FIG. 7 shows the paw-withdrawal threshold in mice for 5wt % crystalline CBD solubilized in 5CS formulation compared to crystalline CBD with the same concentration dispersed in olive oil.

[0123] FIG. 8 shows the paw-thickness of inflammated paw in mice for 5 wt % crystalline CBD solubilized in 5CS formulation compared to crystalline CBD with the same concentration dispersed in olive oil.

[0124] FIG. 9 shows the measured ear thickness of DHT-induced rats 24 hours after treatment.

[0125] FIGS. 10A-D are pictures of rats' ears in DHT test: non-treated DHT-induced (FIG. 10A), and naïve rats (FIG. 10B), 24mg/kg BW of 5CS formulation (FIG. 10C), 48mg/kg BW of AX-1 formulation (FIG. 10D).

[0126] FIGS. 11A-11C shows the pharmacokinetics of the profile of CBD in the blood of rats after oral administration delivered form 5CS and In9(6) systems vs. CBD dispersed in olive oil at various dosage of 10, 25, 50 mg CBD/kg body weight, respectively.

[0127] FIGS. 12A-12B shows the pharmacokinetics of the profile of CBD in the blood of rats after oral administration of: AX-1 compared to AX-1(B) (FIG. 12A), and AX-1, SCS, OR201SE and OR103 (FIG. 12B).

[0128] FIGS. 13A-13B show relative changes in CBD concentration as a function of time while incorporate in AX1 and MeOH, and CBD content within simulated gastric fluid (SGF) as a function of time while solubilized within MeOH, 5CS and AX1, respectively.

[0129] FIG. 13C shows cannabinoids degradation as a function of time within simulated gastric fluid (SGF).

[0130] FIGS. 14A-14B show cannabinoid degradation as a function of time within simulated gastric fluid (SGF) of the commercial product RSHO™ (FIG. 14A) and CBD in olive oil (FIG. 14B).

[0131] FIGS. 15A-15B show samples of 5 wt % CBD-loaded 5CS after compounding with mannitol solution at lyophilized state (FIG. 15A) and reconstituted state (FIG. 15B).

[0132] FIG. 16A shows PK profiles of 5 wt % CBD-loaded 5CS in original microemulsion form compared to lyophilized powder.

[0133] FIG. 16B shows PK profiles of 5 wt % CBD-loaded 5CS in original microemulsion form compared to reconstituted lyophilized powder.

DETAILED DESCRIPTION OF EMBODIMENTS

[0134] Formulations and Preparations

[0135] Exemplary microemulsions described herein are provided in Tables 1-1 to 1-5. As noted above, the formulations are self-assembled systems which are formed in a spontaneous manner. Therefore, several compositions of the formulations were prepared by simple mixing of ingredients at 25-70° C. An exemplary process for preparing the formulation involves mixing together the oil, the surfactant and the co-surfactant (and where applicable also a solvent, a co-solvent and/or a phospholipid) until a homogenous, clear (transparent) mixture is obtained. In case the surfactants or oil are solid at room temperature, heating can be applied while mixing to allow full dissolution and formation of the empty formulation.

[0136] The formulation is then slowly added to a cannabinoid source, for example a plant part or pure cannabinoid, to allow appropriate wetting and then mixed and/or homogenized. Another variation of the process includes adding the cannabinoid source stepwise to the empty (un-loaded) formulation until a homogeneous slurry is obtained.

[0137] Solubilization was carried out under heating and/or inert atmosphere, thereby solubilizing the desired cannabinoid, in this case CBD, into the formulation.

TABLE-US-00001 TABLE 1-1 Formulations (all amounts provided in wt %) Formulation Component 5CS 5CS(1) 5CS(2) 5CS(3) 5CS(6) Oil MCT 3.60 3.60 3.60 3.60 3.63 Glycerin — 19.0 11.40 — — Hydrophilic Polysorbate 80 35.37 28.37 30.0 35.37 35.64 surfactant (Tween 80) Cremophor EL castor 42.57 35.57 40.0 42.57 42.9 oil* Co-surfactant Propylene glycol 12.66 7.66 9.2 12.18 12.28 (PG) Solvent Ethanol 0.2 0.2 0.2 0.2 0.17 Oleic acid — — — 0.48 — Phospholipid PC (Phosphatiydyl 5.3 5.3 5.3 5.3 5.28 Choline) Lyso-PC 0.3 0.3 0.3 0.3 — (Lysophosphatydil choline) CBD loading ≤5 ≤2.5 ≤5 ≤5 ≤5 *Polyoxyl 35 castor oil

TABLE-US-00002 TABLE 1-2 Formulations (all amounts provided in wt %) Formulation Component AX1 NL28B NL28B(1) NL28B(2) NL28B(3) NL28B(4) NL28I Oil MCT — — 6.55 — — — — Castor oil — 6.55 — — — — 5.4 R(+)- 5 — — 6.55 — — — Limonene Isopropyl — — — — 6.55 — — myristate Ethyl — — — — — 6.55 — laurate Hydrophilic Tween 80 45 36.34 36.34 36.34 36.34 36.34 23.60 surfactant Cremophor — 37.64 37.64 37.64 37.64 37.64 26.40 EL Co- PG 45 19.47 19.47 19.47 19.47 19.47 44.60 surfactant Solvent Ethanol 5 — — — — — — CBD loading ≤10 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5

TABLE-US-00003 TABLE 1-3 Formulations (all amounts provided in wt %) Formulation Component In9 In9(1) In9(2) In9(3) In9(4) In9(5) In9(6) Oil MCT 11.5 11.5 11.5 11.5 11.5 5.0 5.0 Glycerol 6.5 6.5 6.5 6.5 6.5 6.5 6.5 Hydrophilic Tween 80 35.0 35.0 35.0 35.0 35.0 35.0 35.0 surfactant Cremophor 35.0 34.0 30.0 30.0 30.0 32.0 32.0 EL Co- PG 6.5 6.5 6.5 6.5 4.0 6.5 6.5 surfactant Solvent Ethanol 5.5 5.5 5.5 3.0 3.0 5.0 6.5 Oleic acid — — — — 5.0 2.5 2.5 Transcutol — — — 2.5 — 2.5 2.5 Phospholipid PC — 1.0 5.0 5.0 5.0 5.0 5.0 or surfactant CBD loading ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5

TABLE-US-00004 TABLE 1-4 Formulations (all amounts provided in wt %) Formulation Component MM7(2) 2BR(9:1) 2CA(9:1) 2BR(8:2) Oil R(+)- — 5.0 5.0 10.0 Limonene Castor oil 2.9 — — — Hydrophilic Tween 80 45.0 45.0 60.0 53.3 surfactant Mirj S40 32.9 — — — Co- PG 45.0 45.0 30.0 23.67 surfactant Solvent Ethanol 5.0 5.0 5.0 10.0 CBD loading ≤5 ≤3 ≤3 ≤4

TABLE-US-00005 TABLE 1-5 Formulations (all amounts provided in wt %) Formulation Component 5CS(5) 5CS(7) 5CS(8) CAS(1) CAS(2) CAS(3) CAS(4) Oil MCT 3.63 3.63 3.63 — — — — Ethyl — — — 7.5 7.5 — — caprate Olive oil — — — — — 4.2 5.0 Hydrophilic Tween 80 35.64 35.64 33.03 15 — — 17.14 surfactant Cremophor 42.90 40.32 42.57 — — — — EL Heco40* — — — — 15 — — Labrasol** — — — — — 61 — Co- PG 12.38 10.22 6.3 50 25 — 57.14 surfactant CC497*** — — — — 25 21 — Solvent Ethanol 0.17 0.17 0.17 22.5 — 12.8 15.0 Oleic acid — 4.5 4.5 — — — — Acetic acid — — — — 22.5 — — Phospholipid PC 5,24 5.24 5.24 5 5 — 5.71 Lyso-PC — 0.08 0.08 — — — — CBD loading ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 ≤5 *ethoxy 40 hydrogenated castor oil **Labrafil M1944CS (Oleoyl macrogolglycerides) ***Plurol Oleique CC 497 (Polyglyceryl-3 dioleate)

[0138] Characterization of CBD-Loaded Formulations

[0139] Empty and CBD-loaded (1 wt %) 5CS systems were characterized using several methods in order to elucidate structural changes as well as the effect of CBD on the formulation. Electrical conductivity, rheology measurements, differential scanning calorimetry, dynamic light scattering and others were utilized to identify phase transitions and changes at the molecular level within the systems.

[0140] Dilutability

[0141] As shown in FIG. 1, CBD-loaded 5CS system was diluted by mixing with up to 9% water. The system remains clear and is fully dilutable, without any phase separation.

[0142] Electrical Conductivity Measurements

[0143] Structural transitions of the system as a result of dilution was carried out by electrical conductivity measurements. In order to facilitate measurement, the 5CS systems were diluted with 0.01M solution of NaCl. Measurements carried out at RT (23±2° C., using conductivity meter 730 (Metler Toledo, GmBH, Switzerland) equipped with 180×65mm/0.61 kg electrode (conductivity range of 0.01 μS/cm-1000 mS/cm). The results are depicted in FIG. 2.

[0144] As can be seen in FIG. 2, which present the electrical conductivity of the empty and CBD loaded systems versus water content, no significant effect is seen as a result of the solubilization of CBD; these results indicate that the formulation may be uniquely tailored to entrap the cannabinoid, in this case CBD, into the interface or to the oil core of the formulation. FIG. 2 also demonstrates that incorporation of CBD into the system has no effect on the stability, disruption or physical changes of the system throughout the whole dilution process (ratio).

[0145] Further, FIG. 2 confirms that at low water content (of ca. 20wt % water) the system is of W/O nano-droplets and transforms into bicontinuous phase (sharp increase in the conductivity), and inverts into O/W droplet (sharp decrease in the conductivity) as a result of dilution effect.

[0146] Viscosity Measurements

[0147] Viscosity measurements as a function of the dilution were carried out at RT (25±0.1° C.), using Thermo Haake Rheo Scope 1 equipped with C60/° 1 cone and glass plate (the distance between the cone and the plate during the measurements was 0.022 mm). At each measurement increasing shear rates (0-100 s.sup.−1) were applied for 6 min.

[0148] As seen in FIG. 3, when W/O nano-droplets are formed (ca. up to 20wt % water) the CBD is located in the outer interface close to the oil and does not interfere with the entanglement of the surfactant tails; similarly, when the system inverts into O/W nano-droplets, the CBD has no effect on the entanglement of the surfactant since it located mostly in the oil core.

[0149] However, significant differences in viscosity are identified between 30 to 50 wt % water between the CBD-loaded system and the empty system. Without wishing to be bound by theory, the CBD molecules interfere with the entanglements between the surfactant's lipophilic tails only at the bicontinuous region where the system is mostly composed of interfaces weakening these interactions, thereby resulting in lower viscosity values compared to the empty (non-loaded) formulation.

[0150] Differential Scanning Calorimetry (DSC)

[0151] The melting/freezing temperature of the water changes as a function of the water molecules environment. Therefore, changes in such temperature may be used to characterize the interaction of water molecules with other species in the system. In order to follow these changes, sub-zero calorimetric measurements were carried out. 8-12 mg samples of 5CS system with different water dilutions, unloaded and 1 wt % CBD-loaded, were cooled from 25° C. to −100° C. and then heated back to 25° C. both at a rate of 5° C./min (using a Mettler Toledo DSC 822). Between cooling and heating the sample kept at an isotherm of −100° C. for 20 min. All measurements were carried out against empty perforated pan as a reference. Melting temperatures and enthalpy of transition for the different samples are presented in Table 3.

TABLE-US-00006 TABLE 3 melting temperatures and melting enthalpies of water within 5CS, empty and CBD-loaded systems Water Empty system 1% CBD-loaded system content T.sub.m ΔH.sub.m T.sub.m ΔH.sub.m (wt %) (° C.) (J/g) (° C.) (J/g) 30 −26.0 1.39 −27.3 1.25 40 −17.9 28.05 −18.9 33.05 50 −8.5 46.1 −12.6 47.95 60 −4.8 65.80 −6.3 96.15 70 −3.8 127.21 −4.3 136.89 80 −2.1 162.56 −2.8 186.96 90 −0.8 206.96 −1.8 241.99

[0152] Beyond 30 wt % water no endothermic peaks appeared, meaning that the water are tightly bound and are found predominantly in the core of the droplet. Above 30% water, the melting temperature and enthalpy increases as water is released from the droplets; in high water concentations (i.e. high dilutions) most of the water is free. Yet from the values of the ΔH.sub.m it is clear that even at 90 wt % water not all the water is free since the ΔH.sub.m of free water is ca. −280 Eg.

[0153] The thermal behavior of the systems indicates that at low water contents the (0-40%) the water is bound to the surfactants and freezes at −30-(−20)° C. At higher water contents the water graduate becomes freer and freezes at higher temperatures closely to 0° C. (above 60 wt % water). That is to say that above 60 wt % of water the continuous phase is the water. At lower water contents (30-50) the water creates continuous domains together with oil continuous domains to create the so called bicontinuous mesophase. Beyond 30 wt % dilution the water is tightly bound to the polyethoxylated head group of the surfactants. The major diffrances between the empty and loaded system are reflacting the freedom of water to move (mobility)—once the CBD is entraped into the core it is assocaited with the head group of the surfactanet, and as a result more water molecules are free to be mobile.

[0154] Dynamic Light Scattering (DLS)

[0155] The oil droplet size of water-diluted formulations was determined by DLS measurements, as well as analysis of drop diffusion coefficient of oil in water. The results of the DLS measurements are provided in Table 4.

TABLE-US-00007 TABLE 4 Droplet size and droplet diffusion coefficient of 5CS, empty and 1 wt % CBD-loaded systems 1% CBD-loaded Empty system system Water Droplet Diffusion Droplet Diffusion content diameter coef. diameter coef. (wt %) (nm) (μm.sup.2/s) (nm) (μm.sup.2/s) 70 11.3 ± 0.2 40.2 11.6 ± 0.15 40.0 80 10.1 ± 0.2 48.7 10.3 ± 0.25 44.1 90 10.1 ± 0.2 48.7 10.3 ± 0.2  48.6

[0156] Typically, solubilization of guest molecules within the formulation causes the swelling of the droplets and increases their diameter. In the 5CS system, the results show that the effect of the solubilization is not significant. This may be due to the relatively low concentration of CBD within the microemulsion.

[0157] The diffusion coefficients of the systems at different dilutions are correlative with drops size—the larger the diameter the slower the drop diffusivity.

[0158] Self-Diffusion NMR (SD-NMR)

[0159] In order to determine the structure of the oil droplets (or micelles) of the formulations, self-diffusion NMR analysis was carried out. SD-NMR is able to locate each component within the NSSL via measurements of its diffusion coefficient. Rapid diffusion (>100×10.sup.−11 m.sup.2s.sup.−1) is characteristic of small molecules, free in solution, while slow diffusion coefficients (<0.1×10.sup.−11 m.sup.2s.sup.−1) suggest low mobility of macromolecules or bound/aggregated molecules.

[0160] NMR measurements were performed with a Bruker AVII 500 spectrometer equipped with GREAT 1/10 gradients, a 5mm BBO and a 5mm BBI probe, both with a z-gradient coil and with a maximum gradient strength of 0.509 and 0.544 T m.sup.−1, respectively. Diffusion was measured using an asymmetric bipolar longitudinal eddy-current delay (bpLED) experiment, or and asymmetric bipolar stimulated echo (known as one-shot) experiment with convection compensation and an asymmetry factor of 20%, ramping the strongest gradient from 2% to 95% of maximum strength in 32 steps. The spectrum was processed with the Bruker TOPSPIN software. NMR spectra were recorded at 25±0.2° C. The components were identified by their chemical shift in 1H NMR.

[0161] FIGS. 4A-4B show the diffusion coefficients (Dx) of the various components for unloaded and 1 wt % CBD-loaded formulations, respectively.

[0162] As noted above, the formulations of this disclosure are constituted by oil droplets which solubilize CBD surrounded by surfactants and co-surfactants. When in the concentrate form (i.e. in the absence of water), the system is arranged in a reverse micelle structure, and when mixed with small amounts of aqueous media, hydrated and solvated surfactants are formed. Upon further dilution with aqueous phase oil-in-water (O/W) nanodroplets entrapping into their oil core the CBD molecules are formed. When diffusion coefficients of CBD and the surfactant are of a similar order of magnitude (when measured in the microemulsion system), the CBD will remain entrapped within the oil core during the structural transformations of the system (i.e. the changes in structure due to dilution); this is a result of the interactions (physical complexation) between the CBD and the surfactants and/or co-surfactants, thus stabilizing the formulation and preventing undesired release of the CBD from the oil core. Release of the CBD from the formulation will occur upon interaction of the droplets with target biological membranes after administration to the subject to be treated.

[0163] FIGS. 3A and 3B indicate that the mobility of all the components are not significantly affected by the solubilization of the CBD in the nanodroplets. Although CBD's chemical shift could not be detected by this NMR technique, the fact that no change was measured in all other components indicated that CBD is completely solubilized throughout the entire dilution process. The mobility of the surfactant is very low, indicating that the CBD is interacting with the surfactant and is in the proximity of the surfactant at the interface.

[0164] Stability of Formulations with CBD from a Plant Source

[0165] 5CS and In9(6) formulations (see Table 5-1) were loaded with 5 wt % CBD and incubated at three different temperature (4, 25 and 40° C.) under different conditions (without protection, with the addition of 600ppm a-tocopherol acetate and under nitrogen atmosphere). Both the concentrate and a diluted microemulsion (80% water) were tested.

TABLE-US-00008 TABLE 5-1 Formulations for stability tests Formulation 5CS Formulation In9(6) Component wt % Component wt % Oil MCT 3.6 MCT 5 Oleic acid 2 Hydrophilic Polysorbate 80 35.37 Polysorbate 80 35 surfactant (Tween 80) (Tween 80) Cremophor EL 42.57 Cremophor EL 32 castor oil* castor oil* Glycerol 6.5 Co-surfactant Propylene glycol 8.46 Propylene glycol 9 (PG) (PG) Solvent — — Ethanol 5.5 Phospholipid Phosal 50 PG** 10 Phosphatidylcholine 5 *Polyoxyl 35 castor oil **Phosal 50 PG composed of 1.5-2.5% wt ethanol, >500 ppm ethylenemethylketone, 0.5 wt % water, 33.8-41.2 wt % propylene glycol, <50.0 wt % phosphatidylcholine, >6 wt % lyso-phosphatidylcholine

[0166] The visual appearance of the samples were recorded after 30 days of incubation. The results are detailed in Table 5-2.

TABLE-US-00009 TABLE 5-2 Stability of CBD-loaded formulations Extraction Incubation 5CS In9(6) Conditions temperature Concentrate 80% dilution Concentrate 80% dilution No  4° C. Stable Stable Stable Stable protection 25° C. Stable Stable Stable Stable 40° C. Yellowish N/A Yellowish N/A 600 ppm  4° C. Stable Stable Stable Stable α-tocopherol 25° C. Stable Stable Stable Stable acetate 40° C. Yellowish Stable Yellowish Stable Nitrogen  4° C. Stable Stable Stable Stable atmosphere 25° C. Stable Stable Stable Stable 40° C. Stable Stable, Yellow Stable Stable, Yellow

[0167] As clearly seen, the CBD-loaded formulations are stable over a wide variety of conditions, namely most of the tested samples remained transparent, without any indication of phase separation or precipitation.

[0168] Stability of Pure CBD Solubilized in AX-1 and 5CS Formulations

[0169] Crystalline CBD was solubilized in a concentration of 5 wt % in AX-1 and 5CS formulations under various conditions: addition of 1000ppm of vitamin E acetate, under passive diffusion of nitrogen or under no special treatment. All samples were kept at three different temperatures of 4° C. ,25° C. and 40° C., four samples of each formulation/treatment for an examination at four time-points including 0 (initial), 15, 30 and 60 days. All samples (2 mL) were kept at 4 ml vails with coordinated labeled .Some of the samples were passively purged with nitrogen. At the predetermined times of sampling, the suitable samples were tested for their appearance and were analyzed by HPLC to determine CBD concentration and presence/absence of degradation products .

[0170] No significant changes in CBD concentration of AX1 and 5CS concentrates was detected after 60 days for all storage temperatures, as seen in FIGS. 5A and 5B, respectively.

[0171] Stability LumiFuge™ Tests

[0172] To determine long term stability of formulations, a rapid measurement was carried out using LUMiFuge™ analytical centrifugation. LUMiFuge analysis enables to predict the shelf-life of a formulation in its original concentration, even in cases of slow destabilization processes like sedimentation, flocculation, coalescence and fractionation. During LUMiFuge measurements, parallel light illuminates the entire sample cell in a centrifugal field; the transmitted light is detected by sensors arranged linearly along the total length of the sample-cell. Local alterations of particles or droplets are detected due to changes in light transmission over time. The results are presented in a graph plotting the percentage of transmitted light (Transmission %) as a function of local position (mm), revealing the corresponding transmission profile over time. CBD-loaded AX1formulations in concentrate form and with 85% water dilution were tested in comparison to the commercial product “Plus CBD oil” by CS Science (formulations tested as is, without any further treatment).

[0173] FIGS. 6A-6C show the change in sample transmission as a function of time. As seen, in both AX-1 formulations (concentrate and diluted form) the samples were stable through the whole analysis time, showing no changes in transmission (FIGS. 6A, 6B respectively). The ‘Plus CBD’ product showed phase separation already in early stage of measurements (FIG. 6C), with significant sediments.

[0174] Thus, while the CBD in an oil formulation was not stable and is predicted to separate and segregate over time, the formulations of this disclosure are stable at 3000 rpm and even after 17 hours of centrifugation. These conditions simulate minimum 2 years of storage.

[0175] In-Vivo Studies

[0176] Paw Withdrawal Test

[0177] Response to pain and anti-inflammatory activity in mice of the CBD-loaded formulations of this disclosure were assessed by oral administration of 5CS formulation loaded with 5 wt % CBD compared to CBD dispersed in olive oil.

[0178] Various doses of CBD were administered in the range of 5, 10, 25 and 50 mg/kg per dose. Paw-withdrawal was assessed by pricking the paw of the mice at varying loads and recording the withdrawal reflex response. FIG. 7 shows the paw-withdrawal threshold in mice for 5%-CBD 5CS formulation compared to CBD in olive oil. FIG. 8 shows the paw-thickness of inflammated paw in mice for 5%-CBD 5CS formulation compared to CBD olive oil extract.

[0179] As seen from FIG. 7, in all dosages tested, mice administered with the CBD-loaded formulation of the present disclosure showed higher tolerance to pain immediately after administration (2 and 24 hours), and at least comparable tolerance to pain to that in the oil-samples for a period of 6 hours from administration. This attests to the improved release, permeation and performance of CBD in the system after administration.

[0180] Further, as seen in FIG. 8, mice administered with the formulation of the present disclosure showed a more significant reduction in paw thickness in all dosages tested as compared to identical dosages of CBD in olive oil. Namely, the formulations of the present disclosure have an improved anti-inflammatory activity as compared to standard CBD in oil.

[0181] Delayed-Type Hypersensitivity (DTH)

[0182] CBD was shown to reduce inflammation response and pain-effected by inflammatory reaction. Without wishing to be bound by theory, inflammation reduction is achieved by various mechanisms, including agonist and antagonist binding to CB1 receptors, adenosine receptors and other GPCRs, involving the reduction of inflammatory cytokines and chemokines levels, such as IL-2, IL-6, TNF-α, MCP-1, etc.

[0183] The therapeutic effect of oral administration of CBD-loaded formulations of this disclosure as anti-inflammatory agents. The CBD effect was evaluated using rat model of inflammation—Delayed Type Hypersensitivity (DHT) model. In this test, the reduction in ear swelling after inflammation-induction following treatment was measured.

[0184] The belly of male rats (average weight 250g) was shaved and challenged 10 times with 500 μl of 2% oxazolone (400 mg oxazolone dissolved in 16 ml acetone and 4 ml mineral oil). The next day (referred to herein as day 1), 500 μl of CBD formulation oral treatment was given via gavage. On day 6, the ear thickness of the rats was measured using a caliper.

[0185] Rats were challenged with another dose of 50 μl of 0.5% oxazolne, and a second oral treatment of 500 μl CBD formulation was administered 2-hours after challenge. The ear thickness was measured again 12 and 24 hours after challenge, and blood samples were taken for serum preparation.

[0186] Samples composition: two doses were administered of crystalline CBD in AX-1 with a dose of 24 mg/kg BW and 48 mg/Kg BW (BW=Body Weight), compared to control of Naïve rats and rats with DTH-induction that were not given any treatment.

[0187] As seen in FIGS. 9 and 10A-D, a significant reduction in ear thickness and inflammatory appearance (redness and edema) as a result of the treatment with crystalline CBD solubilized in AX-1 was obtained compered to DTH-induced rats that were not treated. The anti-inflammatory effect of crystalline CBD solubilized in AX-1 is more significant than that seen for Ethanol extractions with both dose regiments. While the Naive rats showed no redness or swelling, the DTH-challenged rats that were not treated showed an inflammatory and swelling reaction. Rats treated with AX-1 showed relatively significant reduction in swelling and redness of the treated rats.

[0188] Pharmacokinetic Profile-1

[0189] The pharmacokinetics of the profile of CBD in the blood of rats after oral administration 5CS formulation was assessed in comparison to CBD dispersed in olive oil at various dosage of 10, 25, 50 mg CBD/kg body weight. 60 male rates (SD), weighing 230-250g were randomly allocated into the study groups as shown in Table 6. Rats were administered orally via gavage with test formulations.

TABLE-US-00010 TABLE 6 pharmacokinetic test design Treatment Blood Number Dose sampling Group of rats Formulation (mg/kg bw) Regimen (hr) 1 5 5CS 5 PO 0.5, 2, 4, 2 5 10 8, 12, 24 3 5 25 4 5 50 5 5 In9(6) 5 PO 0.5, 2, 4, 6 5 10 8, 12, 24 7 5 25 8 5 50 9 5 Olive oil 5 PO 0.5, 2, 4, 10 5 10 8, 12, 24 11 5 25 12 5 50

[0190] As can be seen from FIGS. 10A-10C, the CBD levels in the blood derived from formulation 5CS and In9(6) within half an hour after oral administration are by up to 16-fold higher than the levels obtained from oil dispersion. These results indicate a very fast absorption and high levels of permeation. After 4 hours the absorption of the CBD in oil reaches its maximum levels (Tmax). It can also be seen that a strong permeation is achieved with formulations of this disclosure with low level of CBD (10 mg/kg), while significantly higher dosage is required for obtaining the same level in the blood when CBD is dispersed in oil required.

[0191] Pharmacokinetic profile-2

[0192] PK assessment for additional formulations were carried out for formulations detailed in Tables 7-1 and 7-2.

TABLE-US-00011 TABLE 7-1 Additional formulations for PK assessment Formulation AX-1 Formulation AX-1(B) Component wt % Component wt % Oil Limonene 5 triacetin 5 Hydrophilic Polysorbate 80 45 Polysorbate 80 45 surfactant (Tween 80) (Tween 80) Co-surfactant Propylene glycol 45 Propylene glycol 45 (PG) (PG) Solvent Ethanol 5 — — Phospholipid — — Phosphatidylcholine 5

TABLE-US-00012 TABLE 7-2 Additional formulations for PK assessment Formulation OR103(2) slow release Formulation OR210SE Component wt % Component wt % Oil Triacetin 5 MCT 5 Hydrophilic Labrasol 25 L-1695- sucrose 60 surfactant Cremophor EL 35 mono/dilaurate castor oil* Co-surfactant Propylene glycol 20 Propylene glycol 20 (PG) (PG) Solvent Isopropyl alcohol 5 Isopropyl alcohol 5 Phospholipid Phosal 50 PG** 10 Phosal 50 PG** 10 *Polyoxyl 35 castor oil **Phosal 50 PG composed of 1.5-2.5% wt ethanol, >500 ppm ethylenemethylketone, 0.5 wt % water, 33.8-41.2 wt % propylene glycol, <50.0 wt % phosphatidylcholine, >6 wt % lyso-phosphatidylcholine

[0193] PK study in rats was carried out to measure the levels of CBD in the bloodstream after oral administration of 25 mg/kg BW (Body Weight) comparing formulations: AX-1 original to AX-1(B). The PK profile of CBD showed similar kinetics for both formulations, as can be seen in FIG. 12A. Therefore, one can replace D-limonene and EtOH by components which are less bitter and are still pharmacologically permitted for administration to improve patient compliance.

[0194] Similarly, PK study in rats was carried out to measure the levels of CBD in the bloodstream after oral administration of 25 mg/kg BW given OR210SE or OR103(2) compared to 5CS and AX-1.

[0195] As seen in FIG. 12B, OR210SE shows a better PK profile, absorbing much higher levels of CBD to the blood stream after oral administration compared to AX-1 and 5CS formulation (all were administered in their concentrated form). The Cmax after administrating OR210SE was observed at 30 min with relatively high concentration compared to AX-1 (˜900 ng/mL vs. 550 ng/mL respectively). Formulation OR103(2) exhibits a more delayed absorption of CBD with a Cmax of between 2-4 hr from administration. This formulation also shows relatively high levels of CBD reaching the bloodstream. OR103(2) and OR210SE may thus be suitable for delayed release formulations.

[0196] Stability of CBD in Stimulated Gastric Fluid (SGF)

[0197] Since oral administration of CBD is known to showed incidents of side effects that might be contributed to the degradation of CBD to THC with the exposer to gastric fluid, CBD stability was tested at simulating gastric fluid environment when solublized in AX(1) and 5CS.

[0198] Stock solutions of 3% CBD in MeOH, AX1 and 5CS were prepared. A medium of stimulating gastric fluid (SGF) was prepared by dissolving sodium chloride (0.2 w/v %) and hydrochloric acid (0.1M) in DDW, and incubated at 37° C.

[0199] For MeOH solution sodium dodecyl sulfate (1 w/v %) was added to the SGF. 500 ml of SGF mediums was contained in an appropriate Erlenmeyer flask. At time 0-1 ml of each CBD stock solution was added to the SGF. The mixture was vigorously shaken at a water bath warmed to 37° C. and immediately 1 ml of the solution was sampled and replaced with an equal volume of preheated SGF medium. Similarly, same volume was sampled at 5, 10, 15, 20, 30, 45, 60, 75, 90, 120, 150 and 180 minutes. Each 1 mL sample was immediately neutralized to a pH of 7 to 9, with 980 μL of 0.1M sodium hydroxide solution and 3 ml MeOH, and pH levels were tested. All samples were kept at 4° C. until HPLC analysis.

[0200] For MeOH solution and AX1 system additional samples were taken every 30 min and injected directly into the HPLC without further treatment. This was to determine that naturalization do not affect the profile seen. The measured CBD concentration was divided to the initial concentration (C/Co) at each time point.

[0201] FIGS. 13A-13B show the changes in CBD content as a function of time in both neutralized and non-neutralized samples. CBD in MeOH as a suspending medium showed significant degradation over time. Degradation had begun very fast, starting already after 5 minutes. Within 30 min 68% of the molecule decomposes, and after 2 hours less than 4% CBD were left. The decomposition of the CBD had resulted in 7 peaks detected using HPLC-UV analysis. 4 unknown peaks, termed ‘Unk’ and three peaks were identified as Δ.sup.8-THC, Δ.sup.9-THC and CBN. However, when CBD was loaded within both 5CS and AX1 no degradation was observed (C/Co remained 1). CBD levels were stable and constant, showing no decomposition products, even after 3 hours of measurements. The samples that were measured after neutralization or immediately after sampling showed similar results, indicating the accuracy of the method.

[0202] According to previous reports, at acidic environment CBD decomposes mainly to THC and some additional minor related cannabinoids. HPLC analysis showed a total of 7 degradation products, including Δ.sup.9-THC, Δ.sup.8-THC and CBN, detected at different times.

[0203] The trend of the descending CBD peak area is shown in FIG. 13C. Simultaneously the peaks area of related degradation products increases, even though it seems some of them also decomposed (compound “unk 2”) within time, while other starts to rise at that time point (‘unk 5’ and ‘unk 3’).

[0204] From the results, it is concluded that administration of CBD in methanol is expected to result in very fast transformation of CBD into THC due to the acidic environment, which is may lead to undesired psychoactive adverse effects. In contract, the CBD solubilized in the 5CS and In9(6) systems is well protected against transformation to THC even after 180 minutes after exposure to the acidic gastric fluid.

[0205] In comparison, the CBD profile in SGF with a commercial product (‘RSHO’™—containing CBD dissolved in vegetable oil) and CBD dissolved in pure olive oil were evaluated. RSHO profile of degradation in SGF is shown in FIG. 14A, while FIG. 14B shows the CBD in olive oil profile of degradation in SGF.

[0206] As opposed to AX-1 and 5CS in which CBD remains stable when exposed to SGF for 180min, the CBD in the commercial product or in olive oil degrades relatively fast within 30min after exposure. Thus, the formulations described herein provide a ‘protective shield’ for CBD to be absorbed directly when administrated orally into the bloodstream, and not its degradation products, such as THC or other cannabinoids.

[0207] Compounding

[0208] Lyophilization and Resuspension

[0209] CBD-loaded formulations 5CS and AX-1 were compounded for lyophilization as detailed below.

[0210] Concentrate samples of 2.5 wt % CBD-loaded AX1 and 5 wt % CBD-loaded 5CS formulations were diluted (10 times) with the following solutions:

[0211] Dextrin (10-20 w/v %)

[0212] Lactose (10-20 w/v %)

[0213] Mannitol (10-20 w/v %)

[0214] Maltodextrin (10-20 w/v %)

[0215] Erythritol (10-30 w/v %)

[0216] Sorbitol (20-70 w/v %)

[0217] The diluted samples were frozen by liquid nitrogen and lyophilized for at least 24 hr. After freeze-drying, powder of solid particles was obtained (FIG. 15A).

[0218] Next, the CBD loaded particles were re-dispersed in water (10-90% WT) to give the reconstructed microemulsion (FIG. 15B). The formulations had completely regained their original transparent homogeneous appearance, showing no phase separation or precipitation of the CBD.

[0219] In order to determine if the nano-sized droplets had retained their structure and size, reconstituted powder of 5CS diluted with mannitol was measured for its droplet size via DLS instrument, as shown in Table 8.

TABLE-US-00013 TABLE 8 Droplet size before lyophilization and after reconstitution After lyophilization and Water content Before lyophilization reconstitution (wt %) (Z-average; nm) (Z-average; nm) 70 11.6 12.01 80 10.3 10.8 90 10.2 10.4

[0220] Similar droplet size was observed seen before lyophilization (original formulation) and after reconstitution with different water ratios.

[0221] The lyophilized powder was introduced into capsules customized in their size for oral administration in rats (TROPAC CAPSULES). The CBD in the bloodstream was evaluated after oral administration compared to the liquid concentrate formulation with the same dose of 10 mg/kg BW. The PK profile of the lyophilized powder and that of the liquid concentrate formulation was similar, as shown in FIG. 16A, showing no effect of the freeze-drying of 5CS to powder, as was foreseen by the DLS results.

[0222] Moreover, the lyophilized powder and its reconstituted sample results in a similar kinetic profile and CBD amount reaching the blood stream (FIG. 16B). This result indicates that there is no effect on the performance and/or bioavailability of the CBD by hydration of the powder.

[0223] As lyophilization and reconstitution did not hinder from the properties of the formulation, it is possible to administer the formulation either in powder or in liquid form, depending on the end-user/patient preference.

[0224] Co-Solubilization with Other Active Components

[0225] Docohexanoic acid (DHA): DHA is an omega-3 fatty acid naturally found throughout the body and is most abundant in the cerebral cortex, retina and heart. Therefore, DHA is essential for the growth and functional development of the brain, showing improvements in learning ability, cognitive behavior and reduced depression. Decrease in DHA consumption is associated with cognitive decline during aging and with onset of sporadic Alzheimer disease.

[0226] In addition, DHA is known to help reduce triglycerides in the blood, decreasing thrombosis and preventing cardiac arrhythmias. Epidemiological studies have shown a strong correlation between fish consumption with high concentration of DHA and reduction in sudden death from myocardial infarction.

[0227] The opposite effects of DHA are also seen and studied with inflammation, particularly with rheumatoid arthritis (RA), and with asthma. DHA has a positive effect on diseases such as hypertension, arthritis, atherosclerosis, depression, adult-onset diabetes mellitus, myocardial infarction, thrombosis, and some cancers.

[0228] It can mainly be obtained from our diet including fish oil or algae, but has very limited bioavailability and therefore should be consumed in high levels and intensity to reach sufficient levels in the body.

[0229] Solubilizing CBD and DHA is not an easy task. Using 5CS and AX-1 formulations, a relatively high concentration of both CBD and DHA in 1:1 ratio (50mg/mL CBD and 50mg/mL DHA and higher) was achieved, resulting in a transparent, stable formulation with nano-sized droplets. This system, although “carrying” a very large amount of total active molecules was still fully dilutable. The system can solubilize any desired ratio of CBD:DHA. This dual molecule system can result in a multifunctional therapeutic effect. Moreover, the DHA added to the composition, being a poly-unsaturated long fatty acid, may act as a bioavailability enhancer improving the delivery CBD.

[0230] Curcumin: Curcumin is a small molecule that is the prototypical ‘curcuminoid’ having similar effects to other polyphenols. It is known as a very potent anti-inflammatory, anti-cancer molecule. It has also been demonstrated as a molecule that helps reduce cognitive decline associated with aging, reduce lipid and plaque levels in arteries and reduce the risk of diabetes. However, it has a very poor oral bioavailability. Combining both CBD and curcumin can have an increase effect on the reduction of inflammation and additional dual beneficial therapeutics affect. Both curcumin and CBD were successfully co-solubilize in 5CS formulation at a concentration of 60 mg/mL CBD and 50 mg/mL curcumin, and 50 mg/mL CBD and 15 mg/mL curcumin in AX-1 formulation. The resulting formulation with both active molecules are transparent with an orange appearance (curcumin coloring effect) showing no phase separation or precipitation.

[0231] Flavoring

[0232] 2.5% CBD-loaded formulations were tested for the possibility to add flavoring agents, such as mint, tea w/lemon, tropical, citrus, cranberry-pomegranate. The diluted formulations were transparent and stable after preparation.

[0233] Further, AX1 samples were prepared with monk fruit (Siraitia grosvenorii) powder and monk fruit juice and flavors (oil base and water base). Both monk fruit powder and monk fruit juice were compatible with AX1 concentrate. In case of flavors, the addition of oil-based flavors resulted in phase separation in contrast to water-based flavors which kept the samples transparent and stable.

[0234] For 5CS, samples were prepared with monk fruit powder and monk fruit juice and flavors (oil base and water base). Only monk fruit powder resulted in stable systems, however in order to completely dissolve the powder extra PG was added (10% of final product). Both water-based and oil-based flavors were compatible.

[0235] Thus, addition of flavoring and other additives does not adversely affect the formulation, allowing to mask the bitter taste in both diluted and concentrated forms.

[0236] Encapsulating into Soft Gel Capsules

[0237] To permit another form of oral administration, 5CS formulation was encapsulated in soft gel capsules. The soft gels were found to be intact after long storage without showing any leakage or damage to the coating, resulting in no weight loss or humidity in the bottle.