Several liposomal cannabinoids and uses thereof

Abstract

The present application relates to a number of liposomal cannabinoids and uses thereof. A prolonged release formulation of several cannabinoids is provided. In one embodiment, the formulations comprise a plurality of liposomes having a lipid membrane and an in-liposome aqueous core wherein the liposomes comprise an embedded cannabinoid and at least one dispersant of the cannabinoid, the dispersant being not a cyclodextrin (CD) compound; or the liposome comprises an embedded cannabinoid, at least a portion of the cannabinoid being embedded within the lipid membrane, and wherein the lipid membrane comprises a molar ratio between the cannabinoid and the one or more liposome-forming lipids, the molar ratio ranging from 1 to 10. Several methods of preparation and several uses of the several formulations for prolonging delivery of the several cannabinoids and several methods of treatment using the same are also disclosed.

Description

[0001] This application is a divisional application of application number CN 202080078712.9 (PCT application number is PCT / IL2020 / 051068), application date October 1, 2020, and invention name “Several liposomal cannabinoids and their uses”. Technical Field

[0002] The present disclosure relates to several liposomal cannabinoids. Background Art

[0003] References believed to be relevant to the background of the presently disclosed subject matter are listed below:

[0004] International patent application publication number WO2017203529;

[0005] International patent application publication number WO2001003668;

[0006] U.S. Patent Application Publication No. US20170044092;

[0007] International patent application publication number WO2018145213;

[0008] U.S. Patent Application Publication No. US20180193399;

[0009] U.S. Patent No. US 9,095,555;

[0010] U.S. Patent No. US1,011,7883;

[0011] U.S. Patent Application Publication No. US20170281701;

[0012] U.S. Patent Application Publication No. US20180318237;

[0013] U.S. Patent Application Publication No. US20180271924A1;

[0014] U.S. Patent Application Publication No. US20170107280;

[0015] International patent application publication number WO2017191630;

[0016] U.S. Patent Application Publication No. US20180303791;

[0017] U.S. Patent Application Publication No. US20180042845;

[0018] U.S. Patent Application Publication No. US20180185324;

[0019] U.S. Patent Application Publication No. US20180289665;

[0020] U.S. Patent No. US9655910;

[0021] U.S. Patent No. US8242178;

[0022] U.S. patent application publication number US20180221304.

[0023] The citation of the above references herein should not be construed as meaning that these references are in any way relevant to the patentability of the presently disclosed subject matter.

[0024] Few publications describe the use of cannabidiol (CBD) associated with various liposomes, for example: WO2017203529 describes several compositions comprising a combination of cannabidiol (CBD) or a derivative thereof and hyaluronic acid or a salt thereof; a phospholipid, and optionally a physiologically acceptable carrier. The cannabidiol can be incorporated into several liposomes formed by the several phospholipids. The composition is described for the treatment of inflammatory joint diseases, or pain or inflammation associated with such diseases. The composition is formulated for local injection.

[0025] Other publications describing liposomal cannabidiol include WO2001003668, describing pulmonary delivery of liposome-entrapped cannabinoids; US20180318237 describing topical administration of cannabinoids, which may be in the liposomes; WO2017191630 describing the use of cannabidiol (possibly in liposomes) for reducing a steroid dose and treating inflammatory and autoimmune diseases; US20180303791, US20180042845, and US2018018532 4 describes the use of cannabinoids for treating multiple myeloma, wherein the cannabinoids may be in liposomes; US9655910 describes the use of cannabinoids (possibly in liposomes) for treating addiction; US8242178 describes the use of cannabidiol (possibly in liposomes) for treating autoimmune hepatitis; and US20180221304 describes complex mixtures containing cannabinoids for treating mast cell-related or basophil-mediated inflammatory diseases, wherein liposomes are suggested as a means of local delivery. Summary of the invention

[0026] The present disclosure provides a prolonged release formulation, which comprises a plurality of liposomes, wherein the plurality of liposomes have a lipid membrane and an aqueous core within the liposome, wherein the liposome comprises one or more liposome-forming lipids, an embedded cannabinoid (e.g., cannabidiol (CBD) or a functional homologue thereof and at least one dispersant of a cannabinoid (e.g., PG, HSA, IVIg), and the at least one dispersant is not a cyclodextrin (CD) compound. The core within the liposome may also include cyclodextrin (CD).

[0027] The present disclosure also provides an extended release formulation comprising a plurality of liposomes having a lipid membrane and an intraliposomal aqueous core, the lipid membrane comprising one or more liposome-forming lipids, wherein the liposomes comprise an embedded cannabinoid, at least a portion of the cannabinoid being embedded in the lipid membrane, and wherein the lipid membrane comprises a molar ratio between the cannabinoid and the one or more liposome-forming lipids, the molar ratio being in the range of 1 to 10.

[0028] The present disclosure also provides a method of treatment, comprising the following steps: administering a therapeutically effective amount of an extended release formulation to a subject in need thereof, wherein the extended release formulation comprises a plurality of liposomes, wherein the plurality of liposomes have a lipid membrane and an intraliposomal aqueous core, wherein the liposomes comprise one or more liposome-forming lipids, an encapsulated cannabinoid compound and at least one dispersant for the cannabinoid, wherein the at least one dispersant is not a cyclodextrin (CD) compound.

[0029] In addition, the present disclosure provides a method of treatment, comprising the steps of administering to a subject in need of treatment an extended release formulation comprising a plurality of liposomes, the plurality of liposomes having a lipid membrane and an intraliposomal aqueous core, the lipid membrane comprising one or more liposome-forming lipids, wherein the liposomes comprise an embedded cannabinoid, at least a portion of the cannabinoid being embedded in the lipid membrane, and wherein the lipid membrane comprises a molar ratio between the cannabinoid and the one or more liposome-forming lipids, the molar ratio being in the range of 1 to 10.

[0030] In some embodiments, the formulation further comprises an embedded cyclodextrin (CD) compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to better understand the subject matter disclosed herein and to illustrate how it can be implemented in practice, several embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

[0032] Figures 1A to 1Care several microscopic images (Zeiss SN 221209, x200 magnification) of various cannabidiol formulations including liposomal cannabidiol (F1, Figure 1A )、Liposomal cannabidiol-HSA 50mg / ml; ( Figure 1B ), Liposomal cannabidiol-HSA 100mg / ml( Figure 1C ).

[0033] FIG. 2A to FIG. 2B Several graphs showing plasma cannabidiol concentrations (ng / ml) following IV administration of a 12 mg / kg dose with various IM formulations (n=3, mean ± SD, no outliers from two independent in vivo studies).

[0034] FIG. 3A to FIG. 3B are several graphs showing the absolute cannabidiol (mg) released in the muscle after intramuscular injection of different formulations (n=3, mean±SD, no outliers) ( Figure 3A ) and the percentage of cannabidiol released from the muscle after intramuscular injection of different preparations (n=3, mean±SD, no abnormal values) ( Figure 3B ).

[0035] Figure 4 is a microscopic image of DMPC:DPPC-cannabidiol liposomes (Zeiss SN 221209, x200 magnification). DETAILED DESCRIPTION

[0036] The present disclosure is based on the unexpected discovery that the presence of cannabidiol embedded in a liposome bilayer affects (reduces / slows down) the release rate of cannabidiol from the liposome, thereby allowing a prolonged delivery of cannabidiol from the liposome formulation. The present invention also discovered that the presence of a dispersant (capable of uniformly dispersing cannabidiol) in the intraliposomal aqueous environment (with or without the presence of cannabidiol in the intraliposomal aqueous environment) can also reduce the release rate of cannabidiol from the liposome.

[0037] Therefore, according to a first aspect, the present disclosure provides a prolonged release formulation comprising a liposome having a lipid membrane and an intraliposomal aqueous core, wherein the liposome comprises one or more liposome-forming lipids, an embedded cannabinoid compound and at least one cannabinoid dispersant, and the at least one cannabinoid dispersant is not (i.e. is not) a cyclodextrin (CD) compound.

[0038] According to a second aspect, the present disclosure provides an extended release formulation, comprising a liposome, the liposome having a lipid membrane and an intraliposomal aqueous core, the lipid membrane comprising one or more liposome-forming lipids, wherein the liposome comprises an embedded cannabinoid or a functional homologue thereof, at least a portion of the cannabinoid being embedded in the lipid membrane, and wherein the lipid membrane comprises a molar ratio between the cannabinoid and the one or more liposome-forming lipids, the molar ratio being in the range of 1 to 10.

[0039] The present disclosure also provides a method of treating a condition requiring prolonged delivery of a cannabinoid compound using the above-described extended release liposomal formulation, the method thus comprising administering the extended release formulation to the subject.

[0040] The several formulations disclosed herein comprise at least one cannabinoid. In the context of the present disclosure, when referring to a cannabinoid, it should be understood to include a single compound or a combination of several cannabinoid compounds (i.e., the term as used herein includes a single or several such compounds). In some embodiments, the combination of several cannabinoids comprises several components of the plant extract, i.e., a plurality of cannabinoids and optionally several plant flavonoids and several terpenoids.

[0041] In some examples, the cannabinoid is or comprises cannabidiol (CBD).

[0042] In some other examples, the cannabinoid is or comprises tetrahydrocannabinol (THC) (Delta9-THC and / or Delta8-THC).

[0043] Other cannabinoids that fall within the scope of the present disclosure include cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerol monomethyl ether (CBGM), cannabinoids (CBC), cannabinone (CBCN), cannabichromene acid (CBCA), cannabichromene (CBCV), cannabichromene acid (CBCVA), isotetrahydrocannabinol (iso-THC), cannabinol (CBN), cannabigerolic acid (CBNA), cannabinol methyl ether (CBNM), cannabinol C4 (CBN-C4), cannabinol C2 (CBN-C2), cannabinol C1 (CBN-C1), cannabichromene (CBND), cannabichromene (CBE), cannabichromene A (CBEA), and cannabinoids (CBEA). -A), cannabidiol B (CBEA-B), cannabicycline (CBL), cannabicyclic acid (CBLA), cannabicycline (CBLV), cannatriol (CBT), cannabitriol (CBTV), ethoxycannabitriol (CBTVE), cannabidiol (CBV), cannabidiol (CBVD), tetrahydrocannabinol (THCV), cannabigerol (CBDV), cannabidiol (CBGV), cannabigerolic acid (CBGVA), cannafuran (CBF), dehydrocannabidiol (DCBF), cannabinol (CBR), each of which constitutes a separate embodiment of the present disclosure.

[0044] In some embodiments, the cannabinoid is cannabidiol or a combination comprising cannabidiol and any one or more of the cannabinoids listed above.

[0045] In some preferred embodiments, the cannabinoid in the formulation is cannabidiol.

[0046] In the context of the present disclosure, the term cannabidiol compounds includes cannabidiol and its functional homologues. When referring to a cannabidiol functional homologue, it should be understood as a compound with similar physicochemical properties to cannabidiol.

[0047] In some embodiments, the functional homologue of cannabidiol is a chemical analogue of cannabidiol comprising at least one benzene ring and a logP greater than 4.

[0048] In some examples, a functional homologue of cannabidiol includes a structural homologue (including isomers) of cannabidiol, which is similar to cannabidiol and lacks the psychoactivity of tetrahydrocannabinol (THC).

[0049] In some embodiments, the cannabidiol compound is a natural phytocannabinoid.

[0050] In some embodiments, the cannabidiol compound is a synthetic cannabidiol homolog.

[0051] Several non-limiting examples of several cannabidiol compounds include 2-[(1R,6R)-6-Isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzo-1,3-diol (Cannabidiol, CBD), synthetic cannabidiol-dimethylheptyl (CBD-DNH), phytoCANNABINOIDs Cannabidivarin (CBDV), cannabidivarinolic acid (CBDVA), cannabidiol monomethyl ether (CBDM) [Paula Morales, Patricia H. Reggio and Nadine Jagerovic "An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Cannabidiol" Front Pharmacol." 8:422, (2017)].

[0052] In some embodiments, the active ingredient is cannabidiol, whose chemical name is 2-[(1R,6R)-6-Isopropenyl-3-methylcyclohexyl-2-en-1-yl]-5-pentylbenzo-1,3-diol.

[0053] The cannabinoid (preferably cannabidiol compound) is embedded in / combined with the several liposomes. In the context of the present disclosure, when referring to the embedding of the compound in the liposome, it should be understood as defining any form of physical or chemical binding between the cannabinoid and the liposome itself. However, it should be clear that only due to the physical binding of the several phospholipids in the form of liposomes, there is no chemical binding between the cannabinoid and the phospholipid itself. Can be by embedding cannabinoid in the aqueous core / medium within the liposome, and / or at least partially embedded in the lipid membrane (for example, due to the hydrophobicity of the cannabinoid), and / or associated with the outer surface of the liposome (for example, by several physical forces).

[0054] The amount of cannabinoid entrapped in the liposomes can be determined using commercial chromatography techniques. In some examples, the concentration of cannabinoids is determined using a high performance liquid chromatography (HPLC) / UV method.

[0055] In some embodiments, the cannabinoid embedded in the lipid membrane is determined by methods known in the art. For example, but not limited to, the ratio between the one or more liposome-forming lipids and the cannabidiol in the lipid membrane can be determined by differential scanning calorimetry (DSC).

[0056] To calculate the intralipidic concentration of cannabinoids, the aqueous intralipidic entrapment volume is also required and can be calculated as previously described [Bangham AD, et al., (1965) J MoI Biol. 13(1):238-52].

[0057] In some embodiments, the amount of cannabinoids and preferably cannabidiol compounds entrapped by the liposomes is at least 30 mg / ml; sometimes, at least 40 mg / ml; sometimes, at least 50 mg / ml; sometimes, at least 60 mg / ml; sometimes, at least 70 mg / ml; sometimes, at least 80 mg / ml; sometimes, at least 90 mg / ml; sometimes, at least 100 mg / ml; sometimes, at least 110 mg / ml; sometimes, at least 120 mg / ml; sometimes, at least 130 mg / ml; sometimes, at least 140 mg / ml; sometimes, at least 150 mg / ml; sometimes, at least 160 mg / ml; sometimes, at least 170 mg / ml; sometimes, at least 180 mg / ml; sometimes, at least 190 mg / ml; or even at least 20 mg / ml.

[0058] In some embodiments, the amount of the cannabinoid and preferably the cannabidiol compound entrapped by the liposome is at most 400 mg / ml; sometimes, at most 350 mg / ml; sometimes, at most 330 mg / ml; sometimes, at most 310 mg / ml; sometimes, at most 300 mg / ml; sometimes, at most 280 mg / ml; sometimes, at most 260 mg / ml; sometimes, at most 240 mg / ml; sometimes, at most 220 mg / ml; sometimes, at most 200 mg / ml; sometimes, at most 190 mg / ml; sometimes, at most 180 mg / ml; sometimes, at most 170 mg / ml; sometimes, at most 160 mg / ml; sometimes, at most 150 mg / ml; sometimes, at most 140 mg / ml; sometimes, at most 130 mg / ml; sometimes, at most 120 mg / ml.

[0059] In some embodiments, the amount of cannabinoids and preferably cannabidiol compounds entrapped by the liposomes is in the range of 30 mg / ml to 400 mg / ml, sometimes in the range of 30 mg / ml to 350 mg / ml; sometimes, in the range of 30 mg / ml to 350 mg / ml; sometimes, in the range of 30 mg / ml to 350 mg / ml; sometimes, in the range of 30 mg / ml to 350 mg / ml; sometimes, in the range of 30 mg / ml to 350 mg / ml; sometimes, in the range of 30 mg / ml to 200 mg / ml; sometimes, in the range of 50 mg / ml to 250 mg / ml; sometimes, in the range of 40 mg / ml to 180 mg / ml; sometimes, in the range of 40 mg / ml to 250 mg / ml; sometimes, in the range of 30 mg / ml to 120 mg / ml; sometimes, in the range of 40 mg / ml to 150 mg / ml; sometimes, in the range of 50 mg / ml to 300 mg / ml; or in any range within the upper and lower limits of the concentration determined above.

[0060] In some embodiments, the molar ratio of cannabinoid to lipid is determined.

[0061] In some embodiments, the cannabinoid compound / lipid molar ratio is between 1-10, sometimes between 1-9, sometimes between 1-8, sometimes between 1-7, sometimes between 1-6, and sometimes between 1-5.

[0062] A unique feature of the present disclosure is the presence of a cannabinoid, and preferably a cannabidiol compound, in combination with at least one cannabinoid dispersant that is not a cyclodextrin (CD) in the intraliposomal compartment. This is unique because the solubility of cannabinoids is very low, such as the solubility of cannabidiol in aqueous solution (the predicted log P of cannabidiol is 7.03), which is achieved using different dispersants. Without being bound by theory, it is believed that the dispersant (i.e., not a cyclodextrin compound, but can be combined with a cyclodextrin compound) when present in the liposomes in the intraliposomal aqueous core maintains an amount of the plurality of cannabinoids in a dissolved or uniformly dispersed form, thereby improving the sustainability of the cannabinoids within the liposomes.

[0063] In the context of the present disclosure, the term "cannabinoid dispersant" is understood to include any chemical entity that promotes or enhances the dispersibility of the cannabinoid(s) (one or more combinations of cannabinoids) in the liquid medium used to load the cannabinoids into the liposomes (preferably, but not exclusively, by passive loading). Without being bound by theory, the cannabinoid dispersant physically binds to the cannabinoids so as to be embedded in the liposomes in the form of a non-covalent complex.

[0064] In some examples, the dispersant is a solubilizer (also referred to as a solubilization enhancing agent). When referring to a solubilizer, it should be understood to include at least one non-cyclodextrin compound. Therefore, in the context of the present disclosure, the term "non-cyclodextrin solubilizer" should be understood as any solubilizing compound that is not a cyclodextrin, but it can be combined with a cyclodextrin as an additional solubilizing compound.

[0065] Several solubilizing agents are known to be useful for increasing drug solubility, particularly when using insoluble drugs or drugs with low solubility. In some examples of the present disclosure, cannabinoids are added to both the lipid phase and the aqueous phase, and it is therefore believed that the cannabinoid compound is distributed between the lipid phase (lipid membrane) and the aqueous liposome internal phase, thereby providing several pools of two different active ingredients (i.e., cannabinoids, such as cannabidiol compounds).

[0066] In other words, without being bound by theory, it is believed that the dispersant maintains the cannabinoid in the aqueous medium within the liposomes and promotes the controlled (specifically, prolonged, e.g., even up to 3 weeks) release of the active ingredient (e.g., cannabidiol compound, from the liposomes).

[0067] There are different types of solubilizers. Solubilizers can be co-solvents, i.e. substances added in small amounts to the main solvent (organic solvent or water) to increase / improve the solubility of poorly soluble compounds; for example but not limited to polyethylene glycols (PEG), such as PEG300, PEG400, propylene glycol (PG), N,N-dimethylacetamide (DMA), ethanol; or they can be considered surfactants, such as but not limited to Tween80 (Polyoxyethylene (20) sorbitan monooleate), Cremophor (propane-1,2,3-triol:oxirane (1:1)), or they can be considered as complexing agents, such as members of the cyclodextrin family of compounds.

[0068] In some embodiments, the solubilizing agent is a co-solvent. A preferred co-solvent is polyethylene glycol (PEG). Another preferred co-solvent is propylene glycol (PG).

[0069] The dispersant may not be a solubilizing agent. In some embodiments, the dispersant is a protein selected based on its ability to disperse the cannabinoid, preferably cannabidiol, in the aqueous medium in which it is dissolved.

[0070] In some embodiments, the dispersed protein is a serum protein.

[0071] In some examples, the serum protein is albumin.

[0072] In some examples, the serum protein is human serum albumin (HSA).

[0073] In some embodiments, the serum protein is a globulin.

[0074] In some embodiments, the serum protein is an immunoglobulin.

[0075] The dispersant and the cannabinoid are bound to each other via non-covalent bonds. In some embodiments, the dispersant and the cannabinoid form a physical complex that allows the cannabinoid to be released from the dispersant under appropriate conditions. Thus, in some examples, the dispersant and the cannabinoid are non-covalently bound to each other.

[0076] In some examples, the formulation includes a combination of two or more dispersing agents.

[0077] In some examples, the combination of two or more dispersants includes at least one cyclodextrin (CD) compound.

[0078] In some examples, the combination of dispersants includes two or more such compounds, none of which is a cyclodextrin compound.

[0079] As mentioned above, the plurality of liposomes may also include a cyclodextrin compound. Cyclodextrin compounds are considered to be cyclic oligosaccharides composed of a plurality of (α-1,4)-linked α-D-glucopyranose units having a lipophilic central cavity and a hydrophilic outer surface. In the context of the present disclosure, the cyclodextrin may be a naturally occurring cyclodextrin, as well as derivatives of naturally occurring cyclodextrins. Natural cyclodextrins include α-, β- or γ-cyclodextrins (αCD, βCD or γCD) composed of six, seven and eight pyranose units, respectively. When referring to derivatives of natural cyclodextrins (also included under the general term "cyclodextrin compounds"), it should be understood as any cyclic oligosaccharide composed of (α-1,4)-linked α-D-glucopyranose units having a lipophilic central cavity and a hydrophilic outer surface.

[0080] In some examples, the cyclodextrin compound is 2-Hydroxypropyl-β-cyclodextrin (HPβCD).

[0081] In some examples, the cyclodextrin compound is 2-hydroxypropyl-γ-cyclodextrin (HPγCD).

[0082] In some examples, the cyclodextrin compound is solfobutyl ether cyclodextrin (SBE).

[0083] In a preferred example, the cyclodextrin is HPβCD or HPCD for short.

[0084] The formulation also includes a plurality of liposomes.

[0085] The liposomes are prepared a priori, and the liposomes have at least one liposome-forming lipid. In the context of the present invention, the term "liposome forming lipids" mainly refers to glycerophospholipids or sphingomyelins that form vesicles (such as but not limited to liposomes) in water, as further discussed below.

[0086] When referring to glycerophospholipids, it is understood that lipids have a glycerol backbone in which at least one, preferably both, hydroxyl groups in the head group are substituted by one or both of an acyl group, an alkyl or alkenyl chain, a phosphate group, or any combination thereof, and / or derivatives thereof, and may contain a chemically reactive group at the head group (e.g., an amine, acid, ester, aldehyde, or alcohol) to provide the lipid with a polar head group. The sphingomyelins are composed of a ceramide unit having a phosphorylcholine moiety attached to position 1, and are therefore in fact N-acylsphingosine. The phosphorylcholine moiety in sphingomyelin constitutes the polar head group of sphingomyelin.

[0087] In some other examples, the lipid forming the liposome is di-lauroyl-sn-glycero-2phosphocholine (DLPC). In some examples, the lipid forming the liposome is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some examples, the lipid forming the liposome is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some examples, the lipid forming the liposome is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some examples, the lipid forming the liposome is 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine. In some examples, the lipid forming the liposome is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some examples, the lipid forming the liposome is 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine. In some examples, the lipid forming the liposome is 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC). In some embodiments, the lipid forming the liposome is 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine. In some embodiments, the lipid forming the liposome is 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-ditricosanoyl-sn-glycero-3-phosphocholine.In some examples, the lipid forming the liposome is 1,2-dilignoceroyl-sn-glycero-3-phosphocholine. In some examples, the lipid forming the liposome is 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine. In some examples, the lipid forming the liposome is 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC). In some examples, the lipid forming the liposome is 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). In some examples, the liposome-forming lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or di-lauroyl-sn-glycero-2phosphocholine (DLPC).

[0088] In some examples, the liposome-forming lipids comprise at least hydrogenated soy phosphatidylcholine (HSPC).

[0089] In a preferred example, the liposome-forming lipid comprises or consists of hydrogenated soybean phosphatidylcholine (HSPC).

[0090] In some examples, the liposome-forming lipid comprises at least 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).

[0091] In some examples, the liposome-forming lipid comprises at least 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

[0092] In a preferred example, the liposome-forming lipid comprises or consists of a combination of DMPC and DPPC. In some examples, the DMPC:DPPC molar ratio of the two liposome-forming lipids is about 45:55. Without limitation, formulations containing DMPC:DPPC are suitable for non-human (veterinary) use.

[0093] In some embodiments, the liposomes comprise a sterol, such as cholesterol.

[0094] In some embodiments, when cholesterol is present in the liposome, it is in an amount not exceeding 4 mol %.

[0095] In some additional or other embodiments, the liposome comprises a lipid polymer, such as a lipid derivatized with polyethylene glycol (PEGylated lipid).

[0096] The plurality of liposomes can be of any shape or size.

[0097] In some examples, the plurality of liposomes are a plurality of multilamellar vesicles or a plurality of oligolamellar vesicles.

[0098] In some embodiments, the plurality of liposomes are a plurality of multilamellar vesicles.

[0099] In some other examples, the plurality of liposomes are a plurality of unilamellar vesicles, preferably a plurality of large unilamellar vesicles.

[0100] The plurality of liposomes may be small, medium, large or even giant. When referring to small liposomes, it is understood that they have an average size in the range of about 20nm-100nm; when referring to medium-sized liposomes, it is understood that they have an average size in the range of about 100nm-200nm; when referring to large liposomes, it is understood that the average size is greater than about 200nm; when referring to giant liposomes (usually giant single-layer or multi-layer vesicles), it is understood that it refers to liposomes greater than 1mm.

[0101] In some examples, the plurality of liposomes are a plurality of multilamellar vesicles (MLVs). In some examples, the multilamellar vesicles have a size distribution with a minimum size of equal to or greater than 100 nm.

[0102] In some examples, the formulation comprising the plurality of liposomes is in a dry form. Specifically, although not exclusively, the plurality of liposomes are lyophilized.

[0103] In some other examples, the formulation comprises the plurality of liposomes maintained in a medium, referred to herein as an "external medium". The external medium can be any composition suitable for accommodating the plurality of liposomes therein. In some examples, the external medium is a medium suitable for storage of the plurality of liposomes, while in some other examples, the external medium is a medium suitable for administration of the plurality of liposomes, such as a physiologically acceptable carrier.

[0104] In some examples, generally when the external medium is one suitable for administration, the external medium may include a cannabinoid. The cannabinoid may be the same as or different from the cannabinoid encapsulated by the liposomes.

[0105] The combination of the cannabinoid and the dispersant allows the formation of the extended release formulation, preferably in a controlled manner. In the context of the present disclosure, when reference is made to "controlled release" or "extended release", it should be understood to mean a controlled release over a period of time. The period of time includes at least a few days, sometimes at least 3 days; sometimes, at least 4 days; sometimes, at least 5 days; sometimes, at least 6 days; sometimes, at least 7 days; sometimes, at least 8 days; sometimes, at least 9 days; sometimes, at least 10 days; sometimes, at least 11 days; sometimes, at least 12 days; sometimes, at least 13 days; sometimes, at least 14 days; sometimes, at least 15 days; sometimes, at least 16 days; sometimes, at least 17 days; sometimes, at least 18 days; sometimes, at least 19 days; sometimes, at least 20 days; sometimes, at least 21 days, and even more than 30 days. The term "extended release" includes any form of controlled release other than immediate release (e.g., more than 50% release within the first 24 hours), and includes extended / extended release and / or delayed release. The extended release can be determined by an in vitro release assay as described in Example 3. Release of ≤70%, sometimes ≤60%, sometimes ≤50% after 2 hours of incubation with 50% serum can be considered as extended release.

[0106] The present disclosure also provides a physiologically acceptable carrier in the formulation suitable for administration by injection or infusion.

[0107] In the context of the present invention, a physiologically acceptable carrier refers to any carrier that can be used to prepare a pharmaceutical preparation, which is generally safe, non-toxic and biologically or otherwise undesirable. In some examples, the physiologically acceptable carrier is a water-based solution suitable for administration by injection. In some examples, the physiologically acceptable carrier suitable for systemic administration includes aqueous and non-aqueous isotonic sterile injection / infusion solutions, which may contain antioxidants, buffers, antibacterial agents, and the intended recipient of the solute that makes the preparation isotonic with blood. In some examples, the carrier is any one or combination of saline, buffered solution, sugar solution (glucose, sucrose, etc.), etc. In some examples, the carrier may also include a thickener, a stabilizer, and a preservative.

[0108] In some embodiments, administration is by intramuscular (im), intra-peritoneal (ip), intravenous (iv), and subcutaneous (sc) injection.

[0109] In a preferred embodiment, the liposomal formulation is for intramuscular injection. Intramuscular injection exhibits the advantage of a prolonged / extended release profile compared to intravenous injection of non-liposomal cannabinoid formulations.

[0110] In some examples, administration is performed to a mammalian subject.

[0111] In some examples, administration is performed to a human subject.

[0112] In some other embodiments, administration is to a non-human (ie, veterinary) subject.

[0113] The amount of the plurality of cannabinoid compounds in the plurality of liposomes is designed to be sufficient to provide a therapeutic effect when the formulation is administered to a subject.

[0114] An amount sufficient or effective to achieve a therapeutic effect upon administration is understood to include at least one therapeutic effect known to be achieved by or associated with the several cannabinoid compounds, especially cannabidiol.

[0115] Without limitation, the therapeutic effect may be any one or combination of treating / improving / alleviating pain and / or inflammation, as well as any other therapeutic effect known to be associated with the administration of specific cannabinoid compounds (particularly cannabidiol).

[0116] The amount of cannabinoid delivered by the disclosed liposomal formulations depends on various parameters known to those skilled in the art and can be determined based on appropriately designed clinical trials (dose range studies), and those skilled in the art will know how to appropriately conduct such trials to determine the effective amount. The dosage depends primarily on the type and severity of the disease to be treated, as well as the treatment regimen (administration method), the sex and / or age and / or weight of the subject to be treated, etc.

[0117] The liposomes in the formulation and the formulations themselves can be characterized by any technique or parameter known in the art of liposome formulations. This includes, but is not limited to, liposome size and / or size distribution (e.g., using dynamic light scattering (DLS)), polydispersity index (PDI), zeta potential, pH of the dispersion using a pH meter, etc.

[0118] The present disclosure also provides a method of administering the plurality of cannabinoids to treat a subject, the method comprising the step of administering the liposome formulation disclosed herein to a subject in need of such treatment.

[0119] In view of the above, in the context of the present disclosure, when referring to treatment performed by the formulation or several liposomes disclosed herein, it should be understood to include ameliorating undesirable symptoms associated with the disease, preventing their manifestation before these symptoms appear, slowing the progression of the disease, slowing the worsening of symptoms, enhancing the onset of remission of the disease, slowing the irreversible damage caused by the progressive chronic stage of the disease, delaying the onset of the progressive stage, reducing the severity or curing the disease, improving survival or recovering more quickly from the disease, preventing the disease from occurring, or a combination of two or more of the above.

[0120] As used herein, the forms "a", "an" and "the" include singular and plural references unless the context clearly dictates otherwise. For example, the term "cannabinoid" includes one or more cannabinoids.

[0121] Furthermore, as used herein, the term "comprising" is intended to indicate that the liposomes include the cannabinoid and the dispensing agent, but do not exclude other ingredients, such as physiologically acceptable carriers and excipients and other agents. The term "consisting essentially of" is used to define, for example, a number of liposomes that include the listed elements, but do not include other elements that may be important for the delivery of the cannabinoid. "Consisting of" means excluding trace elements of the above other elements. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0122] In addition, all numerical values, for example, when referring to the amount or range of the several elements constituting the several liposomes and the formulations containing the said components, are approximate values ​​that vary (+) or (-) up to 20%, sometimes up to 10% from the stated numerical values. It should be understood that all numerical names are preceded by the term "about", even if not always explicitly stated.

[0123] The present invention will now be described by several non-limiting examples implemented according to the present invention. It should be understood that these examples are intended to be illustrative and non-restrictive in nature. Obviously, many modifications and variations of these examples are possible according to the above teachings. Therefore, it should be understood that within the scope of the appended claims, the present invention can be implemented in a manner different from the specific description below in a variety of possible ways.

[0124] Several examples illustrate

[0125] Example 1 - Several cannabidiol liposome formulations

[0126] Liposome preparation and characterization

[0127] Material

[0128] Hydroxypropyl-β-cyclodextrin (HPCD) was obtained from roquette

[0129] Hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Lipoid GmbH (Ludwigshafen, Germany).

[0130] Cannabidiol (CBD) - obtained from THC Pharm (Batch: CBDAPI1802)

[0131] Anhydrous ethanol-purchased from Merck

[0132] Solubilizers: polyethylene glycol (PEG) 300, propylene glycol (PG), Tween 80 and dimethylacetamide (DMA) were purchased from Merck, and Cremophor was obtained from Sigma.

[0133] method:

[0134] Liposome preparation

[0135] Different types of phosphatidylcholines with different acyl chain compositions were used and tested. These included HSPC (mainly stearoyl, C18), DPPC dipalmitoyl, C16 DMPC (di-myristoyl, C14) and DOPC (di-oleoyl, C18:1) without or with cholesterol (5% or 10%).

[0136] Since several of the formulations containing DOPC produced less favorable liposomes, these were excluded from further studies.

[0137] The different liposomes prepared are shown in Table 1.

[0138] Formulation F1 was prepared as follows: HSPC (S PC-3, lipid, batch: 525600-2180662-01 / 042) and cannabidiol (batch: CBDAPI1802) were weighed in a vial. Anhydrous ethanol (Merck) was added and the vial was placed in a water bath at 65°C until the solution was clear. Then, one milliliter of an isotonic aqueous solution (e.g., 5% glucose) was placed in the water bath. Once the lipid phase became clear, it was added to warm water at 65°C while stirring at 65°C for 30 minutes.

[0139] Several formulations containing cannabidiol in the aqueous phase were prepared using a lipid phase similar to that prepared for F1. The aqueous phase was prepared by mixing all components of a particular aqueous phase and then adding a concentrated cannabidiol ethanol solution (700 mg / ml). The ethanol solution was slowly added under stirring and then heated for a short time in some cases. Once the aqueous phase was clear or well dispersed, the lipid phase was slowly added at 65°C and stirring was continued at 65°C for 30 minutes.

[0140] Release assay

[0141] The release of cannabidiol from the liposomes was determined in the presence of 25% bovine serum and 25% sucrose at time zero, 1 hour (in some cases), and 24 hours after incubation at 37° C. At each time point, total cannabidiol and free cannabidiol were determined as described below.

[0142] Total cannabidiol determination

[0143] The liposomal cannabidiol was diluted 20-fold in 25% serum and 25% sucrose, which was further diluted in methanol and analyzed by HPLC under conditions known in the art to detect cannabidiol by HPLC.

[0144] Free cannabidiol detection

[0145] Liposomal cannabidiol was diluted 20 times in 25% serum and 25% sucrose. The dilution was centrifuged and the liposomes floated on the top of the clear phase. The lower clear phase (free cannabidiol) was diluted with methanol and analyzed by HPLC.

[0146] Lipid concentration

[0147] Lipid concentrations were determined by a modified Bartlett method and in some cases by HPLC with an evaporative light scattering (ELSD) detector.

[0148] result

[0149] Several preparations contained cholesterol in the lipid phase.

[0150] Several cannabidiol preparations containing cholesterol in the lipid phase were prepared. Cannabidiol was dissolved in the lipid phase. The lipid phase in all test formulations was 125 mg / ml (including cannabidiol), and cannabidiol was 70% mole of lipid phase content. The formulation contained HSPC and DMPC, did not contain cholesterol, and contained 5% and 10% mole cholesterol. These preparations contained cannabidiol only in the lipid phase (not in the aqueous phase) and are described in Table 1.

[0151] The results showed that the release curve of HSPC was slightly slower than that of DMPC liposomes. Cholesterol increased the release of cannabidiol from liposomes, and this effect was more pronounced for DMPC.

[0152] Therefore, other formulations were prepared with HSPC in the lipid phase (125 mg / ml lipid phase, 70 molar % cannabidiol) and different aqueous phase compositions that allowed for the dissolution or dispersion of cannabidiol in the aqueous phase.

[0153] Table 2 shows two cannabidiol formulations in which cannabidiol is in an aqueous phase containing only HPCD. These formulations produced similar loadings (in terms of D / L ratio) and release profiles (as described in Table 1) to the F1 formulation. This may be the result of the ability of the several law cannabidiol concentrations (law CBD concentrations) to be loaded into an aqueous phase containing only HPCD (8 mg / ml).

[0154] Table 3 describes cannabidiol formulations with HPCD and several surfactants (cremphor EL and Tween 80) in the aqueous phase, allowing 21 mg / ml cannabidiol to be dispersed. These formulations do not result in higher D / L ratios, and their release profiles cannot be determined because they result in a large number of small liposomes that are not separated by our release method.

[0155] Table 4 shows the formula with HPCD and 25% PEG 300 in aqueous phase, allowing 14mg / ml cannabidiol to disperse. Wherein the HPCD content of two kinds of formulas (A39 and A42) is different, causing D / L ratio to be significantly higher, and release curve is slower. These preparations are compared with the liposome with the same aqueous composition without cannabidiol in aqueous phase, causing D / L ratio to be much lower, and release curve is faster. It has been proved that containing a large amount of cannabidiol in aqueous reservoir can slow down release rate.

[0156] Table 5 describes several cannabidiol formulations with HPCD and 10-15% PG in the aqueous phase, allowing 21 mg / ml cannabidiol to be dispersed. For each formulation, a control formulation with the same aqueous phase composition but without cannabidiol was also prepared. The results show that in all cases, the release rate of the formulation containing cannabidiol in the aqueous phase was slower, and the release was more obvious when the D / L ratio was significantly higher than the control.

[0157] Without being limited thereto, it appears from the data presented herein that the addition of cannabidiol to the aqueous phase, either by the addition of a co-solvent (dispersant) or a surfactant, helps to delay the release of cannabidiol from the liposomes.

[0158] In addition, but not limited to this, the data provided in this article show that in the presence of cholesterol, the addition of cannabidiol to the lipid phase in an amount greater than 4 mol%, such as 5-10 mol%, results in a rapid release of cannabidiol from the liposomes (see Table 1, the aqueous phase is DDW).

[0159] In addition, but not limited to this, the data provided herein show that when cannabidiol liposomes are prepared with cannabidiol only in the aqueous phase, the lipid phase is composed only of lipids, such as HSPC (without cannabidiol), and the release rate of cannabidiol from the liposomes is also very fast (78% release after 24 hours). Without being bound by theory, this may be due to the lack of membrane stability when the lipid membrane does not contain cannabidiol or a certain amount of cholesterol.

[0160] It can be concluded from the findings disclosed herein that cannabidiol in the lipid membrane stabilizes the membrane, thereby allowing the controlled (extended) release of cannabidiol from the liposomes.

[0161] Table 1 - Composition and characterization of liposome-cannabidiol containing cholesterol

[0162]

[0163]

[0164] Table 2 - Liposomal cannabidiol formulations with cannabidiol in lipid phase (HSPC) and aqueous phase, containing cannabidiol and HPCD

[0165]

[0166] Table 3 - Liposomal cannabidiol formulations with cannabidiol in lipid phase (HSPC) and aqueous phase, containing cannabidiol, HPCD and surfactant (cremophor or tween 80)

[0167]

[0168] Table 4 - Liposomal cannabidiol formulations with cannabidiol in the lipid phase (HSPC) and the aqueous phase, containing HPCD and PEG 300 with and without cannabidiol.

[0169]

[0170]

[0171] Table 5 - Liposomal cannabidiol formulations with cannabidiol in the lipid phase (HSPC) and the aqueous phase, containing HPCD and PG with and without cannabidiol.

[0172]

[0173]

[0174] a- The stated concentration is not reliable. Probably due to non-uniform sampling.

[0175] Example 2 - Liposomes containing cannabidiol (CBD)-HSA

[0176] A liposome formulation (liposome-cannabidiol (CBD)-HSA) containing cannabidiol (CBD)-HSA as the aqueous phase was developed. For the formulation, the aqueous phase was prepared by dispersing cannabidiol (CBD) in 5%, weighing cannabidiol in a vial and adding a 5% HSA solution. The dispersion was stirred at 4°C for at least two days until a uniform suspension was obtained, and no particles were observed on the vial wall. The dispersion was added to the heated HSPC powder and stirred at 65°C for 15 minutes. An analytical method was developed that could distinguish liposome-bound cannabidiol and HSA-bound cannabidiol. It was found that although the volume of liposomes in the suspension was lower than the additional liposome volume, most cannabidiol was liposome-bound. Table 6 below shows the liposome cannabidiol percentages in different liposome-cannabidiol-HSA formulations. The affinity of cannabidiol to liposomes and HSA was therefore tested as detailed below.

[0177] Two formulations were prepared:

[0178] The volume ratio of cannabidiol (CBD)-HSA preparation to empty MLV (40 mg / ml HSPC in 5% glucose) was 1:1

[0179] The volume ratio of F1 formula (cannabidiol (CBD) is only present in the several membrane lipids) to HSA solution is 1:1.

[0180] The mixture was placed in an incubator at 37°C and shaken at 50 rpm for 2 hours. Table 7 shows the results. The F1 preparation incubated with HSA showed that only 1% of the total cannabidiol was transferred from the liposomes to the HSA. In the case of cannabidiol-HSA incubated with empty MLV, 35% of the cannabidiol was transferred to the liposomes, indicating that the affinity of cannabidiol for lipids is much higher. These results are consistent with the results obtained with the liposome-cannabidiol-HSA preparation (Table 6), indicating that the cannabidiol is mainly liposomal.

[0181] Adding HSA to the several liposomes enabled us to achieve a high D / L molar ratio in the several liposomes.

[0182] Table 6 - Distribution of cannabidiol in different liposome-cannabidiol-HSA formulations

[0183]

[0184]

[0185] Table 7 - Affinity of cannabidiol for lipids and HSA

[0186]

[0187] The release of cannabidiol in different formulations was tested in 50% adult bovine serum. In an HPLC vial, 50mg of the preparation was weighed and 950ul of 50:50 serum: glucose 5% solution was added. The mixture was vortexed and placed in an incubator and shaken for 2 hours at 37°C and 50rpm. After diluting 25 times in methanol, the total cannabidiol content of the mixture was tested. The remaining mixture was transferred to an Eppendorf and centrifuged (30 minutes, 14,000rpm, 4°C), and the upper phase was diluted 10 times in methanol and analyzed by HPLC. The in vitro release of liposomes-cannabidiol-HSA preparations with different cannabidiol contents was tested and described in Table 8. The in vitro release test showed that the release rate was slower as the concentration of cannabidiol in the preparation increased.

[0188] Table 8 - Release of cannabidiol in 50% serum

[0189]

[0190]

[0191] Example 3 - In vivo PK study of cannabidiol formulations

[0192] Two in vivo studies were conducted to investigate plasma distribution and residual cannabidiol in muscle after IM injection of different cannabidiol formulations: the first study tested four formulations for up to 3 days, and the second study tested four formulations for up to 3 weeks. A detailed description of each study is provided below.

[0193] The first study

[0194] Preparation and characterization of formulations

[0195] Details of the materials used to prepare the formulations are summarized in Table 9. All formulations were prepared under aseptic conditions in a biological hood using an autoclave to ensure sterile preparations.

[0196] a. Free cannabidiol in PG: Cannabidiol at a concentration of 50 mg / g PG was prepared and vortexed until a clear solution was obtained.

[0197] b. F1: Liposomal preparation of cannabidiol, wherein cannabidiol is dissolved only in the membrane phospholipids of the liposomes: Cannabidiol and HSPC are dissolved together in ethanol at 65°C (lipid phase) until a clear solution is obtained. The lipid phase is added to a 5% glucose solution at 65°C while stirring and stirred for 30 minutes (at 65°C). The multilamellar liposome (MLV) preparation obtained is then washed with a 5% glucose solution until the weight molar osmotic pressure concentration of the preparation is isotonic.

[0198] c. F-HPCD-PEG: Liposomal formulation of cannabidiol, wherein cannabidiol is dissolved in the liposome membrane phospholipids and is also dispersed in the aqueous phase within the liposome using solubilizers: HPCD and PEG 300. Cannabidiol and HSPC are dissolved in ethanol (lipid phase) at 65°C until a clear solution is obtained. The aqueous phase is prepared by adding a cannabidiol solution in ethanol to a solution containing 27% (w / w) HPCD and 10% (w / w) PEG 300 at 65°C. The aqueous phase is almost transparent. The lipid phase is added to the aqueous phase at 65°C while stirring and left to stir for 30 minutes (at 65°C). The obtained preparation is then washed with a 5% glucose solution until the weight molar osmotic pressure concentration of the preparation is isotonic.

[0199] d. Liposome-cannabidiol-HSA: Cannabidiol was first dispersed in a 5% HSA solution. The dispersion was added to the heated HSPC and stirred at 65°C for 15 minutes.

[0200] Table 9 - Materials

[0201]

[0202] Formulation Characterization

[0203] Cannabidiol Testing

[0204] Total and free cannabidiol content was determined by HPLC. The chromatographic conditions used were based on the USP method for dronabinol and are summarized in Table 10.

[0205] Sample preparation for analysis varies for each formulation and is described below.

[0206] All formulations had similar total cannabidiol concentrations. Specifically, 10-20 mg of the formulation was weighed into a 10 ml volumetric flask. Methanol was added to the line. After vortexing, the samples were centrifuged and the upper phase was analyzed.

[0207] The free cannabidiol content of F1 and liposomal cannabidiol-HSA was tested: 200 μl of the preparation was placed in an Eppendorf and centrifuged at 40°C, 14,000 rpm for 30 minutes. The clarified upper phase was then diluted 10 times with methanol, then vortexed and centrifuged (14,000 rpm, 10 minutes, 40°C). The upper phase was subjected to HPLC analysis.

[0208] Albumin bound CBD of liposomal cannabidiol-HSA preparation is quantified: This method is developed to be able to separate liposomes and albumin bound cannabidiol. For this reason, an isotonic medium is used to allow density-based separation. The culture medium is prepared with 1.5g glucose (Sigma, D9434, batch 119K0042) and 10g Ficoll 400 (Sigma, F-4375, batch 29C-0095) dissolved in 50ml DDW (volume flask). The medium osmotic pressure is 290mOsm / kg. 50mg preparation is placed in an Eppendorf tube and 1.5ml culture medium is added. The test tube is vortexed and then centrifuged (4°C, 30 minutes, 14,000rpm). The upper phase of the tube is cut open to remove all liquid containing precipitate remaining in the lower half. The precipitate is transferred to another Eppendorf and 1ml methanol is added. After vortexing and centrifugation, the upper phase is diluted 10 times with methanol.

[0209] IV formulations in Cremophore: Ethanol were tested for total cannabidiol concentration as above. Appearance was checked after dilution with saline to follow the behavior of the formulation for injection and to ensure that there was no precipitation. The formulation was diluted 10-fold with saline and the appearance was recorded after 1 hour (time to allow injection after formulation preparation).

[0210] Table 10: Chromatographic conditions for cannabidiol determination

[0211]

[0212]

[0213] Release test

[0214] The release of cannabidiol in different formulations was tested in 50% adult bovine serum. In an HPLC vial, 50 mg of the formulation was weighed and 950 ul of a 50:50 serum: glucose 5% solution was added. The mixture was rotated and placed in an incubator at 37°C and 50 rpm and shaken for 2 hours. After diluting 25 times in methanol, the total cannabidiol content of the mixture was tested. The remaining mixture was transferred to an Eppendorf, centrifuged (30 minutes, 14000 rpm, 4°C), the upper layer was diluted 10 times in methanol, and HPLC analysis was performed.

[0215] Particle size measurement

[0216] Particle size was determined using a Coulter LS130.

[0217] Osmotic pressure

[0218] Osmolality was measured by the freezing point method using an Advanced Instruments, Model 3320 Osmometer.

[0219] Lipid concentration

[0220] Lipid concentrations were determined by HPLC / ELSD method.

[0221] Microscope observation

[0222] The several formulations were observed under an optical microscope (Zeiss SN 221209). Few fields were observed and one representative picture was taken for each formulation.

[0223] Injectability

[0224] Fill a one-mL syringe with 0.3-0.5 mL of formulation. Attach a 25G needle to the syringe and measure the amount of formulation injected that is not stuck. This process is repeated three times.

[0225] Sterile

[0226] Hadassah Aliquot's Microbiology Department tested one vial of each formulation. Aliquots from each vial were plated on blood agar and chocolate agar and placed in incubators at room temperature and 37°C.

[0227] In vivo study protocol

[0228] A total of 36 12-week-old female BALB / C mice were injected intramuscularly with a single dose of each formulation (9 mice per group) at an injection volume of ~50 μl (~2.5 ml / kg for 1 injection site). F1, which has a lower cannabidiol concentration, was injected at 2 injection sites with a total injection volume of approximately 100 μl. To ensure accurate dosing, the syringe was weighed before and after injection and the actual injection volume was recorded.

[0229] At the time points detailed below, 3 mice per group were euthanized with CO2 and terminal blood was immediately collected from the retro-orbital sinus in labeled 0.5 ml K3EDTA blood collection tubes (Mini Collect, Greiner-bio-one, Austria). Blood was centrifuged at 2000 g for 10 min and plasma was then extracted and collected in labeled tubes and frozen at -20°C immediately after collection. Samples were then stored at -80°C pending analysis.

[0230] After blood collection, quadriceps muscles were collected into pre-weighed 15 ml tubes.

[0231] The time points for blood and muscle collection were: 2 hours, 24 hours and 72 hours after injection.

[0232] Bioanalytical Testing

[0233] Determination of cannabidiol in plasma

[0234] Cannabidiol was used as an internal standard (IS) from spiked cannabigerol (CBG, 1 mg / ml methanol solution, Sigma, Cat. C-141-1), and then the plasma was diluted five times with acetonitrile. After vigorous vortexing and centrifugation, the upper phase was analyzed. The final IS concentration in the sample was 100 ng / ml.

[0235] Plasma extracts were analyzed by LC-MS method. Specifically, LC-MS / MS analysis was performed on a Sciex (Framingha, MA, USA) Triple Quad TM The concentrations were calculated based on a calibration curve of cannabidiol in plasma in the range of 1-1,000 ng / ml, with an IS of 100 ng / ml.

[0236] The cannabidiol spiking solution used to prepare the plasma calibration curve was prepared in acetonitrile. CBG was prepared in methanol.

[0237] Determination of cannabidiol in muscle (injection site)

[0238] Muscles were surgically removed and their weights were recorded. Thereafter, 2 ml of 15% collagenase solution (Sigma, C7657) was added and the tubes were incubated overnight at 37°C. After incubation, 8 ml of acetonitrile was added, vortexed and centrifuged. The upper phase was subjected to HPLC analysis. Chromatographic conditions are shown in Table 10.

[0239] The concentration of cannabidiol in each muscle was calculated based on a calibration curve of cannabidiol in acetonitrile.

[0240] The cannabidiol preparations spiked into muscle were compared to those spiked into acetonitrile, and the recovery of cannabidiol from muscle was determined for each preparation.

[0241] result

[0242] The formulations were characterized for their total cannabidiol and HSPC content and their molar ratio, particle size, and microscopic appearance. Except for F1, which had a cannabidiol content of 30 mg / g, all formulations had cannabidiol concentrations in the range of 50-60 mg / g. Table 11 summarizes these results.

[0243] The prepared preparation was injected intramuscularly into mice. The weight of the syringe was recorded before and after the injection to accurately calculate the injection dose.

[0244] Table 12 summarizes the plasma and muscle concentrations obtained. This study clearly shows that cannabidiol is retained at the injection site (muscle) for more than 72 hours. During this period, the depot in the muscle releases cannabidiol into the plasma at a rate that depends on the formulation used.

[0245] Table 11 - Formulation Characterization

[0246]

[0247]

[0248] Table 12 - Plasma and muscle concentrations obtained

[0249]

[0250]

[0251] NC = Not Calculated

[0252] The second study

[0253] Preparation and characterization of formulations

[0254] Materials and Methods

[0255] Material

[0256] Table 13 summarizes the detailed information about the materials used for formulation preparation.

[0257] Table 13: Material details

[0258]

[0259]

[0260] method

[0261] Preparation

[0262] All formulations were prepared under sterile conditions in a biological hood using autoclaved equipment to ensure sterile preparations. Three formulation types were used for intramuscular (IM) pharmacokinetic (PK) studies.

[0263] CTRL-PG: Control formulation of cannabidiol dissolved in propylene glycol (PG). Cannabidiol was prepared at a concentration of 50 mg / g PG and vortexed until a clear solution was obtained.

[0264] F1: Liposomal preparation of cannabidiol, wherein cannabidiol is dissolved only in the membrane phospholipids of the liposomes. Cannabidiol and HSPC are dissolved together in ethanol (lipid phase) at 65°C until a clear solution is obtained. The lipid phase is added to a 5% glucose solution at 65°C while stirring and left to stir for 30 minutes (at 65°C). The multilamellar liposome (MLV) preparation obtained is then washed with a 5% glucose solution until the weight molar osmotic pressure concentration of the preparation is isotonic.

[0265] Liposome-Cannabidiol-HSA: Liposomal formulation of cannabidiol, in which cannabidiol is first dispersed in HSA, and then cannabidiol-HSA is passively embedded in liposomes. Cannabidiol is first dispersed in 5% HSA solution. The dispersion is added to heated HSPC and stirred at 65°C for 15 minutes.

[0266] IV formulation: The formulation for IV administration is a 10 mg / g cannabidiol formulation dissolved in a 50:50 solution of Cremophor:Ethanol. The formulation is diluted 10-fold with saline prior to injection to a concentration of 1 mg / ml. The diluted formulation is used within 1 hour of preparation.

[0267] Hereinafter, a formulation is defined as the amount of cannabidiol / ml of solution containing 50 mg of protein. Thus, for example, "liposomal cannabidiol / HSA 50 mg / ml" means a liposomal formulation containing 50 mg of cannabidiol and 50 mg of HSA.

[0268] Formulation Characterization – As described in the first study

[0269] In vivo study protocol

[0270] IV Administration

[0271] A total of 18 12-week-old female BALB / C mice were injected intravenously with a single dose of 10 mg / kg cannabidiol formulation in cremophor:ethanol.

[0272] At the time points detailed below, 3 mice were euthanized with CO2 and terminal blood was immediately collected from the retro-orbital sinus in a labeled 0.5 ml K3EDTA blood collector (Mini Collect, Greiner-bio-one, Austria). The blood was centrifuged at 2000 x g for 10 minutes, and then plasma was extracted and collected in labeled tubes and frozen at -20°C immediately after collection. The samples were then stored at -80°C pending analysis.

[0273] Blood collection time points: 2 minutes, 1 hour, 4 hours, 8 hours, 24 hours and 48 hours.

[0274] Instant messaging management

[0275] A total of 36 12-week-old female BALB / C mice were injected intramuscularly with a single dose of the IM formulation. Nine mice were injected per formulation. The syringes were weighed before and after injection to accurately record the exact volume and dose received by each mouse. Details of the injection volume and estimated dose for each group are summarized in Table 14.

[0276] Two mice were not injected with formulation and served as controls for body weight (BW) changes over time.

[0277] At the time points detailed below, 3 mice per group were euthanized with CO2 and terminal blood was immediately collected from the retro-orbital sinus in labeled 0.5 ml K3 EDTA blood collection tubes (Mini Collect, Greiner-bio-one, Austria). The blood was centrifuged at 2000 x g for 10 minutes, and then plasma was extracted and collected in labeled test tubes and frozen at -20°C immediately after collection. The samples were then stored at -80°C pending analysis.

[0278] After blood collection, quadriceps muscles were collected into pre-weighed 15 ml tubes.

[0279] Blood collection time points: 72 hours, 1 week and 3 weeks after injection.

[0280] The body weight of mice was recorded before administration and before euthanasia. Mice sacrificed at the 3-week time point were also weighed two weeks after administration.

[0281] Table 14: Injection volume and estimated dose per group

[0282]

[0283] result

[0284] This example defines the pharmacokinetic profiles of three particle-based cannabidiol formulations versus a solution of cannabidiol in propylene glycol (PG) following IM or IV administration (administered at a dose of 12 mg / kg, which based on literature has been shown to be efficacious in several animal models1-3).

[0285] formula

[0286] Liposomal cannabidiol-HSA formulations were prepared by hydrating HSPC with cannabidiol-HSA dispersion at 65° C. The obtained liposomes were spherical and homogeneous, as can be observed by microscopic images ( Figure 1B to Figure 1C ).

[0287] The average liposome diameter of the 50 mg / ml preparation was 8.1 μm, and the average diameter of the 100 mg / ml preparation was 6.7 μm.

[0288] The cannabidiol concentration in the several preparations is the expected concentration (based on calculation). The 100 mg / ml preparation presents a high molar drug to lipid (D / L) ratio of 3.05. The cannabidiol in these preparations appears to be distributed between the liposomal cannabidiol (in the film and the internal aqueous phase) and the albumin (cannabidiol-HSA) outside the several liposomal bodies.

[0289] The formulation characterization is provided in Table 15 and the particle sizes are summarized in Table 16.

[0290] Table 15: Formulation Characterization (IM Administration)

[0291]

[0292] *Binding after 2 hours in 50% serum

[0293] **Free means that the entire volume of the formulation is immediately / freely injected from the syringe

[0294] Notably, all formulations were also found to be sterile, i.e., no microbial growth was detected in any of the formulations tested.

[0295] Cremophor: cannabidiol formulations in ethanol for IV administration were also characterized. The concentration of cannabidiol in the concentrate was 11.7 (mg / ml). After dilution with saline, the solution remained clear for at least 1 hour.

[0296] Table 16: Particle size (average value, d10, d50 and d90)

[0297]

[0298] * Span = (D90-D10) / D50

[0299] The distribution of cannabidiol between liposomes (membrane core and liposome inner core) and cannabidiol-HSA in these formulations was determined and is summarized in Table 17.

[0300] Table 17: Distribution of cannabidiol in liposome-cannabidiol-HSA formulations

[0301]

[0302] Table 17 shows that although the liposome volume is lower than the extra liposome volume, most of the cannabidiol is liposomal (86%-91%), and a relatively small portion is bound to HSA outside the liposome (9%-14%). It is therefore assumed that most of the HSA-bound cannabidiol is transferred to the lipids that form the liposomes. The observations are consistent with the cannabidiol partitioning between HSA and lipids described in Table 6 of Example 2.

[0303] After 2 hours of cultivation in the presence of 50% serum, the free cannabidiol concentrations were 33 mg / ml and 53 mg / ml for the 50 mg / ml and 100 mg / ml preparations, accounting for 70% and 57% release (100% binding = % release). The release rate is closer to the rate obtained by F1 liposomes. F1 is a liposome preparation in which the cannabidiol is dissolved by the several membrane lipids, and the cannabidiol may only be located in the liposome membrane. Due to the washing steps required to remove ethanol from the preparation, the cannabidiol concentration in the preparation is 21.3 mg / ml, which is lower than other preparations. The appearance of F1 under the microscope shows small round particles, which are relatively far apart from each other ( Figure 1A The average diameter was 9.2 μm (Table 16). The release rate in serum was 49%, similar to the liposome-HSA formulation (Table 15).

[0304] The reference IM formulation used was a solution of cannabidiol in propylene glycol. Another reference included a group of mice injected intravenously with a 12 mg / g dose (Cremophore: an ethanol formulation diluted with saline before injection).

[0305] PK Introduction

[0306] The PK profiles obtained following IV administration of a 12 mg / kg cannabidiol dose are summarized in Table 18.

[0307] Table 18: Plasma cannabidiol profiles following IV administration of a 12 mg / kg dose

[0308]

[0309] *BLOD – below limit of detection

[0310] Table 18 shows that the cannabidiol concentration dropped rapidly from 8,856 ng / ml at 5 minutes to 9.5 ng / ml at 8 hours after administration. At later time points (24 hours and 48 hours), the cannabidiol concentration was below the limit of detection (BLOD).

[0311] The plasma concentrations obtained following IM administration are summarized in Tables 19A-19B and FIG. 2 .

[0312] Table 19A: Cannabidiol Plasma Concentrations Across IM Formulations

[0313]

[0314] * Plasma concentrations normalized to dose (ng / ml / mg / kg)

[0315] **Mean ± SD plasma concentrations normalized to dose (ng / ml / mg / kg) #Injection error assumed (therefore not included in mean calculation)

[0316] ##Outliers. Defined as values ​​that are twice as high as the group mean.

[0317] Table 19B: Cannabidiol Plasma Concentrations Across IM Formulations

[0318]

[0319]

[0320] * Plasma concentrations normalized to dose (ng / ml / mg / kg)

[0321] **Mean ± SD plasma concentrations normalized to dose (ng / ml / mg / kg)

[0322] Table 19A to Table 19B and Figure 2A It was shown that plasma concentrations after administration and up to 3 weeks after administration for all IM formulations were within the range of the IV curve obtained between 1 hour and 8 hours after administration. This suggests prolonged delivery by the IM injection formulations. Figure 2B Also included are the PK data from the first study (referred to as the “Prior Study” in the figure) for the same formulation tested in the second study, but from time points T=2 hours and 24 hours, thus providing us with a complete overview of the cannabidiol plasma profile following IV or IM administration.

[0323] Interestingly, although high levels of plasma cannabidiol were obtained immediately after administration following IV administration of therapeutic doses of cannabidiol, plasma levels declined within a few hours; however, for IM administration of high doses of cannabidiol, initial levels of plasma cannabidiol were similar to the IV dose (e.g., after 2 hours), and then for all IM formulations, the levels remained essentially high for at least 3 weeks.

[0324] In addition, for all formulations, the decline in plasma levels was very slow, less than an order of magnitude over 3 weeks. This slow decline, compared to the rapid decline of the IV formulation, indicates that the final slope of the IM curve is not elimination-dependent, but absorption-dependent, indicating that the formulations continue to release cannabidiol from the muscle over this long period of time.

[0325] Table 20 summarizes the residual cannabidiol content in muscle compared to the initial cannabidiol administered to each mouse. FIG. 3A to FIG. 3B The average cannabidiol content released by each muscle group compared to the initial cannabidiol administered is shown.

[0326] Table 20 - Cannabidiol muscle concentration (N = 3 per group)

[0327]

[0328]

[0329]

[0330] a-Percentage of cannabidiol released from muscle = 100 x (administered cannabidiol - cannabidiol in muscle) / (administered cannabidiol)

[0331] b- Calculate the estimated daily cannabidiol dose (mg / kg) for a 20g mouse

[0332] = Daily release of cannabidiol / 0.02kg

[0333] *Outliers. Not included in the calculation of the average

[0334] #May be an injection problem. Not included in the calculation of the average

[0335] NC was not calculated due to abnormal values ​​of muscle and plasma concentrations

[0336] At the time point of 1 week, it is found that there are differences between the groups that high dose groups release more.At the time point of 3 weeks, these differences are not found.The cannabidiol amount of muscle release is standardized to the number of days after application, to estimate the cannabidiol amount released every day, thereby estimate the cannabidiol dosage (assuming 20g mice) that reaches circulation every day.When plasma level is standardized to the daily dose of this estimation, average standardized value is similar between each group, and in the scope of 1.7-4.5ng / ml / mg / kg.These values ​​are similar to the IV values ​​obtained after 4 hours to 8 hours after application (table 18), and prove that plasma concentration depends on the cannabidiol of muscle release, therefore may be controlled by preparation.According to % cannabidiol release data, can calculate the cannabidiol reserves in muscle for every kind of formula.Free cannabidiol and liposome preparation have released most of cannabidiol (not including the unexplained low value of liposome cannabidiol-HSA 100mg / ml, 3 weeks) at 3 week time points.

[0337] Table 21 - Pharmacokinetic parameters obtained after IV administration of cannabidiol at a dose of 12 mg / kg

[0338]

[0339]

[0340] Pharmacokinetic analysis was performed for both IV and IM administration. Table 21 gives the IV PK parameters obtained. Cannabidiol has a rapid half-life of 1.68 hours, with a dose-normalized AUC exposure of 417 hours*ng / ml / mg / kg. Pharmacokinetic analysis after IM administration was performed on the three formulations that were also injected during the first study, allowing for 5 time points for each formulation (2 hours, 24 hours, and 72 hours from the previous study and 1 week and 3 weeks from the current study). The PK analysis of the combined data set is shown in Table 22. F1 and free cannabidiol in the PG resulted in the highest AUC. This is consistent with the finding that for both groups, most of the cannabidiol in the muscle was released within 3 weeks (70% and 84%, respectively, Figure 3B ), resulting in AUC normalized to that of IV injection (388 hours*ng / ml / mg / kg and 293 hours*ng / ml / mg / kg, respectively). The normalized AUC value for the liposome-cannabidiol-HSA 50 mg / ml formulation was lower (167 hours*ng / ml / mg / kg), corresponding to a lower percentage of muscle release obtained with that formulation (68%, Figure 3B ).

[0341] Table 22 - Pharmacokinetic parameters obtained after IM administration of cannabidiol formulations injected in this (second) study and in the first study

[0342]

[0343]

[0344] discuss

[0345] The PK curves after IV administration of a 12 mg / kg dose were compared with four formulations of cannabidiol long-acting formulations administered by the IM route. The plasma curves of the IM injection formulations showed that the plasma levels were within the IV plasma curve range for at least 3 weeks after injection. The liposome-cannabidiol-HSA and F1 formulations contained ≥30% of the injected dose, while the muscle of the free cannabidiol group contained only 14%. The fact that the cannabidiol plasma levels of the IM formulations maintained similar plasma concentrations observed with the IV effective dose indicates that these formulations may allow for the prolonged in vivo cannabidiol effect. The differences in the PK curves allow for the selective design of preferred formulations for specific desired release curves.

[0346] Example 4 - Preparation of DMPC / DPPC-cannabidiol liposomes

[0347] A cannabidiol liposome formulation in DMPC:DPPC with a molar ratio of 45:55 was prepared.

[0348] Material:

[0349] 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC): lipid, product number 556200 (lot number: 556200-2190329-01)

[0350] Dipalmitoylphosphatidylcholine (DPPC): Lipid Product No. 556300 (Batch No.: 556300-2170149-01)

[0351] Cannabidiol (CBD): THC Pharmaceuticals

[0352] result:

[0353] The liposomes formed contained a combination of DMPC:DPPC in a molar ratio of 45:55. The lipid phase composition is detailed in Table 23.

[0354] Table 23: Lipid Phase Composition of DMPC / DPPC-Cannabidiol Liposomes

[0355]

[0356] Specifically, histidine (0.155% w / v)-mannitol (4% w / v) buffer (HMB) at pH 6.5 was used as the aqueous phase of the formulation. The lipid phase and the aqueous phase were preheated at 55°C. The lipid phase was then added to the aqueous phase and stirred at 55°C for 15 minutes. Ethanol was removed from the formulation by centrifugation at 4°C after 4 cycles of washing with histidine mannitol buffer.

[0357] Figure 4 The appearance of the formulation under a microscope is shown (magnification 200x). The average size of the liposomes was 5.55 μm and the total cannabidiol concentration was 25.3 mg / ml.

Claims

1. A prolonged release preparation, characterized in that The extended release formulation comprises a plurality of liposomes, wherein the plurality of liposomes have a lipid membrane and an intraliposomal aqueous core, wherein the lipid membrane comprises one or more liposome-forming lipids, wherein the liposome comprises an entrapped cannabinoid, at least a portion of the cannabinoid being entrapped in the lipid membrane, wherein the lipid membrane comprises a molar ratio between the cannabinoid and the one or more liposome-forming lipids, the molar ratio being in the range of 1 to 10; wherein the plurality of liposomes are obtained by a method comprising: dissolving a first portion of a cannabinoid in a lipid phase, dissolving or dispersing a second portion of the cannabinoid in an aqueous phase, and adding the lipid phase containing the first portion of the cannabinoid to the aqueous phase containing the second portion of the cannabinoid while stirring the aqueous phase; The cannabinoid release rate of the plurality of liposomes is slower than the cannabinoid release rate of the plurality of liposomes obtained by the method of dissolving or dispersing the cannabinoid only in the aqueous phase or only in the lipid phase.

2. The extended release preparation according to claim 1, characterized in that At least a portion of the cannabinoid is embedded within the aqueous core of the liposome.

3. The extended release preparation according to claim 1 or 2, characterized in that The cannabinoid includes or is cannabital.

4. The extended release preparation according to claim 1, characterized in that The extended release formulation comprises at least one dispersing agent, and the at least one dispersing agent is not a cyclodextrin compound.

5. The extended release preparation according to claim 4, characterized in that The dispersant is selected from a surfactant, a co-solvent, and a protein.

6. The extended release preparation according to claim 4, characterized in that In addition to the dispersing agent, the extended release formulation also contains a cyclodextrin compound.

7. The extended release preparation according to claim 1 or 2, characterized in that The extended release preparation is used as an injectable preparation.

8. The extended release preparation according to claim 1 or 2, characterized in that The extended release formulation is used in a treatment of a mammalian subject by injection.

9. The extended release preparation according to claim 1 or 2, characterized in that The mammalian subject is a human subject.

10. A method for preparing a prolonged release preparation, characterized in that: The method comprises the following steps: dissolving a first portion of the cannabinoids in a lipid phase; dissolving or dispersing a second portion of the cannabinoids in an aqueous phase; The lipid phase comprising the first portion of the cannabinoid is added to the aqueous phase comprising the second portion of the cannabinoid whilst stirring the aqueous phase.

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