Liposomal cannabinoids and uses thereof

Abstract

The present disclosure provides sustained-release formulations of cannabinoids. The formulations include liposomes having a lipid membrane and an intraliposomal aqueous core, the liposomes containing an entrapped cannabinoid and at least one dispersing agent for the cannabinoid, the dispersing agent being a cyclodextrin (CD) compound or other than the entrapped cannabinoid, at least a portion of the cannabinoid being entrapped in the lipid membrane, the lipid membrane having a molar ratio of the cannabinoid to the one or more liposome-forming lipids ranging from 1 to 10. Methods for preparing and using the formulations for sustained delivery of cannabinoids, as well as therapeutic treatments using the same, are also disclosed.

Description

[Technical Field]

[0001] The present disclosure relates to liposomal cannabinoids.

[0002] The following references are considered relevant as background to the subject matter of this disclosure. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Patent Application Publication No. WO 2017203529 Brochure [Patent Document 2] International Patent Application Publication No. WO 2001003668 Pamphlet [Patent Document 3] US Patent Application Publication No. 20170044092 [Patent Document 4] International Patent Application Publication No. WO 2018145213 Brochure [Patent Document 5] US Patent Application Publication No. 20180193399 [Patent Document 6] U.S. Patent No. 9,095,555 [Patent Document 7] U.S. Patent No. 1,011,7883 [Patent Document 8] US Patent Application Publication No. 20170281701 [Patent Document 9] US Patent Application Publication No. 20180318237 [Patent Document 10] US Patent Application Publication No. 20180271924 [Patent Document 11] US Patent Application Publication No. 20170107280 [Patent Document 12] International Patent Application Publication No. WO 2017191630 Brochure [Patent Document 13] US Patent Application Publication No. 20180303791 [Patent Document 14] U.S. Patent Application Publication No. 20180042845 [Patent Document 15] US Patent Application Publication No. 20180185324 [Patent Document 16] US Patent Application Publication No. 20180289665 [Patent Document 17] U.S. Patent No. 9,655,910 [Patent Document 18] U.S. Patent No. 8,242,178 [Patent Document 19] U.S. Patent Application Publication No. 20180221304

[0004] Acknowledgment of the above references herein should not be inferred to mean that they are in any way relevant to the patentability of the subject matter of this disclosure. [Background technology]

[0005] Few publications have described the use of CBD associated with various liposomes. For example, International Publication No. 2017203529 describes a composition comprising a combination of cannabidiol (CBD) or a derivative thereof with hyaluronic acid or a salt thereof, a phospholipid, and optionally a physiologically acceptable carrier. CBD can be incorporated into liposomes formed by the phospholipid. The composition is described for use in treating inflammatory joint diseases or pain or inflammation associated with such diseases. The composition is formulated for local injection.

[0006] Other publications describing liposomal CBD include WO 2001003668, which describes pulmonary delivery of liposome-encapsulated cannabinoids; U.S. Patent No. 20180318237, which describes the topical administration of cannabinoids, optionally in liposomes; WO 2017191630, which describes the use of cannabidiol, optionally in liposomes, to reduce steroid doses and treat inflammatory and autoimmune diseases; and U.S. Patent No. 20180303791, which describes the use of cannabinoids to treat multiple myeloma, where the cannabinoid may be in liposomes. No. 9,655,910, which describes the use of cannabinoids, optionally in liposomes, to treat addiction; U.S. Pat. No. 8,242,178, which describes the use of cannabidiol, optionally in liposomes, to treat autoimmune hepatitis; and U.S. Pat. No. 20180221304, which describes a cannabinoid-containing complex mixture for treating mast cell-associated or basophil-mediated inflammatory disorders, with liposomes suggested as a means for local delivery. Summary of the Invention

[0007] The present disclosure provides sustained-release formulations comprising liposomes having a lipid membrane and an intraliposomal aqueous core, the liposomes comprising one or more liposome-forming lipids, an entrapped cannabinoid (e.g., cannabidiol (CBD)) or a functional homolog thereof, and at least one dispersing agent for the cannabinoid (e.g., PG, HSA, IVIg) that is not a cyclodextrin (CD) compound. The intraliposomal core may also include a CD.

[0008] Also provided by the present disclosure is a sustained release formulation comprising a liposome having a lipid membrane and an intraliposomal aqueous core, the lipid membrane comprising one or more liposome-forming lipids, the liposome comprising an entrapped cannabinoid, at least a portion of the cannabinoid being entrapped in the lipid membrane, the lipid membrane having a molar ratio of the cannabinoid to the one or more liposome-forming lipids in the range of 1 to 10.

[0009] Also provided by the present disclosure is a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a sustained release formulation comprising liposomes having a lipid membrane and an intraliposomal aqueous core, the liposomes comprising one or more liposome-forming lipids, an entrapped cannabinoid compound, and at least one dispersing agent for the cannabinoid that is not a CD compound.

[0010] Further provided by the present disclosure is a method of treatment comprising administering to a subject in need thereof a sustained release formulation comprising liposomes having a lipid membrane and an intraliposomal aqueous core, wherein the lipid membrane comprises one or more liposome-forming lipids, the liposomes comprising an entrapped cannabinoid, at least a portion of which is entrapped in the lipid membrane, and the lipid membrane has a molar ratio of the cannabinoid to the one or more liposome-forming lipids in the range of 1 to 10.

[0011] In some instances, the formulation also includes an entrapped CD compound.

[0012] In order to better understand the subject matter disclosed herein and to illustrate 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: [Brief explanation of the drawings]

[0013] [Figure 1A]Microscopic images (Zeiss SN 221209, 200x magnification) of various CBD formulations, including liposomal CBD (F1, Figure 1A), liposomal CBD-HSA 50 mg / ml (Figure 1B), and liposomal CBD-HSA 100 mg / ml (Figure 1C). [Figure 1B] Microscopic images (Zeiss SN 221209, 200x magnification) of various CBD formulations, including liposomal CBD (F1, Figure 1A), liposomal CBD-HSA 50 mg / ml (Figure 1B), and liposomal CBD-HSA 100 mg / ml (Figure 1C). [Figure 1C] Microscopic images (Zeiss SN 221209, 200x magnification) of various CBD formulations, including liposomal CBD (F1, Figure 1A), liposomal CBD-HSA 50 mg / ml (Figure 1B), and liposomal CBD-HSA 100 mg / ml (Figure 1C). [Figure 2A] 1 is a graph showing plasma CBD concentrations (ng / ml) following IV administration of a 12 mg / kg dose compared to different IM formulations (n=3, mean±SD, no outliers) in two separate in vivo studies. [Figure 2B] FIG. 1 is a graph showing plasma CBD concentrations (ng / ml) following IV administration of a 12 mg / kg dose compared to different IM formulations (n=3, mean±SD, no outliers) in two separate in vivo studies. [Figure 3A] Graphs showing absolute CBD (mg) released from muscle after IM administration of different formulations (n=3, mean ± SD, no outliers) (FIG. 3A) and percent CBD released from muscle after IM administration of different formulations (n=3, mean ± SD, no outliers) (FIG. 3B). [Figure 3B] Graphs showing absolute CBD (mg) released from muscle after IM administration of different formulations (n=3, mean ± SD, no outliers) (FIG. 3A) and percent CBD released from muscle after IM administration of different formulations (n=3, mean ± SD, no outliers) (FIG. 3B). [Figure 4] 1 is a microscopic image (Zeiss SN 221209, 200x magnification) of DMPC:DPPC-CBD liposomes. DETAILED DESCRIPTION OF THE INVENTION

[0014] This disclosure is based on the unexpected finding that the presence of CBD entrapped in the liposomal bilayer affects (decreases / slows) the release rate of CBD from the liposome, allowing for sustained delivery of CBD from liposomal formulations. This disclosure is also based on the finding that the presence of a dispersing agent (which can distribute CBD evenly) in the intraliposomal aqueous environment (with or without CBD in the intraliposomal aqueous environment) can also slow the release rate of CBD from the liposomes.

[0015] Thus, according to a first aspect thereof, the present disclosure provides a sustained release formulation comprising a liposome having a lipid membrane and an intraliposomal aqueous core, the liposome comprising one or more liposome-forming lipids, an entrapped cannabinoid compound, and at least one cannabinoid dispersing agent other than (i.e., not a) cyclodextrin (CD) compound.

[0016] According to a second aspect, the present disclosure provides a sustained release formulation comprising a liposome having a lipid membrane and an intraliposomal aqueous core, the lipid membrane comprising one or more liposome-forming lipids, the liposome comprising an entrapped cannabinoid or functional homolog thereof, at least a portion of the cannabinoid being entrapped in the lipid membrane, the lipid membrane having a molar ratio of the cannabinoid to the one or more liposome-forming lipids in the range of 1 to 10.

[0017] Also provided by the present disclosure are methods of using the sustained release liposomal formulations described above to treat conditions requiring sustained delivery of cannabinoid compounds, and thus comprising administering the sustained release formulation to the subject.

[0018] The formulation disclosed herein comprises at least one cannabinoid.In the context of the present disclosure, when referring to cannabinoid, it should be understood to include a single compound or a combination of cannabinoid compounds (i.e., the term used herein includes a single or multiple such compounds).In some examples, the combination of cannabinoids comprises the components of plant extract, i.e., multiple cannabinoids and optionally plant flavonoids and terpenoids.

[0019] In some examples, the cannabinoid is or includes cannabidiol (CBD).

[0020] In some other instances, the cannabinoid is or includes tetrahydrocannabinol (THC) (Delta9-THC and / or Delta8-THC).

[0021] Other cannabinoids falling within the scope of the present disclosure include cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerol monomethyl ether (CBGM), cannabichromene (CBC), cannabichromanone (CBCN), cannabichromenic acid (CBCA), cannabivalichromene (CBCV), cannabichromevalic acid (CBCVA), isotetrahydrocannabinol (iso-THC), cannabinol (CB), each of which constitutes a separate embodiment of this disclosure. N), cannabinolic acid (CBNA), cannabinol methyl ether (CBNM), cannabinol C4 (CBN-C4), cannabinol C2 (CBN-C2), cannabinol C1 (CBN-C1), cannabinodiol (CBND), cannabielsoin (CBE), cannabielsonic acid A (CBEA-A), cannabielsonic acid B (CBEA-B), cannabicyclol (CBL), cannabicycloic acid cannabinoids selected from the group consisting of tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabigerovarin (CBGV), cannabigerovaric acid (CBGVA), cannabifuran (CBF), dehydrocannabifuran (DCBF), cannabirispol (CBR), or any combination of two or more cannabinoids.

[0022] In some examples, the cannabinoid is or includes a combination of CBD and any one or more of the cannabinoids listed above.

[0023] In some preferred examples, the cannabinoid in the formulation is CBD.

[0024] The term CBD compounds, in the context of the present disclosure, encompasses CBD and its functional homologs. When referring to a CBD functional homolog, it should be understood as a compound that has similar physicochemical properties to CBD.

[0025] In some instances, a CBD functional homolog is a chemical analog of CBD that contains at least one benzene ring and a logP greater than four.

[0026] In some instances, CBD functional homologs include structural homologs (including isomers) of CBD that, like CBD, lack the psychoactive effects of tetrahydrocannabinol (THC).

[0027] In some instances, the CBD compound is a naturally occurring phytocannabinoid.

[0028] In some instances, the CBD compound is a synthetic CBD homolog.

[0029] Non-limiting examples of CBD compounds include those named 2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol (CBD), the synthetic cannabidiol-dimethylheptyl (CBD-DNH), the phytocannabinoid cannabidivarin (CBDV), cannabidivarinolic acid (CBDVA), and 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)].

[0030] In some instances, the active ingredient is CBD, known by its chemical name 2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol.

[0031] The cannabinoid, preferably a CBD compound, is entrapped / associated with the liposome. In the context of the present disclosure, when referring to the entrapment of a compound in a liposome, it should be understood to define any form of physical or chemical association between the cannabinoid and the liposome itself. However, it should be clear that the physical association is due solely to the phospholipid being in the form of a liposome, and there is no chemical association between the cannabinoid and the phospholipid itself. The association can be due to the cannabinoid being encapsulated in the aqueous core / medium within the liposome, and / or due to the cannabinoid being at least partially embedded within the lipid membrane (e.g., due to the hydrophobicity of the cannabinoid), and / or due to the cannabinoid being associated with the outer surface of the liposome (e.g., by physical force).

[0032] The amount of cannabinoid entrapped in the liposomes can be determined using commercially available chromatographic techniques. In some instances, the cannabinoid concentration is determined using high performance liquid chromatography (HPLC) / UV methods.

[0033] In some instances, the cannabinoids entrapped in the lipid membrane are determined by methods known in the art, such as, but not limited to, the ratio of CBD to one or more liposome-forming lipids in the lipid membrane can be determined by differential scanning calorimetry (DSC).

[0034] To calculate the intraliposomal concentration of cannabinoids, the aqueous intraliposomal entrapped volume is also required, which can be calculated as previously described [Bangham AD, et. al. (1965) J MoI Biol. 13(1):238-52].

[0035] In some instances, the amount of cannabinoid, preferably CBD compound, 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.

[0036] In some examples, the amount of cannabinoid, preferably CBD compound, entrapped by the liposomes is up to 400mg / ml, sometimes up to 350mg / ml, sometimes up to 330mg / ml, sometimes up to 310mg / ml, sometimes up to 300mg / ml, sometimes up to 280mg / ml, sometimes up to 260mg / ml, sometimes up to 240mg / ml, sometimes up to 220mg / ml, sometimes up to 200mg / ml, sometimes up to 190mg / ml, sometimes up to 180mg / ml, sometimes up to 170mg / ml, sometimes up to 160mg / ml, sometimes up to 150mg / ml, sometimes up to 140mg / ml, sometimes up to 130mg / ml, sometimes up to 120mg / ml.

[0037] In some instances, the amount of cannabinoid, preferably CBD compound, entrapped by the liposomes is in the range of 30-400 mg / ml, sometimes in the range of 30-350 mg / ml, sometimes in the range of 30-350 mg / ml, sometimes in the range of 30-350 mg / ml, sometimes in the range of 30-350 mg / ml, sometimes in the range of 30-200 mg / ml, sometimes in the range of 50-250 mg / ml, sometimes in the range of 40-180 mg / ml, sometimes in the range of 40-250 mg / ml, sometimes in the range of 30-120 mg / ml, sometimes in the range of 40-150 mg / ml, sometimes in the range of 50-300 mg / ml, or any range within the lower and upper concentration limits identified above.

[0038] In some instances, the molar ratio of cannabinoid to lipid is determined.

[0039] In some examples, the cannabinoid compound / lipid molar ratio is 1-10, sometimes 1-9, sometimes 1-8, sometimes 1-7, sometimes 1-6, and sometimes 1-5.

[0040] A unique feature of the present disclosure is the presence of a cannabinoid, preferably a CBD compound, in the intraliposomal compartment in combination with at least one cannabinoid dispersing agent that is not a cyclodextrin (CD). This is unique due to the very low solubility of cannabinoids such as CBD in aqueous solution (CBD has a predicted log P of 7.03), achieved using various dispersing agents. Without being bound by theory, it is believed that the dispersing agent (which is not a CD compound but may be combined with a CD compound) maintains the amount of cannabinoid in a dissolved or uniformly dispersed form when present within the liposome within the intraliposomal aqueous core, thereby improving the sustainability of the cannabinoid within the liposome.

[0041] In the context of the present disclosure, the term "cannabinoid dispersing agent" should be understood to encompass any chemical entity that promotes or enhances the dispersibility of a cannabinoid (a cannabinoid, or a combination of cannabinoids) in the liquid medium used to load the cannabinoid into liposomes (preferably, but not exclusively, by passive loading). Without being bound by theory, the cannabinoid dispersing agent physically associates with the cannabinoid, thereby causing it to become entrapped within the liposome in the form of a non-covalent complex.

[0042] In some instances, the dispersing agent is a solubilizer (also recognized by the term solubilization-enhancing agent). Reference to a solubilizer should be understood to encompass at least one compound that is not a CD. Thus, in the context of the present disclosure, the term "solubilizer other than CD" should be understood as any solubilization-enhancing compound that is not a CD, but that may be combined with a CD as an additional solubilization-enhancing compound.

[0043] Solubilizers are known to be used to improve drug solubility, especially when using insoluble or poorly soluble drugs. In some examples of the present disclosure, cannabinoids are added to both the lipid phase and the aqueous phase, so that the cannabinoid compound is believed to distribute between the lipid phase (lipid membrane) and the aqueous internal liposome phase, thus providing two different pools of active ingredient (i.e., cannabinoids such as CBD compounds).

[0044] In other words, without being bound by theory, it is believed that the dispersing agent maintains the cannabinoid in the intraliposomal aqueous medium and facilitates controlled (e.g., sustained, for up to three weeks) release of the active ingredient, e.g., the CBD compound, from the liposome.

[0045] There are various types of solubilizers. The solubilizer may be a cosolvent, i.e., a substance added in small amounts to a primary solvent (whether organic solvent or water) to increase / improve the solubility of a poorly soluble compound, such as, but not limited to, polyethylene glycol (PEG), e.g., PEG300, PEG400, propylene glycol (PG), N,N-dimethylacetamide (DMA), ethanol, or may be recognized as a surfactant, such as, but not limited to, Tween 80 (polyoxyethylene (20) sorbitan monooleate), Cremophor (propane-1,2,3-triol:oxirane (1:1)), or may be recognized as a complexing agent, such as a member of the cyclodextrin family of compounds.

[0046] In some instances, the solubilizing agent is a cosolvent. A preferred cosolvent is PEG. Another preferred cosolvent is PG.

[0047] The dispersing agent may be other than a solubilizing agent. In some instances, the dispersing agent is a protein selected for its ability to disperse the cannabinoid, preferably CBD, in the aqueous medium in which it is dissolved.

[0048] In some instances, the dispersed protein is a serum protein.

[0049] In some instances, the serum protein is albumin.

[0050] In some instances, the serum protein is human serum albumin (HSA).

[0051] In some instances, the serum protein is a globulin.

[0052] In some instances, the serum protein is an immunoglobulin.

[0053] The dispersant and the cannabinoid are associated by a non-covalent bond. In some instances, the dispersant and the cannabinoid form a physical complex that allows the cannabinoid to be released from the dispersant under suitable conditions. Thus, in some instances, the dispersant and the cannabinoid are non-covalently bound to each other.

[0054] In some instances, the formulation includes a combination of two or more dispersing agents.

[0055] In some examples, the combination of two or more dispersing agents includes at least a cyclodextrin (CD) compound.

[0056] In some instances, the dispersant combination includes two or more such compounds, none of which is a CD compound.

[0057] As mentioned above, liposomes can also contain CD compounds. CD compounds are recognized as cyclic oligosaccharides composed of (α-1,4)-linked α-D-glucopyranose units, which contain a lipophilic central cavity and a hydrophilic outer surface. In the context of the present disclosure, CDs can be naturally occurring CDs and derivatives of naturally occurring CDs. Natural CDs include α-, β-, or γ-cyclodextrins (αCD, βCD, or γCD), which are composed of 6, 7, and 8 glucopyranose units, respectively. When referring to derivatives of natural CDs (also encompassed under the general term "CD compounds"), it should be understood as any cyclic oligosaccharide composed of (α-1,4)-linked α-D-glucopyranose units with a lipophilic central cavity and a hydrophilic outer surface.

[0058] In some examples, the CD compound is 2-hydroxypropyl-β-cyclodextrin (HPβCD).

[0059] In some examples, the CD compound is 2-hydroxypropyl-γ-cyclodextrin (HPγCD).

[0060] In some examples, the CD compound is Solfobutyl ether (SBE) cyclodextrin.

[0061] In one preferred example, the CD is HPβCD or HPCD for short.

[0062] Formulations also include liposomes.

[0063] Liposomes are pre-formed using at least one liposome-forming lipid. In the context of the present invention, the term "liposome-forming lipid" refers primarily to glycerophospholipids or sphingomyelins that, in water, form vesicles such as, but not limited to, liposomes, as further explained below.

[0064] When referring to glycerophospholipid, it should be understood as a lipid having a glycerol backbone, in which at least one, preferably two, of the hydroxyl groups of the head group are replaced by an acyl chain, an alkyl chain or an alkenyl chain, a phosphate group, or any combination thereof, and / or one or two of these derivatives, and the head group can contain a chemically reactive group (such as amine, acid, ester, aldehyde or alcohol), thereby providing a lipid with a polar head group.Sphingomyelin consists of a ceramide unit with a phosphorylcholine moiety attached at position 1, and therefore is actually N-acylsphingosine.The phosphocholine moiety in sphingomyelin contributes to the polar head group of sphingomyelin.

[0065] In liposome-forming lipids, the acyl, alkyl, or alkenyl chains are typically 12 to about 24 carbon atoms in length and may be fully, partially, or non-hydrogenated naturally occurring, semi-synthetic, or fully synthetic lipids with varying degrees of saturation, which may affect the rigidity of the liposomes thus formed (lipids with saturated chains are typically more rigid than lipids of the same chain length, especially where unsaturated chains with cis double bonds are present).

[0066] In some instances, the liposomes comprise a single type of liposome-forming lipid.

[0067] In some other instances, the liposomes comprise a combination of liposome-forming lipids.

[0068] In some examples, the liposome-forming lipid is a phospholipid. If the liposome-forming lipid is a phospholipid, its amount in the liposome can be determined as organophosphate by a modified Bartlett method [Shmeeda H, Even-Chen S, Honen R, Cohen R, Weintraub C, Barenholz Y. 2003. Enzymatic assays for quality control and pharmacokinetics of liposome formulations: comparison with nonenzymatic conventional methodologies. Methods Enzymol 367:272-92]. Lipids can also be tested using the ELSD / HPLC method described herein, in a non-limiting example that forms an integral part of the present disclosure.

[0069] In some instances, the liposome-forming lipid is a choline-type phospholipid, such as diacylglycero-phosphocholine, where the acyl, alkyl, or alkenyl chain is as defined above.

[0070] In some other examples, the liposome-forming lipid is di-lauroyl-sn-glycero-2-phosphocholine (DLPC). In some examples, the liposome-forming lipid is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some examples, the liposome-forming lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some examples, the liposome-forming lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some examples, the liposome-forming lipid is 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some examples, the liposome-forming lipid is 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC). In some examples, the liposome-forming lipid is 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-dibehenoyl-sn-glycero-3-phosphocholine 1,2-ditricosanoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1,2-dilignoceroyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine. In some examples, the liposome-forming lipid is 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC). In some examples, the liposome-forming lipid is 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). In some examples, the liposome-forming lipid is 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC) or di-lauroyl-sn-glycero-2-phosphocholine (DLPC).

[0071] In some instances, the liposome-forming lipids include at least hydrogenated soy phosphatidylcholine (HSPC).

[0072] In one preferred example, the liposome-forming lipids comprise or consist of hydrogenated soy phosphatidylcholine (HSPC).

[0073] In some instances, the liposome-forming lipids include at least 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).

[0074] In some instances, the liposome-forming lipids include at least 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

[0075] In a preferred example, the liposome-forming lipid comprises or consists of a combination of DMPC and DPPC. In some examples, the two liposome-forming lipids are in a DMPC:DPPC molar ratio of about 45:55. Without limitation, DMPC:DPPC-containing formulations are suitable for non-human (veterinary) use.

[0076] In some instances, the liposome comprises a sterol, such as cholesterol.

[0077] In some instances, if cholesterol is present in the liposome, it is in an amount of 4% molar or less.

[0078] In some additional or alternative examples, the liposome comprises a lipopolymer, such as a polyethylene glycol-derived lipid (PEGylated lipid).

[0079] Liposomes can be of any shape or size.

[0080] In some instances, the liposomes are multilamellar vesicles or oligolamellar vesicles.

[0081] In some instances, the liposomes are multivesicular vesicles.

[0082] In some other instances, the liposomes are unilamellar vesicles, preferably large unilamellar vesicles.

[0083] Liposomes can be small, medium, large, or even giant. When referring to small liposomes, they should be understood to have an average size in the range of about 20 nm to 100 nm. When referring to medium-sized liposomes, they should be understood to have an average size in the range of about 100 nm to 200 nm. When referring to large liposomes, they should be understood to have an average size greater than about 200 nm. When referring to giant liposomes (typically giant unilamellar vesicles or multivesicular vesicles), they should be understood to refer to those larger than 1 μm.

[0084] In some instances, the liposomes are multilamellar vesicles (MLVs). In some instances, the MLVs have a size distribution with a minimum of 100 nm or greater.

[0085] In some instances, the formulation comprising the liposomes is in a dried form, particularly, but not exclusively, the liposomes are lyophilized.

[0086] In some other instances, the formulation comprises liposomes held within a medium, herein referred to as an "external medium." The external medium can be of any composition suitable for holding the liposomes therein. In some instances, the external medium is suitable for storage of the liposomes, and in some other instances, the external medium is suitable for administration of the liposomes, e.g., a physiologically acceptable carrier.

[0087] In some instances, typically when the external vehicle is suitable for administration, the external vehicle may include a cannabinoid, which may be the same as or different from the cannabinoid entrapped by the liposome.

[0088] The combination of the cannabinoid and the dispersing agent preferably allows for the formation of a sustained-release formulation in a controlled manner. In the context of this disclosure, when "controlled release" or "sustained release" is mentioned, it should be understood to mean controlled release over a period of time. A period of time includes at least several 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, or even more than 30 days. The term "sustained release" encompasses any form of controlled release other than immediate release (e.g., when more than 50% is released within the first 24 hours), including extended / sustained release and / or delayed release. Sustained release can be determined by the in vitro release assay described in Example 3. A release of ≦70%, sometimes ≦60%, sometimes ≦50% in 50% serum after 2 hours of incubation can be considered sustained release.

[0089] The present disclosure also provides within the formulation a physiologically acceptable carrier suitable for administration by injection or infusion.

[0090] In the context of the present invention, a physiologically acceptable carrier generally refers to any carrier useful for preparing a pharmaceutical formulation that is safe, non-toxic, and not biologically or otherwise undesirable. In some examples, the physiologically acceptable carrier is an aqueous solution suitable for administration by injection. In some examples, physiologically acceptable carriers suitable for systemic administration include aqueous and non-aqueous isotonic sterile injection / infusion solutions that may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient. In some examples, the carrier is any one or combination of saline, buffer, aqueous sugar solution (dextrose, sucrose, etc.), etc. In some examples, the carrier may also include thickeners, stabilizers, and preservatives.

[0091] In some examples, administration is by any one of intramuscular (im), intraperitoneal (ip), intravenous (iv) and subcutaneous (sc) injection.

[0092] In one preferred example, the liposomal formulation is for IM injection, which has demonstrated a sustained / extended release profile that offers advantages over IV injection of non-liposomal cannabinoid formulations.

[0093] In some examples, the administration is to a mammalian subject.

[0094] In some examples, the administration is to a human subject.

[0095] In some other instances, administration is to a non-human (ie, veterinary) subject.

[0096] The amount of cannabinoid compound within the liposome is designed to be sufficient to provide a therapeutic effect upon administration of the formulation to a subject.

[0097] An amount sufficient or effective to achieve a therapeutic effect upon administration should be understood to include at least one therapeutic effect known to be achieved by or associated with cannabinoid compounds, particularly CBD.

[0098] Without limitation, the therapeutic effect may be any one or combination of treating / ameliorating / reducing pain and / or inflammation, as well as any other therapeutic effect known to be associated with the administration of certain cannabinoid compounds, particularly CBD.

[0099] 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 ranging studies), which those skilled in the art would know how to properly conduct to determine an effective amount, which will depend, among other things, on the type and severity of the disease being treated, as well as the treatment regimen (mode of administration), the sex and / or age and / or weight of the subject being treated, etc.

[0100] The liposomes within the formulation, and the formulation itself, may be characterized by any technique or parameter known in the art of liposome formulations, including, but not limited to, liposome size and / or size distribution (e.g., using dynamic light scattering (DLS)), polydispersity index (PDI), zeta potential, measuring dispersion pH using a pH meter, etc.

[0101] The present disclosure also provides methods of treating a subject with a cannabinoid, the methods comprising administering the liposomal formulations disclosed herein to a subject in need of such treatment.

[0102] In view of the above, in the context of the present disclosure, reference to treatment with the formulations or liposomes disclosed herein should be understood to encompass ameliorating undesirable symptoms associated with the disease, preventing the onset of such symptoms before they occur, slowing the progression of the disease, slowing the worsening of symptoms, enhancing the onset of periods of remission of the disease, slowing irreversible damage caused by the progressive chronic stage of the disease, delaying the onset of the progressive stage, reducing the severity of or curing the disease, improving survival or more rapid recovery from the disease, preventing the onset of the disease, or a combination of two or more of the above.

[0103] 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.

[0104] Furthermore, as used herein, the term "comprising" is intended to mean that the liposome includes a cannabinoid and a delivery agent, but does not exclude other elements, such as physiologically acceptable carriers and excipients and other drugs. The term "consisting essentially of" is used, for example, to define a liposome that includes the recited elements, but excludes other elements that may be of essential significance to the delivery of the cannabinoid. Thus, "consisting of" is intended to mean excluding more than trace amounts of such other elements. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0105] Furthermore, for example, when referring to amounts or ranges of components constituting liposomes and formulations containing liposomes, all numerical values ​​are approximations that may vary (+) or (-) up to 20%, and sometimes up to 10%, from the stated value. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about."

[0106] The present invention will now be described by way of non-limiting examples that have been implemented in accordance with the present invention. It should be understood that these examples are intended to be illustrative in nature, and not limiting. Many modifications and variations of these examples are obviously possible in light of the above teachings. It should therefore be understood that, within the scope of the appended claims, the present invention may be implemented in a myriad of possible ways other than as specifically described herein below.

[0107] Description of the embodiment Example 1 – CBD liposomal formulation Liposome preparation and characterization material Hydroxypropyl-β-cyclodextrin (HPCD) was obtained from rocket. Hydrogenated soybean phosphatidylcholine (HSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were obtained from Lipoid GmbH (Ludwigshafen, Germany). Cannabidiol (CBD) was obtained from THC Pharm (batch: CBDAPI1802). Absolute ethanol was purchased from Merck. Solubilization enhancers: polyethylene glycol (PEG) 300, propylene glycol (PG), Tween 80 and dimethylacetamide (DMA) were purchased from Merck, Cremophor was obtained from Sigma.

[0108] method: Liposome preparation Various types of phosphatidylcholines differing in acyl chain composition were used and tested, including cholesterol-free or cholesterol-containing (5 or 10%) HSPC (mostly stearoyl, C18), DPPC dipalmitoyl, C16 DMPC (dimyristoyl, C14), and DOPC (dioleoyl, C18:1).

[0109] Formulations containing DOPC produced less favorable liposomes and were therefore excluded from further investigation.

[0110] The various liposomes prepared are detailed in Table 1.

[0111] Formulation F1 was prepared as follows: HSPC (S PC-3, Lipoid, batch: 525600-2180662-01 / 042) and CBD (batch: CBDAPI1802) were weighed into a vial. Absolute ethanol (Merck) was added to the vial, and the vial was placed in a water bath at 65°C until the solution became clear. 1 ml of an isotonic aqueous solution (e.g., 5% dextrose) was then placed in the water bath. As soon as the lipid phase became clear, it was added to the warmed water while stirring at 65°C for 30 minutes.

[0112] A formulation containing CBD in the aqueous phase was prepared using the same lipid phase as that prepared for F1.The aqueous phase was prepared by mixing all the components of the specific aqueous phase, and then adding a concentrated CBD ethanol solution (700 mg / ml).The ethanol solution was slowly added while stirring, and then heated briefly if necessary.As soon as the aqueous phase became clear or uniformly dispersed, the lipid phase was slowly added at 65°C, and stirring was continued at 65°C for 30 minutes.

[0113] Release assay CBD release from liposomes was determined at 0, 1 hour (in some cases), and 24 hours after incubation in the presence of 25% bovine serum in 25% sucrose at 37° C. At each time point, total and free CBD were determined as described below.

[0114] Total CBD Assay Liposomal CBD 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 for detection of CBD by HPLC.

[0115] Free CBD Assay Liposomal CBD was diluted 20-fold with 25% serum and 25% sucrose. This dilution was centrifuged, and the liposomes were floated on top of the clear phase. The lower clear phase (free CBD) was diluted with methanol and analyzed by HPLC.

[0116] lipid concentration Lipid concentrations were determined by a modified Bartlett method and in some cases by an HPLC method with an evaporative light scattering (ELSD) detector.

[0117] result Formulations containing cholesterol in the lipid phase. CBD formulations containing cholesterol in the lipid phase were prepared. CBD was solubilized in the lipid phase. The lipid phase of all formulations tested was 125 mg / ml (including CBD), and CBD was 70% molar of the lipid phase content. Formulations included HSPC and DMPC with no cholesterol, and with 5 and 10% molar cholesterol. These formulations contained CBD only in the lipid phase (not in the aqueous phase). These formulations are listed in Table 1.

[0118] The results show that HSPC liposomes exhibit a slightly slower release profile compared to DMPC liposomes. Cholesterol increased the release of CBD from liposomes, and this effect was more pronounced with DMPC.

[0119] For this reason, additional formulations were prepared using HSPC in the lipid phase (125 mg / ml lipid phase, 70% molar CBD) and an aqueous phase of different composition that allowed for solubilization or dispersion of CBD in the aqueous phase.

[0120] Table 2 shows two CBD formulations with CBD in an aqueous phase containing only HPCD. The formulations yielded loadings (in terms of D / L ratio) and release profiles similar to formulation F1 (listed in Table 1). This is likely a result of the low CBD concentrations that could be loaded into an aqueous phase containing only HPCD (8 mg / ml).

[0121] Table 3 describes CBD formulations with HPCD and surfactants (cremphor EL and Tween 80) in the aqueous phase that allowed for the dispersion of 21 mg / ml of CBD. These formulations did not enhance the D / L ratio and resulted in a significant amount of small liposomes that were not separated by our release method, making it impossible to define their release profile.

[0122] Table 4 shows formulations with HPCD and 25% PEG 300 in the aqueous phase that allow for a 14 mg / ml CBD dispersion. Two formulations with different HPCD content (A39 and A42) substantially increased the D / L ratio and slowed the release profile. Comparing these formulations to liposomes with the same aqueous composition but without CBD in the aqueous phase resulted in a much lower D / L ratio and a faster release profile, demonstrating that having a substantial CBD fraction in the aqueous reservoir slowed the release.

[0123] Table 5 lists CBD formulations with HPCD and 10-15% PG in the aqueous phase, which allowed for a CBD dispersion of 21 mg / ml. For each formulation, a control formulation with the same aqueous phase composition but without CBD was also prepared. The results showed that in each case, the formulations with CBD in the aqueous phase released slower, with release being more pronounced when the D / L ratio was substantially higher than the control.

[0124] Without being bound, it is clear from the data presented herein that the addition of CBD to the aqueous phase, made possible by the addition of a co-solvent (dispersant) or surfactant, helps to delay the release of CBD from the liposomes.

[0125] Furthermore, without being bound, the data presented herein demonstrates that adding CBD to the lipid phase in the presence of cholesterol in amounts greater than 4% molar, e.g., 5-10% molar, results in rapid release of CBD from liposomes (see Table 1, where the aqueous phase is DDW).

[0126] Furthermore, without being bound, the data presented herein reveal that when CBD liposomes were prepared with CBD in the aqueous phase only and the lipid phase consisted solely of lipids, e.g., HSPC (no CBD), the release of CBD from the liposomes was rapid (78% free after 24 hours). Without being bound by theory, this may be the result of a lack of membrane stabilization when the lipid membrane does not contain CBD or some amount of cholesterol.

[0127] One conclusion that can arise from the findings disclosed herein is that CBD within the lipid membrane stabilizes the membrane, thereby allowing for the controlled (sustained) release of CBD from the liposomes. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0128] Example 2 – Liposomes containing CBD-HSA A liposomal formulation containing CBD-HSA as the aqueous phase was developed (liposomal-CBD-HSA). For this formulation, the aqueous phase was prepared by weighing CBD into a vial and adding a 5% HSA solution to disperse the CBD at 5%. The dispersion was stirred at 4°C for at least two days until a homogenous suspension was obtained, with no particles observed on the vial walls. This dispersion was added to warmed HSPC powder and stirred at 65°C for 15 minutes. An analytical method was developed that could distinguish between liposome-bound and HSA-bound CBD. Although the volume of liposomes in suspension was lower than the excess liposome volume, the majority of the CBD was found to be liposome-bound. Table 6 below shows the liposomal CBD (%) in various liposomal-CBD-HSA formulations. Therefore, the affinity of CBD for liposomes and HSA was tested as detailed below.

[0129] Two formulations were prepared: A 1:1 volume ratio CBD-HSA preparation containing empty MLVs (40 mg / ml HSPC in 5% dextrose) F1 formulation with HSA solution in a 1:1 volume ratio (CBD is present only within membrane lipids).

[0130] The mixture was placed in a 37°C incubator with shaking at 50 rpm for 2 hours. The results are shown in Table 7. The F1 formulation incubated with HSA showed only 1% of the total CBD transferred from the liposomes to the HSA. In the case of CBD-HSA incubated with empty MLVs, 35% of the CBD was transferred to the liposomes, indicating a much higher affinity of CBD for lipids. These results were consistent with those obtained for the liposomal-CBD-HSA formulation (Table 6), indicating that the CBD was primarily liposomal.

[0131] The addition of HSA to the liposomes made it possible to reach a high D / L molar ratio within the liposomes. [Table 6] [Table 7]

[0132] CBD release from various formulations was tested in 50% adult bovine serum. 50 mg of formulation was weighed into an HPLC vial and 950 μl of a 50:50 serum:5% dextrose solution was added. The mixture was vortexed and placed in a shaking incubator at 37°C and 50 rpm for 2 hours. After diluting 25-fold with methanol, the mixture was tested for total CBD content. The remainder of the mixture was transferred to an Eppendorf and centrifuged (30 minutes, 14,000 rpm, 4°C), and the upper phase was diluted 10-fold with methanol and analyzed by HPLC. Several liposome-CBD-HSA formulations with different lipid and CBD content were tested for in vitro release and are listed in Table 8. In vitro release studies showed that increasing the CBD concentration in the formulation resulted in slower release. [Table 8]

[0133] Example 3 - In vivo PK study of CBD formulations Two in vivo studies were conducted to examine the plasma profile and residual CBD in muscle following IM injection of various CBD formulations: the first study tested four formulations for up to three days, and the second study tested four formulations for up to three weeks. A detailed description of each study can be found below.

[0134] First test Formulation preparation and characterization Details regarding the materials used in the preparation of the formulations are summarized in Table 9. All formulations were prepared under sterile conditions in a biological hood using autoclave equipment to ensure sterile preparation. a. Free CBD in PG: CBD was prepared at a concentration of 50 mg per 1 g of PG and vortexed until a clear solution was obtained. b.F1: Liposomal formulation of CBD in which CBD is solubilized exclusively within the membrane phospholipids of the liposomes: CBD and HSPC were solubilized together in ethanol at 65°C (lipid phase) until a clear solution was obtained. The lipid phase was added to a 5% dextrose solution at 65°C while stirring and left stirring for 30 minutes (at 65°C). The resulting multilamellar liposome (MLV) formulation was then washed with a 5% dextrose solution until the osmolality of the preparation was isotonic. cF-HPCD-PEG: A liposomal formulation of CBD in which CBD was solubilized within the liposomal membrane phospholipids and also dispersed in the intraliposomal aqueous phase using solubilizers HPCD and PEG 300. CBD and HSPC were solubilized in ethanol at 65°C until a clear solution was obtained (lipid phase). The aqueous phase was prepared by adding an ethanolic solution of CBD to a solution containing 27% (w / w) HPCD and 10% (w / w) PEG 300 at 65°C. The aqueous phase was nearly clear. The lipid phase was added to the aqueous phase at 65°C with stirring and left stirring for 30 minutes (at 65°C). The resulting formulation was then washed with 5% dextrose solution until the osmolality of the preparation was isotonic. d. Liposome-CBD-HSA: CBD was first dispersed in a 5% HSA solution, and this dispersion was added to heated HSPC and stirred at 65°C for 15 minutes. [Table 9]

[0135] Formulation characterization CBD assay Total and free CBD content was determined by an HPLC method. The chromatographic conditions used were based on the USP method for dronabinol and are summarized in Table 10.

[0136] Sample preparation for analysis varies for each formulation and is described below. Total CBD concentrations were similar for all formulations. Specifically, 10–20 mg of formulation was weighed into a 10 ml volumetric flask. Methanol was added up to the line. After vortexing, the sample was centrifuged and the upper phase was analyzed. For F1 and liposomal CBD-HSA, the free CBD content was tested: 200 μl of the formulation was placed in an Eppendorf and centrifuged at 14,000 rpm for 30 min at 40°C. The clear upper phase was then diluted 10-fold with methanol, followed by vortexing and centrifugation (14,000 rpm, 10 min, 40°C). The upper phase was analyzed by HPLC. In liposomal CBD-HSA formulations, albumin-bound CBD was quantified: This method was developed to allow for the separation of liposomal and albumin-bound CBD. For this purpose, an isotonic medium was used, which allows for density-based separation. The medium was prepared using 1.5 g of dextrose (Sigma, D9434, batch 119K0042) and 10 g of Ficoll 400 (Sigma, F-4375, lot 29C-0095) solubilized in a 50 ml volumetric flask. The osmolality of the medium was 290 mOsm / kg. 50 mg of the formulation was placed in an Eppendorf tube and 1.5 ml of medium was added. The tube was vortexed and then centrifuged (4°C, 30 min, 14,000 rpm). The upper phase of the tube was cut off, and any remaining liquid at the bottom containing the precipitate was removed. The precipitate was transferred to another Eppendorf tube, and 1 ml of methanol was added. After vortexing and centrifugation, the upper phase was diluted 10-fold with methanol. Cremophore: IV formulations in ethanol were tested for total content as described above for total CBD concentration. Appearance after dilution with saline was examined to track formulation behavior for injection and ensure the absence of precipitation. The formulation was diluted 10-fold with saline, and appearance was recorded after 1 hour (the allowable time for the formulation to be injected after preparation). [Table 10]

[0137] Release assay CBD release from various formulations was tested in 50% adult bovine serum. 50 mg of formulation was weighed into an HPLC vial and 950 μl of a 50:50 serum:5% dextrose solution was added. The mixture was vortexed and placed in a shaking incubator at 37°C and 50 rpm for 2 hours. After a 25-fold dilution with methanol, the mixture was tested for total CBD content. The remainder of the mixture was transferred to an Eppendorf, centrifuged (30 minutes, 14,000 rpm, 4°C), and the upper phase was diluted 10-fold with methanol and analyzed by HPLC.

[0138] Particle size measurement Particle size was determined using a Coulter LS 130.

[0139] Osmolality Osmolality was measured by the freezing point method using an Advanced instrument, Model 3320 osmometer.

[0140] lipid concentration Lipid concentrations were determined by HPLC / ELSD method.

[0141] Microscopic observation The formulations were observed under an optical microscope (Zeiss SN 221209). Few fields were observed and representative photographs were taken for each formulation.

[0142] Injectability A 1 ml syringe was filled with 0.3 to 0.5 ml of the formulation. A 25 G needle was attached to the syringe, and the amount of unbound formulation injected was determined. This process was repeated three times.

[0143] sterility One vial from each formulation was tested by Hadassah's Microbiology Unit. Aliquots from each vial were plated onto blood agar and chocolate agar and placed in room temperature and 37°C incubators.

[0144] In vivo testing protocol A total of 36 12-week-old female BALB / C mice were injected IM with a single dose of each formulation (9 mice / group) with an injection volume of approximately 50 μl for all formulations (approximately 2.5 ml / kg per injection site). The low-CBD F1 was injected at two injection sites with a total injection volume of approximately 100 μl. To ensure the correct dose was administered, syringes were weighed before and after injection, and the actual injected volume was recorded.

[0145] At the time points detailed below, three mice from each group were euthanized using CO2, and peripheral blood was immediately collected from the retro-orbital sinus into labeled 0.5 ml K3EDTA blood collection tubes (Mini Collect, Greiner-bio-one, Austria). After centrifugation at 2000 g for 10 minutes, plasma was extracted, collected in labeled tubes, and frozen at -20°C immediately after collection. Samples were then stored at -80°C until analysis.

[0146] After blood collection, quadriceps muscle was collected into a pre-weighed 15 ml tube.

[0147] Blood and muscle sampling time points were 2 hours, 24 hours and 72 hours post-injection.

[0148] Bioanalytical Assays Assay for CBD in plasma CBD was extracted from plasma samples spiked with cannabigerol (CBG, 1 mg / ml in methanol, Sigma, catalog C-141-1), used as an internal standard (IS), followed by a 5-fold dilution of the plasma with acetonitrile. After vigorous vortexing, the samples were centrifuged, and the upper phase was analyzed. The final IS concentration in the samples was 100 ng / ml.

[0149] Plasma extracts were analyzed by LC-MS using a Sciex (Framingham, MA, USA) Triple Quad™ 5500 mass spectrometer coupled with a Shimadzu (Kyoto, Japan) UHPLC system. Concentrations were calculated based on a calibration curve of CBD in plasma ranging from 1 to 1,000 ng / ml with an IS of 100 ng / ml.

[0150] CBD spike solutions for generating plasma calibration curves were prepared in acetonitrile, while CBG was prepared in methanol.

[0151] Assay for CBD in the muscle (injection site) The muscle was surgically removed and its weight was recorded. 2 ml of 15% collagenase solution (Sigma, C7657) was then added, and the tube was incubated overnight at 37°C. After incubation, 8 ml of acetonitrile was added, vortexed, and centrifuged. The upper phase was analyzed by HPLC. The chromatographic conditions are listed in Table 10.

[0152] The concentration of CBD in each muscle was calculated based on a standard curve of CBD in acetonitrile.

[0153] After spiking the CBD formulations into muscle, the recovery of CBD from muscle was determined for each formulation compared to spiking into acetonitrile.

[0154] result The formulations were characterized for total CBD and HSPC content and their molar ratios, particle size, and microscopic appearance. The CBD concentration in all formulations ranged from 50 to 60 mg / g, except for F1, which had a CBD content of 30 mg / g. The results are summarized in Table 11.

[0155] The prepared formulations were injected IM into mice, and the syringe weights were recorded before and after injection to accurately calculate the volume of the injected dose.

[0156] Table 12 summarizes the plasma and muscle concentrations obtained. This study clearly demonstrated that CBD is retained at the injection site (muscle) for over 72 hours. During this time, the intramuscular depot releases CBD into the plasma at a rate that depends on the formulation used. [Table 11] [Table 12-1] [Table 12-2]

[0157] Second test Formulation preparation and characterization material and method material Details regarding the materials used in the preparation of the formulations are summarized in Table 13. [Table 13]

[0158] method Preparation of formulations All formulations were prepared under sterile conditions in a biological hood using an autoclave to ensure sterile preparation. Three formulation types were used in intramuscular (IM) pharmacokinetic (PK) studies.

[0159] CTRL-PG: Control formulation of CBD solubilized in propylene glycol (PG). CBD was prepared at a concentration of 50 mg / g PG and vortexed until a clear solution was obtained.

[0160] F1: A liposomal formulation of CBD in which CBD is solubilized exclusively within the membrane phospholipids of the liposomes. CBD and HSPC were solubilized together in ethanol at 65°C (lipid phase) until a clear solution was obtained. The lipid phase was added to a 5% dextrose solution at 65°C while stirring and left stirring for 30 minutes (at 65°C). The resulting multilamellar liposome (MLV) formulation was then washed with a 5% dextrose solution until the osmolality of the preparation was isotonic.

[0161] Liposome-CBD-HSA: A liposomal formulation of CBD in which CBD was first dispersed in HSA, followed by passive encapsulation of the CBD-HSA in liposomes. CBD was first dispersed in a 5% HSA solution. This dispersion was added to heated HSPC and stirred at 65°C for 15 minutes.

[0162] IV Formulation: The formulation used for IV administration was a 10 mg / g CBD formulation solubilized in a 50:50 Cremophor:ethanol solution. This formulation was diluted 10-fold with saline prior to injection to achieve a diluted concentration of 1 mg / ml. The diluted formulation was used within 1 hour of preparation.

[0163] In the following, formulations are always defined by the amount of CBD per ml of solution containing 50 mg of protein, so for example, "liposomal CBD / HSA 50 mg / ml" refers to a liposomal formulation containing 50 mg of CBD and 50 mg of HSA.

[0164] Formulation characterization - as described for study 1 In vivo testing protocol IV administration A total of 18 12-week-old female BALB / C mice were injected IV with a single dose of a 10 mg / kg CBD formulation in cremophor:ethanol.

[0165] At the time points detailed below, three mice were euthanized using CO2, and peripheral blood was immediately collected from the retro-orbital sinus into labeled 0.5 ml K3EDTA blood collection tubes (Mini Collect, Greiner-bio-one, Austria). After centrifugation at 2000 x g for 10 minutes, plasma was extracted, collected in labeled tubes, and frozen at -20°C immediately after collection. Samples were then stored at -80°C until analysis.

[0166] Blood sampling time points: 2 minutes, 1 hour, 4 hours, 8 hours, 24 hours and 48 hours.

[0167] IM administration A total of 36 12-week-old female BALB / C mice were injected IM with a single dose of the IM formulation; 9 mice per formulation. Syringes were weighed before and after injection to allow for accurate recording of the exact volume and therefore the dose each mouse received. Details regarding the injection volume and estimated dose for each group are summarized in Table 14.

[0168] Two mice were not injected with the formulation and served as controls for changes in body weight (BW) over time.

[0169] At the time points detailed below, three mice from each group were euthanized using CO2, and peripheral blood was immediately collected from the retro-orbital sinus into labeled 0.5 ml K3EDTA blood collection tubes (Mini Collect, Greiner-bio-one, Austria). After centrifugation at 2000 x g for 10 minutes, plasma was extracted, collected in labeled tubes, and frozen at -20°C immediately after collection. Samples were then stored at -80°C until analysis.

[0170] After blood collection, quadriceps muscle was collected into a pre-weighed 15 ml tube.

[0171] Blood sampling times: 72 hours, 1 week and 3 weeks after injection.

[0172] Body weights of mice were recorded before treatment and before euthanasia. Mice sacrificed at 3 weeks were also weighed 2 weeks after treatment. [Table 14]

[0173] result This example defined the pharmacokinetic profiles of three particle-based CBD formulations versus a propylene glycol (PG) solution of CBD after IM or IV administration (based on the literature, a 12 mg / kg administration dose that was effective in several animal models).

[0174] formulation Liposomal CBD-HSA formulations were prepared by hydrating HSPCs with the CBD-HSA dispersion at 65 °C. The resulting liposomes were spherical and uniform, as observed by microscopic imaging (Figures 1B-1C).

[0175] The mean diameter of the liposomes was 8.1 μm for the 50 mg / ml formulation and 6.7 μm for the 100 mg / ml formulation.

[0176] The CBD concentrations in the formulations were as predicted (based on calculations). The 100 mg / ml formulation exhibited a high molar drug-to-lipid (D / L) ratio of 3.05. The CBD in these formulations appeared to be distributed between the liposomal CBD (both the membrane and the internal aqueous phase) and the extraliposomal albumin (CBD-HSA).

[0177] Formulation characterization is shown in Table 15 and particle size is summarized in Table 16. [Table 15]

[0178] It should be noted that all formulations were also found to be sterile, i.e., no microbial growth was detected in any of the formulations tested.

[0179] A CBD formulation in Cremophor:ethanol for IV administration was also characterized. The CBD concentration in the concentrate was 11.7 mg / ml. After dilution with saline, the solution remained clear for at least 1 hour. [Table 16]

[0180] The distribution of CBD between the liposomes (both the membrane and the intraliposomal core) and the CBD-HSA within these formulations was determined and is summarized in Table 17. [Table 17]

[0181] Table 17 shows that although the liposome volume was lower than the extraliposomal volume, the majority of the CBD was liposomal (86-91%), with a relatively small fraction bound to extraliposomal HSA (9-14%). Therefore, it was believed that the majority of the CBD bound to HSA was transferred to the liposome-forming lipids. This observation is consistent with the partitioning of CBD between HSA and lipids described in Table 6 of Example 2.

[0182] After 2 hours of incubation in the presence of 50% serum, the free CBD concentrations were 33 and 53 mg / ml for the 50 mg / ml and 100 mg / ml preparations, accounting for 70 and 57% of the release (100 - % bound = % released). This release rate was close to that obtained for F1 liposomes. F1 was a liposomal formulation in which CBD was solubilized by membrane lipids and presumably located exclusively in the liposomal membrane. The CBD concentration in this formulation was 21.3 mg / ml, lower than the other formulations due to the washing steps required to remove ethanol from the formulation. Microscopic appearance of F1 showed small, round particles that were relatively far apart (Figure 1A). The average diameter was 9.2 μm (Table 16). Release in serum was 49%, similar to that of the liposome-HSA formulation (Table 15).

[0183] The reference IM formulation used was a solution of CBD in propylene glycol. Additional references included a group of mice administered IV at a 12 mg / g dose (Cremophore:ethanol formulation diluted with saline prior to injection).

[0184] PK Profile The PK profile obtained after IV administration of a 12 mg / kg CBD dose is summarized in Table 18. [Table 18]

[0185] Table 18 shows that CBD concentrations declined rapidly from 8,856 ng / ml 5 minutes after administration to 9.5 ng / ml 8 hours later. At later time points (24 and 48 hours), CBD concentrations were below the limit of detection (BLOD).

[0186] The plasma concentrations obtained after IM administration are summarized in Tables 19A-19B and FIG. [Table 19] [Table 20]

[0187] Tables 19A-19B and Figure 2A show that plasma concentrations after administration of all IM formulations, and for up to 3 weeks after administration, were within the range of the IV profile obtained from 1 to 8 hours post-dose. This indicates sustained delivery of the formulations via IM injection. Figure 2B also includes PK data from the first study (referred to in the figure as "Previous Study") for the same formulation tested in the second study, but includes PK data from the T=2 and 24 hour time points, providing a complete overview of the CBD plasma profile after IV or IM administration.

[0188] Interestingly, after IV administration of an IV therapeutic dose of CBD, high plasma CBD levels are obtained immediately after administration, but plasma levels decline within hours. However, with IM administration of a high CBD dose, initial plasma CBD levels are similar to those of the IV dose (e.g., after 2 hours), and levels then remain essentially high for at least a 3-week period for all IM formulations.

[0189] Furthermore, the decline in plasma levels was very slow, with less than a single order of magnitude observed over the three-week period for all formulations. This gradual decline, compared to the rapid decline of the IV formulation, demonstrates that the terminal slope of the IM profile is absorption-dependent rather than elimination-dependent, indicating that the formulations continuously release CBD from muscle over this extended period.

[0190] Table 20 summarizes the residual CBD content in muscle compared to the initial CBD administered to each mouse, and Figures 3A-3B show the average CBD released from muscle per group compared to the initial CBD administered. [Table 21-1] [Table 21-2]

[0191] At 1 week, differences were observed between groups, with the high-dose group showing relatively greater release. These differences were not observed at 3 weeks. The amount of CBD released from muscle was normalized for the number of days since dosing to estimate the amount of CBD released per day and, therefore, the dose of CBD reaching the circulation per day (assuming 20 g per mouse). When plasma levels were normalized to this estimated daily dose, the mean normalized values ​​were similar between groups, ranging from 1.7 to 4.5 ng / ml / mg / kg. These values ​​were similar to the IV values ​​obtained 4 to 8 hours after dosing (Table 18), demonstrating that plasma concentrations depend on CBD released from muscle and can therefore be controlled by the formulation. From the %CBD release data, the intramuscular CBD reservoir can be calculated for each formulation. Free CBD and the liposomal formulation released the majority of CBD at 3 weeks (liposomal CBD-HSA 100 mg / ml, 3 weeks, not including unexplained lower values). [Table 22]

[0192] Pharmacokinetic analysis was performed for both IV and IM administration. Table 21 shows the resulting IV PK parameters. The half-life of CBD was rapid at 1.68 hours, resulting in a dose-normalized exposure of 417 h*ng / ml / mg / kg. Pharmacokinetic analysis after IM administration was performed on the three formulations also injected during the first study, allowing for five time points for each formulation (2, 24, and 72 hours from the previous study and 1 and 3 weeks from the current study). PK analysis of the combined data set is found in Table 22. F1 and free CBD in PG yielded the highest AUC. This is consistent with the finding that for these two groups, the majority of intramuscular CBD was released by the 3-week period (70 and 84%, respectively; Figure 3B), yielding AUCs normalized to dose values ​​similar to those obtained for IV injection (388 and 293 h*ng / ml / mg / kg, respectively). The normalized AUC value for the liposomal-CBD-HSA 50 mg / ml formulation was even lower (167 h*ng / ml / mg / kg), corresponding to the relatively low % release from muscle obtained for this formulation (68%, respectively; Figure 3B). [Table 23]

[0193] Consideration The PK profiles after IV administration of a 12 mg / kg dose were compared with four formulations of long-acting CBD administered via the IM route. The plasma profiles of the IM injection formulations showed plasma levels within the range of the IV plasma profile for at least three weeks after injection. The liposomal-CBD-HSA and F1 formulations contained ≥30% of the injected dose, compared with 14% remaining in the muscle of the free CBD group. The fact that the CBD plasma levels of the IM formulations maintained plasma concentrations similar to those observed for the IV effective dose suggests that these formulations may enable sustained CBD effects in vivo. Differences in PK profiles allow for selective design of preferred formulations for specific desired release profiles.

[0194] Example 4 - Preparation of DMPC / DPPC-CBD liposomes A liposomal formulation of CBD in a 45:55 molar ratio of DMPC:DPPC was prepared.

[0195] material: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC): Lipoid, catalog number 556200 (lot: 556200-2190329-01) Dipalmitoylphosphatidylcholine (DPPC): Lipoid Catalog No. 556300 (Lot: 556300-2170149-01) CBD:THC Pharma

[0196] result: 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. [Table 24]

[0197] Specifically, a histidine (0.155% w / v)-mannitol (4% w / v) buffer (HMB) at pH 6.5 was used as the aqueous phase of this preparation. The lipid and aqueous phases were preheated to 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 after four cycles of washing with the histidine-mannitol buffer by centrifugation at 4°C.

[0198] Figure 4 shows the appearance of the formulation under a microscope (200x magnification). The average liposome size was 5.55 μm and the total CBD concentration was 25.3 mg / ml.

Claims

1. 1. A sustained release formulation for administration by injection or infusion comprising a physiologically acceptable carrier carrying liposomes, the liposomes having a lipid membrane and an intraliposomal aqueous core, and containing an entrapped cannabinoid, hydroxypropyl-β-cyclodextrin, and at least one dispersing agent, the at least one dispersing agent being selected from human serum albumin, polyethylene glycol (PEG), and propylene glycol (PG); A sustained-release formulation wherein the molar ratio of the cannabinoid in the lipid membrane to the lipid forming the liposome is in the range of 1 to 10.

2. 10. The sustained release formulation of claim 1, wherein the liposomes contain entrapped cannabidiol CBD or a functional homolog thereof.

3. 3. The sustained release formulation of claim 1 or 2, wherein at least a portion of the cannabinoid is entrapped within the intraliposomal aqueous core.

4. 4. The sustained release formulation of claim 1, wherein the dispersing agent is entrapped within the intraliposomal aqueous core.

5. 5. The sustained release formulation of claim 1, wherein the dispersing agent is selected from polyethylene glycol (PEG) 300 and propylene glycol (PG).

6. 6. The sustained-release formulation of claim 1, wherein the hydroxypropyl-β-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin (HPβCD).

7. 7. The sustained release formulation of claim 1, wherein the formulation is for intramuscular (IM) injection, intravenous (IV) injection, or subcutaneous administration.

8. 1. A method of treatment comprising administering to a non-human subject in need of treatment a sustained release formulation comprising liposomes having a lipid membrane and an intraliposomal aqueous core, said liposomes comprising an entrapped cannabinoid, hydroxypropyl-β-cyclodextrin, and at least one dispersing agent, said at least one dispersing agent being selected from human serum albumin, polyethylene glycol (PEG), and propylene glycol (PG), said administration being by injection or infusion; A method wherein the molar ratio of the cannabinoid in the lipid membrane to the lipids forming the liposomes is in the range of 1 to 10.

9. 9. The method of claim 8, wherein the liposome comprises entrapped cannabidiol (CBD) or a functional homolog thereof.

10. 10. The method of claim 8 or 9, wherein at least a portion of the cannabinoid is entrapped within the intraliposomal aqueous core.

11. 11. The method of claim 8, wherein the dispersing agent other than CD is entrapped within the intraliposomal aqueous core.

12. 12. The method of any one of claims 8 to 11, wherein the administration comprises intramuscular injection, intravenous injection, or subcutaneous administration.

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